Convergence of these two categories of genes in an individual pathogen would deteriorate the antimicrobial resistance (AMR) crisis furthermore. Here, tigecycline-resistant Enterobacterales strains were isolated and detected with carbapenemase genes, characterized by antimicrobial susceptibility testing, PCR, conjugation assay, whole genome sequencing, and bioinformatics analysis. Three tigecycline-resistant isolates consisting of one plasmid-mediated tet(X4)-bearing Escherichia fergusonii and two chromosomal tet(X6)-bearing Proteus cibarius were recovered from chicken feces. The tet(X4) was located on a conjugative IncX1 plasmid pHNCF11W-tetX4 encoding the identical structure as reported tet(X4)-bearing IncX1 plasmids in Escherichia coli. Among two P. cibarius strains, tet(X6) was located on two similar chromosomal MDR regions with genetic contexts IS26-aac(3)-IVa-aph(4)-Ia-ISEc59-tnpA-tet(X6)-orf-orf-ISCR2-virD2-floR-ISCR2-glmM-sul2 and IS26-aac(3)-IVa-aph(4)-Ia-ISEc59-tnpA-tet(X6)-orf-orf-ISCR2-glmM-sul2. Apart from tet(X6), P. cibarius HNCF44W harbored a novel transposon Tn6450b positive for blaNDM–1 on a conjugative plasmid. This study probed the genomic basis of three tet(X)-bearing, tigecycline-resistant strains, one of which coharbored blaNDM–1 and tet(X6), and identified P. cibarius as the important reservoir of tet(X6) variants. Emergence of P. cibarius encoding both blaNDM–1 and tet(X6) reveals a potential public health risk.The emergence of  Carbapenem and tigecycline are regarded as vital antimicrobials reserved for clinical use due to their broad antibacterial spectrum. However, the increasing spread of carbapenemase-encoding genes has rendered carbapenem-resistant Enterobacteriaceae (CRE) infection a great threat . Plasmidl origin . This untet(X) genes were mainly found in Bacteroides species and bacteria derived from environmental microbiota (tet(X) variants are being identified from the pathogens of animal, food, and human origins (tet(X) is expanding from commensals to pathogens (tet(X) and carbapenem-resistant genes in Enterobacterales may be inevitable. Acinetobacter strains coharboring tet(X) and blaNDM–1 were identified from samples of waterfowl and dairy cows recently (tet(X) and blaNDM–1 is unknown, which constitutes a more severe concern. To cover this knowledge gap, we performed a tigecycline-resistant Enterobacterales screening pilot program and identified a Proteus cibarius coharboring both tet(X6) and blaNDM–1, implying that wide surveillance of Enterobacterales conferring resistance to carbapenems and tigecycline among bacteria should be performed worldwide.Initially, crobiota . Alarmin origins , which iathogens . Thus, trecently , highligtet(X) and carbapenem-resistant genes were determined by PCR with reported primers . The species of purified tigecycline-resistant isolates was identified by 16S rRNA gene sequencing and further confirmed by analyzing WGS data. The  primers .tet(X)-positive tigecycline-resistant strains against 13 antibiotics was performed based on the broth microdilution method with Escherichia coli ATCC 25922 as the quality control and interpreted according to CLSI guidelines  of idelines . The tigidelines .E. coli C600 (Rifr) as the recipient to investigate the transferability of tet(X) and blaNDM–1. The donor and recipient strains were mixed at a ratio of 1:4 on a filter, followed by culturing on LB agar plates at 30 and 37°C, respectively, overnight and screening on MacConkey plates containing tigecycline (4 mg/L) or meropenem (2 mg/L) with rifampicin (300 mg/L). Transconjugants were confirmed with PCR. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) was utilized to characterize the plasmid profiles.A conjugation assay was conducted using rifampicin-resistant de novo assembled sequences were annotated by RAST1. Antibiotic resistance genes (ARGs) and plasmid replicon types were determined using the ResFinder and PlasmidFinder2. BRIG and Easyfig were used to compare the plasmid and MDR structures (Genomid DNA (gDNA) was purified with the TIANamp Genomic DNA Kit and quantified by Qubit 4 Fluorometer. Short-read Illumina sequencing and long-read Nanopore sequencing were combined to generate complete genome sequences . The de ructures .tet(X6) variant, TA-cloning and AST were performed. Briefly, the new variant, together with its natural promoter sequence, was amplified by PCR using primers HNCF44W-F/AGCGAACAAGAATATGACTTTACT and HNCF44W-R/CGCCTTTCTGTTTTATAGATTCAT, cloned into a pMD19-T vector and transformed chemically into E. coli DH5α. The resistance phenotype of tet(X6) was tested by measuring the MICs of different tetracycline antibiotics. Phylogenetic analysis of Tet(X) amino acid sequences were conducted in MEGA X using the neighbor-joining method .The complete sequences obtained in this study have been deposited in the GenBank database under BioProject number tet(X)-positive Enterobacterales strains were isolated from 16 samples (18.8%), which consisted of Escherichia fergusonii HNCF11W carrying tet(X4), P. cibarius HNCF43W, and HNCF44W positive for a tet(X6) variant. Notably, HNCF44W also carried the blaNDM–1 gene. Antimicrobial susceptibility testing revealed that the three tigecycline-resistant strains were resistant to multiple antibiotics, including kanamycin, florfenicol, amoxicillin, and tetracyclines, showing high-level resistance to tigecycline (tet(X4)-bearing E. fergusonii HNCF11W, and one plasmid of ca. 140 kb existed in tet(X6)-bearing P. cibarius HNCF44W, but no plasmid existed in P. cibarius HNCF43W (tet(X) and blaNDM–1, complete genome sequences of the three strains were finished successfully.Three tigecycline-resistant, ecycline . In addi HNCF43W . To uncotet(X4)-bearing E. fergusonii harbored one chromosome ; one p0111-like plasmid pHNCF11W-130 kb  harboring aadA1, floR, tet(A), dfrA14, blaTEM–209, blaOXA–10, cmlA1, arr-3, and qnrS1; and one typical IncX1 plasmid pHNCF11W-tetX4  encoding tet(X4) in the genetic structure of IS26-abh-tet(X4)-ISCR2-virD2-floR-△ISCR2 coupled with aadA2, lnu(F), tet(A), and blaSHV–12. A BLASTn search of pHNCF11W-tetX4 against the nr database retrieved other tet(X4)-bearing plasmids p1916D18-1 , p1916D6-2 , and pYY76-1-2  of swine and cattle origins, sharing nearly identical sequences (tet(X4)-bearing plasmids were mobilizable and spread in Enterobacterales of animal origin (tet(X4) was initially found in plasmids of IncFIB and IncQ1 types (tet(X4) between different Enterobacterales.The equences . Both pHl origin . AlthougQ1 types , IncX1 pQ1 types , which pblaNDM–1-bearing plasmid pHNCF44W_NDM-1 of 139,490 bp in size. A conjugation assay failed to recover transconjugants positive for tet(X6). The tet(X6) gene was located on large MDR regions (IS26-aac(3)-IVa-aph(4)-Ia-ISEc59-tnpA-tet(X6)-orf-orf-ISCR2-virD2-floR-ISCR2-glmM-sul2 and IS26-aac(3)-IVa-aph(4)-Ia-ISEc59-tnpA-tet(X6)-orf-orf-ISCR2-glmM-sul2, respectively) embedded in chromosomes of both P. cibarius strains (tet(X6)-bearing elements and reported plasmids p19110F47-2 (CP046044) and pJXP9 (MK673549) (tet(X6)-bearing MDR regions differed by virD2-floR-ISCR2, which was a potential mobile segment (CR2 is the major mobile element associated with tet(X3), tet(X4), and tet(X5) (tet(X6) could be embedded in ICEs or other MDR regions, all of which harbor ISCR2 (tet(X6) to other plasmids or chromosomes.HNCF43W harbored a 3,965,977 bp chromosome. HNCF44W contained a 3,929,176 bp chromosome and one  strains , and theK673549) . The two segment . ISCR2 i tet(X5) . Recentlor ISCR2 , suggestblaNDM–1 gene in HNCF44W was located on an MDR plasmid pHNCF44W_NDM-1 carrying resistance genes aac(6′)-Ib, arr-3, qacE1, sul1, sul2, aph(3′)-la, mph(E), msr(E), qnrA1, aadA1, aadA2, dfrA1, dfrA12, floR, aac(3)-IVa, and lnu(F). No plasmid replicon was identified, but pHNCF44W_NDM-1 was very similar to plasmid plas1.1.2 (CP047114) from Proteus mirabilis of swine origin; it differed mainly in the blaNDM–1-bearing region -bearing chromosomal segment, rendering this a Tn6450b-bearing plasmid with the potential ability to capture tet(X6) under certain circumstances, which warrants further surveillance.The g region . The blablaNDM–1 , designa50b here . Other stet(X6) variants from diverse bacteria (tet(X6) variant carried by HNCF44W and HNCF43W is the same as that reported in P. cibarius from swine (tet(X6) in the cloning vector pMD19-T-tet(X6) in E. coli DH5α conferred resistance to tetracycline (64 mg/L) and tigecycline (16 mg/L) with 32-fold increments compared to that of DH5α-pMD19-T, indicating that this tet(X6) variant can confer tigecycline resistance (tet(X6) variant encoded 378 amino acids and showed 84.7, 66.3, 84.9, 79.9, 87.8, 93.7, and 98.7% amino acid sequence identity to Tet(X), Tet(X1), Tet(X2), Tet(X3), Tet(X4), Tet(X5), and Tet(X6), respectively  variants were found in Proteus and Acinetobacter spp., analysis of the NCBI database shows that E. coli positive for tet(X6) existed as early as 2003 in Denmark (JWJM01000174), indicating the transmission of tet(X6) occurred previously. Furthermore, chromosomal tet(X6)-bearing ICEs and MDRs are found in Proteus, Acinetobacter, and Myroides phaeus (tet(X6) in Proteus, Acinetobacter, and E. coli also emerged according to published reports (tet(X6) is at the stage of being expanded via different mobile elements. This highlights that tet(X6) variants would be the next possible prominent tet(X) allele, approaching the widespread situation of tet(X4) in E. coli (tet(X) by different allele numbers is not recommended because many of the alleles are 85–100% aa identical3, but an identical tet(X) naming system may not benefit research communications and AMR surveillance, especially as different tet(X) alleles possess different resistance phenotypes, genetic contexts, hosts, and evolutionary routes as more research reveals. For example, tet(X4) was found to be more common in E. coli than tet(X6) owing to the fact that tet(X4) has merged into conjugative plasmids adapted in E. coli. Also, the plasmids coharboring both blaNDM–1 and tet(X) in Acinetobacter spp. (tet(X3)- or tet(X6)-bearing plasmids, which could make it difficult to decipher the accurate different genetic contexts if only tet(X) represented the two alleles simultaneously. To better investigate the molecular epidemiology, genomic evolution, and functional analysis and avoid confusion in tet(X) designations, a clear nomenclature scheme acceptable to researchers worldwide should be proposed as soon as possible. Because mcr genes exist in certain bacterial species as housekeeping genes and certain alleles leap over species boundary limits to Enterobacterales via mobile elements like Tn6330 (tet(X) genes may behave similarly because they exist as core genes in certain bacteria and transfer to Enterobacterales by mobile elements like ISCR2. Thus, the mcr nomenclature scheme may be a good example when it comes to tet(X) designation (tet(X) allele numbering system to facilitate research communications.Phylogenetic analysis of Tet(X) indicated that of all reported Tet(X6) variants clustered in a clade, most related to Tet(X5) . Althougs phaeus . Plasmid reports , implyin E. coli . Nomenclter spp.  could bee Tn6330 , tet(X) ignation . More retet(X)-bearing Enterobacterales strains of chicken origin and found that one P. cibarius coharbored both blaNDM–1 and tet(X6). To the best of our knowledge, this is the first report of convergence of tet(X6) and blaNDM–1 genes in Enterobacterales, highlighting the importance of continuous surveillance of tigecycline- and carbapenem-resistant Enterobacterales of animal origin, which may act as the potential reservoir of clinical Enterobacterales resistant to tigecycline and carbapenems.To conclude, we characterized three The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/RL and ZW: conceptualization, writing – review and editing, and supervision. YaL and RL: methodology. YaL and QW: investigation. QW, KP, and YuL: data curation and visualization. YaL: writing – original draft preparation. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous work in C. elegans has shown that SPAS-1 disassembles microtubules in an ATP-dependent manner . Another study shows that SPAS-1 plays a role in synapse remodeling in motor neurons . Here we describe the expression of SPAS-1 in L4 hermaphrodite larvae using the transcriptional reporter, axrEx8[Pspas-1:mCh]: A) Low magnification (10X objective) image of an entire L4 larva. mCherry expression is driven by the spas-1 promoter (sequence provided). Expression is observed in the nerve ring, ventral cord, vulva, and tail ganglia. Expression is also detected in the intestine (asterisks). B) High magnification (100X objective) image of SPAS-1 expression in the developing vulva of an L4 hermaphrodite. C) High magnification (100X objective) image of nerve ring expression. D-F) SPAS-1 expression in the tail ganglia (TG), ventral cord (VC), and mechanosensory neurons (PLM and AVM shown here). Mechanosensory neuron expression is confirmed by overlaying GFP and mCherry fluorescence in axrEx8 [Pspas-1:mCh]; zdIs5 [Pmec-4:GFP + lin-15 (+)] animals. Arrows point at the following mechanosensory neurons: PLM=Posterior Lateral Microtubule cell, AVM=Anterior Ventral Microtubule cell.SPAS-1 is expressed in several neuronal types as well as in the intestine and developing vulva in L4 larvae. These findings complement expression data by Matsushita-Ishiodori who focused on SPAS-1 expression in embryos.Our work shows that axrEx8; zdIs5.Transgenes: axrEx8 [Pspas-1:mCh] transcriptional fusion construction details: 480bp upstream of the spas-1 ATG cloned into Pdonr4-1 gateway vector. Expression clone built by LR multisite recombination to drive mCherry expression. Strain REB22, injection concentration 20ng/ul.spas-1 promoter sequence: provided by the authors and included in the transgene details.