Circular RNAs have shown important regulatory roles in cardiovascular diseases, containing atherosclerosis (AS). We intended to explore the role of circ_0004104 in AS using oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cells and its associated mechanism.Real-time quantitative polymerase chain reaction and Western blot assay were conducted to analyze RNA levels and protein levels, respectively. Cell viability, apoptosis, angiogenic ability and inflammatory response were assessed by 3--2,5-Diphenyltetrazolium Bromide (MTT) assay, flow cytometry, capillary-like network formation assay and enzyme-linked immunosorbent assay, respectively. Cell oxidative stress was assessed using commercial kits. Dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA-pull down assay were performed to verify the intermolecular interaction.ox-LDL exposure up-regulated the level of circ_0004104 in HUVECs. ox-LDL exposure suppressed cell viability and angiogenic ability whereas promoted the apoptosis, inflammation and oxidative stress of HUVECs partly through up-regulating circ_0004104. MicroRNA-328-3p (miR-328-3p) was confirmed as a target of circ_0004104. MiR-328-3p interference largely reversed circ_0004104 silencing-mediated effects in HUVECs upon ox-LDL exposure. MiR-328-3p interacted with the 3′ untranslated region of tripartite motif 14, and circ_0004104 positively regulated TRIM14 expression by sponging miR-328-3p. TRIM14 overexpression largely overturned miR-328-3p accumulation-induced influences in HUVECs upon ox-LDL exposure.Circ_0004104 knockdown attenuated ox-LDL-induced dysfunction in HUVECs via miR-328-3p-mediated regulation of TRIM14.The online version contains supplementary material available at 10.1186/s12872-021-02012-7. Many types of cells are involved in the pathogenetic process of atherosclerosis (AS), containing vascular endothelial cells and smooth muscle cells . EndotheCircular RNAs (circRNAs) are endogenous RNAs without 5′ or 3′ polarity , 5. CircMicroRNAs (miRNAs) reversely modulate gene expression by interacting with the 3′ untranslated region (3′UTR) of messenger RNAs (mRNAs), causing translational repression or degradation of mRNAs , 12. DysTripartite motif 14 (TRIM14) is one of the members of TRIM family . TRIM14 We initially explored the role of circ_0004104 in AS cell model. Subsequently, the working mechanism of circ_0004104 was explored through bioinformatic analysis and rescue experiments.2.Human umbilical vein endothelial cells (HUVECs) acquired from Chinese Academy of Medical Sciences, Shanghai institute Cell Bank  were cultivated in Dulbecco’s modified Eagle’s medium  plus 10% fetal bovine serum  and 1% antibiotics (Gibco) under 37℃ humidified atmosphere with 5% COHUVECs were exposed to 100 μg/mL ox-LDL  for 24 h to establish AS cell model as previously reported , 14.−ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference, while the fold change of miR-328-3p was calculated using the 2−ΔΔCt method with U6 as reference.RNA samples were isolated using Trizol reagent . Complementary DNA (cDNA) was synthesized using the miScript Reverse Transcription kit  and RevertAid First Strand cDNA Synthesis Kit . cDNA was amplified via the SYBR™ Green PCR Master Mix (Invitrogen). The primers purchased from Sangon Biotech  were shown in Table RNA samples (2 μg) were incubated with 3 U/μg RNase R , and RNA levels were determined by RT-qPCR.Transcription inhibitor Actinomycin D  was added to the culture medium, and RNA levels were examined by RT-qPCR at specific time points.Ectopic expression plasmid of circ_0004104 (circ_0004104), pLCDH-cir empty vector (vector), small interfering RNA against circ_0004104 (si-circ_0004104), negative control of siRNA (si-NC), TRIM14 overexpression plasmid (TRIM14) and empty vector (pcDNA) were purchased from Sangon Biotech, and mimics of miR-328-3p (miR-328-3p), miR-NC, inhibitor of miR-328-3p (anti-miR-328-3p) and anti-miR-NC were acquired from Genepharma . All oligonucleotides or plasmids were transfected into HUVECs with Lipofectamine 3000 reagent (Invitrogen).At specific time points, HUVECs were incubated with MTT reagent (Sigma) for 4 h. Afterwards, a total of 200 μL dimethyl sulfoxide  was added to dissolve the formazan products after discarding cell supernatant. The absorbance (490 nm) was determined by the microplate reader .HUVECs were simultaneously stained with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) of the Cell Apoptosis Detection Kit (Qiagen). The apoptotic percentage of HUVECs was evaluated by the flow cytometer .4 cells/well). After culturing for 48 h, the average number of branches of each node was analyzed.HUVECs were plated onto Matrigel (BD Biosciences)-pre-coated 96-well cell culture plates . Protein samples (35 μg) were loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane . After sealing with 5% bovine serum albumin , immunoblot assay was applied through incubating the membrane with the diluted primary antibodies and the horse radish peroxidase (HRP) conjugated secondary antibody (Abcam). Immuno-reactive signals were determined by the enhanced chemiluminescent (ECL) chromogenic substrate (Beyotime). The primary antibodies contained anti-Cleaved-caspase 3 , anti-vascular endothelial growth factor A , anti-TRIM14  and anti-GAPDH .The culture supernatant of HUVECs was collected to assess the release of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) using commercial Human TNF-α/IL-1β Quantikine ELISA Kit .Cell oxidative stress was analyzed through measuring the production of superoxide dismutase (SOD) and malondialdehyde (MDA) using their corresponding commercial kits .http://starbase.sysu.edu.cn) was utilized to predict circ_0004104-miRNAs interactions and miR-328-3p-mRNAs interactions.StarBase database  to generate circ_0004104 wt and TRIM14 3′UTR wt. Meanwhile, mutated counterparts were constructed to generate circ_0004104 mut and TRIM14 3′UTR mut. HUVECs were seeded onto 12-well plates and co-transfected with luciferase plasmids and miR-NC or miR-328-3p. After 48-h transfection, the relative luciferase intensities were determined using the Dual-Luciferase Reporter Assay Kit (Promega).RIP experiment was employed to confirm the binding relation between circ_0004104 and miR-328-3p with Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). Cell extracts were prepared using RIP lysis buffer, and anti-Argonaute2  or anti-Immunoglobulin G -pre-coated magnetic beads were incubated with cell lysates. The levels of enriched RNAs were measured by RT-qPCR.Cell lysates (2 μg) were incubated with 100 pmol Bio-miR-NC, Bio-miR-328-3p-mut or Bio-miR-328-3p-wt. The reaction mixture was then incubated with 100 μL agarose beads (Millipore) for 1 h. The retrieved RNAs were measured by RT-qPCR.t-test (two groups) or one-way analysis of variance (ANOVA) (more than two groups). Differences were identified as statistically significant with the P value of less than 0.05.All experiments were repeated for three times. Statistical analysis was carried out using GraphPad Prism 7.0 software . Data were represented as mean ± standard deviation (SD). The differences were analyzed by Student’s Among several AS progression-associated circRNAs, including circ_0004104 , circ_00Transfection with circ_0004104 ectopic plasmid increased the level of circ_0004104 by five times in HUVECs Fig. a. ox-LDLRT-qPCR confirmed the high silencing efficiency of si-circ_0004104 in HUVECs Fig. a. As shoThe putative binding sites between miR-328-3p and circ_0004104 predicted by StarBase database were shown in Fig. Transfection with anti-miR-328-3p resulted in about 60% reduction in miR-328-3p level in HUVECs Fig. a. MiR-32The potential binding sequence between miR-328-3p and TRIM14 predicted by StarBase was shown in Fig. Western blot assay showed that the transfection efficiency of TRIM14 plasmid was high in HUVECs Fig. a. MiR-32AS results in severe cardio/cerebral-vascular disorders, containing coronary heart disease and stroke . The phe“MiRNA sponge” mechanism is an important way by which circRNAs function in human diseases , 32. ForMiRNAs could regulate gene expression through repressing translation or degrading target mRNAs . StarBasIn conclusion, our study demonstrated that circ_0004104 contributed to ox-LDL-induced injury of HUVECs partly through targeting miR-328-3p/TRIM14 axis. Blockage of circ_0004104 might be a potential strategy to attenuate the abnormal phenotypes of vascular endothelial cells in AS.Additional file 1: Figure 1. The expression of AS progression-associated circRNAs in HUVECs upon ox-LDL exposure. RT-qPCR was applied to analyze the levels of circ_0004104, circ_0001879, circ_0001445, circ_0001599, circ_0010283 and circ_0007478 in HUVECs induced by ox-LDL. This experiment was performed for three times with three technical repetitions. *P < 0.05, **P < 0.01. Student’s t-test was utilized to analyze the differences.


Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP. Human occupational exposures to 4-aminobiphenyl (ABP) led to excess incidence of urinary bladder cancer with sufficient evidence for listing as a Group 1 carcinogen . ABP alsN-hydroxylated by cytochrome P450 to N-hydroxy-ABP (N-OH-ABP) followed by O-acetylation catalyzed by arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) to N-acetoxy-ABP which is highly unstable resulting in DNA adducts. DNA adducts not repaired can result in mutations leading to cancer  are present in the NAT1 coding exon. As previously reviewed , a mock empty vector, or NAT1 possessing a single SNP containing vector pCR3.1 and 80 ng pCMV SPORT-βgal (Invitrogen) plasmid. Transfected cells were harvested after 48 h and then removed, washed, and pelleted by centrifugation. Cell pellets were disrupted in lysis buffer  by sonication on ice. Cell debris was precipitated at 15,000 X g for 15 min at 4°C. No NAT1 activity was detectable in mock-transfected cells. To account for possible differences in transfection efficiency, COS-1 cell lysates were assayed for β-galactosidase activity with O-nitrophenyl β-D-galactopyranoside (The human NAT1 coding exon was amplified by polymerase chain reaction (PCR) using plasmid pESP-3 containing escribed . COS-1 cescribed . As descescribed , construranoside .ABP N-acetyltransferase reactions were conducted as previously described . BrieflyN-OH-ABP O-acetyltransferase assays were conducted as previously described . BrieflyNAT1*4 and SNP variants were tested for significance using one-way ANOVA followed the Dunnett multiple comparison tests.ABP N-acetyltransferase and N-OH-ABP O-acetyltransferases activities carried out at various substrate concentrations were subjected to Michaelis-Menten kinetic analysis and the apparent Km and Vmax were calculated using the Michaelis-Menten program in Graphpad Prism . Differences in apparent Km and Vmax between reference allele The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase catalyzed by recombinant human NAT1 expressed in COS-1 cells. As shown in p > 0.05) affected apparent Km towards ABP or N-OH-ABP.As shown in https://www.ncbi.nlm.nih.gov/snp/rs4986782?vertical_tab=true#frequency_tab). 560G > A (rs4986782) (R187Q) is the most frequent SNP in the NAT1 coding exon with a global frequency of 1.75% but a 13-fold higher frequency (23.8%) has been reported in Lebanese resulting in nearly 50% of a Lebanese population heterozygous for the allele  (E261K), and 787A > G (rs72554611) (I263V) caused slight but significant reduction in ABP N-acetyltransferase and of N-OH-ABP O-acetyltransferase activities that likely is relatively unimportant in risk assessment. Previous studies following recombinant expression of NAT1 in yeast reported that 781G > A (rs72554610) (E261K) caused slight reductions in N-acetylation of the arylamine carcinogens 2-aminofluorene  (R187Q) was identified as a reduced N-acetylation phenotype based upon its effects on the N-acetylation of subjects . In the reviewed , the R18reviewed  and COS-reviewed . R187Q adecrease in the affinity of human recombinant NAT1 for p-aminobenzoic acid  (R187Q) exhibit higher maximum concentrations and overall area under the plasma level versus time curve drug levels irrespective of dosing regimen  SNP caused a 6–10-fold oic acid . Followi regimen , consistn allele .increase (p < 0.05) in affinity for both ABP and N-OH-ABP. None of the SNPs in the NAT1 coding exon affect V93, K100, I106, F125, L209, S215, V216 or F217 residues in the active site that may be important for ABP access  (R187Q) with higher incidence of lung cancer nearly reached statistical significance while an inverse association with the NAT1*10 allele achieved statistical significance (560G > A (rs4986782) (R187Q) also has been associated with higher prevalence of lung cancer in cigarette smokers . In a suificance . Thus thNAT1*14 alleles are reduced function alleles, but in contrast to p-aminobenzoic acid, the current study showed that the 560G > A (rs4986782) (R187Q) caused a 4 to 5-fold increase (p < 0.05) in affinity for both ABP and N-OH-ABP. The increase in affinity for ABP and N-OH-ABP shows that in altering the site for substrate binding, 560G > A (rs4986782) (R187Q) can cause opposite effects on substrate affinity. The findings are consistent with an increased risk for urinary bladder cancer and perhaps also lung cancer in individuals possessing NAT1*14 alleles following lower levels of exposure to ABP as would be expected in active and particularly passive exposure following use of cigarette and other tobacco products.In conclusion, the current study confirms and expands upon previous studies that have characterized the effects of SNPs in the NAT1 coding exon on N-acetylation of ABP and O-acetylation of N-OH-ABP. Our findings confirm previous studies reporting that


Taken as a whole, this study emphasizes that optimized modifications in the C-terminal part of 26RFa are mandatory to design selective and potent peptide agonists for human QRFPR.26RFa is a neuropeptide that activates the rhodopsin-like G protein-coupled receptor QRFPR/GPR103. This peptidergic system is involved in the regulation of a wide array of physiological processes including feeding behavior and glucose homeostasis. Herein, the pharmacological profile of a homogenous library of QRFPR-targeting peptide derivatives was investigated in vitro on human QRFPR-transfected cells with the aim to provide possible insights into the structural determinants of the Phe residues to govern receptor activation. Our work advocates to include in next generations of 26RFa Finally, methylene shortening of the phenylalanine side chain led to the inactive phenyl-glycine (Phg)-containing [Phg22]26RFa(20–26) , [Phg24]26RFa(20–26)  and [Phg26]26RFa(20–26)  diastereoisomers, probably due to the lack of interactions with hydrophobic residues in the core of the hQRFPR binding pocket.Replacement of all three phenylalanine residues of 26RFa peptide . Accordigy model . In ordeHO cells , as prevoncerned . Similarreported . Howeverin vitro A–C. The (20–26) does not encompass any secondary amide bonds within its backbone. Thus, nine analogs with N-methyl-phenylalanine (NMePhe), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) or N-benzyl-glycine (NPhe) were synthesized. These modifications were aimed at analyzing the significance of H-bond contributions of the amide NHs. Alkylation of these NHs without loss of agonistic activity would facilitate further analog design. N-methylation of Phe22  and Phe26  nullified the activity of the parent compound  did not impair the whole activity of LV-2021 (1). The corresponding Tic-containing analogs (28–30) did not totally confirm these data since [Tic24]26RFa(20–26)  and [Tic26]26RFa(20–26)  were devoid of effect while [Tic22]26RFa(20–26)  was 5-fold more potent than 26RFa(20–26)   behaved like the Tic-containing counterparts, and [NPhe22]26RFa(20–26)  was almost as potent as LV-2211 -4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), a marine Cyanobacterium symploca peptide naturally occurring γ-amino acid [34–36) had no positive effect on the potency of the parent heptapeptide  D. It is  LV-2211 E. At thiing site . An incrino acid  (34–36) apeptide .22]26RFa(20–26), [DPhe24]26RFa(20–26) and [DPhe26]26RFa(20–26) results in a complete loss of the [Ca2+]i response 26RFa(20–26) , 26RFa(20–26)  and 26RFa(20–26)  did not confirm that the Phe residues of 26RFa(20–26)  participate in the bioactive conformation of the peptide or that some mandatory features are absent of these analogs  and LV-2188 (39) which antagonized 45–46% of the agonistic response with, respectively, modest and very low IC50 . The Phe22 residue appears to be the most permissive as it tolerated N-substitution like in [Tic22]26RFa(20–26)  which was 5-folds more potent than the lead peptide. Conversely, most modifications of Phe24 and Phe26 did not improve the activity with the exception of para substituents of lesser size than that of a tertbutyl group. Indeed, [Pcp24]26RFa(20–26) , [Pcp26]26RFa(20–26)  and [pNO2Phe26]26RFa(20–26)  were 2-, 3.5- and 3.3-fold more potent than 26RFa(20–26) . Although the amide NH of Phe24 residue was probably not involved in H-bond, our results suggest that some flexibility of the peptide backbone is required at this point. Noteworthy, not only the H-bond capacity, but other features were also impacted by these modifications such as the side chain presentation geometry, amide cis/trans isomerization equilibrium, and/or β-sheet potential of the analog with a wide range of steric, electronic and hydrophobic characteristics.To summarize, the three phenylalanine units exhibit differential contributions to the biological activity of 26RFahQRFPR, the Gly-Gly motif of 26RFa(19–26) seems to adopt an extended conformation [(20–26) and one of the most interesting results has been achieved by introducing a 4-carboxymethylpiperazine (Cmpi) unit in place of the native Gly-Gly pair which leads to the 5-fold more potent analog [Cmpi21]26RFa(21–26) (LV-2043) than the reference heptapeptide [(20–26) residues (52) and LV-2175 (55) containing 2-piperazino-2-[(4-fluoro)phenyl]acetic acid (Cmpi(4-FPhg)) and 2-piperazino-2-[(2-fluoro)phenyl]acetic acid (Cmpi(2-FPhg)) respectively, demonstrated similar [Ca2+]i-mobilizing activity in hQRFPR-transfected cells compared to the lead peptide, while other derivatives exhibited slightly  or detrimentally lower potency (22 residues do neither improve the ability of [Cmpi21]26RFa(21–26) to activate hQRFPR nor reverse the 26RFa-evoked effect.The variety of conformations characterized in enkephalins  and otheormation  generallapeptide . Herein,residues  and evalresidues . In this potency . AltogetN,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Christof Senn Laboratories  or PolyPeptide . Rink amide 4-methylbenzhydrylamine (MBHA) resin was from Novabiochem  and N-methylpyrrolidone (NMP), dimethylformamide (DMF) and dichloromethane (DCM) were from Biosolve . N,N-Diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), triisopropylsilane (TIS), tert-butylmethylether (TBME) were supplied from Sigma-Aldrich . Acetonitrile was from Fisher Scientific  and α-cyano-4-hydroxycinnamic acid (CHCA) matrix from LaserBioLabs .All Fmoc-amino acid residues and building blocks and O-benzotriazol-1-yl-2O (99.5/0.25/0.25) mixture for 120 min at room temperature. After filtration, crude peptides were precipitated by addition of TBME, centrifuged and recovered by elimination of the supernatant (3 folds). Peptides and derivatives were purified by reversed-phase HPLC (RP-HPLC) on a 2.2 × 25 cm Vydac 218TP1022 C18 column  using a linear gradient  of acetonitrile/TFA (99.9/0.1) at a flow rate of 10 mL/min. The purified peptides were then characterized by MALDI-TOF mass spectrometry on a UltrafleXtreme  using CHCA as a matrix. Analytical RP-HPLC, performed on a 0.46 × 25 cm Vydac 218TP54 C18 column (Grace), showed that the purity of all compounds was >97.3%. All peptides and derivatives were synthesized by the solid phase methodology on Rink amide MBHA resin using a Liberty microwave-assisted automated peptide synthesizer  and the standard manufacturer’s procedures at 0.1 mmol scale as previously described . All FmohQRFPR CHO cells were obtained as previously described 26RFa(20–26), highlighting a limited room to accommodate aromatic residues around the Phe aryl group. Although combination of multiple point-effective modifications does not necessarily translate into an additive or synergic effect, we have explored each position of 26RFa(20–26) for complete optimization of its sequence. Future challenges will be to convert several of these point modifications to low molecular weight 26RFa analogs for metabolic disorder, obesity or diabetes therapies. We are confident that, by utilizing subtle amendments, we can design 26RFa(20–26)-based compounds with nanomolar potency, functional selectivity and in vivo bioavailability.The C-terminal extremity of 26RFa was previously identified as a pivotal region to modulate its signaling at hQRFPR . In this


Scientific Reportshttps://doi.org/10.1038/srep26900, published online 09 June 2016Correction to: This Article contains errors.The data used for Supplementary Fig. Supplementary Fig. 2.Effect of PPK-2 deletion on colony morphology (A) and biofilm formation (B) ofM. tuberculosis. (A) Colony morphology of various M. tuberculosis strains was determined by plating 10-fold serial dilutions on MB-7H11 plates. (B) Biofilm images of the standing cultures of wild type, ppk-2 mutant and ppk-2 complemented strains in polystyrene coated 6-well plates in Sauton’s medium. The images depicted are representative of two independent experiments.


