Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors. To counteract viral infection, interferon induces the expression of antiviral effectors collectively termed Interferon Stimulated Genes (ISGs). Different effector genes restrict different viruses through a variety of mechanisms. Identifying the particular ISGs that restrict specific viruses can uncover viral vulnerabilities and inform therapeutic strategies. Here, we developed a CRISPR-activation (CRISPRa) experimental strategy for screening hundreds of pooled ISGs for antiviral activity in parallel while accounting for the ability of some ISGs to inhibit cell cycle and to promote cell death. We applied this approach to detect ISGs that restrict SARS-CoV-2, the virus that causes COVID-19. In a series of controlled experiments, we identified multiple ISGs that counteract SARS-CoV-2 infection, and further validated a subset of these ISGs through focused CRISPRa and cDNA expression studies. Our results validate several previously identified ISGs, and identify multiple novel antiviral effectors with activity against SARS-CoV-2. Overall, our work demonstrates an experimental system for the controlled assessment of ISG antiviral activities, and expands the present understanding of innate antiviral defense against SARS-CoV-2. Interferons (IFN) act as key mediators of the host response to viral pathogens by establishing a general antiviral state through the coordinated induction of hundreds of interferon stimulated genes (ISGs) in infected and uninfected “bystander” cells . ISGs enin vitro [A robust IFN response is critical for host defense against novel respiratory viruses to which immune memory from prior exposure has not been established . As suchin vitro –16, the in vitro . These fin vitro . Howeverin vitro . Dysreguin vitro . Additioin vitro –24. Impoin vitro . Taken tin vitro systems. Lymphocyte antigen 6 complex, locus-E (LY6E) was identified as a SARS-CoV-2 restriction factor in ISG ectopic cDNA expression screens [BST2 (encoding Tetherin), an anti-HIV effector that prevents the release of nascent virions [The specific mechanisms by which IFN restricts SARS-CoV-2 have not been fully characterized. To date, of the hundreds of ISGs, only a handful have been found to restrict SARS-CoV-2 infection in different  screens . A trans screens . In addi virions , has als virions . TRIM25, virions . During  virions ,32.Studies evaluating the antiviral potential of individual ISGs are often implemented through the ectopic expression of ISG cDNA libraries followed by viral challenge to screen for those ISGs that confer resistance ,29,33,34CRISPR-activation (CRISPRa), in which guide RNA (gRNA)-directed endonuclease-deficient Cas9 along with transcriptional activators are targeted to a gene of interest (GOI) to induce expression, offers an alternative to cDNA ectopic expression screens. Advantages include easy to produce gRNA libraries, physiologically relevant expression levels, and multiplexing capabilities . Gene trLY6E [CD74 [TRIM25 [ERLIN1 [). We identified and validated previously undescribed antiviral roles for additional ISGs such as CTSS (Cathepsin S). In agreement with Wickenhagen et al. and Soveg et al., [OAS1 (2′-5′-oligoadenylate synthetase 1) as a SARS-CoV-2 restriction factor capable of inhibiting viral infection and the generation of progeny virus. Taken together, our findings demonstrate the utility of a novel inducible CRISPRa platform for antiviral genetic screens and identify multiple ISGs capable of restricting SARS-CoV-2.Here, we report an ISG-focused CRISPRa screen to identify ISGs that modulate SARS-CoV-2 infection in lung epithelial cells. To mitigate the potential antiproliferative and/or proapoptotic effects of certain ISGs that could impact their library representation, we engineered a Doxycycline (Dox)-inducible CRISPRa system that enables precise temporal control of ISG induction. Using a pooled screen strategy, we tested more than 400 ISGs for effects on SARS-CoV-2 infection in both wildtype cells and isogenic cells engineered to be insensitive to IFN. High ranking antiviral ISG hits included SARS-CoV-2 restriction factors previously identified in recent screens . We ide et al., ,32 we alΔSTAT1 cell lines [ΔSTAT1-SunTag), as well as the STAT1-competent A549 cell line  from which they were derived. After selecting and expanding dual antibiotic-resistant single cell clones, we evaluated their capacity for Dox-inducible, gRNA-targeted gene expression. We transduced A549-SunTag and A549ΔSTAT1-SunTag cell lines with lentiviral constructs expressing gRNAs targeting the promoter region of MX1, a well-characterized ISG that restricts multiple viruses [MX1 mRNA expression was assessed by quantitative RT-PCR (qRT-PCR). In both A549-SunTag and A549ΔSTAT1-SunTag genotypes, Dox induced robust increases in MX1 mRNA in cells expressing MX1 gRNAs . Flow crotected . While wΔSTAT1-SunTag cells to enable productive SARS-CoV-2 infection of our CRISPRa cells. Next, we conducted pilot experiments evaluating SARS-CoV-2 CPE for optimization of screen conditions to balance the strength of selective pressure with its duration for robust detection of hits [ΔSTAT1-SunTag ACE2 cells were transduced with expression constructs for gRNAs targeting LY6E, a known SARS-CoV-2 restriction factor [On cultures expressing LY6E gRNAs exhibited increased viability when infected with SARS-CoV-2. Moreover, A549-SunTag ACE2 cultures exhibited less CPE than A549ΔSTAT1-SunTag ACE2 cultures, implicating additional STAT1-dependent antiviral factors. Based on these data, we approximated optimal infection conditions for ISG screens .To identify ISGs that restrict SARS-CoV-2, we conducted pooled gene activation screens in our engineered CRISPRa A549-SunTag lines. Our general screening strategy was to evaluate the potential of hundreds of individual ISGs to confer resistance to the cytopathic effects (CPE) of SARS-CoV-2. We began by introducing ACE2 expression into A549-SunTag and A549 of hits . A549-Sun factor , or NTGs2 fold-change >2, adjusted p value < 0.05). To focus on antiviral effectors, we excluded known transcription factors [To construct an ISG gRNA library for screening, we merged lists of ISGs tested in previous studies ,4 with a factors , several factors . ISG gRNΔSTAT1-SunTag ACE2 cells, in multiple independently transduced clones, across two independent experiments. A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 cells were transduced with our ISG gRNA library (M.O.I. = 0.1), puromycin selected, and expanded. 48 hours prior to SARS-CoV-2 infection, appropriate cultures were treated with Dox to induce ISG expression . All samples passed quality control metrics for multiple parameters as previously defined [On Mock vs DoxOff Mock and corresponding interaction term in the model).Gene activation screens were conducted in both A549-SunTag ACE2 and A549pression . All exp defined , exhibit defined ,62 to teΔSTAT1-SunTag ACE2 cells, we applied a stringent two-tiered set of filters for gRNAs differentially altered by SARS-CoV-2 infection. “Antiviral” and “proviral” (as determined by MAGeCK-MLE β-score [On infected vs. DoxOff infected coefficients) ISG hits were identified based on statistical significance for infection status (FDR < 0.1), while accounting for the effect of Dox treatment on gRNA abundance (linear model interaction FDR < 0.1). This strategy enabled robust identification of ISGs for which effects on SARS-CoV-2 infection were significantly stronger than their effects in mock infected cultures . We detected 6 and 24 ISGs as “antiviral”  included LY6E, CD74, TRIM25 and IFITM1 (STAT1 genotype included CTSL (encoding Cathepsin L), an entry factor for coronaviruses [CDKN1A (p21) and TNFRSF10A, ISGs with antiproliferative and/or proapoptotic effects [ΔSTAT1-SunTag ACE2 screens (OAS1 (encoding 2′-5′-oligoadenylate synthetase 1). Not surprisingly, our results suggest that STAT1-dependent transcription, perhaps in response to endogenous IFN production in A549-SunTag ACE2 screens, modulates detection of CRISPRa-induced ISG effects on SARS-CoV-2. As illustrated in STAT1 genotypes , but failed to clear significance thresholds in one of the screens. These discrepancies may be due to differences in relative enrichment  of particular ISGs in IFN-responsive versus STAT1-deficient contexts. In sum, in conducting focused CRISPRa ISG screens for cell viability effects in A549 cells infected with SARS-CoV-2, we detected several previously known SARS-CoV-2 restriction factors as well as identified new candidate ISGs with putative anti-SARS-CoV-2 activities.To identify ISGs with an effect on SARS-CoV-2 CPE in A549-SunTag ACE2 or A549 β-score  for DoxOd IFITM1 , each ofaviruses . Additio effects ,66, were effects . Interes screens , we founet al. and Martin-Sancho et al. both employ arrayed expression strategies coupled with flow cytometry or microscopy infection readouts, respectively. Together with our present study, which utilizes a pooled expression approach, ISG-focused screens have evaluated 637 distinct genes in the context of SARS-CoV-2 infection and identified multiple antiviral hits. Each study evaluated different sets of ISGs, due to different inclusion criteria in library construction and/or the availability of sequence-verified cDNA clones for arrayed experiments. Intersecting the ISG libraries of each study revealed a set of 224 ISGs assayed in all three screens, while highlighting that each study tested distinct collections of ISGs  common to all three studies  [To date, two published studies report focused ISG screens for host factors with antiviral activity against SARS-CoV-2 ,31. Wick of ISGs . Somewha studies . A gene- studies . For exa  ,69, wereΔSTAT1-SunTag ACE2 cells were transduced with expression constructs for gRNAs targeting one of eight antiviral ISG hits  or with one of two NTGs, treated with Dox to induce gene expression, and infected with SARS-CoV-2. Compared to screening experiments (M.O.I. = 3), which were designed to select cells resistant to CPE in the context of extensive