FLT3 internal tandem duplication (FLT3-ITD) is a frequent mutation in acute myeloid leukemia (AML) and remains a strong prognostic factor due to high rate of disease recurrence. Several FLT3-targeted agents have been developed, but determinants of variable responses to these agents remain understudied. Here, we investigated the role FLT3-ITD allelic ratio (ITD-AR), ITD length, and associated gene expression signatures on FLT3 inhibitor response in adult AML. We performed fragment analysis, ex vivo drug testing, and next generation sequencing  to 119 samples from 87 AML patients and 13 healthy bone marrow controls. We found that ex vivo response to FLT3 inhibitors is significantly associated with ITD-AR, but not with ITD length. Interestingly, we found that the HLF gene is overexpressed in FLT3-ITD+ AML and associated with ITD-AR. The retrospective analysis of AML patients treated with FLT3 inhibitor sorafenib showed that patients with high HLF expression and ITD-AR had better clinical response to therapy compared to those with low ITD-AR and HLF expression. Thus, our findings suggest that FLT3 ITD-AR together with increased HLF expression play a role in variable FLT3 inhibitor responses observed in FLT3-ITD+ AML patients. FLT3-ITD) represents a large and heterogeneous group of patients associated with unfavorable prognosis4. The recently approved type 1 FLT3 inhibitors have led to improved survival of FLT3-ITD+ AML patients over standard chemotherapy, however, initial responses are often variable and long-term benefits with either monotherapy or combination therapy are rarely achieved8. Hence, further studies are needed for identifying molecular biomarkers associated with variable FLT3 inhibitor responses. The aim of this study was to assess whether (i) FLT3-ITD allelic ratio (AR) and ITD mutation length and/or (ii) transcriptomic features, are associated with ex vivo and clinical responses to FLT3 inhibitors in adult AML. To address this topic and build upon our previously published work on FLT3-ITD+ AML patients carrying a co-operative NUP98-NSD1 gene fusion9, we performed a systematic analysis by integrating ex vivo drug sensitivity and resistance testing (DSRT) with FLT3 mutation fragment analysis, and next-generation sequencing (exome and RNA). These analyses were carried out using 119 samples from 87 adult AML patients (Table Acute myeloid leukemia (AML) with Fms-like tyrosine kinase 3 internal tandem duplication (FLT3) and ITD lengths from 119 gDNA samples collected from 38 FLT3-ITD+ patients and 49 FLT3-ITD- controls. The analysis identified ITD-AR and ITD lengths from all 38 FLT3-ITD+ patients including 22 samples collected at diagnosis (43%) and 29 at relapsed/refractory (R/R) stage (57%). Altogether 29 unique gain-of-function ITD mutations were found ranging from 17 to 213 bp in length (median 45 bp) with the most frequent size being 21 bp. Five patients (13%) carried two distinct ITD mutations and one had bi-allelic ITD (ITD-AR 1.0). In seven patients ITD mutations either appeared or disappeared during disease progression indicating changes in clonal dominance. In the FLT3-ITD+ cohort, ITD-AR varied between 0.016 and 1.0   compared to unmatched diagnostic samples (n = 22) , which indicates that cellular addiction on activated FLT3-signaling increases during disease progression.We initially characterized ITD-AR  group had significantly higher blast counts compared to the ITD-ARlow (< 0.338) group  group compared to the ITDshort (< 45 bp) group (P = 0.0431), while the ITDlong patients also showed elevated blood leukocyte counts (P = 0.0284) compared to ITDshort patients (< 45 bp). No significant difference in overall survival (OS) existed between de novo FLT3-ITD+ patients with either long or short ITD. The survival analysis included both diagnostic and R/R samples, considering that ITD lengths remain unchanged for the majority of patients with FLT3-ITD persistence at disease progression8. Importantly, we found significant difference in OS (P = 0.024) between de novo AML patients having either low or high ITD-AR at diagnosis. The 3-year OS rate was only 17% in ITD-ARhigh compared to 67% in the ITD-ARlow group  regardless of disease stage.We next assessed the impact of ITD-AR and ITD length on leukocyte and BM blast counts, which are frequently increased in oup Fig. . The ITDoup Fig. . In concFLT3-ITD+ and 40 FLT3-ITD-) using a DSRT-assay11. To perform a systematic comparison of FLT3 inhibitors, ten different FLT3 inhibitors with a wide range of selectivity towards FLT3 were included in the analysis  and FLT3-ITD- (Daudi) cell lines and their mixtures in varying proportions using a high-throughput flow cytometry-based DSRT-assay with five FLT3 inhibitors. By measuring relative viabilities of CD19 + (Daudi) and CD19- (MOLM-13) cells and their mixtures after 72 h treatment with five FLT3 inhibitors, we generated experimental evidence showing that increasing ITD-AR leads to stronger response to all five FLT3 inhibitors. Moreover, high sensitivity for specific FLT3 inhibitors guides the selection of highly specific or non-specific FLT3 inhibitors (e.g. gilteritinib and midostaurin) based on the FLT3-ITD-AR , whereas the relative percentage of viable FLT3-ITD- cells (CD19 +) remained unchanged or increased at higher drug concentrations. The strongest responses were observed for FLT3-ITD heterozygous MOLM-13 cells, whereas FLT3-ITD- Daudi cells showed no response to FLT3 inhibitors other than non-selective multikinase inhibitor lestaurtinib, which showed off-target efficacy in FLT3-ITD- cells . Positive association with ITD-AR was found for 15 genes and negative association for nine genes  and ITD-AR  , but not with KLRP1, MDFIC, or NPTX1  and FLT3 WT (n = 364) AML samples from the BeatAML dataset and found significantly (P < 0.0001) higher expression in the FLT3-ITD+ samples  Fig. . As the HLF expression whereas one had low ITD-AR . Based on the preclinical data, FLT3-ITD+ AML patients with high ITD-AR at diagnosis may respond well to gilteritinib, which currently lacks regulatory approval for newly diagnosed AML with FLT3 mutation. We suggest that HLF expression together with ITD-AR should be evaluated further as a potential dual-biomarker approach for treatment selection of FLT3-ITD+ AML.Taken together, the preliminary analyses of FLT3 inhibitor treated patients support that ITD-AR and associated The study was performed with the approval of Helsinki University Hospital Ethics Committee (permit numbers: 239/13/03/00/2010 and 303/13/03/01/2011) and in accordance with the Declaration of Helsinki. A written informed consent was acquired from all patients before sample collection.Supplementary Information.


Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex Song et al. describe an immunoassay method, which is a multiplex, fluorescence-barcoded cell lines system that enables high-throughput screening of antibodies binding to large number of protein variants in a single-tube manner. They demonstrate the utility of this tool by assessing binding of different antibodies to a panel of influenza hemagglutinin antigens and to epitope/domain map CCR2 and CCR5 antibodies. For many multi-subunit or multi-pass transmembrane proteins, the native conformation is preserved only when the protein is properly expressed on the cell membrane2. For this reason, cell-based binding assays using protein antigens expressed on the cell surface are widely used in antibody screening and characterization3. A major limitation in these assays is that they are single analyte, or singleplex methods, and consequently time-consuming, sample-depleting, and costly when used to screen samples with high numbers (>10) of antigens. Although multiplex immunoassays based on the Luminex® technology are well-established, only acellular antigens can be immobilized on detection beads with this platform.Membrane proteins are often critical antigens in humoral responses to infection and physiologically significant in health and disease. They are, therefore, valuable targets in vaccine design and therapeutic antibody development7. Distinct cell populations are labeled with unique signatures of amine-reactive fluorescent dyes of different emission wavelengths and intensities before pooling for exposure to potentially reactive antibody followed by analysis by flow cytometry. The FCB technique improves the throughput of flow cytometry experiments and minimizes staining variability and antibody consumption. Although this FCB approach offers flexibility in barcoding cell samples from different sources, especially primary cells, barcoding, labeling, and quality assurance procedures are required prior to every experiment. In contrast, a panel of reporter cell lines stably barcoded by endogenous expression of fluorescent proteins (FPs) yet capable of expressing select membrane-associated antigens by transduction, would establish a stable and highly reproducible immunoassay platform. Once established, endogenously barcoded reporter cell lines would greatly simplify the FCB approach.Fluorescent cell barcoding (FCB) was developed as a cell-based multiplexing technique® beads. Individual reporter cell sub-lines, each bearing a unique combination of FPs, were subsequently engineered to express different cell surface proteins to permit their pooling in a cell-based multiplex immunoassay. Flow cytometry was then used to de-multiplex signals from pooled reporter cell lines by serial gating to identify FP combinations. In this proof-of-concept study, we established a 16-plex basic panel and tested the feasibility of expanding to a 256-plex panel of reporter cell lines. We successfully applied this platform to multiplex detection of antibody binding to cell panels expressing various influenza A hemagglutinin (HA) trimers or human CCR2b and CCR5, and several domain-swap or point mutants that define specific protein domains/epitopes.Here we describe a cell-based, multiplex immunoassay platform using reporter cell lines with stable barcoding by FPs. Briefly, a basal cell line was engineered to express different combinations of FPs that can be reliably detected and distinguished in different channels on flow cytometers. This resulted in a panel of FP-barcoded reporter cell lines analogous to barcoded Luminex8, simplifying the processes of genetic modification and assay processing. A drawback of K562 as a reporter cell line is its expression of the Fcγ receptor, CD32A, which binds most human IgG subclasses with sufficient affinity to raise background thresholds  but not A/Christchurch/16/2010(H1N1), an A/California/07/2009(H1N1)-like strain. Antibody HC19, specific for HA A/Hong Kong/JY2/1968(H3N2)21, bound to that HA but not to others. This assay may be an alternative to Luminex bead assays in high-throughput screening of antigen-specific and cross-reactive antibodies secreted in clonal cultures of human B cells from influenza-vaccinated subjects22.Influenza HAs are diverse trimeric membrane proteins. Recombinant HA protein preparation can be challenging as some HA ectodomains are unstable and cell surface expression can be a surer option for obtaining native structure when working with unknown or novel influenza strains. Multiplex immunoassays with collections of cell surface-expressed HA antigens may be useful in influenza vaccine design and immunological monitoring. To demonstrate, we prepared a panel of 12 reporter cell lines expressing the HA trimers of diverse influenza A subtypes and successfully validated their structural integrity with several reference antibodies  are key to many biological functions and are an important class of drug targets, constituting