Salmonella (NTS) is the second most common cause of foodborne bacterial gastroenteritis in Australia with antimicrobial resistance (AMR) increasing in recent years. Whole-genome sequencing (WGS) provides opportunities for in silico detection of AMR determinants. The objectives of this study were two-fold: (1) establish the utility of WGS analyses for inferring phenotypic resistance in NTS, and (2) explore clinically relevant genotypic AMR profiles to third generation cephalosporins (3GC) in NTS lineages. The concordance of 2490 NTS isolates with matched WGS and phenotypic susceptibility data against 13 clinically relevant antimicrobials was explored. In silico serovar prediction and typing was performed on assembled reads and interrogated for known AMR determinants. The surrounding genomic context, plasmid determinants and co-occurring AMR patterns were further investigated for multidrug resistant serovars harbouring blaCMY-2, blaCTX-M-55 or blaCTX-M-65. Our data demonstrated a high correlation between WGS and phenotypic susceptibility testing. Phenotypic-genotypic concordance was observed between 2440/2490 (98.0 %) isolates, with overall sensitivity and specificity rates >98 % and positive and negative predictive values >97 %. The most common AMR determinants were blaTEM-1, sul2, tet(A), strA-strB and floR. Phenotypic resistance to cefotaxime and azithromycin was low and observed in 6.2 % (151/2486) and 0.9 % (16/1834) of the isolates, respectively. Several multi-drug resistant NTS lineages were resistant to 3GC due to different genetic mechanisms including blaCMY-2, blaCTX-M-55 or blaCTX-M-65. This study shows WGS can enhance existing AMR surveillance in NTS datasets routinely produced in public health laboratories to identify emerging AMR in NTS. These approaches will be critical for developing capacity to detect emerging public health threats such as resistance to 3GC.Non-typhoidal  PRJNA319593. Supplementary Material is available at Figshare: https://doi.org/10.6084/m9.figshare.16556748.v1 ,12:i:- ST34 where the qnrs1 was detected in 22/26 isolates with inferred reduced susceptibility. Other intermediate combinations included a single plasmid mediated quinolone resistance (PMQR) gene with or without parC[57:T-S] (Table S5). The mph(A) gene was detected in all fourteen concordant isolates that were resistant to azithromycin. Resistance to 3GCs was mediated by blaCMY-2 (n=81), variants of blaCTX-M (n=62), or both (n=4) (Table S5).In total, 94 serovars were detected using S. Senftenberg ST14 was the only lineage where blaSHV-12 and dfrA19 were detected. Isolates from S. Typhimurium ST19 harboured class A β-lactamase genes blaTEM-12 and blaCTX-M-9 in addition to chloramphenicol O-acetyl transferase catA1, blaCARB-2 and tet(G). The latter two genes have been associated with Salmonella genomic island I , gyrA[87:D-Y], parC[80:S-I], parC[57:T-S] was unique to S. Kentucky ST198. Other lineages were notably characterised by a lack of any AMR determinants, such as S. Dublin ST10 and S. Newport ST31.Several lineages were defined by AMR determinants unique to that lineage. For example, island I . Nearly n=128/151) were identified as MDR. The 3GC resistant isolates primarily carried blaCMY-2 (n=68), blaCTX-M-55 (n=32) or blaCTX-M-65 (n=13); accordingly, we specifically investigated AMR profiles in MDR lineages harbouring any of these 3GC resistance determinants. The emergence of these 3GC resistance genes was frequently associated with the presence of distinct plasmid replicons (Table S6).Of the concordant isolates that conferred 3GC cefotaxime resistance, 84.8 %  ISEcp1 and genes blc-sugE  and qnrS1 (n=5/10) of MDR S. Uganda ST684 isolates encoding blaCTX-M-55 did not co-occur with qnrS1 but instead blaCTX-M-55 was co-located on the same contig as blaTEM-215. ∆ISEcp1 was found upstream of blaCTX-M-55 in all but five blaCTX-M-55-positive MDR isolates , p16E080 (NZ_MN647788.1), pVb0499 (NZ_MF627445.1), IncFIB/IncFII: pN16EC0879-1 (CP043745.1), FDAARGOS_446 (CP023947.1), IncHI2/IncHI2A: pMTY18780-1 (AP023198.1), pPJM1 (MN539017.1)).A cluster of eight MDR isolates . The preS. Infantis isolates were shown to carry a MDR IncFIB(K) plasmid which included the blaCTX-M-65 gene -like MDR plasmid detected from a human isolate in the U.S ,12:i: ST34 lineage has been associated with retail meats, particularly pork products, from several Asian countries ,12:i:- ,12:i:- . This ha],12:i:- . FurtherCTX-M-55 , and proIn summary, we demonstrate a high concordance (99.8%) between genotypic and phenotypic AMR profiles in NTS and highlight how WGS data can be utilised to identify emerging AMR NTS in Australia. Our data further highlight the utility of inferring AMR profiles for NTS from WGS data routinely produced in public health laboratories. Our data provide a framework for the ongoing genomic surveillance of AMR in NTS in our setting; and may potentially act as an early warning system for significant changes in the AMR profiles of NTS.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Long non-coding RNAs (LncRNAs) play a vital role in the process of malignant transformation. In breast cancer (BC), lncRNAs field is currently under intensive investigations. Yet, the role of lncRNAs as promising diagnostic and/or prognostic biomarkers and as therapeutic target/tool among BC patients still needs a special focus from the biomedical scientists. In BC, triple negative breast cancer patients (TNBC) are the unlucky group as they are always represented with the worst prognosis and the highest mortality rates. For that reason, a special focus on TNBC and associated lncRNAs was addressed in this review. Colon cancer-associated transcript 1 (CCAT-1) is a newly discovered oncogenic lncRNA that has been emerged as a vital biomarker for diagnosis, prognosis and therapeutic interventions in multiple malignancies and showed differential expression among TNBC patients. In this review, the authors shed the light onto the general role of lncRNAs in BC and the specific functional activities, molecular mechanisms, competing endogenous ncRNA role of CCAT-1 in TNBC. BaxBcl-2 Associated X-ProteinBCBreast CancerBL1Basal-Like 1BL2Basal-Like 2Bmi-1B-Cell Specific Moloney Leukemia Virus Insertion Site 1CAEsCancer-Associated Endothelial CellsCAFsCancer-Associated FibroblastsCARL-5Cancer- Associated Region Long Non-Coding RNA-5CCAT-1Colon Cancer-Associated Transcript 1ceRNACompeting Endogenous RNAChIRP-SeqChromatin Isolation By RNA PurificationcircRNAsCircular RNAsCLIPCrosslinking ImmunoprecipitationCRCColorectal CancerCSCsCancer Stem CellsCTCFCCCTC-Binding FactorEMTEpithelial -To- Mesenchymal TransitionENCODEEncyclopedia Of DNA ElementsEREstrogen ReceptorERK/MAPKExtracellular Signal-Regulated Kinases/Mitogen-Activated Protein KinaseseRNAsEnhancer RNAsGCGastric CancerHCCHepatocellular CarcinomaHER2Human Epidermal Growth Factor Receptor 2HMGA2High Mobility Group At-Hook 2hnRNPA1Heterogeneous Nuclear RNP A1IARCInternational Agency for Research on CancerIMImmunomodulatoryKi-67Proliferation IndexLARLuminal Androgen ReceptorLCLung Cancerlet-7Lethal 7lin-4Lineage-4lincRNAsLong Intergenic ncRNAslncRNAsLong Non-Coding RNAsMMesenchymalMEK/ERK1/2Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase 1/2miRNAsMicroRNAsMREsMicroRNA Response ElementsmRNAMessenger RNAMSLMesenchymal Stem LikeNATSNatural Antisense TranscriptncRNAsNon-Coding RNAsntsNucleotidesPAM50Prediction Analysis of MicroarrayPD-L1Programmed Death-Ligand 1piRNAsPiwi-Interacting RNAsPRProgesterone ReceptorPRC2Polycomb Repressive Complex 2RNase PRibonuclease PRPISeqRNA-Protein Interaction PredictionrRNARibosomal RNAsiRNAsSmall Interfering RNAssncRNAsSmall Or Short Non-Coding RNAssnoRNAsSmall Nucleolar RNAssnoRNPSmall Nucleolar ProteinSNPsSingle-Nucleotide PolymorphismssnRNAsSmall Nuclear RNAsSPRY4Sprouty RTK Signaling Antagonist 4SUV39H1Suppressor Of Variegation 3–9 Homolog 1TILsTumor-Infiltrating LymphocytesTMBTumor Mutational BurdenTMETumor MicroenvironmentTNBCTriple Negative Breast CancerTNMTumor Node MetastasistRNATransfer RNATUSC3Tumor Suppressor Candidate 3WntWingless/IntegratedZFXZinc Finger Protein11.1st century, the discovery of lethal 7 (let-7) miRNA in the nematode Caenorhabditis elegans was published .,49.48,491.2.3cis-lncRNAs that regulate the expression of close proximity genes, and trans-lncRNAs that control distant genes expression . c. c168]. 2In conclusion, identifying the underlying molecular engines fueling BC progression and metastasis is a task that perplexed many researchers worldwide. In this review, we spot the light onto a key player in the game; CCAT-1 lncRNA.CCAT-1 is an upregulated lncRNA in BC and in TNBC patients in particular. This overexpression is associated with several disease parameters such as TNM stage, tumor size, ki-67 and lymph node metastasis. Yet, it was clear that correlation of CCAT-1 and BC stages, severity and resistance to therapy is still to be explored. In this manuscript, a detailed dissection of CCAT-1 lncRNA role in oncology in general and BC in particular was plotted. CCAT-1 was proved to have a key role in modulating several canonical and non-canonical oncogenic signaling pathways in BC. Yet, it is important to spot the light onto the scarcity of literature unraveling the potential role CCAT-1 in immunoncology. Therefore, CCAT-1 lncRNA is a still novel ncRNA that warrants further study with regard its network interactions with miRNAs, target genes, downstream signaling pathways and its correlation with the immunogenic profile of BC in terms of immune ligands, cytokines and interleukins secreted from BC cells.The authors declare no conflict of interest.


Monoclonal antibody (Mab) infusions have reduced hospitalization and mortality among higher risk patients with mild to moderate COVID-19 symptoms. Using an interdisciplinary team approach, we created a clinical team to proactively screen and outreach patients with COVID-19 to equitably offer Mab.From December 28, 2020 - May 3, 2021, a clinical team consisting of an Infectious disease pharmacist and physician, reviewed each outpatient with a positive SARS-CoV-2 PCR test at the Atlanta VA Healthcare System (AVAHCS) daily. The clinical team used the published Emergency Use Authorization criteria to determine eligibility. Eligible patients were prioritized using the Veterans Health Administration (VACO) Index for COVID-19 Mortality, which estimates the risk of 30-day mortality after COVID-19 infection using pre-COVID-19 health status . Eligible patients were contacted via telephone to confirm eligibility and obtain verbal consent. We performed SARS-CoV-2 IgG antibody tests when possible prior to Mab infusion, but results did not preclude Mab receipt. Telehealth follow-up occurred at 1- and 7-days post infusion.Figure 1. Veterans Health Administration COVID-19 (VACO) Index for COVID-19 MortalityOverview of the elements of the VACO index, part 1 of 2.Figure 1 continued. Veterans Health Administration COVID-19 (VACO) Index for COVID-19 MortalityOverview of the elements of the VACO index, part 2 of 2.In total, 1,346 COVID-19 patients were identified; 86 (6%) patients were eligible, and 48/86 (55%) received Mab infusions . The median time from symptom-onset to positive COVID-19 PCR test result was 6 days (0-9) and the median time from positive COVID-19 PCR test result to Mab infusion was 2 days (0-8). SARS-CoV-2 IgG antibodies were detected in 4 of 24 (17%) patients tested. The most common comorbidities were hypertension (73%) and diabetes, (42%) (Table). Five (10%) patients required hospitalization for worsening COVID-19 symptoms post infusion. No deaths occurred.Figure 2. Overview of COVID-19 Monoclonal Antibody (Mab) infusion ProcessSummary of Mab Infusion Screening ProcessTable. Patient Characteristics of Monoclonal (Mab) Infusion Recipients (N = 48)Descriptive Statistics and Findings of Study Data, part 1 of 2Table continued. Patient Characteristics of Monoclonal (Mab) Infusion Recipients (N = 48)Descriptive Statistics and Findings of Study Data, part 2 of 2This approach of combining laboratory surveillance and active screening minimized delay in symptoms onset to Mab infusion, thereby optimizing outpatient treatment of COVID-19 disease. Our approach successfully treated a more diverse patient population compared to clinical trials. Mab infusions overall was well tolerated with few hospitalizations and no deaths in this cohort.All Authors: No reported disclosures


Quadrivalent inactivated influenza vaccines (QIV) induce neutralizing antibodies (Abs) against the viral hemagglutinin (HA). Despite annual update of HA vaccine antigens to match circulating strains, current vaccines provide ~60% vaccine effectiveness (VE). QIV VE can be as low as 10% when circulating strains do not match vaccine HA. The live M2SR (M2-deficient single replication) influenza vaccine candidate has previously shown broad humoral, mucosal and cellular immune responses and protection against multiple influenza A subtypes. Here we show similar properties with the Quadrivalent M2SR (Quad M2SR) against drifted influenza B challenge in comparison to QIV.6 pfu of B/Brisbane/60/2008 (Victoria lineage) influenza virus. Nasal washes were taken for 7 days post-challenge and evaluated for challenge virus by TCID50 assay. Nasal turbinates, trachea and lungs were also evaluated for virus. Ferrets pre-infected with influenza H1N1 and B/Yamagata viruses, were immunized intranasally (IN) with PBS (Mock) or Quad M2SR, or intramuscularly with Fluzone QIV. Serum collected post-vaccination was evaluated for Ab responses. Forty-two days after vaccination, ferrets were challenged IN with 10oC, Fig. 2B) after challenge. In contrast, the Quad M2SR group did not exhibit any significant weight or temperature changes after challenge. Quad M2SR controlled the drifted challenge virus better than QIV as evidenced by significantly lower or absent post-challenge virus titer in nasal washes  and nasal turbinates .Quad M2SR and QIV elicited high serum Abs against the vaccine strain B/Colorado/06/2017  and against the drifted influenza B challenge strain B/Brisbane/60/2008  in ferrets with preexisting immunity. Like Mock, ferrets who received QIV displayed both weight loss  and a rise in temperature  antibody titers for Quad M2SR and QIV against matched Influenza B vaccine strain B/Colorado/06/2017  and drifted strain B/Brisbane/60/2008  on pre-study (Day -3), pre-vaccination (Day 28), and 3 weeks post vaccination (Day 51). The detection limit of the assay  was 15 PRNT50.Figure 2. Post-challenge body weight and temperature changesPercent body weight changes  and average body temperatures changes  following challenge with drifted Influenza B strain B/Brisbane/60/2008 for ferrets vaccinated with Quad M2SR or QIV.Figure 3. Post-challenge virus titers in respiratory tract.Viral titers in nasal washes  and nasal turbinates  collected post-challenge with Influenza B strain B/Brisbane/60/2008 in ferrets vaccinated with Quad M2SR or QIV. No virus was detected in the trachea or lungs. The detection limit of the assay  was 1.5 log10 TCID50/mL and 20 FFU respectively. Virus titer between groups was significant on day 3 of the nasal washes: one-way analysis of variance (ANOVA) with Multiple t tests to compare between groups, #p<0.05,><0.01,><>Despite eliciting similar Ab titers, the Quad M2SR demonstrated superior protection compared to QIV in a drifted influenza B challenge model in ferrets. These results suggest that the intranasal M2SR platform may confer additional advantages over currently available vaccines. Quad M2SR is in late-stage development for testing in a first-in-human clinical study. Lindsay Hill-Batorski, PhD, FluGen (Employee) Yasuko Hatta, DVM, PhD, FluGen (Employee) Michael Moser, PhD, FluGen (Employee) David Marshall, BS, FluGen (Employee) Pamuk Bilsel, PhD, FluGen (Employee)


Cell Death & DiseaseRetraction to: 10.1038/s41419-017-0103-7 published online 23 January 2018The authors have retracted this article because there are anomalies in Figure 5: panel siJun/pc-9 in part F (migration assay) overlaps with panel pc-9/siJun in part G (invasion assay). All authors agree with this retraction.