cell death, infection conditions in validation experiments were reduced slightly (M.O.I. = 2) to retain sufficient viable cells for downstream readouts. Dox-inducible, gRNA-specific CRISPRa-mediated expression for select genes  was confirmed in complementary qRT-PCR experiments  at 24 and 72 hours post-infection. At 24 hours, the fraction of SARS-CoV-2 infected cells  was significantly reduced in both A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 cultures transduced with gRNAs targeting SARS-CoV-2 restriction factors CD74 [LY6E [OAS1 expression in both A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 cultures, while TRIM25 expression demonstrated significant restriction only in A549-SunTag ACE2 cells (CTSS), no additional genes met statistical significance thresholds at the 24 hour time point.To confirm hits identified in the pooled screens, we first conducted “single gene” validation experiments with the CRISPRa system for a subset of candidate antiviral ISGs. A549-SunTag ACE2 and A549eriments . We asseors CD74  and LY6E74 [LY6E  , was recently shown to restrict SARS-CoV-2 infection [ADPRHL2 and GBP1, additional ISGs identified as antiviral screen hits, did not reach statistical significance in CRISPRa validation experiments  was significantly reduced in A549-SunTag ACE2 cultures with activated expression of and OAS1 . We alsoE2 cells . Ectopicnfection . ADPRHL2eriments . Taken tΔSTAT1 ACE2 cells (both without CRISPRa components) were transduced with Dox-inducible cDNA expression constructs for one of CD74, LY6E, CTSS, TRIM25, ADPRHL2 or fLuc . Comparison of protein expression patterns to corresponding CRISPRa cultures and assessments of Dox inducibility properties were carried out by immunoblot analysis of select screen hits , and quantified viral titers by plaque assay over time. Activation of OAS1 expression resulted in a significant decrease in the production of viral progeny, indicating that OAS1 activity functionally restricts SARS-CoV-2 infection  and a shorter OAS1 isoform  that was recently suggested to be the highest expressed isoform of OAS1 in A549 cells [C331A/F332A/K333A) [ΔSTAT1 ACE2 cells. The p42 isoform of OAS1 exhibited similar restriction of infection. Inactivation of OAS1 catalytic activity completely ablated its antiviral effects, suggesting that the mechanism of OAS1-mediated SARS-CoV-2 restriction acts through downstream RNaseL activation. Interestingly, while expression of OAS3 exhibited some restriction of SARS-CoV-2 (significant only in A549 ACE2 cells), other OAS family members did not restrict SARS-CoV-2 infection (OAS2), or may have promoted SARS-CoV-2 infection .OAS1 isoforms have been shown to differ in their antiviral activities . To eval49 cells . To testA/K333A) . We alsoOff mock infected vs DoxOn mock infected, adjusted p value < 0.1, on conditions included target genes for apoptotic signaling  and cell cycle negative regulation (CDKN1A). The small number of enriched gRNAs (i.e. positively selected upon expression) included Transferrin (TF) and Growth differentiation factor 15 (GDF15), both of which have been previously described to promote cell proliferation in A549 cells [ΔSTAT1-SunTag screens included CDKN1A, TNFRSF10A and APOBEC3A, all previously implicated in reducing proliferation [RNF213, MT1H, UBE2L6, JAK2 and PXK, without established roles in cell cycle and/or death pathways  [IFI27 [XAF1 [On and DoxOff cultures in the absence of viral infection, we are able to assess the antiproliferative/proapoptotic effects of each ISG. Indeed, not only did our results include multiple known cell cycle regulators depleted after only 48 hours after Dox induction, but they also included genes  with demonstrable effects on both virus-independent library enrichment/depletion and susceptibility to SARS-CoV-2. While the antiproliferative and proapoptotic effects of IFN are well established [Some ISGs suppress cell proliferation or promote apoptosis . These m1A (p21) , IFI27 [) [IFI27 , XAF1 [827 [XAF1 , and mem27 [XAF1 . Assessi27 [XAF1 –86. Our ablished , a systeTMPRSS2, while endosomal entry is mediated by cathepsins that process the spike protein [CTSL), an entry factor for coronaviruses [CTSS), an ISG in A549 cells [TMPRSS2 [CTSL and CTSS on SARS-CoV-2 infection are surprising and the mechanistic basis for this difference is unclear. CTSS and CTSL have been shown to bind and cleave different polypeptide motifs [CTSS results in suboptimal spike cleavage products that are dysfunctional for viral entry. Alternatively, CTSS may interfere indirectly with spike processing by other cathepsins such as CTSL. Of note, we found that CTSS maintains its restrictive function in A549ΔSTAT1 cells, indicating that its activity against SARS-CoV-2 does not require IFN-induced factors such as CD74, which inhibits SARS-CoV-2 by blocking cathepsin-mediated entry [Coronaviruses can enter cells via two different routes: from the cell membrane or from the endosomal compartment. The route of entry is determined in part by the presence of cellular proteases required for spike protein processing . SARS-Co protein . Catheps protein . Our scraviruses , sensiti49 cells , was ide[TMPRSS2 , making e motifs , raisinged entry .ΔSTAT1-SunTag ACE2 cells. Our experiments further demonstrated that OAS1 catalytic activity is necessary for its effects on SARS-CoV-2. This observation suggests that SARS-CoV-2 may not directly antagonize the generation of 2′-5′A like MERS-CoV, and therefore remains susceptible to RNaseL effector functions [Oligoadenylate synthetase family members are broadly acting ISGs important for innate antiviral defense against multiple viruses . RNaseL,unctions .OAS1 genetic variants were linked to infection and excessive morbidity in the SARS-CoV outbreak [OAS loci with COVID-19 mortality [et al. and Soveg, et al. described an association between a prenylated isoform of OAS1 (p46) and COVID-19 outcomes, further underscoring the importance of OAS1 in SARS-CoV-2 pathogenesis. Both studies demonstrated that prenylated OAS1 is targeted to the endomembranous sites of SARS-CoV-2 replication. They both observed significantly more potent viral restriction by the OAS1p46 isoform than the OAS1p42 isoform, which lacks the CAAX box motif prenylation signal. Interestingly, although A549 cells are capable of expressing only the OAS1p42 isoform [et al. did not detect antiviral activity for OAS1p42 in A549 cells expressing ACE2 and TMPRSS2, while Soveg, et al. observed viral restriction by OAS1p42 that was further enhanced by the prenylated OAS1p46 isoform in 293T cells. Our observation of antiviral activity for both OAS1p42 and OAS1p46 could be a consequence of the assays used, infection conditions, timepoints tested and/or differences in expression levels. It could also be possible that different SARS-CoV-2 lineages are differentially susceptible to OAS1 isoforms .A growing body of genetic, epidemiological, and clinical data support an important role for OAS1 in both SARS-CoV and SARS-CoV-2 host defense. outbreak ,94. Moreortality . Clinicaortality . While tortality ,32. Wick isoform ,38, we fOAS1 has been characterized as proapoptotic [In addition to apparent differences in isoform activity, given the potent effects of OAS1 in our experiments, we were surprised that it had not been detected in several other recently published genome-wide and ISG-focused screens ,47,97,98poptotic , our indWhile our screens were primarily focused on identifying SARS-CoV-2 antiviral factors based on the enrichment of ISG gRNAs in cells protected from virus-mediated CPE, our experimental design enabled parallel identification of ISGs that sensitize cells to CPE based on relative depletion of gRNAs in infected cultures. We identified 12 factors that enhanced SARS-CoV-2 CPE, of which 6 have been previously identified as potential direct interactors with SARS-CoV-2 proteins  –104. Of 2. Vero-E6 cells  were used for propagation of SARS-COV-2 and for plaque-assays. Lenti-X 293T cells (Takara #632180) were used for lentivirus packaging. A549 and A549ΔSTAT1 [All cell lines used in this study were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal-bovine serum (FBS) and 1% Penicillin Streptomycin , and routinely cultured at 37° C with 5% CO49ΔSTAT1 , a kind 49ΔSTAT1 .For Dox treatments to induce gene expression (both CRISPRa cell lines and cDNA ORF expression constructs), cultures were incubated in DMEM 10% FBS supplemented with 5μg/ml Doxycycline (Sigma # 50-165-6938) for 48 hours prior to infection or culture harvest as indicated for each experiment. Infections  were performed for 1 hour at 37°C in the absence of Dox, which was re-added (1μg/ml) upon removal of the virus inoculum. For IFN experiments, cultures were treated with 200U of recombinant human IFNα2b (PBL Assay Science # 11105–1) for 3 hours prior to culture harvest.SARS-COV-2  stocks were grown by inoculating confluent T175 flasks of Vero-E6 cells with SARS-COV-2 isolate (passage 2). Infected cultures were maintained in reduced-serum DMEM (2% FBS) for 3 days, after which medium was collected and filtered by centrifugation  using an Amicon Ultra-15 filter unit with a 100KDa cutoff filter (Millipore # UFC910024). Concentrated virus stocks in reduced-serum DMEM (2% FBS) supplemented with 50mM HEPES buffer (Gibco #15630080) were stored at -80°C.To determine the number of infectious units in each viral stock (IU/ml), target cell lines (A549-ACE2 or A549-SunTag-ACE2) were plated in duplicate in 24 well plates, and were infected with 2-fold serial dilution series of SARS-CoV-2 stocks at 37°C for 1 hour, after which virus was removed and replaced with DMEM supplemented with 10% FBS. At 24 hours post-infection, cultures were harvested and fixed by incubating in 4% Paraformaldehyde (Alfa Aesar #AA433689M) in PBS for 24 hours. The fraction of infected cells was determined by flow cytometry for SARS-CoV-2 N protein (details below). The percentage of infected cells was used to determine the IU/ml values for each viral stock by using the formula:6 Lenti-X 293T cells  in 1 ml of 10% FBS DMEM for 8 