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain UF-8, with an in-frame 12-nucleotide deletion within open reading frame 3a (ORF3a), was isolated from a 78-year-old COVID-19 patient in March 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain UF-8, with an in-frame 12-nucleotide deletion within open reading frame 3a (ORF3a), was isolated from a 78-year-old COVID-19 patient in March 2020. A 78-year-old woman with COVID-19 at the University of Florida hospital’s intensive care unit in March 2020 improved clinically and was discharged 6 days later. The studies were reviewed and approved by the University of Florida Institutional Review Board (IRB).https://www.cdc.gov/coronavirus/2019-ncov/lab/virus-requests.html), generating a quantification cycle (qC) value of 28 for primer set N1. As insufficient vRNA was extracted for direct sequencing using our standard next-generation sequencing (NGS) method, which requires a qC value of ≤20 to obtain adequate coverage, an aliquot of the NP specimen was inoculated onto Vero E6 cells in a T25 flask and incubated at 37°C for virus isolation and the preparation of larger amounts of vRNA for sequencing. The virus required 7 days to attain a virus titer of 2.5 × 106 PFU/ml and induce cytopathic effects (CPEs), whereas other wild-type strains usually attain a titer of ≥3 × 106 PFU/ml and induce CPEs 3 days postinoculation of the cells under the same conditions. RNA purified from the cell growth medium using a QIAamp viral RNA kit was subsequently submitted for NGS  from the patient using a QIAamp viral RNA minikit  tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (vRNA) using the CDC real-time reverse transcriptase PCR (rRT-PCR) diagnostic test for COVID-19  in CLC Genomics Workbench v20.0.3, with default parameters, recovered the near-complete genome sequence, 29,847 nucleotides (nt) with a G+C content of 38% excluding the first 11 nt at the 5′ end and poly(A) tail. The virus was designated strain UF-8. The genomic sequence of UF-8 was confirmed by repeating the NGS process and by site-directed RT-PCR and Sanger sequencing of RNA extracted directly from the NP swab to confirm the 12-nt deletion. Phylogenetic analyses performed as previously described  of the Vero E6 cell host reads were removed using Kraken v2.0 with default parameters. Demultiplexing was automatically performed with the MiSeq software; no trimming was performed. escribed .Importantly, the 12-nucleotide in-frame deletion occurs within open reading frame 3a (ORF3a), which encodes viroporin 3a . ViroporAfter filtering, 2,452 genomes, including 30 new UF isolates (UF1 to UF30), were retained and aligned using MAFFT v7.407 , using tMW221275.1. The SRA accession number for the raw data is SRR13091823.The complete coding DNA sequence (CDS) of SARS-CoV-2/human/USA/UF-8/2020 has been deposited in GenBank under accession number


Background: Sirtuin 3 (SIRT3) has a crucial role in the cardiovascular diseases. Our previous study revealed that SIRT3 knockout (SIRT3KO) promoted cardiac pericyte–fibroblast transition. In this study, we investigated the involvement of pericyte and iron in angiotensin II (Ang-II)-mediated renal fibrosis in the SIRT3KO mice. Methods and Results: NG2-DsRed mice and NG2-DsRed-SIRT3 knockout (SIRT3KO) mice were infused with saline or Ang-II (1000 ng/kg/min) for 4 weeks. Renal fibrosis, iron content and reactive oxygen species (ROS) were measured. Masson’s trichrome staining showed that SIRT3KO enhanced Ang-II-induced renal fibrosis. Immunostaining showed that Ang-II treatment increased the number of NG2-DsRed+ cells in the kidney, and SIRT3KO further enhanced NG2-DsRed+ cells. Moreover, SIRT3KO promoted pericyte differentiation into fibroblasts as evidenced by co-staining NG2-DsRed/FSP-1. Furthermore, DsRed/FSP-1+ and DsRed/transforming growth factor-β1 (TGF-β1)+ fibroblasts were elevated by SIRT3KO after Ang-II infusion. Ang-II-induced collagen I and TGF-β1 expression was also enhanced in the SIRT3KO mice. SIRT3KO significantly exacerbated Ang-II-induced iron accumulation. This was accompanied by an increase in acetyl-p53, HO-1 and FPN expression. Further, SIRT3KO sensitized Ang-II-induced upregulation of p47phox and gp91phox together with increased ROS formation in the kidney. Conclusion: Our study suggests that SIRT3 deficiency sensitized Ang-II-induced renal fibrosis by the mechanisms involved in promoting differentiation of pericytes into fibroblasts, exacerbating iron overload and accelerating NADPH oxidase-derived ROS formation. Hypertension is one of the major causes of renal diseases ,2,3. TheSirtuin 3 (SIRT3), a mitochondrial nicotinamide adenine dinucleotide (NAD)+-dependent deacetylase, plays a key role in hypertension-induced renal injury ,13. ReceHowever, whether pericytes are involved in the pathogenesis of hypertensive renal fibrosis in SIRT3-deficient mice has not been studied yet. In this study, using NG2-DsRed mice to trace pericytes, we aimed to investigate the mechanism of hypertension-induced renal fibrosis involving SIRT3-associated pericyte pathway.All procedures conformed to the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. It was also approved by the Animal Care and Use Committee of University of Mississippi Medical Center (protocol identifier: 1280C). The investigation conformed to the National Institutes of Health  Guide for the Care and Use of Laboratory Animals .Wild-type (WT) control, SIRT3 knockout (SIRT3KO) (Stock#012755) mice and NG2 tracing reporter NG2-DsRedBAC (Cspg4-DsRed) (Stock#008241) mice were obtained from Jackson Laboratory  and were bred by our laboratory. SIRT3KO mice were determined by polymerase chain reaction using the following primers: SIRT3KO common, 5′-CTT CTG CGG CTC TAT ACA CAG-3′; SIRT3KO wild-type reverse, 5′-TGC AAC AAG GCT TTA TCT TCC-3′; SIRT3KO mutant reverse, 5′-TAC TGA ATA TCA GTG GGA ACG-3′. NG2-DsRedBAC mice were determined using the following primers: NG2-DsRed transgene forward, 5′-TTC CTT CGC CTT ACA AGT CC-3′; NG2-DsRed transgene reverse, 5′-GAG CCG TAC TGG AAC TGG-3′; NG2-DsRed positive control forward, 5′-CTA GGC CAC AGA ATT GAA AGA TCT-3′; NG2-DsRed positive control reverse, 5′-GTA GGT GGA AAT TCT AGC ATC C-3′ . SIRT3KO mice were crossed with NG2-DsRedBAC mice. Among these crossed mice, homozygous SIRT3KO mice were selected by PCR firstly. Then, NG2-DsRed-SIRT3KO mice were selected from homozygous SIRT3KO mice for the experiments as previously reported . ExperimFor inducing hypertension, both SIRT3KO and control mice (9 mice/group) were infused with angiotensin (Ang)-II (1000 ng/kg/min) for 4 weeks via subcutaneously implanted Alzet mini-osmotic pumps  under anesthesia .n = 3 mice/group) were fixed in neutral buffered 10% formalin solution  and embedded in frozen optimal cutting temperature compound . Frozen sections and paraffin sections were prepared (8 µm in thickness). Masson’s trichrome staining (paraffin sections) was performed to measure the degree of renal fibrosis (blue). ROS (frozen sections) was measured by dihydroethidium (DHE) staining. Iron content (frozen sections) was measured by Prussian blue staining . The area percentage of fibrosis, iron content and ROS fluorescence intensity were quantified by measuring six random microscopic fields using image-analysis software . The renal tissues  and DsRed antibody  for pericytes, immunostaining of fibroblast-specific protein 1 (FSP-1)  for fibroblasts and immunostaining of TGF-β1 primary antibodies  were also conducted in fresh frozen sections. These immune-stained sections were incubated with second antibodies conjugated with fluorescein isothiocyanate (1:500). A Nikon microscope, Nikon digital camera and Nikon software  were used to capture images. Six random microscopic fields were analyzed using image-analysis software . n = 3–4 mice/group) were collected and homogenized in lysis buffer. The homogenates were centrifuged at 16,000× g at 4 °C for 15 min. A BCA Protein Assay Kit  was used to analyze the protein concentrations. Equal amounts (20 µg) of the protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated with the following primary antibodies overnight: collagen I , TGF-β1 , angiopoietin-1 , angiopoietin-2 , acetyl-p53 , heme oxygenase 1 (HO-1) , ferroportin , PFKFB3 , GLUT1 , p47phox , gp91phox  and β-actin . After washing, the membranes were incubated for 2 h with an anti-rabbit or anti-mouse secondary antibody coupled to horseradish peroxidase . Densitometric analyses were carried out with image acquisition and analysis software (Bio-Rad).Mouse renal cortex tissues  one day prior to the assay. The cells were then washed twice with unbuffered XF base medium (Agilent) and incubated at 37 °C without CO2 for 1 h. Three initial baseline measurements were performed by using the XFe24 Seahorse analyzer followed by the injection of glucose  and three additional measurements. Extracellular acidification rate (ECAR) was calculated in the Wave software (Agilent) and normalized to protein content in each well. Endothelial cells (ECs) were isolated from thoracic and abdominal aorta of WT and SIRT3KO mice and maintained in the EGM-2 medium , as previously described ,16,20. Ep values < 0.05 were considered significant .All analyses were performed using Statistical Package for Social Sciences version 20.0 . Data are expressed as means ± S.E.M. The assumption of normality in comparison groups was determined by normality test. The significance of differences in the means of corresponding values among groups was determined by using the one-way ANOVA. The significance of differences between two values was determined using LSD test. All the statistical tests were two-sided, and Western blot analysis demonstrated that SIRT3 expression was absent in the kidneys of SIRT3KO mice. After infusion of Ang-II for 4 weeks, SIRT3 levels remained unchanged in the kidneys of wild-type (WT) mice compared to WT without Ang-II . Next, wAccumulating evidence suggests an involvement of pericyte–myofibroblast transition in renal fibrosis. Using NG2-DsRed reporter mice, we investigated the potential contribution of NG2-DsRed pericytes in Ang-II-induced renal fibrosis. As shown in We then explored the potential mechanism by which knockout of SIRT3 led to an increase in pericytes in mouse kidneys. Angiopoietins are the key regulators of pericyte recruitment during angiogenesis. Our Western blot analysis revealed that the expression of angiopoietin-1 and angiopoietin-2 was significantly upregulated in the mouse kidneys after Ang-II infusion and this upregulation was further elevated by knockout of SIRT3 C,D. ImmuOur recent study demonstrated that knockout of SIRT3 promoted p53 acetylation, which has been shown to contribute to iron overload in mouse hearts . As shown in Our Western blot analysis also showed that deficiency of SIRT3 resulted in a significant suppression of the endothelial glucose transporter-1 (GLUT1) and glycolytic enzyme PFKFB3 when infused with Ang-II A,B. ThisIn the present study, we revealed that deficiency of SIRT3 deteriorated Ang-II-induced renal fibrosis. SIRT3 knockout mice exhibited more severe renal fibrosis after Ang-II infusion. Using NG2-DsRed mice and NG2-DsRed-SIRT3 knockout (SIRT3KO) mice to trace pericytes, our data further showed that these abnormalities might result from the increased numbers of pericytes, the production of TGF-β1 in pericytes and the differentiation of pericytes into fibroblasts. Interestingly, Ang-II infusion caused an iron overload in the mouse kidneys, and knockout of SIRT3 further promoted iron overload, which might also contribute to the exacerbation of renal fibrosis. Additionally, knockout of SIRT3 caused an impairment of glycolytic gene expression and accelerated NADPH oxidase-derived ROS formation after Ang-II infusion. Renal fibrosis is an important pathological manifestation of hypertensive nephropathy ,22. In tSIRT3 is a NAD+-dependent deacetylase in the mitochondria. In the mitochondria, heme is the main source of synthesized iron ,27,28. EEndothelial dysfunction also plays a crucial role in hypertensive renal injury . Recent In summary, deficiency of SIRT3 sensitized Ang-II-induced renal fibrosis by enhancing Ang-II-induced pericyte recruitment and pericyte–fibroblast transition, by promoting Ang-II-induced iron overload and by accelerating NADPH oxidase-derived ROS formation .