Correction to: J Exp Clin Cancer Res 38, 11 (2019)https://doi.org/10.1186/s13046-018-0997-7Fig. S1b: incorrect sample size listed; correct sample size is 25Fig. S4b: incorrect image used for SUNE1-ARNTL (24 h); correct image is now usedFollowing publication of the original article , the autThe authors provided the journal with their original data. The corrected figures are given here. The corrections do not have any effect on the final conclusions of the paper. The original article has been corrected.Additional file 2: Fig. S1.ARNTL methylation levels in the GSE52068 and GSE62366 nasopharyngeal carcinoma datasets.Additional file 5: Fig. S4. Overexpression of ARNTL had no impact on nasopharyngeal carcinoma cells invasion and migration. (A) Images of Transwell invasion (left) and migration (right) assay with ARNTL-overexpression or Vector-overexpression SUNE1 and HONE1 cells. (B) Images of wound healing assay with ARNTL-overexpression or Vector-overexpression SUNE1 and HONE1 cells.


Monoclonal antibodies (mAbs) encoded by IGHV3-53 (VH3-53) targeting the spike receptor-binding domain (RBD) have been isolated from different COVID-19 patients. However, the existence and prevalence of shared VH3-53-encoded antibodies in the antibody repertoires is not clear. Using antibody repertoire sequencing, we found that the usage of VH3-53 increased after SARS-CoV-2 infection. A highly shared VH3-53-J6 clonotype was identified in 9 out of 13 COVID-19 patients. This clonotype was derived from convergent gene rearrangements with few somatic hypermutations and was evolutionary conserved. We synthesized 34 repertoire-deduced novel VH3-53-J6 heavy chains and paired with a common IGKV1-9 light chain to produce recombinant mAbs. Most of these recombinant mAbs (23/34) possess RBD binding and virus-neutralizing activities, and recognize ACE2 binding site via the same molecular interface. Our computational analysis, validated by laboratory experiments, revealed that VH3-53 antibodies targeting RBD are commonly present in COVID-19 patients’ antibody repertoires, indicating many people have germline-like precursor sequences to rapidly generate SARS-CoV-2 neutralizing antibodies. Moreover, antigen-specific mAbs can be digitally obtained through antibody repertoire sequencing and computational analysis. The worldwide outbreak of coronavirus disease 2019 (COVID-19), caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents a severe health problem. The surface spike (S) glycoprotein of SARS-CoV-2 engages the cellular receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD), which is believed to be a critical target to block viral entry [Recently, a series of RBD-targeted mAbs were isolated from SARS-CoV-2 exposed individuals . MultiplIn this study, we performed antibody repertoire sequencing on 40 samples from 13 COVID-19 patients. Antibodyomics analysis was used to identify shared VH3-53 antibodies elicited by SARS-CoV-2 infection. The development of these shared VH3-53 antibodies was longitudinally tracked. Based on computational analysis, we synthesized repertoire-deduced sequences to produce recombinant mAbs for functional confirmation. The RBD binding activities and neutralizing capabilities of these recombinant mAbs were evaluated by enzyme-linked immunosorbent assay (ELISA), biolayer interferometry (BLI) affinity test, surrogate virus neutralization test (sVNT) , pseudovA total of 13 confirmed COVID-19 patients aged 33 to 81 were enrolled in this study (Table S1). Five blood samples from 5 healthy controls aged 28 to 58 were also included. All patients were hospitalized at Guangzhou 8th People's Hospital, and SARS-CoV-2 infection status was confirmed by RT-PCR. Single  or longitudinal  blood samples were collected during hospitalization and follow-up visits. Four patients were followed for 3 months after discharge.2 incubator.HEK293F is a female human embryonic kidney cell line transformed and adapted to grow in suspension . HEK293F cells were cultured in CD 293 TGE Medium  and place the flask on a shaker platform (120–130 rpm) in the 37°C/5% COPeripheral blood mononuclear cells (PBMCs) were isolated with Opti-Prep lymphocyte separation solution  by following the manufacturer’s instructions. Total RNA of PBMCs was extracted using TRIzol™  according to the manufacturer’s instruction.Dam-PCR using multiplex primers  was performed to amplify the heavy- or light- chain sequences as described previously . Briefly6 cells per mL. Following transfection, cells were maintained in CD 293 TGE Medium  and contained 1/10 CD Feed X supplement  at 37°C in a humidified 5% CO2 incubator rotated at 120 rpm. After 6 days expression, supernatants were harvested and clarified by centrifugation. Subsequent filtered through 0.22-µm filters and incubated with MabSelect  at room temperature for 2 h for antibodies affinity purification. After washing, antibodies were eluted from the MabSelect in chromatography columns using 0.1 M Na-Citrate (pH 3.25) and neutralized with 1M Tris-HCl (pH 8.8). Further purified by centrifugal filtration using Amicon Ultrafilter  in PBS and concentrated and stored at −80°C.The antibody heavy- and light-chain V genes (VH/VL) were synthesized  and were amplified and cloned into human IgG1 expression vector by using Clone Express II One Step Cloning Kit . The equal amounts of heavy- and light-chain plasmids were transfected into HEK293F cells using EZ Cell Transfection Reagent  with cells grown to a density of 1 × 10Coating a 96 well flexible assay plate overnight at 4°C with 100 µL per well of SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein  at 1 µg/mL in PBS. After standard washing, added 200 µL per well of 5% skim milk to block and incubated for 2 hours at 37°C. Washing wells three times with PBS-0.05% Tween 20 (MP), 100 µL semilogarithmic dilutions in PBS of antibodies were added to each well and incubated at 37°C for 2 hours. After washing wells 3 times with PBS-0.05% Tween 20, plates were incubated with 1:5000 dilutions in 5% skim milk of HRP-labelled Goat Anti-Human IgG(H + L)  at 37°C for 1 h. Washing wells 6 times with PBS-0.05% Tween 20, added 100 µL per well of TMB/E Solution (Merck Millipore) and left at room temperature for 15 min. Stop the reaction by adding 50 µL 1 M sulphuric acid and read the OD value at 450 nm.Antibody neutralizing activity was determined by a surrogate virus neutralization test as described previously  using cPBriefly, lentiviral vector pseudotyped by SARS-CoV-2 S protein were produced by co-transfecting the plasmid expressing S protein, which backbone carries an expression cassette for firefly luciferase. Then package this plasmid into 293 T cells to produce pseudotyped lentivirus as previously described . The thrThe affinity between SARS-CoV-2 RBD and antibodies were measured by BLI using GatorTM Label-Free Bioanalysis System (GatorBio). All experiments were performed at 25°C with shaking at 1,000 r.p.m, and anti-human IgG Fc biosensors were pre-equilibrated in Q buffer containing PBS (10mM pH7.4), 0.02%Tween-20, and 0.2%BSA for at least 300 s before use in experiments. 10 μg/mL antibody was loaded onto anti-human IgG Fc biosensors for 120 s and flowed the difference in concentrations of SARS-CoV-2 RBD-His recombinant protein  or buffer as control) for 120 s. Then the both dissociate in Q buffer for 300s. The competitive binding between two antibodies was also measured using the GatorTM Label-Free Bioanalysis System (GatorBio). Anti-His biosensors were pre-equilibrated in Q buffer, then 2 μg/mL SARS-CoV-2 RBD-His recombinant protein  was loaded onto anti-His biosensors for 120s, and flowed 10 μg/mL of the first antibody for 300 s and 10 µg/mL of the second antibody (or buffer as control) for 300 s. We analysed the data by using Gator™ System Analysis.http://www.imgt.org/). Productive sequences that had not stop codons or out-of-frame IGHJ were retained for further analysis.The raw FASTQ files were filtered according to their base qualities. 3′ ends of read sequences with quality scores lower than 20 were trimmed using Trimmomatic v0.36 . FollowiProductive sequences were assigned to antibody clones by grouping sequences according to their V(D)J germline gene usage using MIXCR v3.0.3 . We defihttps://cran.r-project.org/web/packages/cluster/) and the visualization was used the R package igraph (https://cran.r-project.org/web/packages/igraph/index.html). Shared VH3-53-encoded clonotypes presented in this study were further compared with the reported VH3-53-encoded RBD-targeted antibodies.VH3-53-encoded heavy chain sequences were extracted from the total repertoires of each individual and then they were pooled together. Single-linkage clustering was performed by comparing the heavy chain complementarity-determining region (HCDR3) amino acid sequences of each antibody clone to identify shared clonotypes. Shared clonotype refers to antibody clones  that derived from different donors but involving the same V gene, the same J gene, the same HCDR3 length, and HCDR3 amino acid sequences of high identity between donors . Here, whttps://cran.r-project.org/web/packages/stringdist/index.html) and normalized to the length of the overall sequence. Within each repertoire, only sequences with a matching V-gene assignment and light-chain complementarity-determining region (LCDR3) length to each RBD-targeted antibody were considered for identity calculating. Here, we selected a threshold of 100% LCDR3 identity and 95% of overall sequence identity for shared clonotype analysis because of the short LCDR3s.A list of VH3-53-encoded RBD-targeted antibodies was compiled from the recently published literature studies . VK1-9 rFor phylogenetic analysis, the VH3-53 antibody sequences derived from different donors were aligned with each other using MUSCLE v3.8.31  to generhttps://swissmodel.expasy.org/) [https://www.rcsb.org/), which was determined recently [http://zdock.umassmed.edu/) [https://pymol.org/2/).The homology models of VH3-53 antibodies were