hours after which transfection media was removed and replaced with 2ml of 10% FBS DMEM. 3 days post-transfection lentivirus supernatants were collected and centrifuged at 800g for 5 minutes to pellet cell debris, filtered through 45μm PVDF filters (Millipore # SLHVR33RS), and stored at -80°C. Transductions were performed on semi-confluent wells in the presence of Polybrene  using spin-infection . Culture media containing selection antibiotics was added to transduced cultures 24 hours post-transduction.To generate lentiviruses for gRNA or cDNA expression, a mix of 2.5μg of the desired transfer vector, 2μg psPAX2 and 0.8μg of pMD2.G , 14μl Lipofectamine 3000 (ThermoFisher scientific #L3000001) and 20μl of P3000 reagent, was prepared in 250μl of OptiMEM (Gibco #11058021). This transfection mix was added to 1.5x10To generate the plasmids for the inducible CRISPRa system, we re-engineered an existing CRISPRa technology  to enablΔSTAT1-SunTag cells, we transduced A549 or A549ΔSTAT1 cells with lentiviruses encoding the Dox-inducible transactivator component of SunTag (pCW-TRE-scFv-SunTag). Single cell clones were selected with Blasticidin . Next, pCW-TRE-scFv-SunTag single cell clones were transduced with lentiviruses encoding the SunTag nuclease-inactive Cas9 (pHR-PGK-dCas9-SunTag-P2A-HygR) and single cell clones were selected with Hygromycin . Finally, multiple single cell clones of A549-SunTag and A549ΔSTAT1-SunTag, selected for the expression of both components, were tested for gRNA-directed gene activation capabilities with gRNAs targeting CXCR4 and measuring CXCR4 protein expression by flow cytometry as previously described [To generate A549-SunTag and A549escribed .4 target cells  in 12 well plates. Single cell clones were expanded and validated for expression of ACE2 by Western Blot analysis (Abcam #ab15348).To generate ACE2-expressing A549 cell lines, the human ACE2 coding sequence (RefSeq accession NM_001371415.1) was PCR amplified and cloned into the BamHI site of lentiviral vector pHR-PGK (Addgene #79120). Lentivirus was produced as described above and used to transduce 5x10MX1; Hs00895608_m1, CD74; Hs00269961_m1, OAS1; Hs00242943_m1, CTSS; Hs00175407_m1, GAPDH; Hs99999905_m1). Ct values were used for calculation of gene expression using the comparative threshold cycle method [GAPDH expression as loading control, comparing Doxon condition to Doxoff condition.For indicated experiments, A549 cultures were harvested by trypsinization, pelleted, and homogenized in Trizol (Invitrogen #15596026). Total RNA was isolated with the Direct-zol RNA MiniPrep kit (Zymo #R2050), including DNase protocol, according to manufacturer’s instructions. For each sample, 1μg of total RNA was reverse transcribed (Applied Biosystems #4368814) with random hexamers for priming. qRT-PCR was performed with the TaqMan Universal Master Mix (Applied Biosystems #4440038), and TaqMan primer/probe sets for each gene of interest  supplemented with 50mM HEPES and 1% PSN, inoculated to indicated cell cultures, and incubated at 37°C for 1 hour. Infection medium was then replaced with DMEM (10% FBS and 1% PSN) for timepoints indicated in each experiment.ΔSTAT1-SunTag cells were plated in 96-well plates  and infected with SARS-CoV-2. Infections were done in reduced serum DMEM , and virus was left on the cells for the indicated time points. Plates were fixed in 4% Paraformaldehyde at room temperature for a minimum of 24 hours. Cells were then washed twice with 100μl 0.1M sodium tetraborate (Sigma # 221732), stained with 0.5% methylene blue (Sigma # M9140) in 0.1M sodium tetraborate , extensively washed in 0.1M sodium tetraborate, and extracted with 0.1M HCl. Absorbance was measured on a BioTek Cytation plate reader at 595 nm.A549-SunTag and A5492 fold-change >2, adjusted p value < 0.05) after 6 or 48 hours of IFN stimulation in A549 cells [To assemble the list of ISGs targeted by the gRNA library, an established list of ISGs ,4 was co49 cells . To focu49 cells , HLA gen49 cells . The fin49 cells  as descr49 cells . BrieflySTAT1 genotype , 6 x 106 cells were transduced with gRNA library lentivirus (described above) at M.O.I. = 0.1, assuring zero or one gRNA/cell in more than 95% of library cells, and representation of 500 cells/gRNA. Transduced cultures were selected for CROP-seq-opti transduction with Puromycin , and expanded for 14 days. 48 hours prior to SARS-COV-2 infection, gene expression was induced by treating the cells with Dox (5μg/ml). 2 x 106 cells (estimated representation of more that 1500 cells/gRNA) were infected with SARS-COV-2 (M.O.I. = 3). 72 hours post infection, after observation of significant CPE in infected cultures, genomic DNA was extracted from surviving cells for Illumina sequencing library preparation. In total, screens were conducted in two experiments, each experiment including two single cell clones from each STAT1 genotype. In the first experiment, following infection, an additional 4ml of reduced-serum DMEM were added to the cultures, but the virus inoculum was not removed from culture wells. In the second experiment, the virus inoculum was removed from the cells after 1 hour of infection, cultures were washed twice with calcium/magnesium-free PBS, and cultured in DMEM (10% FBS) for 72 hours.For each of 2 clones from each CROP-seq-opti gRNA sequencing libraries were prepared as previously described . Brieflyet al., 2019). Sample x gRNA count tables and quality control matrixes were generated with the MAGeCK count function. Enrichment/depletion analyses were conducted with MAGeCK MLE for each STAT1 genotype. To analyze gRNA enrichment, we used MAGeCK MLE with a design matrix generated by the R (version 4.0.2) model.matrix function, with design formula specified as: ~(dox*virus) + experiment + clone. The resulting generalized linear model included factors for Dox status, SARS-CoV-2 infection status, clone, and experiment, as well as a dox:virus interaction term (used to test for gRNA enrichment/depletion by virus infection differences by dox status). MAGeCK MLE was run with 10 permutations, with normalization to NTG gRNAs. Enrichment/depletion p values were adjusted by the method of Benjamini and Hochberg within the MAGeCK MLE framework. To focus on genes enriched or depleted upon viral infection beyond potential virus-independent effects on library representation, we filtered to include hits with adjusted p value less than 0.1 for both SARS-CoV-2 infection status coefficient (DoxOn SARS-CoV-2 infected vs DoxOn mock infected) and the Dox status:SARS-CoV-2 infection status interaction term. “Antiviral”/“proviral” designations were made based on the sign of the β score for the SARS-CoV-2 infection status coefficient.Illumina BCL sequence files were converted to FASTQ format with the bcl2fastq tool . gRNA enrichment/depletion analyses were conducted with the MAGeCK package , and β score were multiplied by -1 to facilitate enrichment/depletion interpretation within the required model syntax.In analyses for antiproliferative/proapoptotic ISGs, hits were selected as adjusted p value less than 0.1 for the Dox status coefficient  of candFor VSV-GFP experiments, cells were fixed with 4% Paraformaldehyde for 30 minutes at room temperature and analyzed for GFP fluorescence on a Gallios flow cytometer (Beckman-Coulter). For SARS-CoV-2 experiments, cells were fixed with 4% paraformaldehyde at room temperature for a minimum of 24 hours, washed once with PBS and permeabilized with 1X perm-wash buffer (BDBiosciences #554723) for 5 minutes. AlexaFluor 647-conjugated SARS-CoV nucleocapsid (N) antibody  was diluted 1:400 in perm-wash buffer, and added directly to permeabilized samples, which were then incubated at room temperature for 40 minutes in the dark. Samples were washed once with 1X perm-wash buffer, once with calcium/magnesium-free PBS, and acquired on a Gallios flow cytometer (Beckman-Coulter). For all viral infections, analysis was performed with FlowJo software , excluding cell doublets and debris and gating according to mock infected populations . Samplesg x 15 minutes, 4C). Protein amounts were quantified using the Pierce BCA assay (Pierce # 23225). 20–40μg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–12% gradient gel (ThermoFisher Scientific # NW04125BOX), and transferred to a nitrocellulose membrane (ThermoFisher Scientific # IB23001) according to the manufacturer instructions. Membranes were blocked for 1 hour at room temperature with 5% skim milk (Difco # DF0032-17-3) in Tween Tris-Buffered Saline , and blotted with primary antibodies overnight at 4C with gentle agitation. After excessive washing in TBST, membranes were incubated for 1 hour at room temperature with HRP-conjugated secondary antibody. HRP signal was detected using an enhanced chemiluminescence substrate (Pierce # 32106) in a ChemiDoc imaging system (BioRad # 1708265). Antibodies used: Rabbit anti-CTSS ; Rabbit anti-CD74 (CST # 77274); Rabbit anti-OAS2 (CST # 24344); Rabbit anti-OAS3 (CST # 41440); all in 1:1000 dilution in blocking solution. Rabbit anti-OAS1 (CST # 14498) was diluted 1:500. Rabbit anti-OASL (GeneTex # GTX31572) was used at 1:1000 dilution in blocking solution. Rabbit anti-GAPDH was used for loading control (Abcam # ab9485) was diluted 1:10,000. Secondary HRP-conjugated anti-rabbit (CST # 7074S) was diluted 1:2000 in blocking buffer.Cells were lysed in ice-cold RIPA buffer (ThermoFisher scientific # 89900) supplemented with protease inhibitor cocktail (Roche # 11697498001) for 30 minutes on ice and clarified by centrifugation  for 15 minutes. Plates were blocked with 3% Bovine serum albumin  in PBS for 1 hour. AlexaFluor 647-conjugated SARS-CoV nucleocapsid (N) antibody (clone 1C7C7) was diluted 1:2000 in 0.5% BSA in PBS and added to wells for 1 hour incubation. Samples were then washed twice with PBS and stained with 4′,6-diamidino-2-phenylindole  diluted 1:200 in 0.5% BSA in PBS. All steps were done at room temperature. Plates were imaged on a Celigo instrument (Nexcelom Biosciences) and the fraction of infected cells per well was determined using CellProfiler [SARS-CoV-2 infected A549-SunTag ACE2 and A549Profiler .