VS-5584 treatment of 786-O tumor xenografts and RCC cells resulted in feedback upregulation of bromodomain-containing protein 4 (BRD4). Furthermore, BRD4 inhibition (by JQ1 and CPI203), knockdown or complete knockout potentiated VS-5584-induced RCC cell death and apoptosis. Conversely, forced overexpression of BRD4 attenuated the cytotoxicity of VS-5584 in 786-O cells. Collectively, VS-5584 potently inhibits RCC cell proliferation and survival. Its anti-tumor activity is further enhanced by the targeted inhibition of BRD4.Activation of the PI3K/AKT/mTOR pathway promotes the progression of renal cell carcinoma (RCC). This study tested the anti-RCC cell activity of the PI3K/mTOR dual inhibitor, VS-5584. We show that VS-5584 inhibited PI3K/AKT/mTORC1/2 activation in established (786-O and A498 lines) and primary RCC cells, thereby suppressing cell survival, proliferation, migration and cell cycle progression. VS-5584 induced significant apoptosis in RCC cells. A daily single oral dose of VS-5584 (20 mg/kg) significantly inhibited 786-O tumor growth  Renal cell carcinoma (RCC) is the most common renal malignancy globally, causing significant human mortalities each year , 2. In cNovel molecularly-targeted agents are needed for better RCC treatment , 5. In RVS-5584 is a potent dual inhibitor of PI3K and mTOR . It dispBromodomain-containing protein 4 (BRD4), a member of the BET  family , binds aIn cancer cells BRD4 overexpression promotes cell survival, proliferation, and resistance to apoptosis . Recent 50 of VS-5584 was between 1-5 μM  significantly decreased BrdU ELISA OD  reduced the number of migrated 786-O cells  increased the percentage of cells in the G0/G1 phases, while decreasing the percentage of cells in the S and G2/M phases . Testing-O cells . At the in vitro . Treatmein vitro –1G. ThesCell death assay results showed that VS-5584 dose-dependently induced LDH release into the culture medium , indicatThe anti-tumor effects of VS-5584 were tested on the established human A498 RCC cells and two different primary human RCC cells, RCC1 and RCC2 (see our previous studies ). WesterThe ssDNA ELISA OD, an indicator of cell apoptosis, was increased in VS-5584-treated RCC cells . To furtin vivo, nude mice were subcutaneously inoculated with 786-O cells to form xenografts. Tumor growth curve analysis showed that a daily single dose of VS-5584  significantly inhibited 786-O tumor growth -treated 786-O cells in vitro -induced Bcl-2 and c-Myc upregulation . FurtherIn primary RCC cells (“RCC1”), VS-5584 treatment  induced feedback upregulation of BRD4, Bcl-2, and c-Myc . FurtherBecause the pharmacological BRD4 inhibitors (JQ1 and CPI203) might have off-target toxicities, genetic strategies were employed to alter BRD4 expression in 786-O cells. Two lentiviral BRD4 shRNAs, with non-overlapping sequences (“sh-BRD4-S1/S2”), were transfected into 786-O cells. Western blotting results showed that the protein expression of BRD4, as well as the BRD4-regulated c-Myc gene were significantly downregulated by BRD4 shRNA treatment . ImportaTo confirm BRD4 knockdown results, a CRISPR-Cas9-BRD4-KO plasmid was transfected into RCC 786-O cells to completely knockout BRD4 protein in the stable cells. In BRD4-KO cells, no BRD4 protein expression was observed even with VS-5584 treatment  potentiated VS-5584-induced RCC cell death and apoptosis. Furthermore, BRD4 knockdown or knockout enhanced VS-5584-induced cytotoxicity in RCC cells. Conversely, forced overexpression of BRD4 attenuated VS-5584-induced 786-O cell apoptosis.The pharmacological and genetic evidence provided by this study indicate that BRD4 is a VS-5584 drug resistance factor in RCC cells. BRD4 inhibition may be an important strategy to sensitize RCC cells to VS-5584. The observed resistance to a PI3K-Akt inhibitor could be driven by the feedback activation of receptor tyrosine kinases (RTKs) . It has In summary, VS-5584 potently inhibits RCC cell proliferation and survival. Its anti-tumor activity is further enhanced by the targeted inhibition of BRD4.VS-5584, JQ1, and CPI203 were obtained from Sigma-Aldrich . Cell culture reagents were purchased from Gibco . The antibodies were purchased from Cell Signaling Technology . Puromycin was obtained from Sigma-Aldrich.Established human RCC cell lines (786-O and A498) as well as HK-2 human renal epithelial cells were obtained as described previously , 33. The3cells per well). MTT assay was performed to test cell viability, according to the manufacturer’s instructions (Sigma-Aldrich). The MTT optical density (OD) at 590 nm was recorded.Cells were seeded onto a 96-well tissue culture plate  and counted.3 cells per well). The BrdU ELISA kit  was utilized to test cell proliferation in vitro. The BrdU ELISA absorbance at 405 nm was recorded.Cells were seeded onto 96-well tissue culture plates (3 × 105 cells per well). Following the applied treatment, cells were washed, fixed, and incubated with DNase-free RNase and PI. Cells were tested using a FACSCalibur instrument .The propidium iodide  flow cytometry assay was applied to test cell cycle distribution. Cells were seeded onto 6-well tissue culture plates (2 × 104 cells of each condition in 200 μL serum-free medium) were seeded on the upper surfaces of “Transwell” chambers, coated with Matrigel (Sigma) [As described human RCC cells or the HK-1 cells (4 × 10 (Sigma) , 36. The5cells/well) were seeded onto the six-well plates. An EdU (5-ethynyl-20-deoxyuridine) Apollo-488 In Vitro Imaging Kit  [RCC cells or the HK-1 cells   was appl5cells per well). Cell death was examined by measuring the LDH content in the medium, using a 2-step enzymatic reaction LDH assay kit . Percentage of LDH release = LDH released in conditional medium ÷ .Cells were seeded onto 6-well tissue culture plates (2 × 105cells per well). TUNEL In Situ Cell Death Detection Kit  was utilized to quantify the number of TUNEL-labeled apoptotic nuclei.As described previously , cells wAfter the applied treatment, cells were treated with lysis buffer . The totssDNA accumulation is a characteristic marker of cell apoptosis. For each treatment, 30 μg of cell lysate (using the lysis buffer for western blotting) was analyzed. A ssDNA ELISA kit (Roche Diagnostics) was utilized to quantify DNA fragmentation. The ssDNA ELISA absorbance was recorded at 450 nm.5cells per well). Cells were transfected with BRD4 shRNA lentivirus for 24 h. Puromycin (2 μg/mL) was then used to select stable cells (4-5 passages). BRD4 knockdown in the stable cells was confirmed by Western blotting. Control cells were transfected with lentiviral scramble control shRNA (Santa Cruz Biotechnology).Two different lentiviral BRD4 shRNAs, with unique and non-overlapping sequences (“S1/S2”), were provided by Dr. Zhao . 786-O cThe pSUPER-puro-BRD4-GFP expression vector was provided by Dr. Zhao , and wasThe CRISPR/Cas9 BRD4 KO plasmid  was transfected to 786-O cells using Lipofectamine 2000 reagent , and selected with puromycin after 4-5 passages. Control cells were treated with an empty vector with control small guide RNA . BRD4 expression in stable cells was tested by western blotting.s.c.) to the flanks of the nude mice. Within 20 days subcutaneous xenografts were established (around 100 mm3). Mice (n=10 each group) were treated with VS-5584. Mice body weight and bi-dimensional tumor measurements were taken every five days for a total of 35 days [The female nude mice were provided by the Animal Center of Chinese Academy of Science . 786-O cells were injected subcutaneously . Results were compared by one-way analysis of variance (ANOVA) followed by Tukey's test . Values of


Our previous study showed that guanine nucleotide exchange factor T (GEFT) was highly expressed in colorectal cancer (CRC) tissues and CRC patients with high GEFT expression had a poor prognosis, and suggested the close link of GEFT expression and CRC tumorigenesis/metastasis. In this text, the roles and upstream regulatory mechanisms of GEFT in the development and progression of CRC were further investigated.in vivo. GEFT mRNA stability was examined by actinomycin D assay. The relationships of LINC000355, LIN28A, and GEFT were explored by RNA pull down and RIP assays.Expression levels of GEFT mRNA and LINC00355 was measured by RT-qPCR assay. Protein levels of lin-28 homologue A (LIN28A) and GEFT were determined by western blot assay. Cell proliferative, migratory, and invasive capacities were assessed by CCK-8, Transwell migration and invasion assays, respectively. The effect of GEFT knockdown on CRC tumorigenesis was examined by mouse xenograft experiments via LIN28A. LIN28A knockdown weakened the promotive effect of LINC00355 on CRC cell proliferation, migration, and invasion.GEFT was highly expressed in CRC tissues and cell lines. GEFT knockdown inhibited CRC cell proliferation, migration, and invasion, and hindered CRC xenograft tumor growth. GEFT overexpression alleviated the detrimental effects of LINC00355 loss on CRC cell proliferation, migration, and invasion. LINC00355 promoted GEFT expression and enhanced GEFT mRNA stability via LIN28A, deepening our understanding on roles and upstream regulatory mechanisms of GEFT in CRC development and progression.LINC00355 facilitated CRC tumorigenesis and progression by increasing GEFT expression  Colorectal cancer (CRC) is responsible for approximately 10% of all diagnosed malignancy cases and 8.9% of all cancer-related deaths worldwide . PatientGuanine nucleotide exchange factor T (GEFT), also named as