built on the SWISS-MODEL server (sy.org/) . The cryrecently . Dockinged.edu/) , and topp-value of less than .05 was considered statistically significant.Statistical analysis was carried out using GraphPad Prism 8.0 software. Student’s t-test  used for the comparison of VH3-53 germline gene usage in heavy chain repertoires between different time points. A We collected 40 blood samples from 13 COVID-19 patients and performed antibody repertoire sequencing  length, and HCDR3 amino acid sequences of 80% identity between different people . We synthesized these novel VH3-53-J6 heavy chains and co-expressed with the shared VK1-9-J4 light chain in HEK293 cells to produce recombinant mAbs for functional analysis. Antibody binding activity to RBD was measured by enzyme-linked immunosorbent assay (ELISA) . Overall, 23 out of 34 recombinant mAbs showed binding activities to RBD, with rmAb23 (from patient W) having EC50 as low as at 0.1034 μg/mL . Interestingly, several germline VH3-53-J6 heavy chains  have no somatic hypermutations, even they bound to RBD with EC50 < 0.35 μg/mL  was identified in 9 out of 13 SARS-CoV-2 exposed individuals, accounting for over 2/3 of COVID-19 patients. This shared VH3-53-J6 clonotype was derived from convergent gene rearrangements and was evolutionarily conserved. These VH3-53-J6 antibodies have no or few somatic hypermutations, suggesting that many people may have preprogramed in their germline sequence for rapid generation of RBD-targeted antibodies. Importantly, we were able to identify antigen-specific mAbs from millions of antibody sequences by mining antibody repertoires of COVID-19 patients. The repertoire-deduced VH3-53 heavy chain sequences that we identified can bind to RBD and have neutralizing activities, validating our finding of RBD-specific antibodies via repertoire sequencing and bioinformatic analysis.This study highlights the prospect that antibody repertoire sequencing and computational analysis could identify RBD-specific mAbs among COVID-19 patients. Compared with the conventional antibody cloning technology, more than one million antibody sequences can be obtained in a parallel antibody repertoire sequencing, making in-depth studies of the repertoire possible. In addition, it allows to track the certain antibody lineages, such as the VH3-53-encoded antibodies. Although bulk sequencing loss, the information of paired heavy chain and light chain, it could be combined with single-cell 5′V(D)J sequencing or traditional single-cell RT-PCR technology.5GMDL) is highly enriched in acute dengue samples [Convergent antibody rearrangements were also observed in acute human dengue infection and human responses to influenza vaccination. Convergent antibody HCDR3 sequence (ARLDY samples . While a samples . These s samples ,37.An important finding of this study is that the highly shared VH3-53 antibodies elicited in different COVID-19 patients target to the same epitope on SARS-CoV-2 RBD. The VH3-53 antibodies we identified from the repertoires have neutralizing activities through direct blocking of the hACE2–RBD interaction. These VH3-53 antibodies recognize the same epitope region as previously reported RBD-binding mAbs, B-38, CV30, and CC12.3. Antibodies that share the same germline gene usage and similar molecular interactions with antigens have been observed in other viral infections, including Ebola, Zika, and influenza, as well as chronic HIV-1 infection. The VH3-15/VL1-40-based class of antibodies targeting the NPC1-receptor-binding site on Ebola GP has been reported as a common response in individuals infected with the Ebola virus or vaccinated with Ebola virus vaccine rVSV-ZEBOV . The VH3Another important finding from the repertoire analysis is that the VH3-53 antibodies have very limited or even no somatic mutations. So far, most reported SARS-CoV-2-specific mAbs have no or few somatic hypermutations ,12. NotaWith the ongoing global spread of SARS-CoV-2, several viral variants emerged worldwide. Specifically, the B.1.1.7, B.1.351, and P.1 lineages that originated in the UK, South Africa, and Brazil, respectively, are characterized by the accumulation of mutations in spike protein, especially in the RBD. Three mutation K417N, E484K and N501Y in RBD of spike raised the most of concern . NotablyTaken together, we revealed a convergent antibody response encoded by VH3-53-J6 clonotype to SARS-CoV-2. The VH3-53 antibodies provide a benchmark for evaluating SARS-CoV-2 and vaccine-induced antibody responses. Furthermore, our study demonstrated that antigen-specific mAbs can be digitally obtained by identifying shared clonotypes through antibody repertoire sequencing and computational analysis.Click here for additional data file.


Early passage (6-12) pHSCs were plate-adhered and TGF-b-treated (10ng/mL) to maximally activate their pro-fibrogenic genes, collagen 1α1 (Col1A1), tissue inhibitor of metalloproteinase 1 (TIMP1), or α-smooth muscle actin (ACTA2). CDAHFD and SD pHSCs were then treated for 48 hours with increasing doses of BMS-22 or MVC (range: 0.3-120ng/mL) to determine (1) the degree of attenuation of the pro-fibrogenic response as measured by qPCR of fibrogenic genes ; (2) enhancement of a fibrolytic response as measured by qPCR of matrix metalloproteinases (MMP) 2, 9 and 13 genes; and (3) pHSC migration using the scratch assay. Cell viability and CCR2 and CCR5 gene expression in response to escalating doses of antagonists were also measured. RESULTS/ANTICIPATED RESULTS: Plate- and TGF-b activated CDAHFD pHSCs had a 2-fold greater, dose-dependent attenuation of their pro-fibrogenic activity in response to BMS-CCR2-22 and MVC, when compared with plate- and TGF-b activated SD pHSCs, as measured by reductions in collagen 1α1 (Col1A1) and α-smooth muscle actin (ACTA2) gene expression. TIMP1 gene expression was unaffected by drug treatment for 48 hours. Cell viability was not affected up to doses of 30ng/mL of each drug. pHSCs also demonstrated a dose-dependent increase in CCR2, CCR5 and MMP-9 gene expression in response to surface receptor antagonism. Migration assays comparing CDAHFD and SD pHSCs in response to escalating doses of MVC and BMS-22 are ongoing and expected to demonstrate a significantly decreased migratory capacity of CDAHFD pHSCs than SD pHSCs in response to therapy, reflecting the increased susceptibility of the “fat-exposed” pHSCs to anti-fibrotic therapy than normal pHSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-fibrotic drugs that dampen pro-fibrogenic activities of “fat-exposed” pHSCs are urgently needed. CCR2 and CCR5 antagonists, BMS-22 and MVC, respectively, can selectively dampen the pro-fibrogenic response of fat-exposed pHSCs, and must be considered for future trials in human NASH. CONFLICT OF INTEREST DESCRIPTION: Dr. Jill Smith has a patent licensing agreement with Immune Therapeutics, Inc.OBJECTIVES/GOALS: Non-alcoholic steatohepatitis (NASH) is a leading cause of cirrhosis in the world for which no anti-fibrotic therapies exist. We hypothesized that BMS-22 and maraviroc (MVC), chemokine receptor 2 (CCR2) and 5 (CCR5) antagonists, respectively, would diminish the fibrogenic activity of "fat-exposed" murine pHSCs. METHODS/STUDY POPULATION: pHSCs were isolated from livers of 6 week old male mice following 4 weeks on a NASH-inducing choline-deficient high fat diet  or standard diet (SD) and passaged


The single nucleotide polymorphism (SNP) rs62324212, located in IL21 antisense RNA 1 (IL21-AS1), has been identified as a genetic risk variant associated with systemic lupus erythematosus (SLE). We aimed to probe the characteristics of IL21-AS1 and explore its clinical relevance focusing on T helper subsets and disease activity in patients with SLE.+ T cells were analyzed using PCR and flow cytometry. The association among IL21-AS1, CD4+ T cell subsets, and SLE disease activity was accessed.rs62324212 genotyping was determined using allelic discrimination by quantitative PCR. Gene expression in peripheral blood mononuclear cells and cell surface markers in CD4+ T and B cells, but its expression was decreased in patients with SLE. IL21-AS1 expression was positively correlated with mRNA levels of IL-2 but not IL-21, and it was associated with the proportion of activated T follicular regulatory (Tfr) cells. Furthermore, we observed a significant negative correlation between IL21-AS1 expression and disease activity in patients with SLE .Ensembl Genome Browser analysis revealed that rs62324212 (C>A) was located in the predicting enhancer region of IL21-AS1. IL21-AS1 was expressed in the nucleus of CD4IL21-AS1 has an effect on disease activity through an involvement of IL-2-mediated activation of Tfr cells in SLE. Thus, targeting the IL21-AS1 may provide therapeutic approaches for SLE.The online version contains supplementary material available at 10.1186/s13075-021-02682-w. Since IL21-AS1 also decreased in patients with RA, we assessed the correlation between IL21-AS1 and disease activity in patients with RA. However, no significant correlation was observed between IL21-AS1 expression and disease activity scores such as Disease Activity Score with 28 Joint DAS28) using C-reactive protein (CRP) (DAS28-CRP) and DAS28 using erythrocyte sedimentation rate (ESR) (DAS28-ESR), CRP, ESR, and autoantibodies such as rheumatoid factor and anti-cyclic citrullinated peptide antibody  was evaluated using qPCR. Correlation between IL21-AS1 expression levels and RA Disease Activity Score with 28 joint (DAS28) using CRP (DAS28-CRP) and DAS28 using ESR (DAS28-ESR), CRP level, ESR level, rheumatoid factor level, and anti-cyclic citrullinated peptide antibody level. Data symbols represent individual subjects. Spearman’s test was used for correlation analysis between two variables of interest.Additional file 3: Fig. S3. Expression level of IL21-AS1 in human normal tissues. Expression level of IL21-AS1 was obtained from the Functional Annotation of the Mouse/Mammalian Genome database. CPM: counts per million.