some 34% of US Food and Drug Administration approvals26. The combination of gain- and loss-of-binding readouts across all the ectodomains allowed a semi-quantitative determination of the contribution of each individual ectodomain  in ECL2A domain Fig.  showed tTo expand the established 16-plex reporter cell line panel, additional transductions for more FPs are necessary. Potentially, the accumulations of genetic insertions in the reporter cell genome could impact the expression and processing of preexisting FPs, other transduced proteins (antigens), and impair cell metabolism or proliferation. To address this concern, we generated a reporter expressing a fifth FP, LSSmOrange. Reporter cell lines FP0000 and FP1111, with none or four preexisting FPs, were pooled and transduced to express LSSmOrange. Similar transduction efficiencies and LSSmOrange intensities were achieved in both parental lines  remained capable of the expression, folding, and processing of analyte proteins, a complex membrane protein, human CCR5, was tested as an example analyte. Similar transduction efficiencies and surface CCR5 expression levels were detected based on the four cell lines established above .Homo sapiens sapiens) subject under Duke Institutional Review Board Committee guidelines (IRB Pro00062495). Written informed consent was obtained.Peripheral blood mononuclear cells (PBMCs) were obtained from one human (30. pLV-EF1a-IRES-Puro (Addgene plasmid # 85132) was a gift from Tobias Meyer31. pLB (Addgene plasmid # 11619) was a gift from Stephan Kissler27. pMD2.G (Addgene plasmid # 12259) and psPAX2 (Addgene plasmid # 12260) were gifts from Didier Trono. mScarlet-I-mTurquoise2 (Addgene plasmid # 98839) was a gift from Dorus Gadella32. pLenti-smURFP-T2A-mCardinal (Addgene plasmid # 80348) was a gift from Erik Rodriguez and Roger Tsien33. pMito-miRFP703 (Addgene plasmid # 80000) was a gift from Vladislav Verkhusha15. pLSSmOrange-mKate2 (Addgene plasmid # 99868) was a gift from Marc Tramier34. Plasmid mNeonGreen-C1 was provided by Allele Biotechnology and Pharmaceuticals, Inc., under a license for non-commercial use.pU6-(BbsI)_CBh-Cas9-T2A-BFP (Addgene plasmid # 64323) was a gift from Ralf Kuehn17. Coding sequences for other FPs were cloned from plasmids mentioned above, with EBFP210 from pU6-(BbsI)_CBh-Cas9-T2A-BFP, mTurquoise211 from mScarlet-I-mTurquoise2, mNeonGreen12 from mNeonGreen-C1, mCardinal13 from pLenti-smURFP-T2A-mCardinal, mKate214 and LSSmOrange16 from pLSSmOrange-mKate2, and miRFP70315 from pMito-miRFP703. Coding sequences for cytoplasmic domain truncated mouse CD4 (amino acid (aa) 1–423), CD8a (aa 1–222), and CD86 (aa 1–268) were cloned from splenocyte cDNA samples from one C57BL/6 mouse. Coding sequences for full-length human CCR2b and CCR5, and cytoplasmic domain truncated human CD4 (aa 1–424), CD8a (aa 1–209), CD86 (aa 1–276), and CD154 (aa 1–240) were cloned from PBMC cDNA samples from one healthy donor.DNA template coding FP hmKeima8.5 was synthesized (Genscript) based on published amino acid sequencesDNA templates coding influenza HA antigens A/Christchurch/16/2010(H1N1) (GISAID Accession EPI280344) and A/Texas/50/2012(H3N2) (GenBank Accession KJ942744) were synthesized  based on amino acid sequences in the databases. Plasmid containing coding sequences for influenza HA A/Solomon Islands/3/2006(H1N1)  was provided by Aaron G. Schmidt (Ragon Institute). Plasmids containing coding sequences for other influenza HA antigens used were provided by Stephen C. Harrison , including HA antigens A/Japan/305/1957(H2N2) (GenBank CY014976), A/Hong Kong/JY2/1968(H3N2) (GenBank CY147438), A/American black duck/New Brunswick/00464/2010(H4N6) (GenBank CY138045), A/Viet Nam/1203/2004(H5N1) (GenBank AY818135), A/Taiwan/2/2013(H6N1) (GISAID EPI459855), A/Taiwan/1/2017(H7N9) (GISAID EPI917065), A/northern shoveler/California/HKWF1204/2007(H8N4) (GenBank CY039588), A/Jiangxi/IPB13/2013(H10N8) (GenBank KJ406543), and A/mallard/Wisconsin/10OS3941/2010(H14N6) (GenBank CY133266).® Endo-free Plasmid DNA Mini Kit II, Omega Bio-tek) for mammalian cell transfection. Recombinant sequences in all plasmids used were verified by DNA Sanger sequencing .Standard molecular cloning procedures were followed for plasmid modification and gene cloning. Endotoxin-free plasmids were prepared  were constructed by replacing the U6-loxP-CMV-EGFP-loxP cassettes in plasmid pLB with EF1a promoter, EFS core promoter , or EF1a-IRES-Puro cassettes from plasmid pLV-EF1a-IRES-Puro, respectively. pLB-EXIP was further modified to generate bicistronic vectors pLB-EF1a-IRES-EBFP2, pLB-EF1a-IRES-mTurquoise2, pLB-EF1a-IRES-mCardinal, pLB-EF1a-IRES-mCD4, pLB-EF1a-IRES-mCD8a, and pLB-EF1a-IRES-mCD86 by replacing puromycin-resistant gene in pLB-EXIP with coding sequences for corresponding genes following IRES element. pLB-EFS-IRES-mNeonGreen was generated from pLB-EXIP by replacing EF1a promoter with EFS core protomer and puromycin-resistant gene with mNeonGreen coding sequences.Lentiviral transfer vector plasmids expressing EBFP2, mTurquoise2, mCardinal, mKate2, miRFP703, LSSmOrange, and hmKeima8.5 were generated by cloning of corresponding coding sequences into pLB-EF1a. Lentiviral transfer vector plasmid expressing mNeonGreen was generated by cloning of mNeonGreen coding sequences into pLB-EFS. Human CD4, CD8a, CD86, and CD154 coding sequences  were cloned into pLB-EXIP for puromycin-mediated selection. Human CD8a coding sequences were cloned into pLB-EF1a-IRES-mTurquoise2 and pLB-EF1a-IRES-mCardinal, and human CD86 coding sequences were cloned into pLB-EF1a-IRES-EBFP2 and pLB-EFS-IRES-mNeonGreen. These bicistronic expression vectors can co-express human CD8a/CD86 and FPs, with FPs at a lower expression level driven by IRES element. Coding sequences for influenza HA antigens, human CCR2b, CCR5, and their domain-swapped or point mutants were cloned into pLB-EXIP.HEK 293T and K562 cell lines were purchased from ATCC. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% heat-inactivated HyClone fetal bovine serum (FBS) (Cytiva), 10 mM HEPES buffer, and 55 µM 2-Mercaptoethanol . K562 and derivative cell lines were maintained in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated HyClone FBS, 10 mM HEPES buffer, 1 mM sodium pyruvate, 1× MEM NEAA, 55 µM 2-Mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin . For all K562-derivative cell lines, monoclonal cell lines were established by single-cell sorting (see below) and used in binding assays in this study.CD32A exon 1 were designed with the online tool (http://crispr.mit.edu). sgRNAs used in this study were sgRNA-hCD32A-1 (5′-AGCAGCAGCAAAACTGTCAA-3′), sgRNA-hCD32A-2 (5′-ATGTATGTCCCAGAAACCTG-3′), and a negative control sgRNA (5′-TGTCATGCGTCACTTAGTGC-3′). Corresponding DNA oligos were synthesized and cloned into plasmid pU6-(BbsI)_CBh-Cas9-T2A-BFP. K562 cells were transfected with CRISPR-Cas9 targeting plasmids using Lipofectamine 3000 Transfection Reagent (Invitrogen). Ninety-six hours after transfection, the cells were collected for flow cytometry analysis and single-cell sorting (see below).Single-guide RNAs (sgRNAs) targeting human g for 45 min at 32 °C. For transductions with pLB-EXIP-based vectors, cells were selected with puromycin  between 3 and 7 days after transduction. Seven days after transduction, the cells were collected for flow cytometry analysis and single-cell sorting (see below).Lentiviral transfer vector plasmids were co-transfected into HEK 293T cells with packaging plasmids pMD2.G and psPAX2 using Lipofectamine 3000 Transfection Reagent (Invitrogen). Forty-eight hours after transfection, culture supernatants were collected and filtered through 0.45 µm polyvinylidene difluoride membrane filters (Millipore). K562-derivative cell lines were transduced with the filtered supernatants containing lentiviral vectors by spinoculation at 1000 × 18, S5V2-2919, CH6720, and HC1921 were prepared as recombinant human IgG1 antibodies as described35.Monoclonal antibodies used in this study included the following: FITC-conjugated anti-human CD32A , hCD32-APC , hCD16-PE , hCD32-PE , hCD64-PE , hCD4 , hCD8a , hCD86 , hCD154 , mCD8a-PerCP-eFluor710 , mCD86-PE-Vio770 , mCD4-APC-Fire750 , hCD58-PE , HA-tag , hCCR2 , hCCR2 , hCCR5 , hCCR5 , hCCR5 , hCCR5 , and hCCR5 . Isotype control antibodies included the following: Mouse IgG1 κ-isotype control (Rockland 010-001-330), Human IgG1κ , and Human IgG1 Lambda . Secondary antibodies included the following: Goat Anti-Human IgG-PE (Southern Biotech 2040-09), Goat Anti-Mouse IgG, and Human ads-PE (Southern Biotech 1030-09). Influenza HA-specific antibodies FI6g for 2 min at 4 °C, and resuspended in staining buffer . After incubation with antibodies at 4 °C in the dark for 30 min, cells were washed with staining buffer and resuspended in staining buffer for either secondary staining following the same procedure above or stored on ice for flow cytometry analysis or single-cell sorting. For cell counting, CountBright™ Absolute Counting Beads (Invitrogen) were added into cell suspensions prior to flow cytometry analysis.Cultures of K562 and derivative cells were collected, centrifuged at 300 × Flow cytometry analysis was carried out using either BD FACSCanto II cytometer (Duke Cancer Institute Flow Cytometry Shared Resource) or BD LSR II cytometer (The Duke Human Vaccine Institute (DHVI) Research Flow Cytometry Facility). Single-cell sorting was performed with BD Aria II (The DHVI Research Flow Cytometry Facility). The bulk cell line after transfection or transduction were stained with corresponding antibodies and single cells expressing FP or antigen of interest were sorted into 96-well flat-bottom plates containing 100 µl/well of the complete RPMI medium above supplemented with 20% heat-inactivated FBS. Nine days after sorting, healthily proliferating cell clones were transferred into 24-well plates for further expansion. Three days later, individual monoclonal cell lines were validated for the expression of FP or antigen of interest and a single clone with a uniform expression level was selected for further engineering or used as a reporter cell line in immunoassays.FlowJO (Version 10.7.2), Chromas (Version 2.6.6) and Microsoft Office (Version 1808) were used to analyze data and prepare figures for publication.The reproducibility of flow cytometry detections of the expression of FPs and cell surface molecules was confirmed by two or three independently repeated experiments as indicated in figure legends and shown as Further information on research design is available in the Supplementary InformationReporting Summary