ΔSTAT1 SunTag cells, expressing gRNAs targeting either OAS1 or NTG, were pretreated, or not, with Dox , and infected with SARS-CoV-2 (M.O.I. = 0.1). After 1 hour, virus inoculum was removed and replaced with DMEM/10% FBS supplemented with or without Dox. Every 24 hours post infection, 100μl of the culture supernatants was collected, serially diluted, and used to infect 2x105 Vero-E6 cells plated in a 24 well plates . After 1 hour, inoculum was removed and replaced with overlay media consisting of Minimum Essential Media  supplemented with 0.8% SeaPlaque Agarose (Lonza # 50104), 4% FBS and 1% PSN pre-warmed to 37°. At 72 hours post-infection, Vero-E6 cells were fixed in 4% Paraformaldehyde at room temperature for 24 hours, washed twice with PBS and stained with 1% Crystal violet (Sigma # C0775) for 15 minutes. Viral titer was determined by calculating infectious units/ml.A549Unless otherwise indicated, error bars indicate standard deviation from the mean (SD) of at least 3 biological replicates . For flow cytometry and immunofluorescence experiments, statistical significance was determined with one-sided paired ratio Student’s t-test, with p values < 0.05 considered to be significant  Pilot experiment using Methylene Blue assay to assess SARS-CoV-2 CPE under different infection conditions. A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 cells, expressing LY6E gRNA or a non-targeting gRNA (NTG) were infected with SARS-CoV-2 at indicated M.O.I., fixed at indicated time points, and stained with methylene blue. Values indicate percent OD595 absorption relative to time point = 0 (set to 100%). (B) CRISPRa screen quality metrics: sequencing reads per sample. Values indicate number of reads sequenced for each sample in the pooled screens. Percentage values (light fill) for reads that fail to map to gRNA sequences in the ISG library reference. (C) CRISPRa screen quality metrics: Normalized read count distribution per sample. Log10 transformed read count for each sample normalized to the count of non-targeting guides.(TIF)Click here for additional data file.S2 Fig(A) Venn diagram generated from intersecting lists of ISGs from the libraries of Wickenhagen et al. [et al. [(B) Similar to A, highlighting shared and distinct antiviral hits. (C) Comparison of candidate antiviral hits from published studies and this study. Z scores of infections were calculated from published datasets and ranked. Black square: antiviral hit. White square: not antiviral hit. Empty square: not tested/no data available.n et al. , Martin- [et al.  and our (TIF)Click here for additional data file.S3 Fig(A) qRT-PCR analysis demonstrates effective CRISPRa gene induction in A549-SunTag ACE2 cells. Values indicate fold change expression (Doxon relative to Doxoff) for indicated genes  in A549-SunTag ACE2 cells expressing either corresponding gRNA or NTG. Fold change values calculated using the ΔΔCt method with GAPDH as normalization control. Undetectable Ct value for Doxoff condition of cells expressing NTG and probed for CD74 expression was set to 40 to enable fold change calculation. (B) qRT-PCR mean threshold cycle (Ct) values for CD74, OAS1, CTSS and GAPDH in A549-SunTag ACE2 cells expressing gRNA against gene of interest  or NTG (diamond) in Doxon and Doxoff cells. Error bars indicate ± SD Ct value. (C) Representative flow cytometry histograms for SARS-CoV-2 N protein in A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 transduced with indicated gRNAs, treated (red) or not treated (gray) with Dox, at 24 hours post-infection with SARS-CoV-2 (M.O.I. = 2). (D) Percent of infected (SARS-CoV-2 N protein positive) cells quantified across biological replicates (n = 3) for experiments described in (C). Values denote percent of infected cells in Doxon cultures relative to paired Doxoff cultures. Points represent individual biological replicates, black lines indicate mean values of biological replicates for each indicated ISG gRNA. Red points indicate statistical significance (p < 0.05) as determined by paired ratio Student’s t-test. (E) Representative immunofluorescence images for SARS-CoV-2 N protein and DAPI in A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 transduced with indicated gRNAs and treated with Dox, at 72 hours post-infection with SARS-CoV-2 (M.O.I. = 2). (F) Percent of infected (SARS-CoV-2 N protein positive) cells quantified across biological replicates for experiments described in (E). Values denote percent of infected cells in Doxon cultures relative to paired Doxoff cultures. Points represent individual biological replicates, black lines indicate mean values of biological replicates for each indicated ISG gRNA. Statistical significance as in (D).(TIF)Click here for additional data file.S4 Fig(A-B) Immunoblot analysis of CD74 (A) and Cathepsin S (B) expression in A549-ACE2 cells transduced with lentivirus encoding indicated ORFs compared to A549-SunTag ACE2 cells expressing guides targeting the activation of CD74, CTSS or a non-targeting guide. Cells were incubated with Dox to induce gene expression for 48 hours prior to processing for immunoblot analysis with the indicated antibodies.(TIF)Click here for additional data file.S5 Fig(A) ISGs ranked by the inverse of their β-scores for the Dox status coefficient (DoxOff mock infected vs DoxOn mock infected). Significantly  enriched/depleted gRNAs are highlighted in green/purple respectively. (B) Venn diagram of significantly depleted (i.e. candidate antiproliferative/proapoptotic) ISG hits from (A) in A549-SunTag ACE2 and A549ΔSTAT1-SunTag ACE2 screens.(TIF)Click here for additional data file.S6 FigRepresentative gating strategy for identifying SARS-CoV-2 infected cells.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Growth hormone secretagogue receptor 1a (GHS-R1a) is widely distributed in brain including the hippocampus. Studies have demonstrated the critical role of hippocampal ghrelin/GHS-R1a signaling in synaptic physiology, memory and cognitive dysfunction associated with Alzheimer’s disease (AD). However, current reports are inconsistent, and the mechanism underlying memory modulation of GHS-R1a signaling is uncertain. In this study, we aim to investigate the direct impact of selective increase of GHS-R1a expression in dCA1 excitatory/inhibitory neurons on learning and memory.aav-hSyn-DIO-hGhsr1a-2A-eGFP virus in dCA1 region of vGlut1-Cre or Dlx5/6-Cre mice respectively. Virus-mediated GHS-R1a upregulation in dCA1 neurons was confirmed by quantitative RT-PCR. Different behavioral paradigms were used to evaluate long-term memory performance.Endogenous GHS-R1a distribution in dCA1 excitatory/inhibitory neurons was assessed by fluorescence in situ hybridization. Cre-dependent GHS-R1a overexpression in excitatory or inhibitory neurons was done by stereotaxic injection of +) and in inhibitory interneurons (GAD67+). Selective increase of GHS-R1a expression in dCA1 pyramidal neurons impaired spatial memory and object-place recognition memory. In contrast, selective increase of GHS-R1a expression in dCA1 interneurons enhanced long-term memory performance. Our findings reveal, for the first time, a neuronal type-specific role that hippocampal GHS-R1a signaling plays in regulating memory. Therefore, manipulating GHS-R1a expression/activity in different subpopulation of neurons may help to clarify current contradictory findings and to elucidate mechanism of memory control by ghrelin/GHS-R1a signaling, under both physiological and pathological conditions such as AD.GHS-R1a is distributed both in dCA1 excitatory pyramidal neurons (αCaMKIIThe online version contains supplementary material available at 10.1186/s13041-021-00866-8. Ghrelin is the only identified orexigenic gastric hormone that promotes feeding, and is critical for metabolism regulation in both human and rodents . It has Studies have highlighted intriguing contradictory roles that ghrelin and GHS-R1a play in regulating multiple neuronal functions such as learning and memory, other than nutrient sensing and metabolic control . For insIt is important to note that GHS-R1a displays two uncommon features that may greatly contribute to its functional complexity, extremely high constitutive activity  and multaav-hSyn-DIO-hGhsr1a-2A-eGFP) or control virus (aav-hSyn-DIO-eGFP) was delivered in dCA1 of Vglut1-Cre or Dlx5/6-Cre male mice (3–4 month old) respectively to selectively increase GHS-R1a expression in excitatory or inhibitory neurons in dorsal hippocampus. GFP fluorescence in dCA1 region indicated successful viral transfection and virus-mediated GHS-R1a expression in pyramidal neurons or interneurons 3 weeks after injection (Fig. Endogenous GHS-R1a distribution in both excitatory and inhibitory dCA1 neurons was confirmed by fluorescent in situ hybridization assays (Fig. aav-hSyn-DIO-hGhsr1a-2A-eGFP virus exhibited poor spatial memory (Fig. aav-hSyn-DIO-eGFP virus injection. In contrast, we found that Dlx5/6-Cre mice receiving aav-hSyn-DIO-hGhsr1a-2A-eGFP virus displayed better spatial memory (Fig. The effect of increasing GHS-R1a expression on hippocampus-dependent learning and memory performance was evaluated 4 weeks after viral injection. We found that selective GHS-R1a upregulation in dCA1 excitatory pyramidal neurons impairs hippocampus-dependent memory processes. Specifically, Vglut1-Cre mice transfected with The hippocampus is a complex network tightly regulated by interactions between excitatory pyramidal neurons and inhibitory interneurons. Although represent a minority in the hippocampus, interneurons play a critical role in shaping network activities . HoweverIn conclusion, our findings reveal, for the first time, that elevated GHS-R1a expression selectively in dCA1 excitatory/inhibitory neurons differentially regulates memory encoding. It also suggests a causal relationship between hippocampal GHS-R1a expression and memory.Additional file 1. Selectively increasing GHS-R1a expression in dCA1 excitatory/inhibitory neurons have opposite effects on memory encoding.