Rho guanine nucleotide exchange factor 25 (ARHGEF25) and p63RhoGEF, can stimulate the activation of RhoA, Rac1, and Cdc42 GTPases in different cells . In addiin vitro and hampered HCC xenograft tumor growth in vivo by binding with RBP heterogeneous nuclear ribonucleoprotein L (HNRNPL), where the CASC9-HNRNPL complex could co-regulate the expression of genes associated with AKT signaling pathway (It is well known that the regulation of gene expression and function can be mediated by plenty of RNA binding proteins (RBPs), which have the binding activity with diverse RNAs including coding and non-coding RNAs , 13. RBP pathway . LncRNA  pathway .In this text, the effects of GEFT knockdown on proliferation, migration, and invasion of CRC cells and CRC xenograft tumor growth were examined. Also, lncRNAs and related RBPs that could regulate GEFT expression and functions in CRC were further delved.CRC tumor tissues and adjacent normal tissues were collected from 60 patients with CRC who underwent surgical resection. The detailed information of clinical samples was presented in our previous article  and Tab2 atmosphere. FHC cells were cultured in DMEM/F12 medium (Thermo Scientific) containing 25 mM HEPES , 10 ng/ml cholera toxin , 0.005 mg/ml insulin (Sigma-Aldrich), 0.005 mg/ml transferrin (Sigma-Aldrich), 100 ng/ml hydrocortisone (Sigma-Aldrich), 20 ng/ml human recombinant EGF (Sigma-Aldrich), and 10% FBS (Thermo Scientific) at 37°C in a 95% air / 5% CO2 atmosphere.CRC cell lines  and a normal human colon epithelial cell line (FHC) were purchased from American Type Culture Collection . SW480 cells were cultured in Leibovitz’s L-15 medium  supplemented with 10% fetal bovine serum  at 37°C in a 100% air atmosphere. HT-29 and HCT-116 cells were grown in McCoy's 5A (Modified) medium (Thermo Scientific) containing 10% FBS at 37°C in a 95% air / 5% COSmall interference RNAs (siRNAs) targeting GEFT, LIN28A, LINC00355 and the negative control were synthesized by GenePharma Co., Ltd. . GEFT, LIN28A, and LINC00355 overexpression plasmids were customized from Sangon Biotech Co., Ltd. . Cell transfection was conducted using Lipofectamine 3000 reagent (Thermo Scientific) following the protocols of manufacturer. SiRNA target sequences were as follows: 5’-CGTGATGGTTGATAGCTAA-3’for LIN28A#1; 5’-GGGTTGTGATGACAGGCAA-3’ LIN28A#2; 5’-GGCTCAGCTATTCATCAAA-3’ for GEFT#1; 5’-CCGAGACTATTTCTTGCAA-3’ for GEFT#2; 5’-CCTCTCTGTTGAGAGCTAA-3’ for LINC00355#1; 5’-GGACTGTAAACTAGTTCAA-3’ for LINC00355#2.Total RNA was extracted from CRC tissues, normal tissues, CRC cell lines, and FHC using Trizol reagent (Thermo Scientific). cDNA was synthesized using M-MLV Reverse Transcriptase (Thermo Scientific) and subsequent real-time quantitative PCR reactions were performed using SYBR™ Green PCR Master Mix (Thermo Scientific) and specific primers on ABI 7500 Real-Time PCR System . GAPDH functioned as the housekeeping gene to normalize the expression of LINC00355, MIR31HG, and GEFT. Quantitative PCR primers were presented as follows: 5’-CCAACAGGAAGCAAGCACAG-3’ (forward) and 5’-CTAACACTTTGGGCAGCGTTT-3’ (reverse) for LINC00355, 5’-CTCAAGGCCAGTGTAGAGCC-3’ (forward) and 5’-TGCTGCATGGAACATGACCT-3’ (reverse) for MIR31HG, 5’-CCCAAGTCAGAGCATGTGGT-3’ (forward) and 5’-CCCTCAAATCCCCGCAATCT-3’ (reverse) for GEFT, 5’-TTGCCCTCAACGACCACTTT-3’ (forward) and 5’-TGGTCCAGGGGTCTTACTCC-3’ (reverse) for GAPDH.At 24 h after transfection, actinomycin D  was added into media of transfected cells. At the indicated time points  post ActD addition, RNA was extracted and GEFT mRNA level was determined by RT-qPCR assay.Western blot assay was performed using the standard experimental procedures. Proteins (30 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Primary antibodies against LIN28A , GEFT , GAPDH , and goat-anti-rabbit secondary antibody conjugated with horseradish peroxidase  were used in the western blot assay. Protein signals were detected using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific).Wild type GEFT 3’UTR region was cloned into pGL3-Basic firefly luciferase vector by Hanbio Biotechnology Co., Ltd. , and the recombinant plasmid was named as WT GEFT 3’UTR reporter. Also, mutant (MUT) GEFT 3’UTR reporter carrying the mutant binding motif was also constructed by Hanbio Biotechnology Co., Ltd. HCT-116 cells transfected with pcDNA3.1 empty vector or LIN28A overexpression plasmid were co-transfected with pRL-TK Renilla luciferase plasmid, pGL3-Basic luciferase vector/construct. At 48 h post transfection, luciferase activities were measured by Dual-Luciferase Reporter Assay  following the protocols of manufacturer.Cell proliferative ability was assessed at the indicated time points after transfection using the Cell Counting Kit-8 (CCK-8) assay kit  according to the instructions of manufacturer.Cell migratory and invasive abilities were measured using the 24-well Transwell insert chambers  with the filtration membrane pore size of 8 µm. Prior to cell invasion assays, the membranes were coated with Matrigel . Transfected cells in serum-free medium were seeded in the upper chambers and medium supplemented with 10% FBS was added to the lower chambers. After 24 h of incubation, non-migrated or non-invaded cells were removed using a cotton swab and migratory or invasive cells were fixed, stained, imaged, and counted under a light microscope.The binding potential between LIN28A and GEFT/LINC00355 in transfected or un-transfected HCT-116 cells was measured through RIP assay at 48 h post transfection using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit  together with IgG  or LIN28A antibody  according to the instructions of manufacturer. LINC00355 or GEFT mRNA level enriched by IgG or LIN28A antibody were determined by RT-qPCR assay.Biotin-labeled sense or antisense GEFT 3’UTR, biotinylated sense or antisense LINC00355 were customized from Sangon Biotech Co., Ltd. . RNA pull-down assay was carried out in whole-cell lysates of HCT-116 cells using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific) referring to the instructions of manufacturer. LIN28A protein level pulled down by biotin-labeled transcripts was examined by western blot assay.The oligos for sh-GEFT were: 5’-CCGGAAGGCTCAGCTATTCATCAAACTCGAGTTTGATGAATAGCTGAGCCTTTTTTTG-3’ (forward oligo) and 5’-AATTCAAAAAAAGGCTCAGCTATTCATCAAACTCGAG TTTGATGAATAGCTGAGCCTT-3’ (reverse oligo). The sh-GEFT oligos were constructed into pLKO.1 vector. Lentiviruses expressing shRNAs against GEFT (sh-GEFT) and control lentiviruses (sh-con) were obtained from Hanbio Biotechnology Co., Ltd. . HCT-116 cells were infected with sh-con or sh-GEFT lentiviruses. At 72 h after lentivirus infection, cells were screened out for 7 days using 1 μg/ml puromycin to establish stably transfected cell lines with or without GEFT knockdown.Our animal experiments were approved by the Animal Care and Use Committee of our hospital and performed with the standard experimental procedures. BALB/c nude mice  were purchased from Laboratory Animal Center of Zhengzhou University  and raised for 1 week under standard conditions to allow them to adapt to the environment. Mice were randomly divided into sham, sh-con, or sh-GEFT group with six mice in each group.6), HCT-116 cells infected with sh-con lentiviruses (2 × 106), or HCT-116 cells infected with sh-GEFT lentiviruses (2 × 106) were subcutaneously inoculated into the left hip areas of mice in sham, sh-con, or sh-GEFT group, respectively. Sham group referred to xenograft tumors derived from normal HCT-116 cells. The sh-con or sh-GEFT group represented xenograft tumors derived from HCT-116 cells infected with sh-con or sh-GEFT lentiviruses, respectively. Tumor volume was monitored using calipers and calculated using the formula: V = 0.50 × length × width2, where length represents longest tumor diameter and width represents the corresponding perpendicular diameter. Tumors were resected and weighed on day 25 after cell injection.Normal HCT-116 cells  and Tukey’s post-hoc test. P < 0.05 represents that difference was statistically significant.Data were analyzed using GraphPad Prism software 5.0  and expressed as mean ± standard deviation. Difference of two group data was analyzed using Student’s Firstly, RT-qPCR assay showed that GEFT mRNA level was notably increased in CRC tumor tissues (n = 60) compared to adjacent normal tissues (n = 60)  and a scramble control siRNA (si-con) were designed and synthesized. Knockdown efficiency analysis revealed that the transfection of si-GEFT#1 or si-GEFT#2 led to notable reduction of GEFT expression in SW480 and HCT-116 cells compared with si-con group  were downloaded from The Cancer Genome Atlas (TCGA) database. Next, differentially expressed lncRNAs in CRC tumor tissues versus adjacent normal tissues were identified. Among the top 100 up-regulated lncRNAs  EZF1-AS1 , 23, LINEZF1-AS1 , 25, LINEZF1-AS1 –29, LINCEZF1-AS1 , MIR31HGEZF1-AS1 ] have beNext, CRC tumor tissues were divided into LINC00355 high expression group  and low expression group  with the mean value of LINC00355 expression in CRC tumor tissues as the cutoff point. Survival analysis revealed that CRC patients with high LINC00355 expression had a poor overall survival Table 1.versus normal tissues. Combined with the gene differential expression data, seven RBPs were found to be differentially expressed in CRC versus normal tissues among these 44 common RBPs (i.e. ACGCG)  via recruiting c-myc to AR promoter region .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.