Enterobacterales. Therein, bacterial killing observed with meropenem (MEM) in vivo was concordant with its pharmacodynamic profile using MIC values determined in zinc-depleted media compared with conventional cation-adjusted Mueller-Hinton broth (CAMHB). This study aims to evaluate the exposure-response relationship of MEM against VIM- and NDM-harboring P. aeruginosa (PSA) using the murine thigh infection model and zinc-depleted MICs.Prior investigations evaluating the predictive value of zinc-depleted media for MBL-susceptibility testing have focused on in vivo (neutropenic murine thigh infection model) and in vitro (broth microdilution). The 24h murine thigh study was conducted with treatment groups receiving a humanized MEM 2g q8h (3h infusion) dose. Six different zinc-limited media were prepared by the addition of EDTA at concentrations ranging from 3 to 300 mg/L to CAMHB. MEM MICs were determined in triplicate in conventional CAMHB and zinc-limited media. Time > MIC values (generated in each zinc-depleted media) were then plotted against the change in 24h bacterial density count in an Emax model.MBL-harboring PSA isolates  were tested both 10 CFU/thigh for NDM and VIM isolates, respectively. MEM resulted in -0.09 CFU reduction to +3.69 CFU growth against NDM isolates. MEM resulted in -2.59 CFU reduction to +4.81 CFU growth against VIM isolates. All MEM MICs in conventional CAMHB were >64 µg/mL for NDM and ranged from 8 to >64 µg/mL for VIM isolates. Increasing EDTA concentrations resulted in several-fold MIC reductions and on average, a larger magnitude of reduction was observed among VIM- (6-fold) compared with NDM-harboring PSA (4-fold) in CAMHB-EDTA 300 mg/L relative to CAMHB. For both NDM- and VIM-harboring PSA, an Emax model with MICs generated in CAMHB+EDTA 30 mg/L (r2 = 0.88) provided the highest correlation with MEM in vivo activity compared with CAMHB (r2 = 0.55).Average 0 h bacterial densities were 5.21 ± 0.40 and 5.13 ± 0.81 login vivo efficacy of meropenem against MBL-harboring PSA, and addition of EDTA (30 mg/L) to CAMHB appears to be a viable option for in vitro testing of these organisms.Results indicate that MIC values generated in conventional CAMHB do not appropriately characterize the David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase


Aspergillus niger, one of the most common and important fungal species, is ubiquitous in various environments. A. niger isolates possess a large number of cryptic biosynthetic gene clusters (BGCs) and produce various biomolecules as secondary metabolites with a broad spectrum of application fields covering agriculture, food, and pharmaceutical industry. By extensive literature search, this review with a comprehensive summary on biological and chemical aspects of A. niger strains including their sources, BGCs, and secondary metabolites as well as biological properties and biosynthetic pathways is presented. Future perspectives on the discovery of more A. niger-derived functional biomolecules are also provided in this review. Aspergillus, one sizeable genus belonging to Aspergillaceae family, comprises as many as 492 species registered on the database of the National Center for Biotechnology Information (NCBI) to date. Its section Nigri is an important group of species, and the A. niger aggregate represents its most complicated taxonomic subgroup with eight morphologically indistinguishable taxa  results indicated that each WT A. niger strain harbors at least 20 cryptic SM BGCs, including PKS, NRPS, NRPS-like, and their hybrids  were detected till 2020. On the basis of chemical structures, these chemicals are grouped into five types: pyranone, alkaloid, cyclopentapeptide, polyketide, and sterol and, respectively, introduced as follows. , as many as 166 aterials .)A. niger, including γ-naphthylpyradones (1–31), α-pyranones (32–56), and γ-pyranones (57–60). A. niger–derived naphthylpyradones are sorted into two classifications: monomers and dimers, with linear and angular naphtho-γ-pyrone. Fonsecin (1) is one of the most frequently isolated γ-naphthylpyradone produced by several A. niger strains from various sources, including terrestrial soil (3) and TMC-256C1 (8) also effectively inhibited the IL-4 driven luciferase (2) and nigerasperone A (4) exhibited weak bioactivity against luciferase and DPPH (5) was purified from strain IFB-E003 endophytic on Cynodon dactylon Linn.  with potent radical scavenging activity  consist of two monomers with linear and/or angular structure(s). It is interesting that most of these bis-naphtho-γ-pyrones were produced by symbiotic A. niger strains. Chemical investigation of eight A. niger strains led to isolation of the same SM aurasperone A (10) (15) had potent radical scavenging activity against DPPH with an IC50 value of 0.01 μM , two dimeric naphthylpyradones nigerasperones B (29) and C (19) were obtained from strain EN-13 and shown to exhibit a moderate radical scavenging effect on DPPH  (e A (10) , which pe A (10) , strong e A (10) , and xane A (10) . Auraspe 0.01 μM . Marine on COX-2 . In addi on DPPH . Bioassaone (30) .1–3 and 8 by successive catalytic reactions in a nonreducing PKS (nrPKS) system. Two of these monomers further dimerized at various carbon positions  and resulted in the formation of dimers 16, 21, 27, and 28. Lately, one nrPKS gene D8.t287 responsible for the biosynthesis of the initial precursor heptaketone was identified and characterized by target gene knockout experiment and UPLC-MS analysis , 7 dicyclics , three tricyclics , and four tetracyclics (52–55). Chemical analysis of an endophytic A. niger strain colonizing in liverwort Heteroscyphus tener (Steph.) Schiffn resulted in isolation of three new amide campyrones A–C (38–40) together with compounds 33 and 34  and B (46) and nigerapyrones A-H  were first discovered from a mangrove plant–derived strain MA-132 (50) and bicoumanigrin (52) were new α-pyranone analogs produced by marine sponge-derived A. niger strains; the former exhibited a powerful and selective inhibitory effect on NFRD (NADH-fumarate reductase) (53), kotanin (54), and 7-desmethyl-kotanin (55) were produced by a number of A. niger strains from various sources, and 53 showed potent inhibitory activity against wheat coleoptile growth at 1 mM but not toxic to day-old cockerels   and the ductase) . Three 8ockerels . Biosyntockerels  (Huttel ockerels . In this57–60) had been detected in SM of A. niger. Among these substances, kojic acid (57) is the most common product with weak antimicrobial property (59) and tensidol B (60), one new benzyl γ-pyranone nigerpyrone (58) was discovered from a mutant strain FGSC A1279 ΔgcnE and was found to have potent and selective activity against Candida parapsilosis  are a family characterized by pyrano  pyrrole skeleton, and their biosynthesis are manipulated by the pyn gene cluster in A. niger  and Ab (63), which 63 showed a strong inhibitory effect on the growth of neonate larvae of the plant pest insect Spodoptera littoralis (65), S (66), and E (67) were important agents with potent radical scavenging activity toward DPPH and superoxide (67) had been first proposed by L-Ser (pynA (PKS-NRPS hybrid synthase), pynI (encode thioesterase), pynC (encode methyltransferase), pynG (encode flavin-dependent oxidase), pynD (encode CYP450), and pynH (encode aspartyl protease). After non-enzymatic reaction, two pyranonigrin E (67) units could be dimerized to form pyranonigrin F (69). One soil-derived A. niger strain was found to produce a new dichlorinated pyrrole pyoluteorin (70), which obviously induced cell cycle arrest and apoptosis in human triple-negative breast cancer cells MDA-MB-231 (71) and B (72) from strain FKI-2342 were potentiators of antifungal miconazole activity  have one benzyl group and possess antimicrobial and cytotoxic properties (chatr 4). Two new α-pyridones aspernigrins A (73) and Bb (74) were isolated from one A. niger strain of marine sponge Axinella damicornis and showed moderate cytotoxicity and a potent neuroprotective effect, respectively, , B (75), and p-fluoro nygerone B (77)  and one cyclic peptide (111), three 2-benzyl-γ-pyridones aspernigrins B-D (80–82) were obtained from the marine strain SCSIO Jcsw6F30, and 81 was found to have potent inhibitory activity toward HIV-1 SF162-infected TZM-bl cells  . In addibl cells .2 (83), B1 (84), and B4 (85) from stains FGSC A1279 and IBT 28144 were carcinogenic (aza gene cluster in strain ATC C1015 was found be responsible for biosynthesis of azanigerone D (86) (70), phenazine-1-carboxylic acid (87) was produced by the soil A. niger strain (88) and nigerazine B (89) were purified from strain ATCC 11414, and their biosynthesis were regulated by the naphthopyrone precursor BGC alb gene cluster (90) displayed a potent effect on the tumor cell lines nasopharyngeal epidermoid KB, cervical carcinoma Hela, and colorectal carcinoma SW1116 (Three fatty amines fumonisins Binogenic . The azae D (86) . In addir strain . Two new cluster . Endophya SW1116 .91–96) had been isolated and characterized from A. niger strains. Fractionation of crude extract of marine strain BRF-074A afforded one furan ester derivative (91), one cerebroside chrysogeside D (93), and two spiro amides pseurotins A (95) and D (96), among which 91 exerted a cytotoxic effect on HCT-116 cell line (92) with free radical DPPH scavenging activity and inhibitory effect on lipoxygenase-I (LOX-1) and rat lens aldose reductase (RLAR) (94) was a new natural Diels–Alder adduct of ergosteroid and maleimide produced by the strain EN-13 from marine alga  . Ergosteine alga .A. niger are cyclic and consist of ten dipeptides (97–106), eight pentapeptides (107–114), and three bis(dipeptide)s (115–117). In addition to α-pyranones 32–34, 38, and 40, four diketopiperazines  were isolated from an endophytic strain of liverwort Heteroscyphus tener (Steph.). Schiffn, and compounds 115 and 116 showed weak activity against the human ovarian carcinoma cancer cell line A2780  (117) and nine monomers (98–106)  were a group of SMs containing structural skeleton of cyclo-D-cysteinyl-D-cysteinyl-L-amino acid-D-amino acid-L-amino acid (107) demonstrated antibacterial (114) exhibited a broad spectrum of biological properties including anti-HIV-1  . Phytoch(98–106) . Compount growth , and 101toxicity . Eight mino acid . Malformacterial  and antiacterial , while mti-HIV-1 , cytotoxti-HIV-1 , anticanti-HIV-1 , and antti-HIV-1 .118–155) are the largest group of SMs produced by A. niger. Citric acid (118) and itaconic acid (119) have been large-scale products in food and pharmaceutical industry for decades  (p-hydroxyphenylacetic acid (129) (130) (131) (147)  dramatically attenuated , carlosic acid methyl ester (124), and agglomerin F (125) (132–146), in which biosynthesis was deduced to be manipulated by the yan gene cluster in strain KB1001 ] together with eight additional genes yanB (encode decarboxylase), yanC (encode CYP450), yanD (encode dehydrogenase), yanE (unknown), yanF (encode oxidase), yanI , yanH (encode CYP450), and yanG (encode prenyl transferase).Polyketides ( decades . Some otol (128) , p-hydroid (129) , gallic 9) (130) , benzoic0) (131) , and asp1) (147) . In comptenuated . By over F (125) . Chemican KB1001  (Bugni en KB1001 . Further147) was the common product of strains NRRL-3 and CFTRI 1105 (86), five highly oxygenated pyranoquinones  were detected in SMs of strain T1 by activation of the aza gene cluster (azaE (encode ketoreductase), azaF (encode acyl:CoA ligase), azaG (encode FAD-dependent oxygenase), azaH , azaI (encode CYP450), azaJ (encode dehydrogenase), and azaL (encode FAD-dependent oxygenase) play important roles. Funalenone (150), one phenalene derivative, was obtained from strain ATCC 11414 whether the albA gene was auxotrophic or not (150) was also found in A. niger mutant ΔgcnE (strain FGSC A1279 lacking epigenetic regulatorgcnE) (157) and TAN-1612 (158) were, respectively, obtained from strains WB2346 and ATC C1015 and shown to be acyclic binding inhibitors of neuropeptide Y receptors  . Two teteceptors .159) and its benzoate (160) were the first steroids isolated from A. niger (161) and B (162) had potent antiproliferative activity against human promyelocytic leukemia (HL60) and human lung carcinoma (A549), with IC50 values of 0.11 and 0.43 μM, respectively, (94), four steroid derivatives (163–166) were discovered from the endophytic strain EN-13 associated with marine brown alga  approach (A. niger–derived SMs (A. niger, construction and breeding of robust strains as well as optimization of their cultivation and fermentation processes should be intensively conducted at various levels (approach  and geneapproach . Moreoveived SMs . In ordes levels .