The lack of such techniques hampers a deeper understanding of the pathophysiological role of BCM in diabetes, the implementation of personalized BCM-based diabetes management, and the development of antidiabetic therapies targeting BCM preservation and restoration. Nuclear medical techniques have recently triggered a major leap in this field. In particular, radioisotope-labeled probes using exendin peptides that include glucagon-like peptide-1 receptor (GLP-1R) agonist and antagonist have been employed in positron emission tomography and single-photon emission computed tomography. These probes have demonstrated high specificity to β cells and provide clear images accurately showing uptake in the pancreas and transplanted islets in preclinical in vivo and clinical studies. One of these probes, 111indium-labeled exendin-4 derivative ([Lys12(111In-BnDTPA-Ahx)]exendin-4), has captured the longitudinal changes in BCM during the development and progression of diabetes and under antidiabetic therapies in various mouse models of type 1 and type 2 diabetes mellitus. GLP-1R-targeted imaging is therefore a promising tool for non-invasive BCM evaluation. This review focuses on recent advances in non-invasive in vivo β-cell imaging for BCM evaluation in the field of diabetes; in particular, the exendin-based GLP-1R-targeted nuclear medicine techniques.Pancreatic beta (β)-cell dysfunction and reduced mass play a central role in the development and progression of diabetes mellitus. Conventional histological β-cell mass (BCM) analysis is invasive and limited to cross-sectional observations in a restricted sampling area. However, the non-invasive evaluation of BCM remains elusive, and practical  The number of patients with diabetes worldwide is on the rise. The estimated prevalence of patients with diabetes 20 years of age or older has risen from 151 million to 463 million from 2000 to 2019, and it is predicted to increase to 700 million by 2045  receptor agonist and antagonist] have been promising tools for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging. A number of these probes, including 111indium-labeled exendin-4 derivative {[Lys12(111In-BnDTPA-Ahx)]exendin-4}, have demonstrated the visualization of the mouse pancreas and its image analysis for BCM quantification and thereby have shown potential for clinical use -cell mass (BCM) and β-cell function involves the onset and progression of diabetes; BCM plays a central role in the pathophysiology of both type 1 and type 2 diabetes mellitus . In typeical use . This reBoth type 1 and type 2 diabetes mellitus are characterized by a progressive reduction in BCM . AlthougIn type 1 diabetes mellitus, pancreatic β cells are predominantly destroyed by autoimmune attacks, which result in markedly reduced BCM and severe insulin deficiency. However, the remaining viable β cells, including non-functional ones, are observed in over 30% of patients with long-duration type 1 diabetes mellitus , 21, 22.Islet transplantation, in which isolated donor islets are infused into the portal vein, has become a promising treatment option for patients with type 1 diabetes mellitus , 25. AltIn type 2 diabetes mellitus, a hypothetic model of BCM changes has been proposed; type 2 diabetes mellitus occurs in response to a reduction in BCM following a temporary compensatory increase in BCM induced by obesity and insulin resistance. BCM subsequently decreases progressively . This hyIn vivo β-cell visualization and quantification require imaging modalities with sufficiently high spatial resolution to image individual islets, and no widely available clinical modality [including computed tomography (CT) and magnetic resonance imaging (MRI)] has satisfied this requirement  , 41, gluSUR1 is a subunit of ATP-dependent potassium channels and is specifically expressed in β cells except in the brain. Given that sulfonylureas are known for their binding affinities to SUR1 and in light of their clinical use experience, a number of radiolabeled sulfonylurea derivatives such as glibenclamide and glipizide have been investigated. However, these probes have failed to achieve sufficient specificity to β cells; low accumulations in the pancreas and high background signals , 58. Alt18F]dihydroxyphenylalanine [(18F)DOPA], has been investigated for nesidioblastosis, with several groups reporting the successful imaging of responsible foci. However, [18F]DOPA was taken up in both endocrine and exocrine pancreatic cells and showed high background signals, which suggests a limited potential use for this probe (D2 and D3 dopamine receptors involve glucose-stimulated insulin secretion in β cells  and haveis probe .11C)5-hydroxytryptophane [(11C)HTP] is a tracer employed for evaluating serotonin biosynthesis and is metabolized by dopamine decarboxylase to (11C)serotonin. Given that serotonin is accumulated in β cells, [11C]HTP PET has been investigated in β-cell imaging, demonstrating substantially reduced accumulation in the pancreas of patients with type 1 diabetes mellitus (11C)HTP PET also demonstrated its potential utility for the longitudinal observation of islet mass in type 1 diabetes mellitus and islet transplantation ((mellitus . (11C)HTantation , 51. Howantation , 61, 62.18F)fluoropropyl-DTBZ has been employed for evaluating BCM in patients with type 1 diabetes, showing lower accumulations in the pancreas compared with those of healthy participants , a ligand of VMAT2, have been actively researched as potential β-cell imaging probes. However, most DTBZ derivatives have demonstrated high exocrine-islet ratios in the pancreas, lacking the specific accumulation in β cells.  have been synthesized and have shown high in vivo stability with a high affinity to GLP-1R  have shown rapid blood clearance and uptake of the radiotracer into the tumor and pancreas, which could be detected by SPECT. 111In-labeled exendin-3, {Lys40[(111In)DTPA]}exendin-3, was subsequently synthesized, given that the high sensitivity of 111In-labeled tracers allowed for the administration of small amounts of tracer to prevent receptor saturation and adverse effects in SPECT (40[(111In)DTPA]}exendin-3 in insulin-expressing cells, with the highest uptake in the pancreas and lungs followed by the kidneys -labeled exendin-3 has also been developed as a probe for PET imaging -labeled exendin (9-39) exhibited good affinity to GLP-1R and high uptake in mice pancreas (125I)BH-exendin (9-39) differed in human and mouse tissues (19 showed comparable affinity in human and rat tissues (111In) diethylenetriaminepentaacetic acid (DTPA) or tetraazacyclododecane tetraacetic acid (DOTA) via aminohexanoic acid linker (Ahx) has been introduced to obtain clearer SPECT images of the pancreas. In accordance with (125I)BH reagents, partial modifications of exendin (111In]DTPA-labeled derivatives (111In-[Lys40(Ahx-DTPA)NH2]exendin benzyl (Bn)DTPA-exendin (9-39) demonstrated higher affinity for GLP-1R compared with its parent compound, exendin (9-39), and clearly visualized INS-1 tumors in mice (111In-[Lys40(Ahx-DTPA)NH2]exendin (9-39), higher levels of tumor accumulation and uptake ratios between tumors and surrounding organs were observed in a biodistribution study involving INS-1 tumor-bearing mice (18F)FB40-exendin (9-39) showed moderate affinity to GLP-1R, visualized the mouse pancreas, and exhibited superior potentials to other 18F-labeled exendin (9-39) derivatives on different conjugating sites (Lys12 and Lys27)  also has been expected as a potent skeleton of GLP-1R-targeted probe for β-cell imaging, and its derivatives have been investigated. One of the considerable advantages of exendin (9-39) is its ability to avoid hypoglycemia due to GLP-1R activation. A preclinical study of pancreas . However tissues . Based o tissues . In addi-39 also ivatives . Howeverivatives , 76. 