Non-Muscle-Invasive Bladder Cancer (NMIBC) tumors, only 30% of patients suffer from muscle invasion and distant metastases. Radical cystoprostatectomy, radiation, and chemotherapy have proven to be efficient in metastatic tumors. However, tumor relapse is observed in a noticeable ratio of patients following the chemotherapeutic treatment. Non-coding RNAs (ncRNAs) are important factors during tumor progression and chemo resistance which can be used as diagnostic and prognostic biomarkers of BCa.Bladder cancer (BCa) is the ninth frequent and 13th leading cause of cancer related deaths in the world which is mainly observed among men. There is a declining mortality rates in developed countries. Although, the majority of BCa patients present In present review we summarized all of the lncRNAs and miRNAs associated with chemotherapeutic resistance in bladder tumor cells.This review paves the way of introducing a prognostic panel of ncRNAs for the BCa patients which can be useful to select a proper drug based on the lncRNA profiles of patients to reduce the cytotoxic effects of chemotherapy in such patients. Bladder cancer (BCa) is the ninth most frequent cancer worldwide with an annual estimated 356,000 new cases and 145,000 deaths . Many famiR-133b and MST1P2 expressions in cisplatin-resistant bladder tumor cell lines, respectively. MiR-133b directly suppressed the SIRT1 expression. MST1P2/miR-133b axis had an important role in cisplatin-resistance of BCa through SIRT1/p53 pathway [HIF1A-AS2 in tissues and cell lines of BCa following the cisplatin treatment which makes bladder tumor cells resistance to cisplatin-triggered cell death. HIF1A-AS2 enhanced survival of tumor cells by upregulating high-mobility group A1 (HMGA1), thereby limiting the transcriptional function of p53 family. It was identified that interaction of p53 with HMGA1 restricted their transcriptional activity on proapoptotic BAX protein [MiR-582-5p has tumor-suppressive functions and reduces the tumor cell proliferation and migration via targeting CDK1, FOXC1, and RAB27a [UCA1 was up regulated in BCa. It acts as an endogenous sponge to down regulate the miR-582-5p which resulted in ATG7 over expression. UCA1 is important for the regulation of proliferation and invasion of BCa cells through modulating UCA1-miR-582-5p-ATG7-autophagy axis. As UCA1 shRNA markedly reduced the expression level of LRP, MRP1, and GST, and significantly overexpressed TOPO-II, it is hypothesized that knockdown of UCA1 decreases chemo resistance [GAS5 down regulation in bladder transitional cell carcinoma which was associated with advance grade and stage. GAS5 also increased doxorubicin-induced apoptosis through BCL-2 suppression [LncRNAs are key regulatory molecules involved in cell proliferation, development, and oncogenesis that achieve their roles through post-transcriptional regulation . They ha pathway . It has  protein . MiR-582d RAB27a –63. ATG7d RAB27a . It has sistance . It has pression .MALAT1 increases the expression levels of MRP1 an MDR1 through STAT3, thereby is responsible for inducing cisplatin-resistance in lung tumor cells [MALAT1 repression caused a better response of BCa cells to chemotherapy and increased cisplatin sensitivity. MALAT1 induced chemo resistance through regulating miR-101-3p/VEGFC axis. Bladder tumor tissue had higher level of MALAT1 compared with normal margins [GHET1 overexpression in BCa, which was positively correlated with advance tumor grade and muscle invasion. GHET1 up regulation was also associated with higher Gemcitabine-chemo resistance in BCa cells. Moreover, GHET1 up regulated the MRP1 in BCa cells, which in turn enhanced their Gemcitabine resistance [FOXD2-AS1 is significantly up regulated in BCa, and via establishing a positive feedback loop with AKT and E2F1 is contributed to increased progression and aggressiveness of bladder tumor cells [FOXD2-AS1 overexpression in gemcitabine resistant bladder tumor cells. Repression of FOXD2-AS1 expression resulted in lower levels of ABCC3 protein, and down regulation of several genes related to inducing drug resistance including MDR1, MRP2, and LRP. Therefore, FOXD2-AS1 regulated gemcitabine-resistance in BCa cells. FOXD2-AS1 indirectly targets the ABCC3 through miR-143 sponging [MiR-143 up regulation suppresses tumor cells proliferation and migration, and triggers apoptosis. It also increases the oxaliplatin sensitivity of tumor cells through targeting IGF-1R [miR-143 expression in bladder tumor cell lines and tissue samples compared with normal margins. There was an inverse association between miR-143 and IGF-1R mRNA expression levels which showed that the miR-143 exerts its tumor-suppressive role through IGF-1R regulation. MiR-143 overexpression significantly inhibited the p-ERK and p-AKT levels. It also attenuated the gemcitabine resistance via IGF-1R suppression [Drug efflux is also another mechanism of tumor drug resistance that can be regulated by different lncRNAs . MALAT1 or cells . It has  margins . Gastric margins . MRP1 issistance . Gemcitasistance , 69. FOXor cells . It has sponging . Insulinsponging . IGF-1R sponging , 73. MiRg IGF-1R . It has pression .UCA1 is frequently up regulated in bladder malignancies and contributed to aggressiveness of bladder tumor cells [UCA1 up regulation has been shown in bladder tumor tissues following cisplatin treatment. UCA1 overexpression was also contributed to up regulation of WNT6 and induction of WNT pathway which promotes cisplatin resistance in tumor cells [UCA1 overexpression, which leads to cisplatin resistance [UCA1 promotes epithelial-mesenchymal transition (EMT) and activates mTOR and ERK pathways, and increases Gefitinib resistance in EGFR-mutant lung carcinoma [UCA1 activates miR-196a-5p via CREB which results in gemcitabine/cisplatin resistance. UCA1 up regulation had a significant association with diminished rate of apoptosis and higher cell survival. UCA1 promotes CREB phosphorylation through AKT pathways. Therefore, UCA1-dependent CREB activation was considered as a key step in miR-196a-5p transcriptional regulation in bladder tumor cells [TUG1 is an oncogenic lncRNA in various cancers [TUG1 knockdown decreased Dox resistance through restraining the activity of Wnt/β-catenin pathway; whereas, TUG1 up regulation was significantly associated with Dox resistance and poor prognosis [CDKN2B-AS is an oncogenic lncRNA in various cancers [CDKN2B-AS in bladder urothelial carcinoma tissues and cell line which was positively associated with advance tumor grade. CDKN2B-AS up regulation was also correlated with Gemcitabine chemo resistance in BCa patients. Suppression of CDKN2B-AS attenuated the Gemcitabine-resistance in 24/Gem cells through inactivation of WNT signaling pathway. Therefore, CDKN2B-AS induced Gemcitabine-resistance via sponging Let-7 for activating WNT signaling pathway [HIF-1α, IL-2, and VEGF [TGFβ1 was overexpressed following Gemcitabine treatment. Moreover, aberrant expression of lncRNA-LET/NF90/miR-145 pathway was mediated by TGFβ1 which eventually increased stemness and chemo resistance. KLF4 and HMGA2 as the miR-145 targets were responsible for miR-145 suppressive effect against the stemness of BCa cell [LnRNAs can also be associated with drug response during tumor progression through regulation of different signaling pathways . Studiesor cells . WNT sigor cells , 78. A sor cells . The WNTsistance , 79. UCAarcinoma . It has or cells . TUG1 is cancers –86. Doxo cancers . It has rognosis . CDKN2B- cancers –90. Gemc cancers . It has  pathway . Althoug pathway . NF90 isand VEGF –96. The BCa cell .DLEU1 is an lncRNA associated with tumor cell aggressiveness and migration [DLEU1 expressions in bladder tumor tissues compared with normal margins. DLEU1 up regulation was also associated with worse prognosis in BCa patients. Moreover, up regulation of DLEU1 enhanced tumor growth and aggressiveness and induced cisplatin resistance through HS3ST3B1 induction. DLEU1 up regulated the HS3ST3B1 via miR-99b suppression. Overexpression of HS3ST3B1 was significantly correlated with shorter survival rates in BCa patients. Furthermore, ectopic HS3ST3B1 expression enhanced tumor growth, invasiveness, and cisplatin resistance [NEAT1 is an oncogenic lncRNA in various cancers and promotes the cell proliferation, migration, and aggressiveness through regulation of various miRNAs. Some studies have demonstrated that NEAT1 attenuates cisplatin chemo resistance [NEAT1 as inducer of cisplatin resistance [NEAT1.1 was down regulated following cisplatin treatment in BCa cells. The p53, OCT4, and c-MYC regulated the expression level of NEAT1 through interacting with its promoter region. There were OCT4, c-MYC, and p53 up regulations in cisplatin-resistant BCa cells. The knockdown of NEAT1 suppressed the proliferation and migration of BCa cells and induced apoptosis following cisplatin treatment [The igration –100. It sistance . NEAT1 isistance , 102, whsistance , 104. Itreatment .miR-22-3p enhanced resistance to chemotherapy in bladder tumor cells through suppressing NET1. NET1 was also markedly up regulated in 5637 cell line compared with H-bc cell line. Moreover, there was an inverse association between NET1 and miR-22-3p expression levels. NET1 was introduced as the direct target of miR-22-3p in chemo resistance of bladder tumor cells [MiR-27a down regulation in bladder tumors can be associated with reduced chemotherapeutic response [Neuroepithelial cell transforming 1 (NET1) is a guanine nucleotide exchange factor for RhoA and is involved in regulating extracellular signal transduction. It has been observed that the or cells . MiR-27aresponse , 105–107miR-27a. The findings have shown a significant correlation between miR-27a overexpression and improved chemotherapeutic outcomes. The carriers of rs11671784 A allele had significantly poorer outcomes after chemotherapy compared with rs11671784 GG homozygote patients. It was also indicated that the miR-27a significantly down regulated the P-glycoprotein [miR-27a up regulation was significantly associated with overexpression of CASP3 and BAX, and BCL-2 down regulation. MiR-27a decreased tumor cells’ resistance to chemotherapy by increased rate of apoptosis. There was a correlation between rs11671784 G/A variation and reduced miR-27a expression which results in increased RUNX-1 expression drug resistance. RUNX-1 up regulation was significantly correlated with reduced bladder tumor drug sensitivity. Therefore, miR-27a/RUNX-1 pathway has a key function in chemo-resistance in bladder malignancies [miR-27a deregulation induced cisplatin resistance in BCa cells via up regulating SLC7A11, followed by increased cystine import and higher intracellular glutathione levels. The results suggested that the miR-27a/27b and SLC7A11 expression levels along with intracellular glutathione levels in BCa tissue could be considered as predictive factor for determining the probability of cisplatin-chemo resistance. Patients with down regulated SLC7A11 showed better response to therapy and had better prognosis [miR-101 and COX-2 expressions in which miR-101 up regulation reduces cisplatin-chemo resistance through COX-2 inhibition [miR-101 in BCa cells resistant to cisplatin. Therefore, miR-101 regulates cisplatin sensitivity in bladder tumor cell lines via targeting the COX-2 [RUNX-1 is a direct target for inducing tumor chemo-sensitivity using oprotein . It has gnancies . Many sognancies . Glutathgnancies . Glutamagnancies . Heterodgnancies , and is rognosis . Cyclooxrognosis , 114. Thhibition . It has he COX-2 .MiR-34a is a potential tumor suppressor miRNA and its down regulation has been reported in various malignancies [miR-34a has been associated with resistance to chemotherapeutic drugs [miR-34a on p53 signaling pathway. Ectopic expression of miR-34a caused apoptosis, cell cycle arrest, and drug response alteration through SIRT-1, CDK6, E2F3, and BCL-2 targeting [miR-34a up regulation and cisplatin sensitivity in BCa. Moreover, miR-34a targets CD44 after cisplatin therapy [miR-34a significantly reduced Epirubicin chemo resistance in bladder tumor cells through targeting TCF1 and LEF1. Therefore, miR-34a up regulation leads to the suppression of WNT signaling pathway while increasing the rate of epirubicin -induced apoptosis [miR-34a expression in gemcitabine-resistant BCa cells. MiR-34a reduced the stemness of chemo resistant BCa cells and increased gemcitabine and cisplatin responses. GOLPH3 was also significantly over expressed in BCa cells, xenograft, and sphere cells resistant to gemcitabine and cisplatin. Moreover, the up regulation of CSC biomarkers including KLF4, SOX2, and CD44 were observed in bladder tumor cells and xenograft. Therefore, miR-34a regulation of GOLPH3 