Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication

doi: 10.1371/journal.ppat.1010113

Figure Lengend Snippet: (A-D) Representative images from transmission electron microscope of TGEV (MOI = 0.1, 24 h) induces membrane structures. There are typical DMVs (diameter ~200 nm) were observed ( A and B ). Electron micrograph of an area with abundant double-membrane spherule (DMSs) ( C ) and irregular coiled membrane formed by ERs expansion ( D ). Scale bar, 1μm or 500 nm. (E-F) Electron micrograph showing mock-infected and TGEV-infected (MOI = 0.1, 24 h) TMEM41B KO cells bearing several monolayer vesicles. Scale bar, 500 nm or 250 nm. (G) Confocal fluorescence microscopy analysis of the co-localization of TGEV-NSP3-HA (indicated in red) and restored expression of TMEM41B-FLAG (indicated in fuchsia) in TMEM41B KO cells. Scale bar, 5 μm. (H) Analysis of the subcellular location of ectopic expression TMEM41B in KO cells mock-infected (above) or infected with TGEV (MOI = 1, 12 h) (bottom). The restored expression of TMEM41B (indicated in red) were co-localization with dsRNA (indicated in green). Scale bar, 5 μm. (I) Assessment of the co-localization of dsRNA (indicated in green) and ectopic expression of TMEM41B-FLAG (indicated in red) in L929 cells during MHV (MOI = 1, 12 h) infection via confocal fluorescence microscopy. Scale bar, 5 μm. (J) A model for the role of TMEM41B in the formation of coronavirus replication organelles. WT, wild-type; KO, knock out; Mock, uninfected cells; TGEV, Transmissible gastroenteritis virus infected cells; MHV, Mouse hepatitis virus infected cells; M, Mitochondria; ER, Endoplasmic reticulum; DAPI, 4’,6-diamidino-2-phenylindole; HA, HA-Tag; FALG, 3×FLAG-Tag; dsRNA, double-stranded RNA.

Article Snippet: ST (CRL-1746) and L929 (CCL-1) cell lines were purchased from ATCC (USA).

Techniques: Transmission Assay, Microscopy, Membrane, Infection, Fluorescence, Expressing, Knock-Out, Virus

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication

doi: 10.1371/journal.ppat.1010113

Figure Lengend Snippet: (A) Alignment of TMEM41B amino acid sequences from pigs, human and mice. The accession Numbers of TMEM41B from NCBI protein database for Sus scrofa is XP_003129438.1 (pTMEM41B), for Homo sapiens is NP_055827.1 (hTMEM41B), and for Mus musculus is NP_705745.3 (mTMEM41B). Protein sequences were aligned using Constraint-based Multiple Alignment Tool (COBALT). The dark star indicates the regions of sequence conservation amongst the three species (letters indicated in red); the blue letters indicate the regions with sequence variation among the three species; the box indicates the VTT domain. (B) Rescue assays for WT, TMEM41B-KO and TMEM41B-KO-rescue L929 cells infected with MHV (MOI = 0.1, 24 hpi). Cell supernatant was collected and assessed for MHV titers using TCID 50 . (C-F) Rescue assays for WT, TMEM41B-KO and TMEM41B-KO-rescue PK-15 cells infected with (C) PDCoV (MOI = 0.01, 18 hpi), (D) JEV (MOI = 0.1, 24hpi), (E) IAV-PR8 (MOI = 0.01, 24 hpi) and (F) VSV (MOI = 0.01, 18 hpi). The cell supernatant was collected and tittered using TCID 50 assays and immunofluorescence assays to detect NP positive cells. (G) Viral titers for TMEM41B-KO, TMEM41B/IFNAR double KO and TMEM41B/IFNAR double KO-TMEM41B-rescue cells infected with VSV (MOI = 0.1, 18 hpi) and cell supernatants was collected and titrated using TCID 50 . (H) Immunofluorescence assays for TMEM41B-KO, TMEM41B/IFNAR double KO and TMEM41B/IFNAR double KO-TMEM41B-rescue cells infected with IAV (MOI = 0.1, 18 hpi) and detection the NP positive cells. WT, wild-type; KO, knockout; hpi, hours post-infection; MHV, Mouse hepatitis virus; PDCoV, porcine deltacoronavirus; JEV, Japanese encephalitis virus; IAV-PR8, PR8 influenza A virus; VSV, Vesicular stomatitis virus. ** P < 0.01; *** P < 0.001; **** P <0.0001. P values were determined by two-sided Student’s t-test. Data are representative of at least three independent experiments.

Article Snippet: ST (CRL-1746) and L929 (CCL-1) cell lines were purchased from ATCC (USA).

Techniques: Sequencing, Infection, Immunofluorescence, Knock-Out, Virus

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication

doi: 10.1371/journal.ppat.1010113

Figure Lengend Snippet: (A) Line graphs showing the body weight of TMEM41B +/− and WT mice during the 3 days post-inoculation with MHV A59 (n = 6 mice per genotype). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significant. P -values were determined by two-way measure. (B) The total weight change of TMEM41B +/− and WT mice during the 3 days post-inoculation of MHV A59 (n = 6 mice per genotype). ** P < 0.01; ns, no significant. P -values were determined by one-way measure. (C) Survival curves for TMEM41B +/− and WT mice infected with MHV A59 (n = 6 mice per genotype). (D and E) Histopathological analysis of the degree of liver damage in TMEM41B +/− and WT mice infection with MHV A59 at 3 dpi. (D) Gross postmortem examination of liver tissue; (E) histological examination of Hematoxylin-eosin (H&E) stained liver tissue. (F) The virus titers of half livers of TMEM41B +/− and WT mice were measured TCID 50 assay with L929 cells (n = 3 mice per genotype). ** P < 0.01. P values were determined by two-sided Student’s t-test. (G) A model illustrating the roles of TMEM41B in the CoV replication cycle. In WT cells, CoV binds to its receptor and enters through clathrin- and caveolin-mediated endocytosis before releasing their genome into the cytoplasm after membrane fusion in the early/late endosome. Subsequently, CoVs Nsps (NSP3, NSP4, and NSP6) act with TMEM41B and other transmembrane host proteins in the formation of ROs. In TMEM41B KO cells, endocytosis is impaired, and the internalization of virions was reduced. Moreover, the formation of DMVs was blocked during membrane elongation leading to the inhibition of TGEV replication so that almost no virions were produced. DMVs, double-membrane vesicles; ER, Endoplasmic reticulum; Mock, uninfected mice; MHV-A549, A549 Mouse hepatitis virus infected mice. WT, wild-type; KO, knockout; TMEM41B +/− mice used in Fig 7 were the F2 generation. Data are representative of at least three independent experiments. Source data are provided as a Source Data file. The experiments were repeated three times with similar results and representative results shown. Scale bar, 200 nm.

Article Snippet: ST (CRL-1746) and L929 (CCL-1) cell lines were purchased from ATCC (USA).

Techniques: Infection, Staining, Virus, Membrane, Inhibition, Produced, Knock-Out

Journal: Molecules

Article Title: Ligand-Targeted Delivery of Photosensitizers for Cancer Treatment

doi: 10.3390/molecules25225317

Figure Lengend Snippet: Examples of ligand-targeted lipid-based NPs for photodynamic therapy (PDT).

Article Snippet: Liposomes , Erythrosine-decyl ester , Biotin , Biotin receptor , _ , In vitro: ATCC ® CCL1.3™ cells , [ ] .

Techniques: In Vitro, In Vivo, Modification