RNH1 prevents inflammation by inhibiting inflammasome activation through controlling caspase-1 protein levels. In COVID-19 patients, RNH1 expression levels were negatively associated with disease severity and inflammation, suggesting a role for RNH1 in SARS-CoV-2–mediated inflammation and pathology. Rnh1−/− mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1–mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP  proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1β production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1β expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in  Saccharomyces pombe (RANGAP1), and other NOD-like receptors (NLRs) (Ribonuclease inhibitor (RNH1) is a cytosolic leucine-rich repeat (LRR) protein; however, it can also be found in the nucleus and mitochondria . The knos (NLRs) . Interess (NLRs)  which foInflammasomes are cytosolic pattern recognition receptors, which sense invading pathogens or endogenous danger molecules that are released by dying cells. This process is critical in eliminating pathogens and initiating tissue repair . Inflammhttps://covid19.who.int). The clinical manifestation of patients with COVID-19 includes hypoxemic respiratory failure, acute respiratory distress syndrome, and myocardial injury, among others  . COVID-1g others . Recent g others . It is tIn this study, we found that RNH1 inhibits inflammasome activation by controlling proteasome-mediated degradation of the downstream effector molecule caspase-1. RNH1-deleted mice displayed higher inflammasome-dependent activation of caspase-1. Interestingly, we also found that RNH1 expression in buffy coat and lung biopsies is negatively related with SARS-CoV-2–mediated severity and inflammation in COVID-19 patients. Collectively, these findings establish RNH1 as a previously unidentified negative regulator of inflammasome activation and indicate its potential role in human inflammatory diseases including SARS-CoV-2–mediated inflammation and pathology.Human RNH1 protein consists of 15 LRR repeats whose sequence and organization are similar to those present in multiple NLRP proteins  Fig 1A)Fig 1A). NLRP3 is the most studied inflammasome and is activated by several pathogens and danger stimuli . To inveActivation of the canonical NLRP3 inflammasome occurs in two steps . The priTo check whether RNH1 is also involved in activation of AIM2 and NAIP/NLRC4 inflammasomes, we stimulated WT and RNH1-KO THP1 cells with their agonists. We used cytosolic dsDNA (pdA:dT) and flagellin to activate AIM2 and NAIP/NLRC4 inflammasomes, respectively. Interestingly, RNH1-KO showed an increase in mature IL-1β production and caspase-1 cleavage compared with WT cells , suggestcaspase-1 and ASC are equivalent in WT and RNH1 KO cells caspase-1Rnh1 leads to embryonic lethality in mice  to understand the in vivo relevance of our results . Administration of polyinosinic:polycytidylic acid (polyI:C) into Rnh1fl/f;Mx1-Tg+ mice leads to cre-recombination and complete deletion of RNH1 protein expression in hematopoietic organs such as the BM, which we refer to as Rnh1−/− mice (Rnh1fl/fl) and Rnh1−/− mice and then stimulated them with inflammasome activators. As observed in cell lines, loss of RNH1 in primary mouse BMDMs led to increased mature IL-1β production and caspase-1 cleavage compared with WT cells upon stimulation with NLRP3, AIM2, and NAIP/NLRC4 agonists  and Rnh1−/− (Rnh1fl/flMx1-Cre+) BM into irradiated CD45.1 congenic mice to exclude Rnh1 deletion effects from non-haematopoietic cells. After 8 wk, we checked reconstitution levels in WT and Rnh1−/− BM transplanted CD45.1 congenic mice. We found comparable reconstitution in WT and Rnh1−/− BM transplanted CD45.1 congenic mice via FACS analysis Rnh1 leadc organs , we trananalysis . Rnh1 wanic mice . In a MS WT mice . Consist WT mice . We nextsurvived . AltogetRecent studies suggest that pronounced inflammation and inflammasome activation in patients with COVID-19 correlates with increased disease severity and poor prognosis . BecauseCOVID-19 demographic data from independent study in the UK.Table S1 Recent studies document the critical role of monocytes and macrophages in SARS-CoV-2 infection . To checRnh1 in mice is embryonically lethal due to marked defects in erythropoiesis. These defects originate from decreased translation of the transcriptional erythropoiesis regulator GATA-1 rather than detectable effects on mRNA levels. This suggests that RNH1 fulfills functions beyond inhibition of ribonucleases  and mediates proteolytic processing of pro-IL-1β and pro-IL-18 while promoting pyroptosis by cleaving gasdermin . Few sturadation .Uncontrolled inflammation mediates immune pathology in several inflammatory diseases, including the COVID-19 pandemic . Recent The data provided here using in vitro experiments and mouse models of inflammation support the role of RNH1 in suppressing inflammation. These experiments suggest that RNH1 is a new negative regulator of NLRP3 inflammasome activation and NFkb signaling. Because inflammasomes were involved in SARS-CoV-2 infection–mediated inflammation and pathology we also studied the role of RNH1 in COVID-19. We found a significant decrease in RNH1 protein expression in ICU COVID-19 patients compared with COVID-19 general ward patients. Furthermore, RNH1 expression is largely absent in the lungs of deceased patients with COVID-19 compared with patients who succumbed to non-viral causes. These results suggest a potential involvement of RNH1 in the severity of disease; however, there are limitations to the study. First, statistically, no significant correlation between the expression of the RNH1 protein and the WHO severity scores of COVID-19 patients. Further studies with a larger number of patient samples are required to clarify the correlation between RNH1 expression and disease severity in COVID-19 patients. Second, our studies have investigated the expression of RNH1 in COVID-19 patients at admission stage but not at other time points, so we do not know how the expression of RNH1 could affect the progression of the disease over time. More prospective studies by analyzing the level of the RNH1 protein at different intervals are necessary to understand the role of RNH1 in disease progression. Third, we cannot exclude an inflammation independent role of RNH1 in COVID-19 disease severity. Further molecular studies with RNH1 mouse models of COVID-19 infection are required to clarify this. Collectively, our studies introduce RNH1 as a new negative regulator of inflammasome activation. However, further studies are warranted to understand the molecular details of how RNH1 regulates the priming and activation signal of the NLRP3 inflammasome and how these mechanisms are dysregulated in human inflammatory diseases, including COVID-19 and sepsis.Rnh1 conditional-knockout (Rnh1fl/fl) mice were generated on a C57BL/6 background. A 13.8 kb region used to construct the targeting vector was first subcloned from a positively identified C57BL/6 BAC clone (RP23: 217N5) using a homologous recombination-based technique. The region was designed in a way that the long homology arm (LA) extends 6.23 kb 3′ to the single LoxP site. The short homology arm (SA) extends about 3.0 kb 5′ to the LoxP/FRT flanked Neo cassette. The single LoxP site is inserted downstream of exon 10, and the Neo cassette is inserted upstream of exon 3. The size of the target region is about 4.6 kb containing exons 3–10 (Rnh1 targeting vector was electroporated into (C57BL/6) embryonic stem (ES) cells. Targeted (C57BL/6 FLP) embryonic stem cells were microinjected into Balb/c blastocysts. Resulting chimeras with a high percentage black coat colour were mated to C57BL/6 WT mice to generate Neo deleted, F1 heterozygous offspring. Germline transmission was confirmed by PCR of tail genomic DNA. Screening of Rnh1fl/fl mice by PCR genotyping was carried out using the following primers on ear genomic DNA: 5′-ACATGGTGTTCTGGG TGTACGGTGG-3′ (forward in the intron region after second exon), 5′-CTGAGTAAGGAC TGCTGGGCTGAG-3′ . This reaction amplifies 314 bp in size for WT allele and 514 bp size for floxed allele (Rnh1fl/fl mice with Mx1-Cre mouse strain (Jackson: 002527) to generate inducible mouse model (Rnh1fl/flMx1-Cre+). To exclude non-haematopoietic deletion, total BM was isolated from WT (Rnh1fl/fl) or Rnh1fl/flMx1-Cre+ mice and transplanted (7 × 106 BM cells) into lethally irradiated CD45.1+ congenic recipient mice  . After 8 wk of reconstitution, Rnh1 was excised by giving three rounds of 200 μg poly(I:C) (Invivogen) using IP injections once in 2 d. We used these mice for MSU induced peritonitis model and LPS lethality model. To detect Rnh1 deletion after Cre-recombination, we used a third primer 5′-AAGACCCATCCAGAGCCGAGG-3′  in above mentioned PCR reaction using DNA form BM cells, which amplifies 365 bp size  and housed for 2 wk before starting the BMT experiments. All animal experiments were approved by the Swiss Federal Veterinary Office under valid authorization (BE39/16). Mice were handled according to Swiss Federal Veterinary Office guidelines under valid authorization. We used the ARRIVE reporting guidelines to report in vivo experiments  were age- and sex-matched. We used littermates for our experiments. All the cages were placed at the same location and NLRP3 agonist LPS and MSU were injected at the same time to reduce potential confounders. Sample size was determined based on previous studies and no animals were excluded from the study . Mice weeriments .2-asphyxiation and the peritoneal cavity was flushed with sterile PBS. The lavage fluid was centrifuged, and supernatants were analysed by ELISA for IL-1β production, and the pelleted cells were analysed for the influx of neutrophils  by BD LSRII flow cytometer (BD Biosciences). Data were analysed by using FlowJo  software.Mice were injected IP with sterile PBS or MSU (1 mg/mouse) to induce peritonitis. After 12 h mice were euthanized by COMice were injected IP with LPS (10 mg/kg) and survival rates were monitored.Healthy human controls and inflammatory disease patients BM biopsies were obtained from Swiss MDS Registry. These biopsies were negative for COVID-19 infection. Biopsies were stained with human RNH1 antibody from Prestige Antibodies Sigma-Aldrich (HPA039223). These studies were approved by the competent local human ethics committee (ID:2017-00734).2. BMDMs were generated by established protocols. Briefly, total BM cells were isolated from the femur and tibias of mice and differentiated into macrophages for 7 d in complete DMEM medium  supplemented with M-CSF (40 ng/ml) .Mouse BMDMs, iMACs , and human THP-1 cells were used for inflammasome activation experiments.THP-1 cells and iMACs were grown in RPMI 1640 GlutaMAX-I medium (Invitrogen) were supplemented with 10% (vol/vol) FBS (Amimed) and 1% of penicillin and streptomycin (PAA Laboratories) and incubated at 37°C and 5% COWe purchased Nigericin (N7143), pdA:dT (poly(dA-dT)•poly(dT-dA)) sodium salt (P0883), Gramicidin A (G5002), PMA (P1585), Actinomycin D and Cycloheximide from Sigma-Aldrich, Ultra-pure flagellin (AG-40B-0025) from Adipogen, ultrapure LPS (LPS-EK), MSU (tlrl-msu), and Pam3Cysk4 (tlrl-pms) from Invivogen.Cloning of full-length caspase-1 and OMI were amplified by PCR and sub-cloned into the mammalian expression vectors pCR3 in frame with the N-terminal flag tags. Full-length RNH1-Flag construct was described previously . To cons6 cells per mL as described previously . THP1 cells were obtained from ATCC, and iMAC cells were provided by Petr Broz (University of Lausanne) (RNH1-KO THP1 cells (human monocytic cell line) were generated as described previously . For RNHausanne) .