is active in bladder CSCs resistant to gemcitabine and cisplatin [miR-34a. MiR-34a enhances apoptosis rate through suppressing BCL-2 expression. CDK6 interacts with CDK4 and CCND1 to form a complex which is fundamental for Rb function and G1/S transition. SIRT-1 is a NAD-dependent deacetylase which targets FOXO, SFRP1, p53, and PGC1 [miR-34a sensitized tumor cells to cisplatin by targeting SIRT-1 and CDK6. The MI-TCC patients resistant toward cisplatin chemotherapy had significantly lower levels of miR-34a expression compared with sensitive patients [miR-34b-3p attenuated chemo resistance in BCa through suppressing CCND2 and P2RY1 [gnancies . Dysreguic drugs , 117–120argeting –123. CD4argeting –126. It  therapy . It has poptosis . Golgi ppoptosis . GOLPH3 poptosis . It has isplatin . Aberranisplatin –131. Theand PGC1 –134. It patients . Cyclin patients , 136. Thpatients , 138. Bipatients , 140. ItMiR-98 was recognized as an important agent in regulating mitochondrial activity, which increases bladder tumor cells resistance toward mitochondrial apoptosis. It was also established that miR-98 targets LASS2 tumor suppressor. There was also an inverse association between miR-98 and LASS2 mRNA levels in bladder tumors. LASS2 functions in negative regulation of mitochondrial activity and has a putative role in mediating chemo-resistance caused by miR-98. Therefore, miR-98 promotes chemo-resistance through targeting LASS2, which enhances mitochondrial fusion and disrupts mitochondrial membrane potential [otential .MiR-101-3p is considered as a tumor suppressor and is down regulated in different malignancies such as BCa, colorectal cancer, and breast cancer [miR-101-3p is a member of the Polycomb-group family involved in transcriptional repression [miR-101-3p down regulations in bladder urothelial carcinoma tissues and cell lines resistant to cisplatin. MiR-101-3p overexpression also suppressed the MRP1 expression level. Therefore, miR-101-3p decreased cisplatin-resistance in bladder urothelial carcinoma through repressing EZH2 and MRP1 [t cancer –144. EZHpression . MRP1 ispression –148. It and MRP1 . Gemcitaand MRP1 .MiR-129-5p is involved in tumor cells drug response via modulation of NOTCH signaling receptor DLK1 [miR-129-5p was correlated with lower sensitivity of BCa cells to gemcitabine therapy; however, overexpression of miR-129-5p inhibits resistance to gemcitabine in BCa cells and promotes their apoptosis via targeting WNT5a [NOTCH signaling pathway has a pivotal role in various cellular processes such as cell cycle, migration, metabolism, and apoptosis , 151. Mitor DLK1 . WNT5a itor DLK1 . WNT5a itor DLK1 –155. Somtor DLK1  and indutor DLK1 . It has ng WNT5a .MiR-193a-3p impedes tumor proliferation and decreases drug resistance through down regulation of various genes such as CCND1, ERBB4, and PTEN [HOXC9 belongs to highly conserved homeobox family of genes, and encodes proteins that function as homeodomain transcription factors playing a crucial role in morphogenesis in all multicellular organisms. It has been revealed that the miR-193a-3p mediated HOXC9 down regulation which resulted in poorer sensitivity of BCa to chemotherapeutic drugs. Oxidative stress and DNA damage response were also influenced by epigenetic suppression of HOXC9 through miR-193a-3p [PSEN1 was directly targeted by miR-193a-3p and executed its impact on the multi-chemo resistance. PSEN1 up regulation rendered H-bc cells more sensitive to chemotherapy-induced cell death. However, RNA interference-mediated repression of PSEN1 gene resulted in lower rates of apoptosis and desensitizing 5637 cell line to chemotherapy-induced cell death [miR-193b-3p. Moreover, miR-193b-3p had regulatory effect on ETS1 and CCND1. MiR-193b-3p was also important for CDDP-triggered cell cycle arrest, cell cytotoxicity, and inhibition of cellular migration. CEBPD/miR-193b-3p axis had key roles in cisplatin response of urothelial carcinoma cells in which CEBPD up regulates the miR-193b-3p and improved cisplatin cytotoxicity in urothelial carcinoma. This process was associated with ETS1 and CCND1 down regulations, cell migration inhibition, cell cycle arrest, and cisplatin-triggered cytotoxicity in NTUB1 cell line [miR-193a-3p is due to its suppressive effects on various genes such as KRAS and c-KIT [miR-193a-3p induced multi-drug resistance in BCa cells through down regulating LOXL4, and thus initiating oxidative stress pathway [miR-193a-3p’s effect on chemo resistance in BCa through regulation of Myc/Max, NF-jB, DNA damage response, and NOTCH pathway [Substantial epigenetic changes along with genetic variations are the origin of all cancerous features . These eand PTEN , 163. HO-193a-3p . Preseni-193a-3p . Studies-193a-3p . It has ll death . Platinull death . C/EBP ill death –168. CEBell line . The oncnd c-KIT , 170. Ly pathway . Hyperme pathway  and dige pathway . SRSF2 b pathway . HIC2 in pathway , 175. It pathway .miR-200 family are potent inhibitors of EMT through inhibiting the expression of ZEB1 and ZEB2 [miR-200b and cisplatin resistance in BCa. Microarray analysis showed that genes associated with CDDP sensitivity or cytotoxicity, such as TNFSF10, ICAM1, and IGFBP3 were induced in the resistant cells as a result of miR-200b/cisplatin treatment [Members of and ZEB2 , 177. IGand ZEB2 . TNFSF10and ZEB2 . It has reatment .miR-203 down regulation and poor prognosis in BCa patients who were under cisplatin-based chemotherapy. MiR-203 up regulation also increased the cytotoxic effects of cisplatin and decreased tumor cell viability through suppressing Survivin and BCL-w. Patients with non-progressive form of BCa had notably higher levels of miR-203 compared with cases with progressive form [Although, Cisplatin is the main drug in BCa combination chemotherapy regimens including GC (gemcitabine and cisplatin) and MVAC , almost half of the MIBC patients do not respond to the cisplatin-based treatment . BCL-w eive form .MiR-214 functions as a tumor suppressor agent and is deregulated in various cancers [miR-214 occurs in BCa which shows marked correlation with higher tumor grade/stage and lymph nodes involvement [MiR-214 down regulates the P53 and PDRG1 in BCa [miR-214 expression in BCa tissues and cell lines. The miR-214 attenuated chemo resistance through apoptosis induction. It was able to down regulate the PARP and CASP-3 levels. It also inhibited AKT phosphorylation. AKT signaling pathways regulates chemotherapy-induced apoptosis via BCL-2 up regulation. Therefore, the impact of miR-214 on drug resistance is mediated through its modulatory function in AKT/BCL-2 axis. The miR-214 decreased chemo resistance in BCa tissues and cell lines by suppressing NTN1 [ cancers –186. Dowolvement . MiR-2141 in BCa . It has ing NTN1 .miR-218 up regulation markedly decreased glucose uptake through GLUT1 targeting. Over expression of miR-218 was also beneficial in attenuating cisplatin resistance in BCa cells [Cisplatin mainly functions through induction of oxidative stress . NeverthCa cells .miR-222 enhances tumor cell proliferation and metastasis by inhibiting the PPP2R2A, TIMP3, and p27 [MiR-222 expression has been linked to the tumor drug response [miR-222 up regulation increased cell proliferation and decreased cisplatin-induced apoptosis in bladder tumor cells through modulation of PPP2R2A/AKT/mTOR pathway. Tumor cells with high levels of miR-222 had activated AKT/mTOR axis. The mTOR or AKT suppression were also beneficial in inhibiting tumor cells’ proliferation and restoring cisplatin sensitivity due to miR-222 up regulation [Aberrant expression of  and p27 –193. MiRresponse . PP2A isresponse , 195–197gulation .Regarding the importance of ncRNAs in regulation of drug response in tumor cells, in present review we have summarized all of the reported ncRNAs which are associated with chemotherapeutic resistance in BCa. It was observed that the lncRNAs were the most reported ncRNAs associated with drug response of BCa. This review paves the way of introducing a prognostic panel of ncRNAs for the BCa patients to improve the selection of an efficient chemotherapeutic strategy based on ncRNA profile of BCa patients.


Streptococcus salivarius DB-B5 was isolated from the supragingival plaque of a healthy female subject. The complete 2.3-Mb genome consists of one circular chromosome, two circular plasmids (including a megaplasmid), and one linear phage-like episome. The genome possesses two separate loci encoding bacteriocins. Streptococcus salivarius DB-B5 was isolated from the supragingival plaque of a healthy female subject. The complete 2.3-Mb genome consists of one circular chromosome, two circular plasmids (including a megaplasmid), and one linear phage-like episome. The genome possesses two separate loci encoding bacteriocins. Streptococcus salivarius is a normal commensal of the healthy human oral microbiome . Paired-end libraries (2 × 300 bp) were constructed using the Nextera XT library kit (Illumina) and sequenced on the MiSeq v3 platform (Illumina) at the Centre for the Analysis of Genome Evolution and Function at the University of Toronto. In parallel, DB-B5 genomic DNA was also extracted using the Wizard genomic DNA purification kit (Promega) and sent to Génome Québec for library preparation and PacBio sequencing. The PacBio library was prepared following the manufacturer’s instructions; DNA was sheared using g-TUBES , and SMRTbell templates were prepared using Express template preparation kit v2.0 reagents (Pacific Biosciences). The library was sequenced without size selection on the PacBio Sequel platform using the Sequel sequencing plate v3.0, single-molecule real-time (SMRT) cells (1M v3), and 10-h movies. Illumina reads were quality filtered using Trimmomatic (v0.38.0) (de novo hybrid assembly was performed using Unicycler (v0.4.8.0) with 1,722,228 Illumina paired-end reads and 629,563 PacBio long reads , with a genome coverage of ∼1,000× , one megaplasmid named pIKMIN-B501 , one small plasmid named pIKMIN-B503 , and one linear phage-like episome named pIKMIN-B502 . The linear ends of pIKMIN-B502 contain inverted terminal repeat sequences similar to those of other linear phages infecting Gram-positive cocci, confirming the likelihood that the linear ends are complete (v0.38.0) . PacBio complete , 12. Theads N50, 9,339 bp,S. salivarius DB-B5 genome was searched for bacteriocins using BAGEL4 (http://bagel4.molgenrug.nl)  . The anaCP054153 (chromosome), CP054154 (pIKMIN-B501), CP054155 (pIKMIN-B502), and CP054156 (pIKMIN-B503). The raw reads from both the PacBio and Illumina sequencing are available under the SRA BioProject accession number PRJNA635659.The complete genome sequences have been deposited in GenBank under the accession numbers


PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3′-UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression, and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p levels. In mice, hepatic miR-483 overexpression increased LDLR levels by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3′-UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3′-UTR (ΔBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic LDL receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL-cholesterol (LDL-C) levels and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3′-UTR of  MicroRNA-483 targeting 3’ untranslated region of PCSK9 mRNA inhibits hepatic PCSK9 expression, resulting in increased LDLR expression and LDL uptake, which ameliorates hypercholesterolemia and atherosclerosis. SREBF2), a membrane-bound transcription factor that regulates multiple genes involved in cholesterol homeostasis, including LDLR (Hypercholesterolemia is a major risk factor for cardiovascular disease. Circulatory levels of low-density lipoprotein cholesterol (LDL-C) are predominantly determined by hepatic clearance of LDL-C via the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol metabolism by targeting the LDLR , 2. PCSKing LDLR . Intraceing LDLR . Once seing LDLR , 6. In cPCSK9   . Howeverlisiran) , 11. In t models .SREBF2 and SREBF1, miR-33 downregulates cholesterol efflux and HDL biogenesis by targeting ABC transporters, i.e., ABCA1 and ABCG1 (microRNAs (miRs) are ubiquitously involved in epigenetic regulation of gene expression by targeting the 3′-UTR of the protein-encoded transcripts. Ample evidence demonstrates that miRs are integral regulatory parts of the transcriptome, including genes involved in regulating cholesterol metabolism. For example, miR-96/182/183 target MED1/FBXW7 in hepatic cells, which regulates the transcription of multiple lipid-regulating and lipid synthesis genes , 14. miRnd ABCG1 . Althougnd ABCG1 , the fulIGF2. The sequences of miR-483-3p/-5p are highly conserved among mammalian