111terparts . (111In) in mice . Compareing mice . As for ing mice , 78. (18d Lys27) . Furtherd Lys27) .40(Ahx-DTPA-111In)NH2]exendin-4 exhibited higher and more stable uptake in the murine pancreas compared with 111In-[Lys40(Ahx-DTPA)NH2]exendin (9-39) and visualized the pancreas with SPECT in healthy participants and patients with type 1 diabetes mellitus (40(Ahx-DTPA-111In)NH2]exendin-4 to detect insulinoma and showed successful visualization and clinical safety in patients with insulinoma (40 [Ahx-hydrazinonicotinamide (HYNIC)-99mTc]NH2}exendin-4 was also developed as an alternative to [Lys40(Ahx-DTPA-111In)NH2]exendin-4 -1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA) (12[(68Ga)NODAGA]}exendin-4 was more stable than {Lys40[(68Ga)NODAGA]}exendin-4 in human blood plasma. In this context, an exendin-4 derivative labeled with 111In via BnDTPA and Ahx attached to the epsilon amino group at the lysine-12 residue, [Lys12(111In-BnDTPA-Ahx)]exendin-4, has been synthesized (111In-labeled exendin-3 derivative (12(111In-BnDTPA-Ahx)]exendin-4 showed higher and more stable murine pancreatic uptake compared with [111In]BnDTPA-exendin (9-39) (in vivo β-cell imaging probes (12(111In-BnDTPA-Ahx)]exendin-4, the pancreas-to-liver uptake ratios increased in a time-dependent manner, and the pancreas-to-kidney uptake ratios remained stable, which enhanced the contrast and allowed for clear visualization of the murine pancreas in SPECT images  . The conthesized . This prrivative . A precln (9-39) , 75. In g probes , 77. ForT images .68Ga, 18F, 64Cu, and zirconium-89 (89Zr)-labeled exendin-4 derivatives have been developed NH2]exendin-4 showed significant uptake in insulinomas developed in RipTag2 mice and proved to be a potential alternative to [Lys40(Ahx-DTPA-111In)NH2]exendin-4 NH2]exendin-4 showed high binding to INS-1 cells and fast internalization kinetics, yielding in vivo tumor visualization in mice bearing INS-1 xenografts exendin-4 showed comparable tumor uptake and reduced kidney uptake in INS-1 mouse xenografts to that of a control agent without the cleavable MVL sequence (40(Ahx-DFO-68Ga)NH2]exendin-4 demonstrated high serum stability and high and specific in vivo accumulation in mice bearing RINm5f xenografts  and 89Zr . Although 64Cu-DO3A-VS-Cys40-exendin-4 showed strong accumulation in INS-1 xenografts and transplanted islets NH2]exendin-4 showed comparable biological performance with [Lys40(Ahx-DFO-68Ga)NH2]exendin-4, whereas [Lys40(Ahx-DFO-89Zr)NH2]exendin-4 revealed long kidney retention times -[Cys40]exendin-4 and [18F]fluoronicotinamide (FNEM)-[Cys40]exendin-4 have improved renal clearance and tumor-to-kidney contrast in mice bearing INS-1 tumors  and BCM  (r = 0.83) (40([111In]DTPA)]exendin-3 showed a distinctively different uptake in resected pancreas between healthy and alloxan-treated diabetic Brown Norway rats. The ex vivo pancreatic uptake of [Lys40([111In]DTPA)]exendin-3 determined by SPECT showed linear correlations with histological BCM (r2 = 0.52) and with BCM based on optical projection tomography (r2 = 0.77) (12(111In-BnDTPA-Ahx)]exendin-4 demonstrated good linear correlations between ex vivo pancreatic uptake and histological BCM in several diabetic model mouse strains such as NOD (r = 0.90) (2 = 0.75)  . Similar = 0.77) . Importa = 0.77) . Moreove = 0.77) . Althoug = 0.77) , 107, DTPA)]exendin-3 showed a good correlation between pancreatic uptake determined by in vivo SPECT/CT and histological BCM in biobreeding diabetes-prone rats with unilateral nephrectomy (r = 0.89)  ]exendin-4 . In vivo SPECT imaging analysis demonstrated good correlation between pancreatic uptake determined by in vivo SPECT scans and histological BCM in NOD (r = 0.89)  . Furtherctively) , 109. Hoctively) , 109. A xendin-4 . The excxendin-4 . Accordi = 0.89)  and db/d = 0.89) , 103. Th40([111In]DTPA)]exendin-3 have been performed on patients with type 1 diabetes mellitus ]exendin-4 has enabled the longitudinal observation of BCM changes in diabetic model mice with and without interventions. In NOD mice, a reduction in pancreatic probe uptake in in vivo SPECT images was longitudinally observed in the mice that developed hyperglycemia, whereas no significant changes in pancreatic probe uptake in the in vivo SPECT images were observed in the mice that did not develop hyperglycemia  and DS-8500a (a G protein-coupled receptor 119 agonist) attenuated the progression of BCM loss in db/db mice (via changes in GLP-1R expression levels on the β-cell membrane surface (12(111In-BnDTPA-Ahx)]exendin-4 in SPECT images could replicate the BCM relationship among these model mice with different glycemic states (12(111In-BnDTPA-Ahx)]exendin-4 maintained a linear correlation with histological BCM, even among diabetic and non-diabetic RCS-10 mice ]exendin-3 showed non-invasive visualization of islet grafts and quantification of islet graft volume on SPECT , the linear correlation between probe uptake and islet graft volume was maintained NH2]exendin-4 SPECT demonstrated focal accumulation in the left forearm at the site of islet transplantation ]exendin-4 is reflected in the in vivo glucose-lowering effects of dulaglutide in diabetic RCS-10 mice, which suggests that the pancreatic uptake value could predict responders and non-responders to dulaglutide therapy ]exendin-4 SPECT is a useful tool for in vivo longitudinal BCM monitoring. Further clinical investigation is necessary to meet the medical needs of BCM evaluation in diabetes. Although various novel chemical modifications and structures of exendin peptides have yet to be investigated, further comparative studies including head-to-head comparisons among the candidate probes and techniques are warranted to standardize non-invasive BCM evaluation methods using GLP-1R-targeted imaging techniques.The last two decades of research have achieved remarkable advances in non-invasive β-cell imaging and BCM evaluation, including the discovery of probe target molecules, the development of suitable radioisotope-labeled chemically modified probes, and the establishment of an analysis method for image-based signals using SPECT and PET. In particular, exendin-4 based derivatives for SPECT and PET appear to be promising candidates for non-invasive BCM evaluations. At least in rodents, [LysTM performed a literature search and wrote and edited the manuscript. HF contributed to the plan and discussion. NI reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Japan Foundation for Applied Enzymology (Front Runner of Future Diabetes Research) (to TM), MSD Life Science Foundation (to TM), and Manpei Suzuki Diabetes Foundation (to NI).NI received clinical commissioned/joint research grants from Daiichi Sankyo, Terumo, and Drawbridge Inc.; speaker honoraria from Kowa, MSD, Astellas Pharma, Novo Nordisk Pharma, Ono Pharmaceutical, Nippon Boehringer Ingelheim, Takeda, Eli Lilly Japan, Sumitomo Dainippon Pharma, and Mitsubishi Tanabe Pharma; scholarship grants from Kissei Pharmaceutical, Sanofi, Daiichi Sankyo, Mitsubishi Tanabe Pharma, Takeda, Japan Tobacco, Kyowa Kirin, Sumitomo Dainippon Pharma, Astellas Pharma, MSD, Eli Lilly Japan, Ono Pharmaceutical, Sanwa Kagaku Kenkyusho, Nippon Boehringer Ingelheim, Novo Nordisk Pharma, Novartis Pharma, Teijin Pharma, and Life Scan Japan.The remaining 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.


In June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases surged in Liberia. SARS-CoV-2 sequences from patients hospitalized during March–July 2021 revealed the Delta variant was in Liberia in early March and was dominant in June, irrespective of geography. Mutations and deletions suggest multiple SARS-CoV-2 Delta variant introductions. Before May 2021, Liberia reported <10 coronavirus disease (COVID-19) cases per day among its population of ≈5 million (https://www.qiagen.com) lysis buffer to extract total nucleic acid and performed PCR by using the Triplex-CII-SARS-Cov-2 rRT PCR assay . We noted sample collection date and site and sex and median age of patients from whom samples were obtained (https://www.roche.com) on first strand cDNA synthesized from 89 RNA samples (https://arborbiosci.com). We sequenced captured libraries on Nextseq 2000 or Nextseq 550 , which yielded 5–8 million 220-bp reads per sample. We mapped reads to a SARS-CoV-2 reference sequence (GenBank accession no. NC_045512) to determine variants , Next-Strain  yielded complete coding sequences with a minimum depth of ≈15× . Using high-throughput sequencing data, we generated consensus fasta sequences of 77 SARS-CoV-2 genomic sequences and further analyzed sequences by using Geneious R10  were Alpha variant (B.1.1.7); 6 (7.8%) were Beta variant (B.1.351); 1 (1.3%) was Iota variant (B.1.526); 6 (7.8%) were Eta variant (B.1.525); and 56 (72.7%) were Delta variant (B.1.617.2) viruses . We idenUsing reference sequence NC_045512 as a baseline, we found 3 Alpha variant–specific amino acid deletions  in the surface glycoprotein of all Alpha variant genomes and 3 Beta variant–specific amino acid deletions  in the surface glycoprotein of all Beta variant genomes. All 56 Delta variant genomes had the 2 variant-specific amino acid deletions, F157del and R158del, and 8 of 9 other Delta variant–specific amino acid substitutions in the surface glycoprotein . The A222V surface glycoprotein mutation was absent in only 2/56 Delta variant genomes, LIB-0226 and LIB-0217, collected from Monteserrado County in May 2021 . The COVID-19 vaccination campaign is ramping up as <30 cases/day are reported in Liberia, but the currently circulating Delta variants are a concern because they contain mutations and deletions in the surface glycoprotein that might influence vaccine efficacy (Before June 2021, only a small percentage of the population was vaccinated in Liberia. The infections we report occurred in unvaccinated persons. The Ministry of Health, Liberia, initiated a vaccination drive in August 2021. By September, ≈130,000 persons, >2% of the population, had received a single dose of the Johnson & Johnson/Janssen vaccine (Additional information on SARS-CoV-2 sequence analysis during COVID-19 case surge, Liberia, 2021.


Following publication of the original article (Sitapara et al. From: The α7 nicotinic acetylcholine receptor agonist GTS-21 improves bacterial clearance in mice by restoring hyperoxia-compromised macrophage functionTo: GTS-21, an α7nAChR agonist, increases pulmonary bacterial clearance in mice by restoring hyperoxia-compromised macrophage functionThe original article has been corrected.


Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly  (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1  and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 , Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.Recently we reported the immune-potentiating capacity of a  Chlamydia trachomatis is an intracellular pathogen and recognizably the most common agent of bacterial sexually transmitted diseases globally, with over 100 million new cases per year  than intranasal (IN) immunization route  biovar] expressed as inclusion forming units (IFU/mL) was purchased from Virusys Corporation . The mouse-derived McCoy fibroblasts cell line and Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and L-Glutamine were both purchased from American Type Culture Collection (ATCC) . PLGA polymer (85:15 poly-lactide: poly-glycolide), dichloromethane (DCM), polyvinyl alcohol (PVA), and mitomycin-C were purchased from Sigma-Aldrich . ELISA MAX™ Deluxe kit for interferon-gamma (IFN-γ), interleukin-2 (IL-2), and interleukin-17 (IL-17) were purchased from BioLegend® Inc. . RPMI-1640 with GlutaMax™ and HEPES, heat-inactivated fetal bovine serum (FBS), ACK lysing solution, and antibiotic-antimycotic were all purchased from Life Technologies . Anti-CD 90.2 magnetic beads and MACS columns were purchased from Miltenyi Biotech . CellTrace™ CFSE (carboxyfluorescein succinimidyl ester) cell proliferation assay kit (C34554) and Remel™ PathoDx™ Chlamydia culture confirmation kit (R62210) were purchased from Thermo Fisher Scientific . The Fc block anti-CD16/32 antibody (BD:553141), fluorochrome-conjugated antibodies: CD3-APC-Cy7 (BD:560590), CD4-PerCP-Cy5.5 (BD:550954), CD62L-APC (BD:553152), CD44-PE (BD:553134), and Opti-EIA sets for interleukin-10 (IL-10) were obtained from BD-Biosciences . Medroxyprogesterone acetate (Depo-Provera) was purchased from Pfizer , and cycloheximide was obtained from EMD Biosciences .The rMOMP  was encaad libitum. Mice were acclimatized for two-weeks before all experimental procedures as approved by Alabama State University Institutional Animal Care and Use Committee (IACUC). Mice used here were a subgroup of our recently published study  were purchased from Charles River Laboratory  and housed under standard pathogen-free and controlled environmental conditions, and provided with food and water ed study . Mice we5 IFU of C. muridarum in sucrose-phosphate-glutamic acid (SPG) buffer  and PBS (5/group) were each injected SC with 2.5 mg of Depo-Provera two-weeks after the last immunization and one week later (day 49) by an intravaginal challenge with 1×10C. muridarum challenge (day 49) and two-weeks after the re-challenge (day 170). All swabs were collected in SPG buffer and stored at -80°C to quantify C. muridarum vaginal load (4/well) in 96-well cell culture plates. Confluent cell monolayers were inoculated with vaginal swab suspensions containing 0.5 µg/mL cycloheximide, centrifuged at 750g for 1 h at RT, and then incubated for 2 h at 37°C in a 5% CO2 humidified atmosphere. After that, fresh media containing 0.5 µg/mL cycloheximide was added to cells and incubated for 30 h. Cells were washed, fixed in 95% ethanol, and stained with a FITC-labeled Chlamydia antibody using the Remel™ PathoDx™ Chlamydia Culture confirmation kit. All swabs were cultured in duplicates, and 25 fields from each well were captured using an automated Nikon confocal microscope .Vaginal swabs were collected from each mouse at three-day intervals up to three-weeks after the first nal load . Briefly2 humidified atmosphere and used as antigen-presenting cells (APCs). Purified T-cells (1×106) and APCs (1×106) co-cultures were stimulated with rMOMP (5 μg/mL) in round bottom-polypropylene tissue culture tubes and incubated for 48 h at 37°C in a 5% CO2 humidified atmosphere. Cell-free culture supernatants were collected by centrifugation and stored at -80°C for cytokines quantification.Spleens were collected from immunized (day 42) and re-challenged mice (day 185) for T-cell stimulation as described , 15. BriCytokines  were quantified in cell-free culture supernatants as described (2 humidified atmosphere. CFSE labeled T-cells (0.5×106) were co-cultured with APCs (0.5×106) and stimulated with rMOMP (5 μg/mL) in round-bottom polypropylene tissue culture tubes and incubated for 120 h at 37°C. After incubation, the cells were harvested and stained using CD3-APC-Cy7, CD4-PerCP-Cy5.5, CD62L-APC, and CD44-PE to evaluate T-cell proliferation and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes. Stained cells were washed, fixed, and data were acquired on a BD LSR II flow cytometer and analyzed using FCS Express 6 FLOW . Gating on CFSE+ T-cells was used for the selection of CD3+CD4+ T-cell populations. Histogram fluorescence intensities were used to quantify the proliferating and resting T-cells amongst the total CFSE+CD3+CD4+ T-cells.Purified T-cells were subjected to CFSE-based proliferation assay, as previously reported , 10. BriPooled sera or vaginal wash samples from each group of mice were used to quantify rMOMP-specific antibodies IgG isotypes (IgG2a and IgG2b (Th1) and IgG1 (Th2)) and mucosal IgA as described , 14, 15.Serum antibody isotypes avidity index (AI) was determined as previously described , 13. ELIC. muridarum neutralization by serum from immunized and re-challenged mice was performed using McCoy cells as previously described (4/well) in 96-well flat-bottom cell culture plates. In a separate 96-well plate, C. muridarum elementary bodies (EBs) at 500 IFU/well were treated with optimally diluted serum (1:100) and incubated for 30 min at 37°C on a slowly rocking platform. Treated-EBs were then added to McCoy cells confluent monolayers and centrifuged at 750g for 1 h, followed by incubation for 2 h at 37°C in a 5% CO2 humidified atmosphere. The medium was replaced with fresh DMEM containing 10% FBS and 0.5 µg/mL cycloheximide and then incubated for 30 h. Untreated-EBs and naïve serum were, respectively, used as positive and negative controls. After incubation, the plates were fixed with 95% ethanol and stained using the Remel™ PathoDx™ Chlamydia culture confirmation kit. All samples were cultured in triplicates, and three fields from each well were captured using an automated Nikon confocal microscope. Experiments were repeated at least two times.escribed . Brieflyin vitro neutralization between and within groups. One-way ANOVA followed by Holm-Sidak was used for mean IFU comparison between groups, and all analyses were performed using GraphPad Prism 8 . P values ≤ 0.05 were considered statistically significant.Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test to compare recovered IFU, cytokines production, and via the SC rather than the IN route  on day 49 to evaluate protection by quantifying chlamydial IFU from vaginal swabs. SC and IN immunization significantly protected (P < 0.05 to 0.001) mice against a challenge as compared to the PBS control mice that consistently had higher recovered IFU  120 days after the first challenge. Both SC and IN immunization significantly (P < 0.0001) enhanced protection after re-challenge , Th2 (IL-10) and Th17 (IL-17) cytokines secreted by purified T-cells from mice after PLGA-rMOMP immunization (pre) and again after challenge and re-challenge (post). As illustrated in P < 0.0001) compared to the PBS controls. Similarly, IFN-γ production was higher (P < 0.0001) in the SC and IN mice (post) relative to the PBS controls. IL-2 was significantly (P < 0.001) produced by T-cells from immunized (SC and IN) mice (pre) and by more than a 2-fold increment (P < 0.0001) after re-challenge (post) compared to the PBS mice (P < 0.0001) after re-challenge (post) (P < 0.01) only after re-challenge of the SC mice (P < 0.0001) in the SC and IN mice (pre) in comparison to the PBS control mice, and all groups of mice after re-challenge (post) as seen in Induction of cell-mediated immunity is pivotal in protection against genital hlamydia . Thus, w+ T-cell-derived memory and effector phenotypes play important roles in vaccine-induced immunity against Chlamydia, as we previously reported (Chlamydia. T-cells were collected from mice after immunization (pre) and CFSE-labeled. Labeled T-cells were co-cultured with APCs followed by stimulation with rMOMP to evaluate T-cell proliferation along with memory and effector phenotypes employing multi-parameter flow cytometry.CD4reported . We hypo+CD3+CD4+ T-cells were high in the SC (46.63% (vi)) and IN (46.32% (x)) immunized mice (pre) as compared to the PBS control (43.93% (ii)). Moreover, there were more proliferating CD4+ T-cells (M1 population) than resting CD4+ T-cells (M2 population) with the SC mice exhibiting higher proliferating CD4+ T-cells (70.03% (vi)) as compared to the IN (66.42% (xi)) and PBS control (66.62% (iii)). Following stimulation of cells with rMOMP an increase of CFSE+CD3+CD4+ T-cells was seen more in the SC (51.26%) than the IN (47.78%) and PBS (45.92%) groups, respectively (+ T-cells in the immunized SC (74.71%) and IN (69.59%) mice but not the PBS control (66.17%)  and effector (CD44high CD62Llow) phenotypes in unstimulated and rMOMP-stimulated cells by gating on the CFSE+CD3+CD4+ T-cell populations. Unstimulated proliferating CD3+CD4+ T-cells from the SC-immunized mice (pre) differentiated into 19.93% memory phenotype (CD44+ CD62L+) as compared with 12.95% and 12.12%, respectively, for the IN and PBS groups (pre)  in SC-mice (30.94%) (+ CD62L-) increased in both the SC (21.18%) and IN (23.20%), compared to the PBS (10.66%) mice . SC immunization induced higher Th2 (IgG1) and lower Th1 (IgG2a and IgG2b) antibody titers, whereas IN immunization only induced a minimal Th2 (IgG1) antibody response Table 2.The binding strength of a specific antibody to a selective epitope enhances the functionality of antibody immune responses. Chlamydial MOMP contains antigenic epitopes that are important in inducing specific antibodies . To deteChlamydia infection by binding on the EBs surface. Therefore, we assessed the serum antibody-mediated neutralization of chlamydial EBs in vitro, using immune (pre) and re-challenge (post) sera from mice. EBs pre-incubated with sera) were added to McCoy fibroblasts and incubated for 30 h to assess infectivity by quantifying the IFU. The results in P < 0.001) than those of the IN and PBS groups (post). Of significance, re-challenge (post) significantly (P < 0.05) enhanced the serum-neutralizing antibodies in the SC but not the IN mice.Neutralizing antibodies play an essential role in Chlamydia cellular infectivity.Further analysis showed that sera from the SC- or IN-immunized mice (pre) neutralized EBs by 40% and 35%, respectively, relative to the PBS mice (pre)   . Similarc routes . As seenChlamydia infections can lead to reproductive morbidities that arise from severe inflammation, especially at the later stages , mice were significantly protected against a reported , 40. How 28 days  and 92 d 28 days  between C. muridarum challenge and re-challenge. The heightened protective chlamydial-specific Th1 cytokines (IFN-γ and IL-2) and CD4+ memory and effector T-cells produced after immunization of mice (SC and IN) corroborate reports of Th1 immune effectors that can clear a C. muridarum vaginal challenge  and effector (CD44high CD62Llow) phenotypes. The expansion of the CD4+ T-cell phenotypes indicates that the effective delivery of the nanovaccine within APCs and presentation via MHC II  and long-term memory formation (Chlamydia in mice. Significantly higher production of systemic Th1 (IgG2a and IgG2b) and Th2 (IgG1) Chlamydia-specific antibodies and their titers were more notable in the SC rather than the IN delivery of the nanovaccine. Consistent with our results is evidence supporting that PLGA delivery of antigens induces higher production of antibodies via the SC- than the IN-immunization route  avidity isotypes whose functionality improved after re-challenge. High rMOMP-specific functional avidity antibodies, especially in the SC-immunized mice, may, in part, explain their better protective efficacy. Previously, we showed that the functionality of antibodies becomes stronger with Chlamydia challenge in mice immunized SC with a MOMP peptide (M278) encapsulated within PLA-PEG nanoparticles  and the National Science Foundation (NSF)-CREST (HRD-1241701) and NSF-HBCU-RISE (HRD-1646729) grants. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.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.