−1) 3 d after infection.Flag-RNH1 was further sub-cloned into retroviral vector pMSCVpuro (Clontech). Retroviral vector pMSCVpuro-Flag–RNH1 was co-transfected with the helper plasmids VSV-G and Hit60 into HEK293T cells using PEI transfection reagent. Culture supernatants containing recombinant viral particles were harvested and used to infect THP1 cells. To establish stable cell lines, THP1 cells were selected with puromycin (5 μg mlRNH1-KO THP1 cells were infected with lentiviruses expressing RNH1 (pLenti CMV Blast GFP-RNH1) or the empty vector pLenti CMV-GFP-Blast  as previously described . After 4−1) 3 d after infection.Various lentiviral shRNA plasmids against RNH1 were purchased from Sigma-Aldrich and lentivirus was generated as previously described . To estaPrecipitated media supernatants or cell extracts were analysed by standard immunoblot techniques. The following antibodies were used: anti-human RNH1 (A9), anti-mouse RNH1 (C10), anti-human ASC (Sc-514414), anti-GAPDH  from Santa Cruz Biotechnology; anti-human IL-1β antibody (12242), anti-IκBα, anti-Phospho-IκBα, anti-GFP (2555) from Cell signaling; anti-human caspase-1 , anti-mouse caspase-1 , anti-NLRP3 (Cryo-2), anti-mouse ASC (AG-25B-0006) from Adipogen; anti-mouse IL-1β antibody (AF-401-NA) from R&D; anti-β-Actin from Abcam.B activation, cells were stimulated at 10, 20, 40, 60, and 180 min by Pam3CSK4 . After stimulation, cell lysates were isolated and analysed via Western blot.WT and RNH1-KOTHP 1 cells were stimulated with TLR2 agonist Pam3CSK4 (500 ng/ml) at different time points 3, 6, and 14 h or at different doses 100, 500, and 1,000 ng/ml for 3 h. For NF-κHEK293T cells were treated with or without MG-132 and transfected with full-length flag-tagged caspase-1 or OMI/HTAR2 plasmids with or without GFP-tagged RNH1 plasmid with the indicated concentration. After 24 h, cells were harvested, and cell lysates were analysed by immunoblot. HEK293T cells routinely tested negative for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit . HEK293T cells were obtained from ATCC.Cell supernatants were analysed for cytokines and Lactate Dehydrogenase (LDH). Human and mouse IL-1β, TNF, and IL-6 cytokine secretion measured by ELISA according to the manufacturer’s instructions (eBioscience). LDH was measured by colorimetric NAD linked assay by in-house developed kit according OPS Diagnostics instructions.2, 1 mM EDTA, 1 mM EGTA, and 320 mM sucrose) by syringing 35 times using a 21-G needle. Cell lysates were centrifuged for 8 min at 1,800g and supernatants were collected (30 μl of the supernatants were kept aside to check ASC expression in the lysates). Supernatants were further diluted two times with lysis buffer and then centrifuged at 2,000g for 5 min. Next, supernatants were diluted with 1 vol of CHAPS buffer  and then centrifuged for 8 min at 5,000g. The supernatant was discarded, and the pellet was resuspended in 50 μl of CHAPS buffer supplemented with 4 mM of disuccinimidyl suberate (Thermo Fisher Scientific) and incubated at RT for 30 min. After incubation, samples were centrifuged for 8 min at 5,000g, and pellets were resuspended in SDS sample buffer without reducing agents and Western blot was performed.We detected ASC oligomerization by Western blot as described previously . BrieflyTo further confirm the ASC specks formation, we performed confocal microscopy as described previously . BrieflyTo study caspase-1 mRNA and protein stability, WT and RNH1-KO THP1 cells were treated with the following inhibitors for 0, 3, and 6 h. Cells were treated with Actinomycin D (10 μg/ml), Cycloheximide (75 μg/ml), proteasomal inhibitor MG-132 (20 μM) to assess transcriptional, translational, and posttranslational changes. After treatment, cells were harvested, and cell lysates were analysed by immunoblot.2O were negative for target and housekeeping genes. The following gene-specific primers (Microsynth) were used. 18S rRNA:5′-GCAATTATTCCCCATGAACG-3′(f), 5′-AGGGCCTCACTAAACCATCC-3′ (r); ASC: 5′-CCT CAG TCG GCA GCC AAG-3′ (f), 5′-CAG CAG CCA CTC AAC GTT TG-3′ (r); CASP1: 5′-TCC TCA GGC TCA GAA GGG AA-3′ (f), 5′-TGC GGC TTG ACT TGT CCA T-3′ (r); RNH1: 5′-GATCTGGGAGTGTGGCATCA-3′ (f), 5′-CTGCAGGACTTCACCCACAG-3′ (r).Total RNA was isolated from WT and RNH1-KO THP1 cells using the QIAGEN RNeasy Kit according to the manufacturer’s protocol. Complementary DNA was generated from total RNA as described previously . The SYBThe SARS-CoV-2 pseudoviral particles expressing COVID-19 spike protein pGBW m4137384: S protein was purchased from Addgene (149543) and the virus particles were produced as described previously . WT and We analysed a total of 28 COVID-19 patients samples from a previously published study (n = 13) , and witPost-mortem (PM) tissues were collected as part of the ethically approved LoST-SoCC study in accordance with the “Declaration of Helsinki” and Human Tissue Authority legislation. The study was approved by the Newcastle North Tyneside 1 research ethics committee 19/NE/0336, IRAS 193937. Non–COVID-19 causes of death were all non-respiratory, non-infectious, obtained from PMs that were done before the pandemic, and lungs were morphologically within normal limits. The original FFPE tissue blocks were taken from representative samples at post-mortem, formalin fixed and processed as per standard diagnostic blocks. For tissue microarray (TMA) construction, representative 3 mm tissue cores were cored out of FFPE tissue blocks and re-blocked within a 4 × 4 format TMA, sectioned and mounted onto glass slides. Slides were dehydrated and endogenous peroxides quenched. Antigen retrieval was done using TRIS buffer pH9 boiled for 15 min. Slides were incubated with a permeabilization buffer (0.3% Triton-X100 in PBS) then blocking buffer (10% FBS in PBS). Anti-RNH1  was added overnight at 1:500, followed by anti-rabbit secondary, ABC amplification and DAB detection (both Thermo Fisher Scientific). Slides were then counterstained with Meyer’s haematoxylin and coverslips applied with aqueous mounting agent and slides imaged using Zeiss axioscan Z1. Images were analysed using Zen Lite software (Zeiss).http://iqtree.cibiv.univie.ac.at/) using ultrafast bootstrap analysis with 1,000 number of bootstrap alignment  were aligned using MAFFT multiple sequence aligner . Sequenclignment . Circulalignment . For strlignment . Sequenct test. A value of P < 0.05 was considered statistically significant. To compare the survival in LPS model, log-rank (Mantel–Cox) test was performed. For correlation studies, Spearman’s rank correlation statistical test was performed. Statistical analyses for COVID-19 patients’ data were performed with R. All other statistical analyses were calculated using GraphPad Prism version 9.All the data were represented as mean ± SEM. Comparison between two groups was performed by a two-tailed allam.ramanjaneyulu@dbmr.unibe.ch.Plasmids and transgenic mice are available upon request. Please write to the lead contact:


R)-4,6-dimethoxy-3-(4-methoxy phenyl)-2,3-dihydro-1H-indanone [(R)-TML 104] is a synthesized analog of the natural product resveratrol sesquiterpenes (±)-isopaucifloral F. The present study aimed to investigate the effects and underlying mechanisms of (R)-TML104 on neointima formation. Our results showed that (R)-TML104 prevented neointima formation based on a carotid artery injury model in mice. Furthermore, (R)-TML104 inhibited platelet-derived growth factor-BB (PDGF-BB)-induced vascular smooth muscle cells (VSMC) phenotypic transformation, evidenced by increased α-smooth muscle actin, reduced VSMC proliferation, and migration. Simultaneously, (R)-TML104 upregulated sirtuin-1 (SIRT1) expression in VSMC. We further uncovered that SIRT1 expression is critical for the inhibitory effects of (R)-TML104 on PDGF-BB-induced VSMC phenotypic transformation in vitro and injury-induced neointima formation in vivo. Finally, (R)-TML104-upregulated SIRT1 inhibited PDGF-BB-induced VSMC phenotypic transformation by downregulating nicotinamide adenine dinucleotide phosphate oxidase 4 expression via decreasing nuclear factor-κB acetylation. Taken together, these results revealed that (R)-TML104 upregulates SIRT1 expression and ameliorates neointima formation. Therefore, the application of (R)-TML104 may constitute an effective strategy to ameliorate neointima formation.Neointima formation is a serious complication caused by mechanical trauma to the vessel. ( Cardiovascular diseases are the major cause of death worldwide . AlthougPhenotypic transformation of the vascular smooth muscle cells (VSMC) plays a vital role in neointima formation and can be triggered by oxidative stress, which stems from the excessive production of reactive oxygen species (ROS) , 6. The R)-4, 6-dimethoxy-3-(4-methoxy phenyl)-2, 3-dihydro-1H-indanone [(R)-TML104], a synthetic analog of resveratrol sesquiterpenes (±)-isopaucifloral F -TML104  and atorvastatin (20 mg/kg) were administered daily by gastric gavage to the model group mice. (R)-TML104 and atorvastatin were both dissolved with saline. Mice were euthanized 28 days post-surgery by an overdose of sodium pentobarbital (150 mg/kg) via intraperitoneal injection.Male C57BL/6J mice  were maintained in a pathogen-free environment. Food and water were freely available under a controlled temperature (24 ± 1°C) with a 12/12 h dark/light cycle. We used a carotid artery injury mouse model, according to previously described protocols (R)-TML104 was synthesized and provided by Dr. Xun Sun's laboratory at the School of Pharmacy . Atorvastatin (MB1021) and resveratrol (MB1199) were purchased from Meilun Bio . BAY 11-7082 (S1523) and N-acetyl-L-cysteine (S0077) was purchased from Beyotime . 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Solarbio .Antibodies against α-smooth muscle actin  and β-Actin (AC026) were purchased from Abclonal . Antibodies against Ac-p65 (ab19870), NF-κB (ab16502), NOX1 (ab131088), NOX2 (ab129068), NOX4 (ab133303), proliferating cell nuclear antigen , cyclin D1 (ab134175), and SIRT1 (ab110304) were obtained from Abcam . PDGF-BB was purchased from R&D . Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 647 (A21235) and Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 555 (A21428) were purchased from Thermo Fisher Scientific .   for morphological analysis. Image-Pro Plus software  was used to determine neointima formation. A mean value was generated from five independent sections of each artery sample.The 5 μm slices were cut from paraffin-embedded blocks and placed on microscope slides. Briefly, the sections were microwaved in the citric acid buffer to retrieve antigens for 30 min. Sections were then permeabilized with 0.1% Triton X-100 for 15 min and blocked with 1% bovine serum albumin for 30 min, incubated with primary antibody at 4°C overnight. The following antibodies were used: PCNA (1:100), cyclin D1 (1:100), α-SMA (1:100), SIRT1 (1:100). Afterward, sections were washed with PBS and incubated with appropriate secondary antibody  for 1 h at room temperature. Nuclei were then stained with DAPI. The images were obtained using a Zeiss LSM880 microscope . The integrated optical density values were obtained using the ImageJ Pro Plus software .2/95% air atmosphere and their identity were confirmed using α-smooth muscle actin antibody.Rat VSMC were enzymatically isolated from the Sprague-Dawley rats according to the protocols previously described . For funR)-TML104 for 24 h. Then, MTT reagent was added into the medium for a further 4 h. Next, the supernatant was then discarded, and dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. The absorbance was measured at 570 nm with a microtiter plate reader .The viability of VSMC was determined with 3--2, 5-diphenyl-2H-tetrazolium (MTT) bromide assay kit . Briefly, the VSMC were plated in a 96-well microplate . After the VSMC were incubated with different concentrations of (R)-TML104 for 4 h and subsequently treated with PDGF-BB for 24 h, and then incubated with EdU for 2 h. Next, the cells were fixed with 4% paraformaldehyde  for 30 min, permeabilized with 0.1% Triton X-100 for 10 min, and the cells were stained with Hoechst 33342 (50 μL/well) for 10 min. The images were captured using fluorescence microscopy . The ratio of EdU-positive cells (EdU-stained cells/Hoechst-stained cells ×100%) was determined using a fluorescence microscope .We used a 5-Ethynyl-2′-Deoxyuridine (EdU) incorporation assay  to detect the proliferation of VSMC. Briefly, VSMC were seeded in 96-well plates. After growing to 60% confluence, the cells were serum-starved for 24 h. After the VSMC were incubated with different concentrations of (R)-TML104 for 4 h and subsequently treated with PDGF-BB for 24 h. The images were taken by light microscopy . Wound healing images were analyzed using ImageJ Pro Plus software.VSMC were seeded in a 6-well plate and scraped with a sterile tip in a straight line. The cells were immediately washed with cold phosphate buffer saline (PBS). After growing to 60% confluence, the cells were serum-starved for 24 h. After the VSMC were incubated with different concentrations of (R)-TML104 for 4 h and subsequently treated with