species in general (https://doi.org/10.1172/jci.insight.143812DS1). miR-483 targets the 3′-UTR of connective tissue growth factor (CTGF), platelet-derived growth factor, tissue inhibitor of metalloproteinase 2, and SMAD4, a key component protein in the TGF-β signaling pathway , are intronic miRs that are encoded together with their parental gene  pathway –20. In v pathway .PCSK9 mRNA. Experimentally, miR-483-5p directly targeted the 3′-UTR of PCSK9 mRNA, which in turn increased hepatic LDLR expression. The circulatory levels of miR-483-5p were lower in humans with elevated LDL-C levels, and miR-483 overexpression in hypercholesterolemic mouse models greatly reduced the serum levels of total cholesterol and LDL-C. This mechanism of cholesterol lowering via intracellular PCSK9 mRNA targeting by miR-483 may have therapeutic efficacy.In this study, we uncovered a mechanism by which miR-483-5p robustly targets the 3′-UTR of Pcsk9 mutant (D377Y) lacking the 3′-UTR region is commonly used to induce hyperlipidemia and experimental atherosclerosis in mouse models . Mice were administered AAV8-PCSK9 or AAV8-PCSK9-3′-UTR, then fed a high-fat diet (HFD) for 12 weeks. The HFD-induced hyperlipidemia was significantly alleviated in mice receiving AAV8-PCSK9-3′-UTR compared with AAV8-PCSK9 -PCSK9 encoding a gain-of-function e models . To explV8-PCSK9 . ConsistV8-PCSK9 . Also, h9-3′-UTR . These ry others , whereasA 3′-UTR . A lucifactivity . Among tactivity . Such inactivity .PCSK9 3′-UTR, we used bioinformatics to locate the binding site of miR-483-5p at the 3′-UTR of both human and mouse PCSK9 mRNA ] or mutant PCSK9 3′-UTR [Luc-PCSK9 (MT)] with mutation of the miR-483-5p targeting site. Pre–miR-483 significantly decreased the luciferase activity of the cotransfected Luc-PCSK9 (WT) in HepG2 cells but only moderately decreased it in cells cotransfected with Luc-PSCK9 (MT) (PCSK9 mRNA were enriched in the miRNA-induced silencing complexes (miRISCs) that contained Argonaute-1 or Argonaute-2  . Howeveronaute-2 . As a po3 target , was alsPCSK9, we used CRISPR/Cas9 gene editing to create a HepG2 mutant cell line (mPCSK9 HepG2) with deletion of the miR-483 targeting site in the PCSK9 3′-UTR    . With th = 0.20) . To this = 0.20) .P = 0.699 vs. near/above optimal, 0.196 vs. borderline high, and < 0.0001 vs. high)  . Consists. high) . When cos. high) . TogethePCSK9 and CTGF, mRNA levels of Pcsk9 and Ctgf were enriched in miRISCs isolated from the livers of mice receiving AAV-483 versus AAV-null  encoding the coding region of mouse Pcsk9 fused with the 3′-UTR or AAV8-PCSK9-3′-UTR with the deleted miR-483 binding site . All groups were fed an HFD for 8 to 10 weeks and treated in parallel with or without AAV-483 or AAV-null , including IL1B, IL6, TGFB1, and monocyte chemoattractant protein 1  were obtained from ATCC . Cells were maintained in DMEM (Gibco) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate at 37°C and 5% COPCSK9 gene were predicted by using miRanda v3.3a with the parameters “-sc 120, -scale 4, -en -10” (Putative miR-483 targeting sites in the 3′-UTR of the -en -10” . The preThe miR mimics (pre-miR) and anti-miRs were purchased from Ambion. HepG2 cells at 50%–70% confluence were transfected with 60 nM pre-miR or anti-miR using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium for 6 hours and then changed to complete growth medium. At 24 hours after transfection, cells were lysed. Equal amounts of MirVana miRNA Mimic Negative Control #1  or anti-miR miRNA Inhibitor Negative Control #1  were used as controls for pre-miR or anti-miR experiments, respectively.PCSK9 3′-UTR containing the miR-483 putative binding site was subcloned  into the pMIR-REPORT vector (Ambion) to generate the pMIR-Luc-PCSK9-3′-UTR WT reporter [Luc-PCSK9 (WT)]. Then the CGG to GCC mutations in the miR-483 binding seed sequence of the PCSK9 3′-UTR were introduced into the Luc-PCSK9 (WT) plasmid by using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). Renilla luciferase plasmid (pRL-TK) was used as the transfection control. Luc-PCSK9 (WT), Luc-PCSK9 (MT), and control pRL-TK plasmids were cotransfected into HepG2 cells by using Lipofectamine 2000 (Invitrogen). At 24 hours after transfection, cells were lysed for luciferase activity measurement with the Dual-Glo Luciferase Reporter Assay Kit (Promega).The 908-bp human 2 UV light, then lysates were incubated with protein G Dynabeads conjugated with anti-Ago1 or anti-Ago2 antibody  at 4°C overnight. The same amount of mouse IgG was used as an isotype control. The immunoprecipitated RNAs and input RNAs were extracted by using Trizol for qPCR detection.HepG2 cells were cross-linked under 400 mJ/cmPcsk9 3′-UTR was subcloned into the FseI site , then the 5′-CTGTCT-3′ miR-483 binding site was deleted  as shown in Pcsk9 coding sequence replaced by mouse pri-mR-483 between AgeI and FseI sites. AAVs and empty control viruses were enveloped by the UCSD Vector Development Core. Various AAV8 viruses (1 × 1012 vector genomes) were administered to mice by tail vein injection.pAAV8/D377Y-mPCSK9 plasmid was obtained from Addgene. Mouse 6 plaque-forming units to each well of a 6-well plate for 2 days. The mixed population of infected HepG2 cells was selected for genotyping.Single-guide RNAs were designed by using an online tool  Supplem. Oligonu(-ΔΔCq) method with β-actin as an internal control. For miR quantification, the TaqMan MicroRNA Assay followed the manufacturer’s protocol (Life Technologies). U6 was detected as the internal control in cultured cells and tissues from animals. To quantify the miR-483 level in serum, a C. elegans miR  was spiked in at 2 nM before RNA extraction, with the level used as the internal control.RNA was isolated from cultured cells, liver tissues, or serum by using TRIzol or TRIzol LS (Invitrogen). Total RNA was reverse-transcribed with use of the PrimeScript RT-PCR Kit (Takara), followed by qPCR with SYBR Green (Bio-Rad) in a Bio-Rad CFX-96 real-time system. Primers used in this study are in Protein extracts from cultured cells or tissues were resolved by SDS-PAGE and transferred to 0.45 μm NC membranes. Western blot analysis was performed with the following antibodies: anti-PCSK9 , anti-LDLR , anti-CTGF , anti–α-tubulin , and anti–β-actin antibody . Horseradish peroxidase-conjugated anti-rabbit , anti-mouse , or anti-goat  were used as secondary antibodies.Monolayers of HepG2 cells were incubated with 50 μg/mL fresh, isolated, nonacetylated human LDL labeled with Dil (Invitrogen) for 1 hour in the dark with serum-free medium. Cells were trypsinized to obtain a single-cell suspension and Dil-LDL uptake was quantified by flow cytometry (BD Accuri C6 flow cytometer). LDL Uptake Assay Kit (Cayman) was used to evaluate LDL uptake. Briefly, cells were incubated with 5 μg/mL LDL-DyLight 550 working solution prepared in serum-free medium for 12 hours, then fixed and imaged under a fluorescence microscope .-/-Ldlr mice (stock no. 002207) obtained from the Jackson Laboratory were maintained under a specific pathogen–free environment .C57BL/6J mice (stock no. 000664) and Mice fed an HFD or chow diet were administered AAV8-null or AAV8-miR-483 with or without AAV8 PCSK9-3′-UTR (WT) or AAV8 PCSK9-3′-UTR (ΔBS) by tail vein injection. After 6–12 weeks, animals were killed, and blood was collected from the left ventricle by using a heparinized syringe fitted with a 25-gauge needle. Serum levels of liver aspartate aminotransferase and alanine transaminase were determined by using the Aspartate Aminotransferase (AST) and Alanine Transaminase (ALT) Detection Kits (Teco Diagnostics). For all experiments, both sexes of mice with the same age and similar weight were randomly assigned to experimental and control groups. Frozen sections of livers and hearts were cut 5 μm thick and stained with 0.5% Oil-red O, then counterstained with hematoxylin. The entire aortic trees were opened longitudinally, stained with Oil-red O, and digitally scanned. Histological analysis and image processing involved use of the Hamamatsu Nanozoomer 2.0HT Slide Scanner. En face lesion area was assessed with WinROOF 6.5 (Mitani Co).PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols. Briefly, cell culture supernatants or serum samples were diluted and incubated in precoated plates for 2–4 hours. After 2 hours of incubation with horseradish peroxidase–conjugated secondary antibody, TMB substrate solution was added and samples were incubated further for 30 minutes before stop buffer was added. Plates were read immediately by using a microplate reader (BioTeK) set at 450 nM.Lipoprotein profiles were determined as described . Brieflyg for 10 minutes, the serum was aliquoted into separator tubes, quickly frozen in liquid nitrogen, and stored at –80°C until use. The lipid profiles were detected by automatic chemical analysis (Hitachi LABOSPECT 008AS).A group of 179 individuals were enrolled from the First Affiliated Hospital of Xi’an Jiaotong University during 2018–2019. The baseline characteristics of human subjects are summarized in t test for parametric data or Mann-Whitney U test for nonparametric data. Experiments with more than 2 groups were compared by 1-way ANOVA with a Bonferroni’s post hoc test for parametric data or Kruskal-Wallis test with Dunn’s multiple comparisons for nonparametric data. All statistical analyses were performed with GraphPad Prism version 5.01; 2-tailed P values of less than 0.05 were considered statistically significant.All results are presented as mean ± SEM. Initially, data were tested for normality and equal variance to confirm the appropriateness of parametric tests. Experiments with 2 groups were compared by 2-tailed Student’s The UCSD animal care personnel maintained all animals in accordance with NIH guidelines, and the IACUC of UCSD approved all experimental procedures . UCSD has an Animal Welfare Assurance document (A3033-01) on file with the Office of Laboratory Animal Welfare and is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The study protocol for humans was approved by the ethics committees of Xi’an Jiaotong University. Written informed consent was obtained from all subjects.JYJS, MH, and JD conceived the original idea and designed the overall experimental plan. JD, MH, J. Li, AP, CW, JZ, YS, WTW, YZ, J. Liu, SCW, and PHH performed experiments. JD and MH interpreted the data and performed statistical analysis. ST, ZYY, and PLSMG provided essential input to the overall research plan. MH, JD, JYJS, ST, and PLSMG wrote the manuscript.


Caenorhabditis elegans behavior, such as sensing environmental gases (oxygen and carbon dioxide), regulation of systemic fat levels and egg laying . To identify factors that control BAG specification, we performed a forward genetic mutagenesis screen using the Pgcy-33::gfp reporter, which is exclusively expressed in the BAG neurons. We isolated a new allele (rp4) that exhibits a loss of Pgcy-33::gfp expression. Using the one-step whole-genome sequencing and SNP mapping strategy  we mapped the genetic lesion to the egl-46 gene, encoding a zinc finger transcription factor homologous to mammalian INSM1/2, which we had previously shown to be important for BAG specification . The new lesion we identified egl-46(rp4) (TGC>TAC) causes a single amino acid change in a highly conserved cysteine residue (C185Y) that lies in the first zinc finger domain of EGL-46, which would be predicted to affect DNA binding. Analysis of Pgcy-33::gfp expression in the rp4 allele reveals that it exhibits the same phenotype as the previously published rp13 deletion allele, which is an out-of-frame deletion that removes the zinc finger domains . Therefore, rp4 acts as a strong loss-of-function/null allele and may be of use to those researchers interested in elucidating additional functions of EGL-46.The BAG neurons control multiple aspects of Pgcy-33::gfp; Pdop-3::rfp was mutagenized using ethyl methanesulfonate. Mutants with decreased GFP expression in the BAG neurons were isolated using the automated COPAS biosorter platform. The one-step whole-genome sequencing and SNP mapping strategy  was used to identify the genetic lesion of the isolated rp4 allele. Phenotypic analysis of Pgcy-33::gfp BAG expression was performed as described previously .In the forward genetic screen, the BAG reporter strain rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp)RJP22 egl-46(rp4); rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp)RJP56 egl-46(rp4); rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp); rpEx2046 (WRM0636bB06) 1ng/µl + Punc-122::gfp 30ng/µl Line 1RJP4585 egl-46(rp4); rpIs3(Pgcy-33::gfp); vsIs33(Pdop-3::rfp); rpEx2047 (WRM0636bB06) 1ng/µl + Punc-122::gfp 30ng/µl Line 2RJP4586 Strains will be available at the CGC.