Our findings provide novel insights on the synergies between hyperlipidemia and hypomethylation in establishing TI and promoting inflammation in NASH and NAFLD progression and novel targets for future therapeutic interventions for NASH and NAFLD, metabolic diseases, transplantation, and cancers.We performed a panoramic analysis on both human nonalcoholic steatohepatitis (NASH) microarray data and microarray/RNA-seq data from various mouse models of nonalcoholic fatty liver disease NASH/NAFLD with total 4249 genes examined and made the following findings: ( Several metabolic diseases significantly drive the development of cardiovascular disease, nonalcoholic fatty liver disease (NAFLD) , and nonInnate immune system inflammation is considered a landmark of NASH pathogenesis. Mouse models of NASH have demonstrated that increased danger/pathogen-associated molecular pattern (DAMP/PAMP) receptor and Toll-like receptor (TLR) signaling  are assoThe secretome can be defined as the portion of total protein secreted by cells to the extracellular space to maintain tissue homeostasis –22. TregInnate immune cells can develop an exacerbated immunologic responses and long-term inflammatory phenotypes following brief exposure to endogenous or exogenous insults , which leads to an enhanced inflammatory response after a second challenge, which is known as trained immunity (TI) , 26–28. Oxidation of cholesterol and phospholipids can lead to lipid peroxidation and cell death –35. FurtDNA methylation plays a significant role in the progression of NAFLD. Previous studies have shown that there are differences in DNA methylation levels of multiple genes between liver samples from NAFLD patients with those from healthy individuals , 40, andTo fill in important knowledge gaps, we performed a panoramic database mining analysis on both human NASH microarray data and microarray data from various NAFLD mouse models and examined a total of 4249 genes by using the strategy we pioneered . We madeThe 10 microarray datasets were collected from the National Institutes of Health- (NIH-) National Center for Biotechnology Information- (NCBI-) Gene Expression Omnibus (GEO) database and analyzed with an online software GEO2R Figures . One datIPA was used to characterize the clinical relevance and molecular and cellular functions related to the identified genes. Differentially expressed genes were identified and uploaded into IPA for analysis. Core and canonical pathway analysis was used to identify molecular and cellular pathways , 15.p values, (3) filter data for fold change ± 1.5, and (4) retrieve expression data for various genes of interest  the high-fat diet (HFD) and methionine- and choline-deficient (MCD) diet model (GSE35961) [2) the high-fat-cholesterol diet- (HFCD-) induced diet model [3) the glycine N-methyltransferase- (GNMT-) KO genetic model, (4) the liver-specific methionine adenosyltransferase 1A- (MAT1A-) KO (GSE63027) [5) two human NASH microarrays  , (2) theet model , (3) theSE63027)  genetic GSE63067  and GSE1GSE63067 ). The quCytokines and chemokines –55 play To determine whether NAFLD mouse models share innate immune cytokines and chemokines with human NASH, we performed Venn diagram analysis. Six innate immune cytokines and chemokines including CXCL10, tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B), lymphotoxin beta , TNFSF10, interleukin 1 receptor antagonist (IL-1RN), and nicotinamide phosphoribosyltransferase (NAMPT) were shared between human NASH and NAFLD mouse models . Two NAFTo ensure a comprehensive analysis of liver inflammation in human NASH and NAFLD mouse models, we collect 2641 canonical secretome genes . The 123To determine whether NAFLD mouse models share canonical secretomic cytokines and chemokines with human NASH, we performed Venn diagram analysis. Seven canonical secretomic cytokines and chemokines including TSLP, CXCL10, CCL28, CCL25, CXCL14, IL21, and IL1RN were shared between NAFLD mouse models and human NASH . By compCaspase-1-deficient mice are protected from high fat-induced hepatic steatosis, inflammation, and early fibrogenesis , suggestTo determine whether NASH/NAFLD upregulated cytokines and chemokines classified into noncanonical secretomes, we collected four types of secretomes with total 11,385 proteins including canonical secretome (2641 proteins) , caspaseTaken together, our results have demonstrated innate immune secretomic cytokines and chemokines suggesting that human NASH and NAFLD mouse models share innate immune mechanisms and there is a significant role of exosomes and caspase-4 secretomic in driving liver and systemic inflammations.β-oxidation I, and leukocyte extravasation, among which leukocyte extravasation was shared by the second human NASH and MCD+HFD, LXR/RXR activation was shared by HFCD, and fatty acid β-oxidation was shared by GNMT-KO and HFCD /retinoid X receptor (RXR) activation, fatty acid β-oxidation I, stearate biosynthesis I, antioxidant action of vitamin C, oxidative phosphorylation, glutaryl-CoA degradation kinase, triacylglycerol biosynthesis, and tricarboxylic acid cycle (TCA) cycle II, among which fatty acid β-oxidation I and stearate biosynthesis I were shared with the first human NASH (GSE17470). The MAT1A-KO (GSE63027) had top upregulated pathways including nicotine degradation II, superpathway of cholesterol biosynthesis, cholesterol biosynthesis I, cholesterol biosynthesis III, mevalonate pathway I, and NAD salvage pathway II, among which nicotine degradation II was shared with the first human NASH.The GNMT-KO (GSE63027) had top upregulated pathways including fatty acid β-oxidation I, among which LXR/RXR activation was shared with the first human NASH, fatty acid β-oxidation II was shared with the first human NASH and GNMT-KO, and nicotine degradation II was shared with MAT1A-KO.The HFCD model (GSE53381) has only four upregulated pathways including LXR/RXR activation and fatty acid κB signaling and neuroinflammation (γ) receptor-mediated phagocytosis, IL-8 signaling, Rac signaling, and glucose 6-phosphate (G6P) signaling, among which IL-8 signaling was shared with the second human NASH.The second human NASH (GSE63067) had top upregulated pathways including NF-ammation . The MCDκB signaling [Taken together, our results have demonstrated that the two human NASH datasets have diversified signaling pathways, which allow us to classify the first one into LRX/RXR activation-related metabolic NASH and the second NASH into NF-ignaling /T helperignaling , 65 inflTo examine the differential roles of caspase-1- and caspase-11-dependent pyroptosis, we collected 90 canonical and 14 noncanonical inflammasome pathway regulator genes and determined the expression changes of these inflammasome regulators in four NAFLD mouse models.MCD+HFD upregulated 18 (20%) including caspase-1 and downregulated 11 (12.2%) out of 90 canonical inflammasome pathway regulators. MAT1A-KO upregulated 5 (5.6%) and downregulated 4 (4.4%) out of 90 canonical inflammasome pathway regulators. GNMT-KO upregulated 1 (1.1%) and downregulated 2 (2.2%) out of 90 canonical inflammasome pathway regulators. HFCD upregulated 7 (7.8%) and downregulated 26 (28.9%) out of 90 canonical inflammasome pathway regulators . In addiTaken together, these results have demonstrated that the MCD+HFD upregulates more canonical inflammasome regulators than the other three NAFLD models. Conversely, the MAT1A-KO upregulates more noncanonical inflammasome regulators including caspase-4 than other models, suggesting that canonical and noncanonical inflammasome pathways are regulated differentially in various NAFLD models. The HFCD downregulates more than upregulates canonical and noncanonical inflammasome regulators, suggesting that HFCD model uses more focused set of regulators in modulating the expression of these regulators.We hypothesized that the expressions of TI enzymes are modulated in NASH and NAFLD to establish metabolic reprogramming and achieve innate immune memory . Three mThe first human NASH upregulated 25 (35.2%) and downregulated 5 (7%) out of 71 glycolysis enzymes genes . The secFurthermore, the first human NASH upregulated 4 out of 24 (16.6%) acetyl-CoA generation enzyme genes. The second human NASH upregulated 1 out of 24 (4.2%) genes. The MCD+HFD upregulated 3 (12.5%) and downregulated 7 (29.2%) out of 24 genes. The MAT1A-KO upregulated 1 (4.2%) and downregulated 4 (16.7%) out of 24 genes. The HFCD upregulated 7 (29.2%) and downregulated 2 (8.3%) out of 24 genes .In addition, the first human NASH upregulated 2 (20%) and downregulated 3 (30%) out of 10 mevalonate pathway enzyme genes. The second human NASH upregulated 1 out of 10 (10%) gene. The MCD+HFD upregulated 7 (70%) and downregulated 1 (10%) out of 10 genes. The MAT1A-KO upregulated 4 (40%) and downregulated 1 (10%) out of 10 genes. The GNMT-KO upregulated 3 out of 10 (30%) genes. The HFCD upregulated 7 out of 10 (70%) genes .