PDGF-BB for 18 h, the transwell membranes were fixed with 4% paraformaldehyde for 15 min. The membranes were stained with a 0.1% crystal violet solution for 10 min. The non-migrating cells on the top surface of the membrane were scraped with a cotton swab. Images were captured using light microscopy to quantify the average number of migrated cells. Five randomly chosen high-power fields (×200) in three independent experiments were used to calculate the average number of migrated cells. The migratory cells were evaluated by ImageJ Pro Plus software.The migration assay was performed using a transwell chamber . Briefly, VSMC were seeded into each well of the upper chamber, and PBS or PDGF-BB were loaded into the bottom chamber. After growing to 60% confluence, the cells were serum-starved for 24 h. After the VSMC were incubated with different concentrations of . Equal amounts of protein were then separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skim milk plus tris-buffered saline for 1 h, the membrane was incubated with a primary antibody. The following antibodies were used: Ac-p65 (1:1000), NF-κB (1:2000), NOX1 (1:2000), NOX2 (1:2000), NOX4 (1:2000), PCNA (1:1000), cyclin D1 (1:1000), α-SMA (1:500), SIRT1 (1:1000), and β-Actin (1:10000) at 4°C overnight. The next day, the membrane was washed three times and then incubated with secondary antibodies (1:5000) for 1 h. Finally, the immunoreactive proteins were visualized using a chemiluminescence reagent . Signals were detected using a chemiluminescence system . The β-Actin loading control was used for quantifying protein expression levels.2O2 levels were detected using a Hydrogen Peroxide Assay Kit  according to the manufacturer's instructions. Briefly, scrape the lysed VSMC with a pipette tip and transfer cell lysate to a microcentrifuge tube. The cells were sufficiently homogenized and then centrifuged at 12 000 g for 5 min at 4°C. The supernatant was then incubated with a detection reagent for 30 min. The H2O2 production was assessed by using a microtiter plate reader.The dye, 2, 7-dichlorofluorescein diacetate  was served as a fluorescence probe to detect intracellular ROS. Briefly, VSMC were incubated with DCFH-DA in a dark container at 37°C for 30 min. The cells were washed three times with PBS and finally analyzed using the FACSCalibu flow cytometry system . The relative mean fluorescence intensity of each sample was analyzed using Flow Jo software version 10 . Intracellular HTo determine the mRNA expression levels of genes, total RNA was isolated from VSMC using TRIzol reagent , and cDNA was synthesized using a Prime Script RT reagent Kit according to the manufacturer's instructions. SYBR Green PCR reagents  were used to determine the relative expression of all gene transcripts by a Real-Time PCR Detection System . The expression of sample genes was quantified by the level of the β-Actin gene. The specific primers of NOX1, NOX2, NOX4, and β-Actin were available in The SIRT1 short hairpin was linearized plasmid and ligated into the pLVX vector. Lentivirus was produced by co-transfection of the SIRT1 lentiviral construct, the packaging plasmid psPAX2, and the envelope plasmid pMD2.G into HEK-293 T cells using Lipofectamine 3000 . The virus supernatant was harvested at 24 h and 48 h after transfection and stored at 4°C until the concentration step. The supernatant was filtered through a 0.45 μM filter and then centrifuged at 100,000 g for 2 h. The collected virus pellet was stored at −80°C. The mature antisense sequences of sh-SIRT1-1 and sh-SIRT1-2 were available in post-hoc test. All statistical analyses were performed using GraphPad Prism . Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001.Data were expressed as mean ± SD. Differences among three or more groups were determined using analysis of variance (ANOVA) followed by Tukey's R)-TML104 affected injury-induced neointima formation, we treated the mice with two doses of (R)-TML104  after injury. (R)-TML104 treatment significantly decreased injury-induced neointimal area (R)-TML104 treatment mitigated injury-induced downregulating α-SMA protein expression and upregulating PCNA and cyclin D1 expression (R)-TML104 at 20 mg/kg exhibited optimal protective effects and we used this dose for subsequent studies. To further confirm the function of (R)-TML104 on neointima formation, we used atorvastatin as a positive control. Interestingly, (R)-TML104 exhibits more prominent beneficial effects on neointima formation than atorvastatin at the same dosage (R)-TML104 could mitigate neointima formation in vivo.To investigate whether -TML104 affects PDGF-BB-induced VSMC phenotypic transformation, we first detected the cytotoxicity of (R)-TML104 on VSMC. The MTT assay showed that (R)-TML104 (1-10 μM) had no significant effect on the viability of VSMC (R)-TML104 concentration-dependently reversed PDGF-BB-induced the expression of α-SMA, PCNA, and cyclin D1. Among the doses examined, (R)-TML104 at 10 μM exhibited optimal inhibitory effects yet no cytotoxic effect and we used this dose for subsequent studies. Meanwhile, the EdU assay showed that (R)-TML104 could inhibit PDGF-BB-mediated VSMC proliferation (R)-TML104 could abolish PDGF-BB-induced VSMC migration -TML104 on VSMC phenotypic transformation, we chose atorvastatin and resveratrol as positive controls (R)-TML104 was similar to that of atorvastatin (R)-TML104 at the same dosage exhibited greater protective effects on these changes than resveratrol. Collectively, these results indicate that (R)-TML104 could inhibit PDGF-BB-induced VSMC phenotype transformation in vitro.To further explore the effects of -TML104 inhibits PDGF-BB-mediated VSMC phenotypic transformation by modulating SIRT1. We then detected the expression of SIRT1 in VSMC in response to PDGF-BB. We found that SIRT1 expression was time-dependently and dose-dependently upregulated by (R)-TML104 treatment (R)-TML104 exerted more significant effects on SIRT1 expression than resveratrol at the same dosage -TML104 treatment (R)-TML104-mediated SIRT1 expression and abolished the inhibitory effects of (R)-TML104 on VSMC phenotypic transformation, as evidenced by increased PCNA expression (R)-TML104 inhibits PDGF-BB-induced VSMC phenotypic transformation via upregulating SIRT1 in vitro.Our results showed that PDGF-BB decreased SIRT1 expression in VSMC, which is restored by -TML104 on neointima formation were mediated by SIRT1 in vivo, we then investigated the expression of SIRT1 in mice. We found that SIRT1 expression was decreased in vascular tissue after injury, which was reversed by (R)-TML104 treatment (R)-TML104-mediated SIRT1 expression and significantly abolished the protective effect of (R)-TML104 on neointima formation (R)-TML104-mediated PCNA, cyclin D1 and α-SMA expression was abolished by genetic SIRT1 knockdown (R)-TML104 inhibits neointima formation by upregulating the expression of SIRT1 in vivo.To investigate whether the inhibitory effects of -TML104 could modulate PDGF-BB-induced NOX expression in VSMC, the expression of NOX1, NOX2, and NOX4 in VSMC was measured. NOX1, NOX2, and NOX4 were all significantly higher both at the protein and mRNA level in VSMC in response to PDGF-BB when compared with control groups (R)-TML104 treatment specifically inhibited the PDGF-BB-induced NOX4 expression, but not NOX1 or NOX2 expression both at the protein and mRNA level (R)-TML104 treatment also markedly reduced PDGF-BB-induced production of ROS -TML104 on VSMC phenotypic transformation, as evidenced by decreased PCNA expression, increased α-SMA expression -TML104 on PDGF-BB-induced VSMC phenotypic transformation (R)-TML104 inhibits PDGF-BB-induced VSMC phenotypic transformation through the NOX4-ROS signaling pathway.To detect the role of ROS in PDGF-BB-induced VSMC phenotypic transformation, VSMC were treated with a ROS scavenger, N-acetyl-L-cysteine . Our results showed that NAC treatment significantly alleviated the PDGF-BB-increased ROS  and H2O22 levels . Meanwhiormation , prolifeormation  and migrormation . CollectR)-TML104-mediated NOX4 expression is regulated by SIRT1 in VSMC. Next, we measured the expression of NOX4 in VSMC by Western blot. It showed that SIRT1 knockdown by siRNA reversed (R)-TML104-mediated NOX4 expression in VSMC (R)-TML104 treatment. We then examined whether (R)-TML104-increased SIRT1 modulated NF-κB acetylation in VSMC. Next, we knocked down the expression of SIRT1 in VSMC by siRNA transfection. Our results showed that SIRT1 knockdown abolished the inhibitory effect of (R)-TML104 on the acetylation of NF-κB -TML104, suppressed PDGF-BB-induced NOX4 expression (R)-TML104-upregulated SIRT1 inhibits PDGF-BB-induced VSMC phenotypic transformation by downregulating NOX4 expression via decreasing NF-κB acetylation.To assess the role of NF-κB in NOX4 expression in VSMC, we used BAY 11-7082 (NF-κB inhibitor) to inhibit NF-κB activation. BAY 11-7082 treatment, similarly to (pression . CollectR)-TML104 could prevent neointima formation in vivo. Furthermore, (R)-TML104 inhibited PDGF-BB-induced VSMC phenotypic transformation in vitro. We also found that SIRT1 expression is critical for (R)-TML104 to exert its protective effects. Finally, (R)-TML104 inhibited PDGF-BB-induced VSMC phenotypic transformation through NOX4 modulation via decreasing NF-κB acetylation. In summary, we found that (R)-TML104 against neointima formation and upregulates SIRT1 expression -TML104 could prevent vascular diseases, which was confirmed by our results showing that (R)-TML104 inhibited PDGF-BB-induced VSMC phenotypic transformation and injury-induced neointima formation. Next, we used atorvastatin as a positive control in vivo (R)-TML104 against neointima formation were better than atorvastatin at the same dosage. We speculate that this superior effect of (R)-TML104 in vivo may be due to the key role of SIRT1, a well-known regulatory target of resveratrol, in the process of neointima formation (R)-TML104.Previous work has demonstrated that resveratrol can protect from vascular disease . We hypo in vivo . Interesormation . The expormation . In lineR)-TML104. Consequently, we concluded that NOX4 down-regulation is responsible for the anti-oxidative effects of (R)-TML104 that confer vascular protection. In contrast, Chandrika showed that NOX4-derived ROS play an inhibitory role in the differentiation phenotypic of diabetic atherosclerosis -TML104-increased SIRT1 inhibited PDGF-BB-induced NOX4 expression in VSMC, whereas SIRT1 knockdown abolished (R)-TML104-mediated inhibitory effects on NOX4 expression. Therefore, the fact that (R)-TML104 inhibits the PDGF-BB-induced expression of NOX4 likely depends on SIRT1 expression in VSMC. Previous studies have highlighted the influence of NF-κB-induced oxidative stress on the modulation of VSMC phenotypic transformation (R)-TML104-reduced oxidative stress was associated with NF-κB activation. We found that (R)-TML104-increased SIRT1 inhibited NOX4 expression by reducing the acetylation status of NF-κB. This result is consistent with a previous report that SIRT1 regulated NOX4 expression by attenuating NF-κB acetylation in pancreatic cancer cachexia -TML104 on other factors-induced VSMC proliferation would be investigated as a follow-up study.PDGF-BB is not the only factor that drives the injury-induced neointima formation , 50. A lR)-TML104-increased SIRT1 expression led to a reduction in NF-κB acetylation, thereby inhibit PPDGF-BB-induced VSMC phenotypic transformation by down-regulating NOX4 expression. Taken together, our findings suggest that (R)-TML104 may be an important therapeutic drug to prevent neointima formation.In summary, our data revealed that , the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province (Grant no: BK20200026), Jiangsu Province Recruitment Plan for High-level, Innovative and Entrepreneurial Talents (Innovative Research Team), Wuxi Social Development Funds for International Science & Technology Cooperation (Grant no: WX0303B010518180007PB), Jiangsu Province “Six Summit Talents” program (Grant no: YY-038), Jiangsu Province Qing Lan Project, the Fundamental Research Funds for the Central Universities (Grant nos: JUSRP221037 and JUSRP22007), and Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province and Wuxi Taihu Talent Project. Shanghai Municipal Committee of Science and Technology (Grant no: 17JC1400200).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.