Journal: Cell host & microbe

Article Title: Genetic ablation of butyrate utilization attenuates gastrointestinal Salmonella disease

doi: 10.1016/j.chom.2018.01.004

Figure Lengend Snippet: Key Resources Table

Article Snippet: Six hours after inoculation, cultures were harvested and stored at −80°C for subsequent RNA extraction. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains Ty2 S. Typhi isolate, Vi + ATCC 19430 IR715 Nalidixic acid-resistant derivative of ATCC 14028s ( Stojiljkovic et al., 1995 ) FF176 IR715 phoN::Tn10d-Cam ( Faber et al., 2016 ) AJB715 IR715 phoN ::Km R ( Kingsley et al., 2003 ) FF357 IR715 Δ ydiD This study DNB4 IR715 phoN ::Km R Δ ydiD This study RC60 IR715 phoN :: viaB fepE ::pGP704 ( Crawford et al., 2013 ) DNB3 IR715 phoN :: viaB fepE ::pGP704 Δ ydiD This study Salmonella Typhi Ty2 ATCC ATCC 19430 Salmonella Typhimurium IR715 Nalidixic acid-resistant derivative of ATCC 14028s ( Stojiljkovic et al., 1995 ) DH5α λ pir F − endA1 hsdR17 (r − m + ) supE44 thi-1 recA1 gyrA relA1 Δ( lacZYA-argF ) U189 φ80lac Δ M15 λpir ( Pal et al., 2005 ) S17-1 λ pir C600::RP4 2-(Tet:: Mu ) (Kan::Tn7) λ pir recA1 thi pro hsdR (r − m + ) ( Simon et al., 1983 ) BL21 (DE3) F- ompT hsdSB (rB-mB-) gal dcm (DE3) ( Studier and Moffatt, 1986 ) Plasmids used in this study pRDH10 ori (R6K) mobRP4 sacRB Tet R Cm R ( Kingsley et al., 1999 ) pWSK29 ori (pSC101) Carb R ( Wang and Kushner, 1991 ) pYDID pWSK29 carrying ydiD gene transcribed from its native promoter This study pET28a Expression vector with a His6-tag, Kan R Novagen pWJ19 pET28a carrying hilD gene transcribed from T7 promoter This study Critical Commercial Assays TRI-reagent Molecular Research Center cat#: RT 111 DNA-free kit Applied Biosystems cat#: AM1906 Aurum Total RNA kit BioRad cat#: 7326820 PowerSoil DNA Isolation Kit Mo-Bio cat#: 12888 Bradford assay BioRad cat#:5000006 Gel extraction kit Qiagen cat#: 28706 EMSA kit Invitrogen cat#: E33075 Lipocalin-2 ELISA R&D systems cat#: DY1857 Experimental Models: Cell Lines T84 colonic carcinoma cell ATCC cat#: CCL-248 RRID: CVCL_0555 Experimental Models: Organisms/Strains Mus musculus CBA/J Jackson Labs cat#: 000656 RRID: IMSD JAX:000656 Mus musculus Gnotobiotic Swiss Webster Bred in-house; originally acquired from Taconic cat#: SW-F and SW-M Oligonucleotides Primers used in this study, see Table S1 This paper N/A Software and Algorithms Prims v7.0a GraphPad N/A Open in a separate window

Techniques: Virus, Expressing, Plasmid Preparation, DNA Extraction, Bradford Assay, Gel Extraction, Enzyme-linked Immunosorbent Assay, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a-c , Ifnar-/- mice (n = 4 per time point) were subcutaneously infected with 1000 p.f.u. of ZIKV and euthanized at 2, 3, and 4 DPI. The brains were subjected to an in-situ RNA hybridization (RNAScope) assay with a probe detecting ZIKV RNAs. Positive signals for the target RNA are shown in dark brown, whereas the counterstaining with hematoxylin is shown in light blue. Images taken with a 20X objective were stitched with Hugin panorama image stitch software. Black, gray, and white arrowheads indicate virus-infected cells, virus, and the absence of ZIKV-specific staining, respectively. Detailed images of the areas outlined with a black box are shown in ai, bi, and ci (cortex and capillaries) and aii, bii, and cii (choroid plexus). Scale bars = 200 μm. d . AG129 mice (n = 3 per group, two experiments) were subcutaneously infected with 1000 p.f.u. of TC-83 and euthanized at 2 days post infection. The TC-83 vRNA was visualized in an RNAScope assay with a VEEV-specific probe. Scale bars = 200 μm. e , Ifnar-/- mice (left, n = 4–6/group) or IFNAR +/- mice (right, the cage mate n = 4) were infected with 1000 p.f.u. of ZIKV subcutaneously, and the brains were harvested at 3 and 4 DPI. The viral loads in the cortex (blue circles), meninges (red squares), and choroid plexuses (green triangles) were determined with a ZIKV-specific qRT-PCR assay and normalized to the expression of GAPDH mRNA in each sample. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison tests. * P = 0.003, ** P = 0.0001.