β-hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase inhibitors) block cholesterol synthesis and mevalonate generation, preventing TI induction [Statins  , of whicArachidonic acid (AA) undergoes oxidative metabolism under enzymatic or nonenzymatic mechanisms. Cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450s (CYPs) mediated enzymatic peroxidation of AA to the respective metabolites. The 12-lipoxygenase (12-LO) and 15-lipoxygenase (15-LO) mediated conversion of AA to 12- and 15-hydroperoxyeicosatetraenoic acid (HpETE) and the downstream glutathione peroxidase- (GPx-) mediated conversion to the respective hydroxyeicosatetraenoic acids (12- and 15-HETE). ROS-induced lipid peroxidation (LPO) of 12- and 15-HpETE results in the generation of 4-hydroxynonenal (4-HNE) and 4-hydroxydodecadeinal (4-HDDE). These reactive aldehydes interact with and inactivate GPx, leading to an increased rate of 12-HpETE and 15-HpETE peroxidation . The nonWe hypothesized that downregulation of lipid peroxidation antioxidant enzymes CYPs and GPXs is inversely associated with upregulation of cytokines and chemokines in NAFLD and that upregulation of proinflammatory lipid peroxidation enzymes COXs and LOXs  is assocThe MCD+HFD upregulated 2 (7.7%) and downregulated 9 (34.6%) COXs . The MATThe MCD+HFD upregulated 6 (13.6%) and downregulated 13 (29.5%) CYP lipid peroxidation antioxidant enzymes (CYP-LPAEs) . The MATThe MCD+HFD upregulated 4 (15.4%) and downregulated 11 (42.3%) AA metabolism enzyme genes . The MATThe MCD+HFD upregulated 4 out of 8 (50%) GPXs . The MATfirst, proinflammatory MCD+HFD upregulates 7.7% COXs, 13.6% CYP-LPAEs, 15.4% AA metabolism enzymes, and 50% GPXs and downregulates 34.6% COXs, 29.5% CYP-LPAEs, and 42.3% AA metabolism enzymes, which are in big contrast with that of less inflammatory HFCD with upregulations of 34.6% COXs, 50% CYP-LPAEs, and 92.3% AA metabolism enzymes and downregulation of 3.8% COXs, 2.3% CYP-LPAEs, and 12.5% GPXs; second, significant upregulation of lipid peroxidation enzymes in HFCD is positively correlated with significant upregulation of TI enzymes and is, however, inversely associated with low-level upregulation of canonical inflammasome regulators (6.7%), noncanonical inflammasome regulators (7.1%), and 2.4% secretomic cytokines; third, no upregulation of GPXs in HFCD  and downregulated 8 (20.5%) NASH/NAFLD-upregulated cytokines and chemokines. In comparison, caspase-1 deficiency upregulated one cytokine CXCL14 (2.6%) and downregulated one cytokine LTB (2.6%) NASH/NAFLD-upregulated cytokines and chemokines . These rfirst, caspase-11 and caspase-1 partially modulate the expressions of cytokines, chemokines, canonical and noncanonical inflammasome pathway regulators, TI enzymes, and lipid peroxidation enzymes; second, in the context of NASH/NAFLD, caspase-11 has stronger capacities than caspase-1 in modulating inflammatory gene expressions; and third, experimental conditions in caspase-11 deficiency [Taken together, our results have demonstrated that, ficiency  and caspficiency  were noti) human NASH and NAFLD mouse models upregulate both cytokines and chemokines and canonical secretome; (ii) pathway analysis indicated that human NASH can be classified into metabolic and immune NASH; MCD+HFD, GNMT-KO, MAT1A-KO, and HFCD can be classified into inflammatory NAFLD, SAM accumulation NAFLD, cholesterol/mevalonate NAFLD, and LXR/RXR-fatty acid β-oxidation NAFLD, respectively; (iii) canonical and noncanonical inflammasome pathways play differential roles in the pathogenesis of NASH/NAFLD; (iv) TI enzymes are significantly upregulated in human NASH and NAFLD mouse models; HFD upregulates TI enzymes more than cytokines, chemokines, and canonical and noncanonical inflammasome regulators; statins promote rather than suppress TI enzyme expression; (v) lipid peroxidation enzyme expression indicated that there are two representative NAFLD models: the MCD+HFD is a proinflammatory cytokine- and canonical and noncanonical inflammasome-upregulated model; in contrast, the HFCD is a lipid peroxidation enzyme- and TI enzyme-upregulated model; and (vi) caspase-11 and caspase-1 partially upregulate the expressions of cytokines, chemokines, canonical and noncanonical inflammasome pathway regulators, TI enzymes, and lipid peroxidation enzymes.In this study, we performed a panoramic database mining analysis on microarray data of both human NASH and NAFLD mouse models. We made the following significant findings:  promotes macrophage infiltration and progression of NASH . MAT1A ain vitro and in vivo experimental models will be needed to verify all the findings. Nevertheless, our findings provide novel insights on the roles of proinflammatory cytokines and chemokines [One limitation of the current study is that, due to the low-throughput nature of verification techniques in all the research laboratories, we could not verify every result we identified , 114. Weemokines , 59 and emokines , 116, ca


Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs.Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11’s interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the  (+)RNA viruses have to co-opt numerous host proteins to support their replication in infected cells. These viruses induce the biogenesis of viral replication organelles (VROs), the sites of replication, in cells. However, what the mechanism of subversion for most of the cytosolic host proteins is not yet dissected. In this paper, the authors used a plant (+)RNA virus, tomato bushy stunt virus (TBSV), to study the role of a cellular proteasomal protein, called Rpn11 (POH1), as a protein interaction hub. They show that knockdown of Rpn11 or retargeting Rpn11 into the nucleus and destruction of actin filaments diminishes TBSV replication in yeast and plant cells. This effect is due to diminished recruitment of pro-viral metabolic enzymes into VROs. Overall, data presented support a novel viral recruitment strategy for cytosolic host factors. Via the small viral replication protein, TBSV targets the cytosolic proteasomal Rpn11 protein interaction hub protein and the co-opted and stabilized actin filaments. The combined and coordinated subversion of Rpn11 and the actin network allows tombusviruses to gain access to abundant cytosolic proteins, such as the glycolytic and fermentation enzymes, which are then efficiently delivered to perform pro-viral functions within the VROs. Positive-strand (+)RNA viruses code for only a small number of genes, therefore, they rely on subverting a long list of host factors to build robust viral replication organelles (VROs) or replication compartments –8. RecenIn addition to membranous compartments and transport vesicles, TBSV also hijacks several cytosolic host factors, such as the heat shock protein 70 (Hsp70), Vps4 and other ESCRT  proteins, translation elongation factors, and a few DEAD-box RNA helicases , 12–18. in vitro and in vivo approaches including the use of yeast (Saccharomyces cerevisiae) model host , 70ts), pol  , which is partially deficient in actin filament depolymerization at semi-permissive temperature, also supported ~3-fold increased ATP production within the TBSV VROs  . We founure Figs  and S6. ROs Figs  and S6. WT yeast . On the WT yeast  and the WT yeast  by ~3-toN. benthamiana plants. Expression of RavK reduced ATP production within TBSV and CIRV VROs by ~3-to-4-fold in N. benthamiana  the syntaxin18-like Ufe1 SNARE protein and Sac1 PI4P phosphatase-positive subdomains within the ER membrane, and (ii) the Rab5-positive early endosomes , 85–87. In summary, we present results that support a novel viral recruitment strategy for cytosolic host factors based on TBSV and CIRV. These viruses target via the small viral replication protein the cytosolic Rpn11 protein interaction hub protein and the co-opted and stabilized actin filaments. The combined and coordinated subversion of Rpn11 and the actin network allows tombusviruses to gain access to abundant cytosolic proteins, such as the glycolytic and fermentation enzymes, which are then efficiently delivered to perform pro-viral functions into the VROs, which represent the site of viral replication. Accordingly, knockdown of Rpn11 or destruction of actin filaments diminishes VRO biogenesis and tombusvirus replication in yeast and plant cells. Other (+)RNA viruses with small number of genes might also exploit similar strategies to maximize the recruitment of host factors with the involvement of limited number of viral proteins into VROs .Additional Materials and Methods are presented in the supplementary information .Parental yeast strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), was purchased from Open Biosystems. SC1 (MATa his3Δ1 leu2Δ trp1Δ289 uraΔ52) yeast strain was purchased from Invitrogen. BY4747-ADH-His92 yeast strain expressing p92 replication protein from the chromosome was made earlier [Plasmids used in this study are described in Legionella VipA effector on tombusvirus replication, BY4741 yeast was co-transformed with pGBK-CUP-Flagp33/Gal-DI72, pGAD-Cup-Flagp92 or pGBK-CUP-Flagp36/Gal-DI72, pGAD-Cup-Flagp95 and one of the following plasmids: pYC-NT vector, pYC-NT-VipA [− media supplemented with 2% glucose and BCS (from VWR) at 29°C for 16 h. Yeast cultures were washed and grown in SC-ULH− supplemented with 2% galactose at 23°C for 10 h. TBSV repRNA replication was induced by adding 50 μM of CuSO4 for 24 h at 23°C. To test the effect of Legionella RavK effector on tombusvirus replication, BY4741 yeast was co-transformed with pGBK-CUP-Flagp33/Gal-DI72, pGAD-Cup-Flagp92 and one of the following plasmids: pYC-NT vector, pYC-NT-RavK, pAG416GAL-ccdB-RavK [32P-labeled DI72 RI/IV as a radioactive probe [6-VipA and His6-RavK proteins.To test the effect of the -NT-VipA , pAG416GcdB-RavK . Yeast cve probe . Total pve probe  and protThe tombusvirus replicase preparations were made as described before with modifications . Briefly− media, whereas transformed Sc1 yeast cultures were plated in SC-ULHT− media. Single colonies were streaked and grown in 20 ml of SC-ULH− (or SC-ULHT− in the case of Sc1 yeast) supplemented with 2% glucose and 100 μM BCS at 23°C for 16 h. Yeast cultures were washed with sterile water and grown in 40 ml of SC-ULH− (or SC-ULHT−) supplemented with 2% galactose and 50 μM of CuSO4 at 23°C for 24 h. Yeast cultures were harvested and incubated in 35 ml of Phosphate Buffer Saline (PBS) containing 1% formaldehyde for 1 h on ice [2, 10 mM KCl) with 0.1% of yeast protease inhibitor. Yeast cells were broken and centrifuged to discard the supernatant. Yeast pellet was solubilized in High Salt TG buffer with 2% Triton X100 and 0.1% of yeast protease inhibitor. The tubes were rotated for 8 h in the cold room. Then, the tubes were centrifuged at 35,000 g for 20 min and the supernatant was transferred to an equilibrated Bio-Rad Bio-Spin chromatography column with 20 μl of FLAG M2 resin (Sigma). The column was rotated for 16 h at 4°C. The column was washed