In vitro and in vivo assays showed that miR-144-3p significantly inhibited proliferation, migration and invasion of CRA cells. In particular, miR-144-3p could suppress EMT process of CRA cells by regulating the cytoskeleton and EMT markers. Bioinformatics analysis indicated that EMT associated transcription factors ZEB1 and ZEB2 were potential targets of miR-144-3p, and miR-144-3p inhibited ZEB1 and ZEB2 expression and was negatively correlated with their expression in CRA. Finally, we confirmed that ZEB1 and ZEB2 down-regulation collaboratively mediated the inhibitory effect of miR-144-3p on proliferation, invasion and EMT of CRA cells. In conclusion, our study provided evidence that miR-144-3p could inhibit CRA cell proliferation, invasion and EMT by targeting ZEB1/2.miR-144-3p is aberrantly expressed in several types of human cancer and functions as a tumor suppressor by inhibiting metastasis. However, the clinical significance and biological function of miR-144-3p in colorectal adenocarcinoma (CRA) have yet to be elucidated. Here we reported that miR-144-3p expression level was significantly down-regulated in CRA tissues compared with matched noncancerous colorectal mucosae tissues. Low miR-144-3p expression was correlated with adverse clinicopathologic characteristics and poor prognosis of CRA patients. Cox regression analysis showed that low miR-144-3p expression was an independent risk factor for DFS and OS in CRA.  Colorectal cancer (CRC) has become the second leading cause of cancer related death, accounting for about 1 in 10 cancer death . DespiteEpithelial-mesenchymal transition (EMT) occurs in embryonic developmental stages  and pathological or physiological processes . EMT is often aberrantly activated in tumor cells to gain invasive ability for metastasis , 7. The miRNAs regulate gene expression by preventing translation or promoting mRNA degradation . AlteratIn this study, we investigated the expression profile and clinical significance of miR-144-3p in CRA patients and explored molecular mechanisms of miR-144-3p in CRA.Fresh frozen colorectal adenocarcinoma tissues (CRAT) and corresponding noncancerous colorectal mucosae tissues (NCMT) harvested from 160 CRA patients at the First Affiliated Hospital of Nanchang University from January 2009 to December 2011 were set as training cohort. In addition, matched NCMTs, CRATs, liver metastatic nodule (LMNs) were obtained from 8 patients with liver-only metastases. Another 140 matched fresh frozen CRATs and NCMTs from CRA patients undergoing radical resection between July 2011 and July 2013 at People's Hospital of Pingxiang were set as validation cohort. None of CRA patients had chemotherapy or radiotherapy before the surgery. This study was approved by Ethics Committee of First Affiliated Hospital of Nanchang University. All participants provided informed consent.Overall survival (OS) indicated the time from the surgery to tumor related death or the last follow-up if the patients survived . DiseaseCancer cell lines HCT116, LoVo, SW480, HT-29, SW620 and colorectal mucosal cell line FHC were provided by American Type Culture Collection. Cell were cultured and cell transfection was performed with miR-144-3p mimic, inhibitor and their corresponding control sequences  by using Lipofectamine .®-Green Master kit (Toyobo)  and cDNA was synthesized by using cDNA kit . PCR analysis was performed by using SYBRTotal proteins were extracted by using RIPA buffer , and the details for Western blotting were shown in The tissue sections were dewaxed, rehydrated, blocked, and then incubated with primary antibodies and horseradish peroxidase conjugated secondary antibody, sequentially . The sections were then stained with 3,3'-diaminobenzidine substrate.The proliferation ability of CRA cells was analyzed by methyl thiazolyl tetrazolium (MTT), EdU proliferation and colony formation analysis  assay, respectively (details in The details of IF staining and flow cytometry of vimentin and E-cadherin were described in 6) were subcutaneously injected into left upper flank of nude male BALB/c mice (4-5 weeks old). Tumor volume was calculated with formula: tumor volume (cm3) = (long axis × short axis2)/2 [in vivo metastatic assay, the spleen was exposed after anesthesia and the incision on left lateral flank in nude mice. One month after intrasplenic injection of 1×106 CRA cells, the mice were euthanized and liver specimens were collected to examine metastatic nodules.CRA cells (5 × 10axis2)/2 . After 6t test or χ2 test. Survival curve was calculated by Kaplan-Meier method. Factors associated with OS and DFS were identified by Cox proportional hazard regression analysis. P < 0.05 indicated significance.All data were analyzed with software SPSS 18 . The differences between two groups were analyzed by The study was approved by the Ethics Committee of the Institutional Review Boards of the First Affiliated Hospital of Nanchang University and Jiangxi Pingxiang People's Hospital, and was performed in accordance with the Declaration of Helsinki and current ethical guidelines. Prior informed consent was obtained from all participants.Patients provided written informed consent for publication. All authors have read and approved of publication of this manuscript.The datasets used and/or analyzed during the current study are openly available.The analysis of 160 pairs of fresh frozen CRATs and corresponding NCMTs from training cohort showed that miR-144-3p level was lower in CRATs than in NCMTs . Further2 fold change of CRAT/NCMT ≤ −1) and high miR-144-3p expression group (log2 fold change of CRAT/NCMT > −1). First, we found no differences in clinicopathologic characteristics for CRA patients between training cohort and validation cohort (P=0.002), pN stage (P=0.002), pT stage (P=0.003), distant metastasis (P=0.004) and lymphatic/microvascular/nerve invasion (P=0.001) (P=0.011), pT stage (P=0.003), pN stage (P=0.027), tumor size (P=0.001), distant metastasis (P=0.018) and lymphatic/microvascular/nerve invasion (P<0.001) (P=0.006) and shorter DFS (P=0.002) compared to patients with high miR-144-3p level (P=0.007) and DFS (P=0.009) compared to patients with high miR-144-3p level  . SimilarP<0.001) . Based o3p level , 1F. CRA3p level , 1H. Fur3p level , 4.Next we explored biological function of miR-144-3p in CRAPCR analysis of miR-144-3p expression in CRA cell lines and colorectal mucosal cell line FHC showed lower expression of miR-144-3p in CRA cell lines than in FHC cell line . In partMTT assay showed that miR-144-3p mimic inhibited Lovo cell proliferation, while miR-144-3p inhibitor significantly promoted HCT116 cell proliferation . MoreoveTranswell migration assays showed that miR-144-3p mimic significantly inhibited Lovo cell migration, while miR-144-3p downregulation in HCT116 significantly increased cell migration . TransweNext, we wondered whether miR-144-3p inhibited CRA metastasis by suppressing EMT. We detected actin cytoskeleton of CRA cells based on F-actin staining, because actin transformation is involved in cell adhesion and migration and EMT , 25. ComNext we attempted to identify the targets of miR-144-3p, and searched four databases, including TargetScan , miRDB , miRTarBZEB forms a mutually inhibitory feedback loop with transcription factors OVOL1/2 . TherefomiR-144-3p mimic cells and downregulated ZEB1/2 expression in HCT116miR-144-3p inhibitor cells (miR-144-3p mimic cells (miR-144-3p mimic cells was completely restored (miR-144-3p inhibitor cells, knockdown of ZEB1 or ZEB2 could partly inhibit enhanced proliferation, which could be fully suppressed by knockdown of both ZEB1 and ZEB2 (miR-144-3p mimic cells (miR-144-3p mimic cells were completely restored after the overexpression of both ZEB1 and ZEB2 (miR-144-3p inhibitor cells, knockdown of ZEB1 or ZEB2 could partly inhibit enhanced migration and invasion (Next, we upregulated ZEB1/2 expression in Lovoor cells , 6B. MTTic cells . Howeverrestored . In HCT1and ZEB2 . Similaric cells . The migand ZEB2 . In HCT1invasion , whereasinvasion .miR-144-3p mimic cells, the upregulation of ZEB1 or ZEB2 alone could partly upregulate vimentin expression and partly inhibit E-cadherin expression (miR-144-3p inhibitor, knockdown of ZEB1 or ZEB2 alone could partly downregulate vimentin expression and upregulate E-cadherin expression, but the effects were better after knockdown of both ZEB1 and ZEB2 (Finally, we examined whether miR-144-3p may inhibit EMT of CRA cells by targeting ZEB1 and ZEB2. In Lovopression , 7B. Whepression , 7B. In and ZEB2 , 7B. Immand ZEB2 , 7D.Next we performed IHC to detect E-cadherin, vimentin, ZEB1 and ZEB2 in serial sections of NCMT and CRAT with high or low miR-144-3p expression level, and found that NCMT and CRAT with high miR-144-3p level had low levels of ZEB1, ZEB2 and vimentin and high level of E-cadherin, while CRAT with low miR-144-3p level exhibited opposite levels of these proteins .This study showed that miR-144-3p level in CRATs was lower compared to matched NCMTs, and was lower in CRATs with advanced TNM stage than in CRATs with early TNM stage. In addition, miR-144-3p level progressively decreased from matched NCMTs, CRATs to LMNs. These results suggest that miR-144-3p may modulate CRA metastasis.miRNA expression level has been wildly utilized to assess tumor pathological stages and clinical prognosis –35. In tStudies have shown that miR-144-3p could inhibit tumor proliferation, invasion and metastasis –38. HoweZEB1 and ZEB2 are EMT-associated transcription factors involved in tumor metastasis –42. Basein vitro and suppressed CRA metastasis in vivo. Furthermore, we revealed the mechanism that miRNA-144-3p inhibited ZEB1 and ZEB2 expression to suppress CRA growth and metastasis. Therefore, our study suggested that miR-144-3p could be a therapeutic target and prognostic marker for CRA.In summary, miR-144-3p was downregulated in CRA and its downregulation was significantly correlated to poor prognosis of CRA patients. Moreover, miR-144-3p inhibited CRA cell proliferation, invasion, and EMT process Supplementary Materials