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Infection, In Situ, Hybridization, RNAscope, Software, Virus, Staining, Quantitative RT-PCR, Expressing, Comparison

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a , Ifnar-/- mice (n = 6–8/time point from two independent experiments) were infected with ZIKV at the footpad as above. The serum and CSF were harvested at 2,3 and 4 DPI as described in the Methods section. The virus titers in the serum (blue circles) and in the CSF (red squares) were measured with a standard focus-forming assay in Vero 76 cells. b-d , Anti-ZIKV IgM or isotype control IgM was administered intrathecally to ZIKV-infected mice at 2 or 3 DPI (n = 6 per group per time point from two independent experiments). The brains were harvested at 6 DPI. b . The viral loads in the right hemisphere of the brains from the isotype control group (anti-fluorescein IgM, blue circles) and Anti-ZIKV IgM (red squares) were determined. c . A representative image of the RNAScope assay for the left hemisphere of the brains (n = 4 per group). d . Clinical scores (0 to 4, normal to severe) of the tested mice at 6 DPI. NEU: neurological symptoms; GRO: grooming; GRI: grimace scale; Act: Activity. n.d.: Not detected.

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Infection, Virus, Focus Forming Assay, Control, RNAscope, Activity Assay

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a, The lateral choroid plexuses from ZIKV-infected Ifnar-/- mice (n = 4 per time point) were harvested at 4 DPI and subjected to whole mount IFA assay. The whole tissue was stained with anti-CD31 antibody (endothelial cells, green), hu-4G2 (ZIKV envelope, red), and Hoechst 33342 (nuclei, blue) and then mounted for evaluation by confocal microscopy Scale bar = 100 μm. ai , A higher resolution image of the inset area of Fig 3 a . Scale bars = 20 μm. b , A representative image of ZIKV-infected cells tightly connected with endothelial cells. Scale bars = 20 μm. c , The lateral choroid plexus of AG129 mice infected with ZIKV (1000 p.f.u. at 4 DPI) were stained with FITC-IB4 (endothelial layer, green), hu-4G2 (ZIKV envelope, red), and Hoechst 33342 (nuclei, blue). A total of 74 images taken with a 20X objective were stitched together with Hugin image stitch software. Scale bar = 500 μm.

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Infection, Staining, Confocal Microscopy, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a-d , Ifnar-/- mice were infected with ZIKV as above, and the choroid plexuses were isolated at 4 DPI. a , Choroid plexuses were stained with antibodies against TTR (choroid plexus epithelial cell maker, green) and ZIKV-E protein (red). left : three layers of confocal images (labeled with Top, Middle, and Bottom) along the Z-axis. right : a three-dimensional reconstruction of the z-stacked image, highlighting the ZIKV-infected cells in the stroma layer of the choroid plexus. The top, middle, and bottom layers showed TTR+/ZIKV-, TTR-/ZIKV+, and TTR+/ZIKV-, respectively. A representative image is shown. b , Choroid plexuses were costained with FITC-IB4 for capillaries (green), anti-PDGFβR antibody for pericytes (purple), and anti-ZIKV-E antibody for the Zika envelope protein (red). Nuclei were stained with Hoechst 3342 stain (blue). The ZIKV-E signal colocalized with PDGFβR. For a and b , scale bars = 20 μm. c-d , ChP (n = 6, two from three mice) were harvested following cardiac perfusion and stained with anti-PDGFβR antibody for pericytes, anti-ZIKV-E antibody for the Zika envelope protein, and Hoechst 33342 for nuclei. (See for detail). The percentage of ZIKV-infected cells in the total ChP stromal cells(c) and PDGFβR (+) populations in the ZIKV (+) cells (d) were enumerated by using Imaris image analysis software. Each symbol represents an individual choroid plexus** P <0.01 (Student’s t-test). e , An electron micrograph showing a pericyte infected with ZIKV on a choroid plexus capillary. Pseudocolors (green and blue) indicate the cytoplasm and the nucleus, respectively, of a choroid plexus endothelial cell (EC) facing the lumen of a capillary (LUMEN). Patches of electron-dense particles (arrowheads) were found in a pericyte next to the EC. f , A higher magnification of the image from e shows that the particles have characteristics typical of flavivirus nucleocapsids. fi , A higher magnification image of the inset area of Fig e , Scale bars: 800 nm in e and 100 nm in f and fi .