with High Salt TG Buffer and the Flag-p33 protein preparation was recovered from the column with 30 μl of SDS loading buffer. β-Mercaptoethanol was added to the eluted sample and boiled for 40 minutes to reverse the crosslink. Purified Flag-p33 preparations were analyzed by western blot and Flag-p33 protein was detected with anti-FLAG antibody. The co-purified proteins were detected with anti-His antibody. Detection with NBIP-BCIP has been described previously [Transformed BY4747-ADH-His92 yeast cultures were plated in SC-ULHh on ice . Formaldeviously . ProteinCo-purification studies were conducted from BY4747-ADH-92 yeast co-transformed with HpGBK-CUP1-Flagp33/Gal-DI72, or HpGBK-CUP1-Hisp33/Gal-DI72 as a control with pRS315-Gal-HisRavK and plasmids UpYES-Pdc1 or UpYES-Adh or UpYC-Rpn11 as above.rpn11-14ts yeast strains were transformed with plasmids HpGBK-CUP1-Flagp33/Gal:DI72, LpGAD-CUP1-Flagp92 and one of the following plasmids UpYES-Pdc1, pYES-Adh1 or pYC2-Fba1. Transformed yeasts were pre-grown in SC-ULH− media supplemented with 2% glucose and 100 μM BCS for 16 h at 23°C (permissive temperature). Yeast cultures were transferred to SC-ULH− media supplemented with 2% galactose for 24 h at 23°C or 32°C (semi-permissive temperature). Then, 50 μM of CuSO4 was added to the yeast cultures for 6 h. Proteins were crosslinked and Flag-p33 protein preparations were obtained from detergent-solubilized membrane fractions using anti-FLAG M2 agarose as mentioned above.The haploid BY4741 and N. benthamiana plants were co-agroinfiltrated with pEarleyGate100-VipA (OD600 0.6) or pGD-FlagVipA (OD600 0.6), together with p19 (OD600 0.2) and CNV (OD600 0.2). Plant samples were harvested 2.5 days post-agroinfiltration [N. benthamiana leaves were co-agroinfiltrated with pGD-FlagVipA (OD600 0.6) and p19 (OD600 0.2). Then, 16 h later, plants were inoculated with TBSV and CIRV sap preparations. Plant samples were harvested 2 and 3 dpi, respectively. To test the effect of RavK expression, N. benthamiana leaves were co-agroinfiltrated with pGD-FlagRavK (OD600 0.6) and p19. The agro-infiltrated leaves were inoculated with TBSV 16 h later. Plant samples were harvested at 2 dpi. Total plant RNA was extracted and tombusvirus RNA accumulation was detected by northern blot with CNV, TBSV and CIRV 32P-labeled probes as described [ltration . To testescribed , 93.N. benthamiana leaves were co-agroinfiltrated with pGD-EGFP vector, pGD-EGFP-Rpn8 (OD600 0.7) or pGD-EGFP-NRS-Rpn8 (OD600 0.7) together with pGD-CNV20KStop (OD600 0.2) and pGD-p19 (OD600 0.2). Total RNA was extracted from infiltrated leaves 2.5 days post agroinfiltration and CNV gRNA levels were analyzed by northern blot [ern blot , 93. Ima600 0.2), pGD-CNV-20K-stop and one of the following pGD-nYFP-Pgk1 (OD600 0.2), pGD-nYFP-PK1 (OD600 0.2), pGD-nYFP-Pdc1 (OD600 0.2), pGD-nYFP-Adh1 (OD600 0.2) were used for agroinfiltration. For the BiFC control, pGD-cYFP vector (OD600 0.2) and one of the plasmids above were used. Plant cells were visualized in the confocal laser microscope 1.5 days after agroinfiltration [Knockdown of Rpn11 expression via VIGS is described in ltration .benthamiana leaves were co-agroinfiltrated with BiFC plasmids pGD-p33-cYFP (OD600 0.2), pGD-CNV-20K-stop (OD600 0.2), pGD-RFP-SKL (OD600 0.2), pGD-RavK (OD600 0.6), pGD-p19 (OD600 0.15) and one of the following pGD-nYFP-Pgk1 (OD600 0.2) or pGD-nYFP-PK1 (OD600 0.2). Plant samples were visualized 50 h after agroinfiltration. Additional BiFC experiments are described in N. YEMK-p92-based biosensor using a confocal microscope [YEMK-p92 orUpCM189-Tet or HpESC-Gal-p33/Gal-DI72, LpGAD-ADH-ATeamYEMK-p92 (or LpGAD-ADH-ATeamRK-p92 as a control). The transformed yeasts were pre-grown in SC-ULH− media supplemented with 2% raffinose at 23°C for 14 h, then transferred to SC-ULH− media supplemented with 2% glucose at 23°C for 4 h.Relative ATP levels in yeast and plant cells were visualized based on the ATeamcroscope , 65. BriYEMK-p95 or UpCM189-Tet, HpGBK-CUP1-p36/Gal-DI72, LpGAD-CUP1-ATeamYEMK-p95 (or LpGAD-CUP1-ATeamRK-p95 as a control). The transformed yeasts were pre-grown in SC-ULH− media supplemented with 2% raffinose and 25μM of CuSO4 at 23°C for 14 h, then transferred to SC-ULH− media supplemented with 2% glucose and 50 μM of CuSO4 at 23°C for 4 h.In the case of CIRV, BY4741 yeasts were transformed with UpCM189-Tet-RavK, HpGBK-CUP1-p36/Gal-DI72, LpGAD-CUP1-ATeamact1-132ts and cof1-8ts yeasts were transformed with HpESC-Gal-p33/Gal-DI72, LpGAD-ADH-ATeamYEMK-p92 or LpGAD-ADH-ATeamRK-p92 as a control. The transformed yeasts were pre-grown in SC-LH− media supplemented with 2% glucose at 23°C for 14 h and then transferred to SC-LH− supplemented with 2% glucose at 23°C or 32°C for 1 h and 3 h. Confocal FRET images were taken with Olympus microscope. FRET was measured using Olympus FLUOVIEW software and ImageJ software. Graphics were done using Prism6 Software [BY4741, Software .N. benthamiana leaves were co-agroinfiltrated with pGD-FlagRavK (OD600 0.06), pGD-p33-ATeamYEMK(OD600 0.02), pGD-p19 (OD600 0.02) or pGD-p33-ATeamYEMK(OD600 0.02), pGD-p19 (OD600 0.02) or pGD-Flag (OD600 0.06). The agro-infiltrated leaves were inoculated with TBSV 16 h later. The images were taken 52 h after agroinfiltration. For CIRV-based experiments, N. benthamiana plants were co-agroinfiltrated with pGD-FlagRavK (OD600 0.06), pGD-p36-ATeamYEMK (OD600 0.02), pGD-p19 (OD600 0.02), pGD-CIRV (OD600 0.02) or pGD-Flag (OD600 0.06), pGD-p36-ATeamYEMK (OD600 0.02), pGD-p19 (OD600 0.02), pGD-CIRV (OD600 0.02). The images were taken 52 h after agroinfiltration and analyzed as describe above [To visualize ATP production within the VROs in plant cells, be above , 21.S1 Text(DOC)Click here for additional data file.S1 Fig6-tagged glycolytic and fermentation enzymes in Rpn11 knockdown versus control VIGS (TRV-MBP-5’) plants. Total proteins in SDS-PAGE were stained with coomassie blue as controls.VIGS-based knockdown of Rpn11 in N. benthamiana. (A) Top Images: phenotypes of Rpn11 knockdown plants. Semi-quantitative RT-PCR shows the Rpn11 mRNA level after VIGS treatment. RT-PCR of tubulin mRNA and ribosomal RNA from the same samples are used as loading controls. (B) Western blot analysis of the ectopically-expressed His(TIF)Click here for additional data file.S2 FigN. benthamiana plants expressing GFP-NRS-Rpn8 were analyzed via confocal laser microscopy 2.5 days post-agroinfiltration. Control experiments included plants expressing GFP-Rpn8. (B) Expression of GFP-NRS-Rpn8 did not change the localization of TBSV p33-BFP replication protein. Control experiments show the partial re-localization of GFP-Rpn8 into p33-BFP foci (pointed by arrows). (C) Expression of GFP-NRS-Rpn8 sequesters RFP-Rpn11 into the nucleus. Bottom image: co-localization of GFP-Rpn8 and RFP-Rpn11 in the cytosol and the nucleus. Scale bar is 10 μm. Each experiment was repeated.(A) RFP-H2B transgenic (TIF)Click here for additional data file.S3 Figrpn11-14ts yeasts together with His6-Tdh3. First panel: Western blot analysis of co-purified His6-Tdh3 with TBSV replicase from detergent-solubilized membrane fraction of yeast cultured at either permissive (23°C) or semi-permissive (32°C) temperatures. The co-purified His6-Tdh3 was detected by western blot with anti-His antibody. Second panel: Western blot shows Flag-affinity purified p33 in the same samples as above with anti-Flag antibody. Third panel: Western blot analysis shows the levels of Flag-p33 in total protein extracts detected with anti-Flag antibody. Fourth panel: Western blots of His6-Tdh3 and His6-p33 in total protein extracts detected with anti-His antibody. Fifth panel: Coomassie-blue stained gel SDS gel of the total protein extracts as loading controls. (B) Flag-p33 and Flag-p92 replication proteins were expressed in WT and rpn11-14ts yeasts together with His6-Tdh2 or His6-RH2 helicase. See further details in panel A.(A) Flag-p33 and Flag-p92 replication proteins were expressed in WT and (TIF)Click here for additional data file.S4 Fig(A) Top row: Transgenic N. benthamiana plants expressing GFP-mTalin actin-binding protein, and co-expressing VipA, p33-BFP and RFP-SKL peroxisomal luminal marker . Second row: Control GFP-mTalin N. benthamiana plants expressing p33-BFP, RFP-SKL were visualized via confocal microscopy. The plants were infected with TBSV 16 h after agroinfiltration. Plant samples were analyzed using confocal microscopy 36 h post-infection. (B) The same experiment as in panel A, except plants did not express viral components. The plants were mock-inoculated. See details in panel A. (C) Transgenic N. benthamiana plants expressing GFP-mTalin, and co-expressing VipA, CIRV p36-BFP and RFP-Tim21 mitochondrial marker . The plants were also agroinfiltrated with pGD-CIRV. Plant samples were analyzed using confocal microscopy 36 h post-infection. Note that we show two sets of images to illustrate the enlarged size of CIRV VROs when VipA is co-expressed in plants. The control image is shown in (TIF)Click here for additional data file.S5 FigN. benthamiana 4 d post-agroinfiltration. The leaves were inoculated with CNV or CIRV or mock-inoculated (C) Western blot analysis of the ectopically-expressed His6-tagged glycolytic Pgk1, His6-PK, His6-Fab2 and fermentation His6-Pdc1 and His6-Adh1 enzymes in RavK (detected via anti-Flag- rabbit antibody) expressing versus control N. benthamiana plants. Asterisk depicts a nonspecific band detected by the anti-Flag- rabbit antibody. Total proteins in SDS-PAGE were stained with Coomassie blue as controls.(A-B) Lack of visible phenotypes of transient RavK expression on the leaves of (TIF)Click here for additional data file.S6 Figts and cof1ts yeasts at the permissive and semi-permissive (32°C) temperatures. The quantitative FRET values of multiple cells are shown in Relative ATP levels produced within the tombusvirus VROs was visualized via expressing ATeam-p92 in WT, act1(TIF)Click here for additional data file.