This paper provides an overview of the factors influencing the biodistribution and targeting properties of affibody molecules and the chemistry of their labeling using positron emitters.Affibody molecules are small (58 amino acids) engineered scaffold proteins that can be selected to bind to a large variety of proteins with a high affinity. Their small size and high affinity make them attractive as targeting vectors for molecular imaging. High-affinity affibody binders have been selected for several cancer-associated molecular targets. Preclinical studies have shown that radiolabeled affibody molecules can provide highly specific and sensitive imaging on the day of injection; however, for a few targets, imaging on the next day further increased the imaging sensitivity. A phase I/II clinical trial showed that  Radionuclide molecular imaging permitting non-invasive quantitative visualization of molecular targets is an attractive alternative to biopsy-based methods for stratifying patients for targeted therapies . The use64Cu (T½ = 12.7 h), 89Zr (T½ = 78.4 h), and 124I (T½ = 100.2 h)  maleimide ([18F]-FBEM) [18F]-N-(4-fluorobenzylidene)oxime ([18F]-FBO) [18F]-fluoro-phenyloxadiazole methylsulfone ([18F]-FPOS) -FBEM) ,57, [18F8F]-FBO) ,46,58, aF]-FPOS)   .18F]-AlF-NOTA was associated with a low level of hepatobiliary excretion. In the case of [18F]-FBO labeling, 40–50% of the activity was found in the intestines and their contents. A quite appreciable level of hepatobiliary excretion was also observed in the case of the labeling of affibody molecules at the C-terminus using [18F]-FPOS [18F]-FBO -FPOS . Interes18F]-FBO . It is w18F]-FBO . A high 111In-labeled affibody molecules was evaluated in mice bearing xenografts with high  and low  HER2 expression levels. Affibody molecules PEP05541 (KD = 116.7 ± 0.1 pM), PEP05838 (KD = 157 ± 4 pM), and PEP07127 (KD = 3804 ± 178 pM) were evaluated -DOTA  from 2+ (not eligible for such treatment) level of expression . However68Ga-labeled ABY-025 affibody molecules demonstrated that an injected activity of 212 ± 46 MBq permits a good imaging quality up to 4 h after injection F− have been installed for manufacturing [18F]-FDG, and delivery logistics have been established. Gallium-68 can be produced from a long-lived 68Ge/68Ga generator, even at PET centers without its own cyclotron. In addition, 68Ga can be produced by cyclotrons using easy-to-operate liquid targets -fluorobenzamido) ethyl) maleimide ([18F]FBEM)  A 56]. T. T18F]-f18F]-fluorobenzaldehyde ([18F]-FBA) with an oxoamine, resulting in oxime formation [18F]-FBA , with subsequent complexation by derivatives of the NOTA chelator [18F]AlF was performed in acetate buffer at pH 4.0 and 90 °C for 15 min, with subsequent purification using an HLB cartridge and buffer exchange using an NAP-5 size-exclusion column. The overall radiochemical yield was 21 ± 6% after the 40-min procedure. The refolding capacity of affibody molecules provided a preserved affinity after labeling in such harsh conditions. [18F]-AlF-NOTA-ZHER2:2395 demonstrated excellent tumor targeting 4 h pi (tumor-to-blood ratio of 145 ± 24), but the bone uptake was twice as high as that of its 111In-labeled counterpart, which indicates a somewhat lower chelate stability. This approach has been applied to label the HER2-binding affibody molecules NOTA-CGGGRDN-ZHER2:234 -AlF-labeling of heat-sensitive proteins at moderate temperatures [18F]-AlF was performed at 37 °C for 15 min and resulted in a radiochemical yield of 20 ± 7%. In rhesus monkeys, [18F]-AlF-RESCA-PEP04314 had a biodistribution similar to the biodistribution of [18F]-AlF-NOTA-PEP04314, but with a somewhat elevated bone uptake, which might indicate a lower label stability.A new acyclic chelator known as RESCA  has beeneratures . The lab18F-labeled 2-cyanobenzothiazole ([18F]-F-CBT)   87]. A . A 18F-l18F]AlF-NOTA, [18F]FBA, and the silicon-fluoride acceptor [18F]p-(di-tert-butyl-fluorosilyl)benzaldehyde ([18F]-SiFA-A). [18F]-SiFA-labeling   C can be 18Al]-AlF-NOTA-, [18F]-FBA-, and [18F]-FPOS chemistry provide the fluorination of affibody molecules with yields compatible with clinical translation. However, the use of [18F]-FBA and [18F]-FPOS is associated with undesirable hepatobiliary excretion. It is worth evaluating if the application of hydrophilic linkers  before the C-terminal cysteine can suppress this excretion pathway.Overall, Ga-(DOTA-A1)-ZHER2:S1 provided a significantly higher tumor-to-lung ratio than [68Ga]Ga-(DOTA-K58)-ZHER2:S1.The incorporation of a radiometal-chelator complex modifies the local charge and lipophilicity of the surface of a protein. This can affect off-target interactions with normal tissues and binding to blood proteins. It was shown for contrast ,41. The us (K58) . All var68Ga-labeled at the N-terminus using NOTA, NODAGA, and DOTA (68Ga]Ga-NODAGA-ZHER2:S1 had the highest tumor-to-blood ratio (60 ± 10) at 2 h pi in comparison with both [68Ga]Ga-DOTA-ZHER2:S1 (28 ± 4) and [68Ga]Ga-NOTA-ZHER2:S1 (42 ± 11). The same variant had the highest tumor-to-liver ratio [68Ga using DOTA  and DOTAGA (single negative charge of the complex)  studies have demonstrated that the labeling strategy  has a strong influence on the contrast and thus on the sensitivity of imaging using affibody molecules.A comparative evaluation of the synthetic affibody and DOTA A–C showeer ratio . These dcomplex) D was eva68Ga]-Ga-ABY-025 can be automatically produced on the Modular-Lab PharmTrace synthesis platform (Eckert & Ziegler), with a non-decay corrected radiochemical yield of approximately 40% Ga-DFO-ZEGFR:2377 provided a higher uptake in tumors and lower uptake in the liver compared with that using [68Ga]-DOTA-ZEGFR:2377 (The acyclic siderophore-derived chelator deferoxamine (DFO) is suitable for the labeling of proteins with 68Ga . Site-spnd 85 °C . An in vGFR:2377 . This cr68Ga only permits this with a minimal margin. As noted above, imaging at time points beyond 3-4 h might provide a better contrast for a number of targets with expression in normal tissues. This rationalizes an exploration of long-lived positron emitters as labels for affibody molecules.Clinical studies suggest that the affibody-based imaging of HER2 expression in disseminated cancer 4 h pi permits better discrimination between tumors with high and low HER2 expression than that at 2 h pi . The hal½ = 12.7 h, positron yield 17.8%) is an actively studied positron emitter because of its facile production using low-energy cyclotrons, commercial availability, and potential for theranostic applications [64Cu resulted in at least a two- to three-fold higher liver uptake of the conjugates in comparison with analogues labeled with 68Ga or 111In  is a positron emitter that offers a broader imaging time window than that of yclotron . Althougon [44Sc . Synthet 98 ± 2% . [44Sc]S½ = 3.3 days, positron yield 23.3%) is gaining increasing attention because it is essentially the only positron emitter suitable for immunoPET with full-length immunoglobulins [89Zr after one hour at room temperature [89Zr]-Zr-DFO-cetuximab, [89Zr]-Zr-DFO-ZEGFR:2377 demonstrated higher tumor-to-organ ratios [89Zr]-Zr-DFO-ZEGFR:2377 than for other EGFR-targeting affibody molecules, and this might be associated with an instability of the complex in vivo. To provide a higher label stability, another siderophore-based chelator, fusarinine C (FSC), was conjugated to ZEGFR:2377 Zr-DFO-ZEGFR:2377 labeled at room temperature, i.e., a higher labeling temperature improved the stability of the [89Zr]Zr-DFO complex. The tumor-to-organ ratios for [89Zr]Zr-FSC-ZEGFR:2377 and [89Zr]Zr-DFO-ZEGFR:2377 (labeled at 85 °C) were approximately equal, but [89Zr]Zr-DFO-ZEGFR:2377 provided a better tumor-to-liver ratio.Zirconium-89  might be a better alternative from a dosimetry point of view. This radionuclide can be produced using low-energy cyclotrons [57Co (T ½ = 275 d) simplifies the preclinical development of 55Co-labeled probes [55/57Co by heating at 60 °C in 0.2 M ammonium acetate buffer at pH 5.5 for 30 min [57Co]Co-NOTA-HEHEHE-ZHER3:06898 are 1.5-2-fold higher at 24 h than at 3 h, and this could improve the sensitivity of imaging [57Co]-Co-DOTA-ZEGFR:2377 provided the best tumor-to-liver ratio among all tested affibody-based EGFR imaging probes -Affinity should be in the low nanomolar range for targets with a high expression, and a subnanomolar affinity is necessary in the case of low expression;A desirable affinity should be achieved by affinity maturation, not by dimerization;Meticulous dose-finding studies should be performed in the case of the physiological expression of a target in normal tissues. Conventional microdosing studies might be misleading due to a much higher normal tissue uptake when compared to imaging using optimal doses;The molecular design of affibody-based tracers should aim to minimize off-target interactions. Labeling approaches resulting in an increased lipophilicity are undesirable, but their negative effect might be compensated for by adding a negatively charged linker .The combination of a radiometal and chelator has an appreciable influence on off-target interactions and thus on the imaging contrast. Several alternative approaches should be tested to select the best one.We believe that the following considerations should be taken into account for the preclinical and clinical development of affibody-based imaging probes:


Dear Editor,https://www.worldometers.info/coronavirus/#countries). Such harsh conditions urged scientists across the world to gear up to develop vaccines and antiviral drugs against COVID-19, which also lead to massive requirement for experimental animals.Since the outbreak of a novel coronavirus disease (COVID-19) in late 2019, it has spread rapidly and developed into a global pandemic. As of August 12, 2020, more than 215 countries and territories around the world have reported more than 20.5 million confirmed COVID-19 cases with over 745,693 deaths  generating transgenic mice bearing hACE2 receptor,4 (ii) establishing adenovirus hACE2 mouse model with recombinant adenovirus expressing hACE2,5 and (iii) adapting the SARS-CoV-2 by serial passages in the respiratory tract of mice.8Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of COVID-19. It has been demonstrated that SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as cellular receptor for entry into target cells. Mouse model is the most commonly used animal model for studying human diseases. However, SARS-CoV-2 fails to invade and replicate in this traditional animal model due to the structural differences in mouse ACE2 (mACE2) compared with human ACE2 (hACE2),Alphavirus, family Togaviridae. Alphavirus replicon particles (VRPs), including VEEV-VRPs, represent efficient vectors for gene delivery and have been applied to studies of vaccine development, gene therapy and cell transduction. They contain self-replicating RNA-encoding viral replicase proteins (nsP1-nsP4) and express the gene of interest in place of viral structural protein genes.9 By providing viral structural proteins in trans, the replicon RNA is packaged into VEEV-VRPs for in vitro and in vivo gene delivery.10 Due to their intrinsic biological properties, VEEV-VRPs offer several advantages with a broad range of susceptible host cells, high expression level of cytoplasmic proteins and easy manipulation of recombinant RNA molecules using cDNA clones.11In this study, we used an alternative strategy to generate a SARS-CoV-2-sensitive mouse model by exogenous delivery of hACE2 with Venezuelan equine encephalitis replicon particles (VEEV-VRP-hACE2) . VEEV is1 infection. The SARS-CoV-2 NP-specific IFA-positive cells were only observed in VEEV-VRP-hACE2-transduced MLE-12 cells but not in the DMEM-treated cells  replicon expressing hACE2 with a C-terminal S-tag was packaged into VRPs using the helper RNAs encoding VEEV capsid and envelope proteins to produce VEEV-VRP-hACE2 , Fig. S1lls Fig. . Similarlls Fig. . These r6 IU VEEV-VRP-hACE2 or DMEM (negative control) in a volume of 80 μL DMEM through intranasal route. After 24 h, mice from different groups were inoculated intranasally with 105 PFU SARS-CoV-2, and then were monitored and weighted daily for 14 days. No obvious clinical symptoms and body weight loss were observed for mice in each group (6–107 copies/g) in both BALB/c and C57BL/6 mice transduced with VEEV-VRP-hACE2 from day 1 to day 5 post infection and decreased to background levels (104 copies/g) on day 9 in transduced BALB/c mice  had a higher neutrophil-to-lymphocyte ratio in the peripheral blood at 2 days post infection (dpi) compared with the mice infected with SARS-CoV-2 (SARS-CoV-2) or transduced with VEEV-VRP-hACE2 only (VRP-hACE2)  Fig. . MoreoveE2) Fig. .To characterize the pathological features caused by SARS-CoV-2 infection in VEEV-VRP-hACE2-transduced mice, histopathological analysis of lung sections from infected mice were performed Fig. . Hematox12 and an inactivated vaccine candidate that had entered clinical trials were chosen to examine their protective effects against SARS-CoV-2 infection using this mouse model. For CB6 protective assay, viral RNAs in the lungs of VEEV-VRP-hACE2-transduced mice were quantified at 3 d after SARS-CoV-2 infection. Consistent with previous study,12 CB6 exhibited strong protective effects against SARS-CoV-2 infection as the viral RNAs of CB6 mAb treated group were more than twenty times lower than those of mock-treated group , and the productive viral replication and visible histopathology in transduced mice that mimic clinical COVID-19 illness allow us to test potential antiviral agents and vaccines using this model.Supplementary information, Methods and Figures