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Infection, Isolation, Staining, Labeling, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a , ZIKV replication in primary pericytes from mouse choroid plexus (IFNAR +/- ). Cells (three independent wells) were infected with ZIKV, PLCal_ZV (MOI = 0.1) and grown in the presence of 5 μg/mL mouse anti-IFNAR-1 neutralizing antibody (clone MAR 5A3, ɑ-IFNAR) or isotype control antibody (clone MOPC-21). The cell culture supernatant was harvested at 5 DPI, and the virus titer was enumerated. * P <0.05 (Student’s t-test) b , Primary human brain vascular pericytes (five independent wells) were infected with ZIKV as in a and then incubated in the presence/absence of a human type 1 IFN neutralizing antibody mixture (ɑ-IFN Aby). The supernatants were harvested every 24 hours for five days, and the virus titers were enumerated with a virus titration assay. ** P < 0.005 (two-way ANOVA). c , Primary human brain vascular pericytes were infected with ZIKV and ZIKV-infected cells were enumerated by using FACS with anti ZIKV-E (clone 4G2). Each dot represents cells from single well. d , Mock- or ZIKV-infected HBVP were stained with antibodies against PDGFβR (red) and ZIKV-E protein (green). Two consecutive confocal layers were projected into a single layer with the Z-project function of ImageJ software (version 2.0.0). e , HBVP infected with ZIKV were stained with antibodies against AXL (green), PDGFβR (magenta) and ZIKV-E protein (red). Scale bars = 20 μm. f and g , HBVP were pre-treated with antibody for three hours then infected with ZIKV (m.o.i. = 1). Three days later, viral RNA ( f ) and progeny virus titers in the supernatants ( g ) were analyzed. **** P < 0.001 (one-way ANOVA).

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Infection, Control, Cell Culture, Virus, Incubation, Titration, Staining, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a , CPs isolated from ZIKV-infected mice were subjected to immunostaining in a whole-mount choroid plexus assay with antibodies against ZO-1 and ZIKV-E. The top and stroma cell layer was imaged with a Zeiss LSM 710 Duo/LIVE 5 confocal microscope along the Z-axis, and the maximum Z-project image was created with Imaris software. b , HIBCPP Transwell model. HIBCPP cells were grown and differentiated into a monolayer in Transwell inserts. Fully developed HIBCPP Transwell inserts (determined by TEER) were placed in wells with various testing conditions (e.g., primary human brain pericytes or conditioned media). c . Fully developed HIBCPP barriers grown in Transwell inserts (n = 3–5 per group) were basolaterally exposed to ZIKV-infected HBVP cells (starting at 1 DPI). As a leakage control, a scratch was made on the HIBCPP cells fully grown in Transwell inserts. Samples from the apical and basolateral chambers were harvested every 24 hours, and the virus titers were determined. d , Fully developed HIBCPP barriers grown in Transwell inserts (n = 4–5 per group) were basolaterally exposed to ZIKV-infected HBVP cells (3 d.p.i) or fresh media spiked with ZIKV (1.0 x 10 5 pfu/well/mL). Twenty-four hours later, the cell supernatant in the apical camber was harvested and the virus amount was enumerated. e , HBVP (blue circles) and HIBCPP (red squares) cells grown on 12-well plates were infected with ZIKV (m.o.i. = 0.1) and then incubated for two days. The virus titers in the supernatants were enumerated with a virus titration assay. f , Expression of Axl and Tyro3 in HBVP and HIBCPP was determined by using realtime q-PCR and normalized to endogenous GAPDH expression.

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Isolation, Infection, Immunostaining, Microscopy, Software, Control, Virus, Incubation, Titration, Expressing

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a-d , Fully developed HIBCPP barriers grown in Transwell inserts (n = 3–4 per group) were basolaterally treated with HBVP co-culture, UV-treated conditioned media from HBVP, or UV-treated cell supernatant from ZIKV-infected HBVP, and normal culture medium. The barrier functions were measured by TEER daily (a), and, at day 3 post treatment, FITC-dextran permeability ( b ) was measured. The transwells were stained with an anti-ZO-1 antibody ( c , a representative image). Cells with cytosolic ZO-1 stain were counted (white arrow heads) and the percentage of the cells were analyzed ( d ). * P = 0.033 (one-way ANOVA); *** P = 0.0002, **** P < 0.0001 (two-way ANOVA).

Article Snippet: The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains PLCal_ZV (Human/2013/Thailand; GenBank: KF993678) and DAK AR 41524 (Mosquito/1984/Senegal; GenBank: KX198134.2).

Techniques: Co-Culture Assay, Infection, Permeability, Staining