ABSTRACT IMPACT: We devised a new method to produce highly potent SARS-CoV2-specific that can be used to treat severely ill patients with Covid-19. OBJECTIVES/GOALS: Neutralizing antibodies against SARS-CoV-2 are thought to offer the most immediate and effective treatment for those severely afflicted by Covid-19. We devised an approach for rapid and efficient generation of human monoclonal antibodies with neutralizing activity against SARS-CoV-2. METHODS/STUDY POPULATION: SARS-CoV-2 S1 spike protein-specific memory B cells were isolated from 12 subjects recovering from infection with that virus. Paired end single index sequencing was performed using up to 10,000 antigen-specific B cells per subject. Antigen-specific B cell clones were identified by unique diversity and joining gene V(D)J rearrangements and the CDR3 regions. VH and VL regions were cloned and the products expressed in 293T/17 cells to generate spike-specific human monoclonal antibodies. RESULTS/ANTICIPATED RESULTS: Forty-three human monoclonal antibodies were produced. Every monoclonal antibody so generated neutralized viruses pseudotyped with Spike protein of the Wuhan-1 strain. Eighteen monoclonal antibodies neutralized pseudotyped viruses with half-maximal inhibitory concentration (IC50s) between 1 pg/mL and 1 ng/mL (6.7 x 10E-15 M to 6.7 x 10E-12 M), exceeding by 10-100-fold the potency of previously reported anti-SARS-CoV-2-neutralizing monoclonal antibodies. Eight monoclonal antibodies neutralized viruses pseudotyped with mutant spike proteins previously identified in clinical isolates, including receptor binding domain mutants and the C-terminal D614G mutant with IC50<6.7 x10E-12M. DISCUSSION/SIGNIFICANCE OF FINDINGS: We show that SARS-CoV-2 evokes high affinity B cell responses. Some B cells produce antibodies that are broadly neutralizing; others produce strain-specific antibodies. However, antigenic variants that would potentially escape control by immunity or vaccination were nonetheless identified.


C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function.Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched  Inside the animal body, many “signals” are exchanged between tissues and cells to ensure healthy growth and physiological conditions. Too much or less signals have been known to cause various diseases such as cancer and diabetes. When developing animals are not fed enough food, their growth rates generally slow down. We found that when nematodes lacking the PTR-18 protein are hatched out of the egg in the absence of sufficient food, they do not appropriately halt their growth. Analyses at the molecular and cellular levels showed that immediately before hatching, PTR-18 re-uptakes a signaling molecule called GRL-7, which is known to promote growth, from the outside to the inside of cells, resulting in the degradation of GRL-7. As a result, worms lacking PTR-18 still contain excess GRL-7 after hatching, which results in unwanted growth even when ample food is not available. Thus, our findings uncover the role of PTR-18 in terminating a signal on time. Humans also possess a protein called PTCHD1, which is structurally similar to the worm’s PTR-18 and has been proposed to cause autistic spectrum disorders and learning disabilities. Our findings may provide a clue to further understand the function of human PTCHD1. C. elegans L1 larvae hatch out of their eggshells with quiescent stem and progenitor cells. Sufficient food supply initiates L1 development by coordinating the release of stem and progenitor cells from quiescence  I, +/szT1 X, VC20514 ptr-3(gk333566) II, VC1110 +/szT1[lon-2(e678)] I, ptr-4(ok1576)/szT1 X, VC1067 ptr-5(gk472) X, VC2301 ptr-6(ok2988) II, MY1, which possesses the polymorphism WBVar01902623(S787Ochre) in the ptr-8 locus, RB1693 ptr-10(ok2106) I, VC20409 ptr-11(gk100342) I, VC40042 ptr-12(gk105502) I, VC40161 ptr-17(gk490736) I, RB2542 ptr-18(ok3532) II, RB2393 ptr-20(ok3263) II, VC3219 ptr-23(ok3663) I, and PD4666 ayIs6 X. Further, the following strain was purchased from the National BioResource Project: FX10640 grl-27(tm10640) IV. RB2058, RB1097, RB2018, RB1999, RB2495, RB2322, RB2112, and FX10640 were outcrossed against N2 six times and designated as YB3786, YB3788, YB3790, YB3289, YB3847, YB3848, YB3793 and YB4020, respectively, prior to experiments, while RB2542 was outcrossed against N2 four times to yield YB2891. grl-10(td193) was constructed by replacing the protein coding region of the grl-10 gene with Self-Excising Drug Selection Cassette (SEC), as described in [grl-10 locus. The introduced SEC was excised from the genome by heat shock, resulting in the td193 allele. pCH102.1 was made by PCR using primers, MF1515 and CH346 and PU6::unc-119_sgRNA plasmid as a template [All nematode strains were cultured according to standard procedures . The strptr-18, grl-5, grl-7 and gel-27 were constructed using the fosmids WRM0613dH03, WRM0631aE09, WRM0615cE01 and WRM0636cB11, respectively, using “recombineering” as described previously [mCherry::3x flag tags were PCR-amplified using pBalu1 and pCH78.1 plasmids, respectively. The primers used are listed in the Key Resources C. elegans was performed as described previously [Fosmid-based reporter genes for eviously . GFP andeviously .venus::3xflag and mCherry tags were inserted into the grl-7 gene using the CRISPR/Cas9 system according to the protocol described in [venus::3xflag and mCherry tags, both of which contain grl-7 homologous arm sequences at both the 5’ and 3’ ends were constructed via PCR using primer pairs MF2984-MF2985 and MF3047-MF3048, respectively. DNA fragments encoding venus and mCherry cDNA were PCR-amplified using primer pairs MF2607-MF2608 and MF3049-MF3050, respectively. These PCR products were subsequently annealed to make “dsDNA hybrid donors”, as described in [grl-7 gene CTATATTTACAATTGACGGT was used to custom-order the grl-7-specific Alt-R CRISPR-Cas9 crRNA. On the other hand, we could not insert venus and HA tags using CRISPR-Cas9 crRNAs targeting to the sequences, CTCCGAACGGATTTTTCAAC, AACCAGTTGAAAAATCCGTT, and CCGTTCACTTTGATAACTTT in the ptr-18 locus.ribed in . Brieflyribed in . Each ofribed in . The parA drop of the starved L1 larvae was placed on polylysine-coated slides, permeabilized using the freeze-cracking method, and fixed in MeOH/acetone at -20°C for 5 min, as described previously . The sliThe C. elegans embryos and adults were mounted on 4% agar pads, as described previously [eviously . FluoresC. elegans was cultured at 20°C in NGM agar plates. Live worms were immobilized with 50 mM levamisole in M9 and mounted on a slide with 2% agarose. The worms were imaged with a DeltaVision OMX Optical Microscope (version 4), Software: DeltaVision OMX softWoRx. Oil 1.518. Objective 60X NA 1.42. Pixel size: x = 0.08, y = 0.08, and z = 0.125 μm. All images were reconstructed using the same parameters. Images were post-processed with OMERO.web 5.3.4-ice36-b69.L1 larvae were anesthetized as described above. Super-resolution imaging was performed using a Zeiss LSM980 quipped with Airysccan2 and the Plan-Apochromat 63x/Oil 1.4 DIC objective. Approximately 150 Z-section images/embryo were acquired using the SR-4Y Multiplex mode at 0.17 μm intervals and processed with ZEN pro software and Fiji.The L1 larvae were prepared and either starved in complete S medium or grown synchronously as described previously . The conE. coli before observation.The synchronized embryos shown in Figs ptr-18 mutant animals after 24 h L1 starvation were prepared as described in C. elegans culture. Newly hatched larvae were first filtered through an L1 harvest filter . Filtered animals were collected via centrifugation in 15 mL polypropylene tubes at 3,500 rpm for 1 min at 4°C, further centrifuged in 1.5 mL microtubes at 15,000 rpm for 1 min at 4°C, and snap-frozen by liquid nitrogen. Proteins were extracted in the urea lysis buffer .Wild-type and  X- 100; ) by soniThe embryos harvested using the alkali/bleach method described above and the resulting hatched larvae were cultured at 10 embryos/μl in 10 mL of cholesterol-, ethanol-free complete S medium in a 15 mL polypropylene tube with rotation at 30–40 rpm at 20°C for 10 days. The larvae were subsequently transferred onto freshly seeded plates, and the number of transferred and recovered larvae was scored after 3 days.rab-5 was conducted using the ORFeome-RNAi v1.1 library [Feeding RNAi targeting  library  accordin library . Empty p library  was usedAll statistical analyses were conducted using the Microsoft Excel software.S1 Figgrl-5, grl-7, and grl-27 does not cause the delay of the timing of P cell activation. Data are presented as mean ± SD. Experiments were repeated three times, and n ≥50 animals were scored for each trial. N.S.: Not statistically significant (Fisher’s exact test).(A) Primordial germ cells, Z2 and Z3 (arrows), were visualized using anti-PGL-1 antibody. The absence of the proliferation of Z2 and Z3 was scored under the Nomarski microscopy after 5-day starvation in complete S media. Experiments were repeated three times, and n ≥35 animals were scored for each trial (see text). Scale bar: 10 μm. (B) Newly hatched L1 larvae were starved in cholesterol/EtOH-free complete S medium, and the viability was assessed after 10 days of culture. Experiments were repeated three times, and n ≥50 animals were scored for each trial. Data are presented as mean ± SD. ****: p <0.0001 (Fisher’s exact test). (C) Simultaneous loss of (PDF)Click here for additional data file.S2 Figdct-5 promoter was only detectable after hatching. The arrow and arrowhead in the left panels indicate the excretory duct and hypodermal cells, respectively. Arrowheads in other panels point to cells indicated below. Types of cells expressing each reporter gene are indicated below each image. Scale Bar: 10 μm. (E) Expression of PTR-18::VENUS and mCherry driven by the hlh-3 promoter. P cells are indicated by arrowheads. Scale Bar: 10 μm. (F) PTR-18::VENUS is expressed in hypodermal but not P cells when driven by the Q system. The constructs introduced are indicated on the left. Note that the expression of PTR-18::VENUS was downregulated below detectable levels in P cells (arrowheads). Scale Bar: 10 μm. (G) Schematic of the hypodermis-specific expression of PTR-18::VENUS. The dpy-7 promoter drives the expression of transcriptional activator QF in both the hypodermal and P cells. However, QS expressed in P cells under the control of the hlh-3 promoter suppresses QF, resulting in the expression of PTR-18::VENUS only in the hypodermal cells.(A) Percentage of animals at each developmental stage after harvesting early embryos. Data are presented as mean ± SD. Experiments were repeated three times, and ≥50 animals were scored for each time point. (B) Hypodermal expression of PTR-18::GFP becomes detectable 11 h after the L1-arrested larvae are fed. Descendants of P cells (arrows) and F, K, and U cells (arrowhead) are indicated. Scale Bar: 10 μm. (C) Seam cells with or without PTR-18::GFP expression are shown in red and white arrowheads, respectively. The arrow indicates hypodermis. Scale Bar: 10 μm. (D) Expression of PTR-18::VENUS driven by the indicated promoters in 3-fold embryos. Expression of PTR-18::VENUS driven by the (PDF)Click here for additional data file.S3 Figgrl-5::mcherry::3xflag reporter gene. Scale Bar: 10 μm. (D) Expression of GRL-5::mCherry::3xFLAG in fed L1 larvae. Photographs show fed L1 larvae at indicated hours after the L1-arrested larvae were fed. Note that GRL-5::mCherry::3xFLAG accumulates at the apical surface of the whole body 11 h after the transfer. Photographs at the bottom are magnified views of the part of the above ones (indicated by the rectangles). All photographs showing GRL-5::mCherry::3xFLAG expression were taken at the same exposure time for comparison. Scale Bar: 10 μm (DIC image) and 1 μm (magnified image). (E) Expression of DPY-7::mCherry in 3-fold embryos (top panels) and newly hatched L1 larvae (bottom panels). The photograph of the L1 larva was taken after 24 h L1 starvation. Scale Bar: 10 μm.(A) Percentages of 3-fold embryos and L1 larvae after harvesting early embryos via the bleach treatment of gravid adults. Data are presented as mean ± SD. Experiments were repeated three times, and n ≥50 animals were scored for each trial. (B) Expression patterns of GRL-7::mCherry::3xFLAG in fed L1 larvae. Images shown are of L1 larvae 1 h (top panels) and 11 h (bottom panels) after the L1-arrested larvae were transferred to the fed condition. The photographs were taken with the focal planes on the apical surface  and the ventral midline (midline). GRL-7::mCherry::3xFLAG localized at the vesicular structure and along the apical membrane are indicated by the arrowhead and arrow, respectively. The right panels are magnified views of the areas within the rectangle in the images on the left. Scale Bar: 10 μm (DIC image) and 2 μm (magnified image). (C) Expression of GRL-5::mCherry::3xFLAG during embryogenesis. Differential interference contrast and fluorescent images of embryos carrying the (PDF)Click here for additional data file.S4 Figgrl-7::mCherry reporter gene co-expressed with ptr-18::gfp derived from a fosmid. Trios of images below show PTR-18::GFP (green), GRL-7::mCherry (red), and both, respectively, from left to right. (A) Maximum intensity Z-projection image of an embryo showing apical distributions of PTR-18::GFP and GRL-7::mCherry. Scale bar: 10 μm. (B) Maximum intensity Z-projection image of an embryo showing apical and vesicular distributions of PTR-18::GFP and apical localization of GRL-7::mCherry. (C) Magnified view from a Z section of an embryo indicated in B. Intensity profiles are determined along the arrow in the merged image. Scale bar: 10 μm. (D) Maximum intensity Z-projection image of an embryo showing predominantly vesicular patterns of PTR-18::GFP and apical and vesicular distributions of GRL-7::mCherry. Scale bar: 10 μm (upper panel) and 1 μl (bottom panel). (E) Magnified view from a Z section of an embryo indicated in B. Intensity profiles are determined along the arrows in the merged image. Note that PTR-18::GFP only localized to GRL-7::Cherry-positive vesicles at the left and middle regions, but not at the right. Intensity profiles are determined along the corresponding arrows in the merged image. Scale bars: 1 μm. (F) PTR-18 and GRL-7 localize to the same compartment. Structured illumination microscopy of L4 worms expressing PTR-18::GFP and GRL-7::mCherry. PTR-18 and GRL-17 populate the same compartment. While PTR-18 is present on the limiting membrane, GRL-7 accumulates inside. The nature of the compartment likely changes during development, comparing the top and bottom panels. N = 8 animals. Arrowheads point to the PTR-18 and GRL-17 positive compartments.Super-resolution images showing the expression of CRISPR-generated (PDF)Click here for additional data file.S5 Figptr-18 L1 larvae. Bottom panels are magnified views of the part of the middle panels (indicated by the rectangles). Scale bars indicate 10 μm (upper panel) and 1 μm (bottom panel). (B) Percentage of animals showing apical GRL-7::mCherry::3xFLAG localization during L1 starvation. GRL-7::mCherry::3xFLAG localization was scored at the indicated time points of L1 starvation in complete S medium. Note that the presence of cholesterol and ethanol in the medium does not affect the phenotype after 24 h L1 starvation  L1 larvae. ptr-18 mutant larvae with or without the indicated transgenes after 24 h L1 starvation are shown. Scale Bars: 10 μm. (E) Percentage of newly hatched, ptr-18(ok3532) mutant larvae showing apical localization of GRL-7::mCherry::3xFLAG in the presence or absence of the transgenes that drives the expression of ptr-18::venus under the control of the P-cell specific hlh-3 promoter. Data are presented as mean ± SD. Experiments were conducted with three transgenic lines (#1 to #3) and repeated three times. n ≥50 animals were scored for each trial. ****: p <0.0001 (Fisher’s exact test). Siblings of line #2 that did not inherit the transgenes were used as controls.(A) GRL-7::mCherry::3xFLAG expression in starved wild-type and (PDF)Click here for additional data file.S6 Figptr-18 mutant L1 larvae showing the vesicular distribution of GRL-5::mCherry::3xFLAG. The bottom panels are magnified views of the area within the rectangle from the images above. Scale bars indicate 10 μm (upper panel) and 1 μm (bottom panel). Photographs were taken with the same exposure time. B) Apical localization of GRL-5::mCherry::3xFLAG was rarely detected in the starved wild-type and ptr-18 mutant L1 larvae. n ≥50 animals were scored for each genotype. Data were collected after 24 h L1 starvation and are presented as mean ± SD. Experiments were repeated three times, and n ≥50 animals were scored for each trial. (C) Wild-type and ptr-18 mutant 3-fold embryos and L1 larvae after 24 h L1 starvation are shown. These animals carry transgenes that express GRL-5::VENUS fusion protein under the control of the native grl-5 promoter. Note that the fluorescence of L1 animals under the GFP filter is derived from gut granules. The presence of the transgene is marked by the co-injected plasmid that expresses mCherry in the hypodermis. Photographs were taken with the same exposure time. Scale Bars: 10 μm. (D) Percentage of starved wild-type and ptr-18 mutant L1 larvae showing GRL-5::VENUS expression. Data were collected after 24 h L1 starvation and are presented as mean ± SD. Experiments were repeated three times, and n ≥50 animals were scored for each trial.(A) Both newly hatched wild-type and (PDF)Click here for additional data file.S7 Figmir-235 and daf-16 does not affect the temporally-regulated internalization of GRL-7::mCherry::3xFLAG. GRL-7::mCherry::3xFLAG expression was assessed after 24 h L1 starvation. n ≥50 animals were scored. Experiments were repeated three times, and ≥50 animals were scored for each trial. Data are presented as mean ± SD. N.S.; Not statistically significant compared to wild type (Fisher’s exact test). (B) Loss of mir-235 in ptr-18 mutant animals enhances the quiescent defective phenotype. Data in ptr-18 mutants. The phenotype was scored after 5-day L1 starvation. Experiments were repeated ≥3 times, and ≥35 animals were scored for each experiment. Data are presented as mean ± SD. ****: p <0.0001 (Fisher’s exact test).(A) Loss of (PDF)Click here for additional data file.