Journal: PLoS ONE

Article Title: Alternative Pathway Inhibition by Exogenous Factor H Fails to Attenuate Inflammation and Vascular Leakage in Experimental Pneumococcal Sepsis in Mice

doi: 10.1371/journal.pone.0149307

Figure Lengend Snippet: Mice infected with 1x10 7 CFU of S . pneumoniae (TIGR4) and sham infected control mice, were all treated with antibiotics at t = 17 h and indicated groups received an injection with hFH or PBS as control (n = 10). At t = 26 h serum mC3a, mC3 and hFH levels were measured by ELISA. Each point depicted indicates one mouse. One infected mice of the PBS treated group reached the humane endpoint at t = 22 h and was excluded from the graphs. Two data points of mC3 are missing, as insufficient serum was available to do all the measurements in all mice. Data are individual complement factor values and were analyzed after logarithmic transformation; the horizontal line represents the median. Dash line indicates lower limit of detection. Comparison between groups were performed by using the non-parametric Mann-Whitney test with Bonferroni correction * p < 0.05 was considered significant. ** p< 0.01, *** p<0.001, ns = not significant.

Article Snippet: In addition, the following complement proteins were measured with ELISA assays; mouse C3a (mC3a) (USCN life science, E90387Mu), mouse C3 (mC3) (Mybiosource, MBS564065) and hFH (Hycult, HK342).

Techniques: Infection, Injection, Enzyme-linked Immunosorbent Assay, Transformation Assay, MANN-WHITNEY

Journal:

Article Title: Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoglobulin Loci

doi:

Figure Lengend Snippet: Survival of CBA/N mice after infection with serotype 3 S. pneumoniae. (A) Survival of mice treated with MAb doses of 10 μg; (B) survival of mice treated with mAb doses of 1 μg. The y axes show percentages of mice surviving the number of days after infection depicted on the x axes. Mice were infected as described in the text after the administration of IgM (depicted by ×), PBS (#), MAb 1.2 (■), or mAb 1.10 (○). The experiments were performed twice, and the results of one experiment are shown. In panel A, IgM (x) and PBS (#) had identical survival.

Article Snippet: Mouse inoculations were performed as follows: groups of 10 mice each received 0.5 ml (1 or 10 μg in sterile saline) of MAb 1.2, MAb 1.10, the human myeloma IgM control (Calbiochem) used as an isotype control as described elsewhere ( 46 ), or PBS intraperitoneally; 1 h later, 0.2 ml (300 CFU in tryptic soy broth) of S. pneumoniae (ATCC strain 10813) was given via intravenous inoculation into the lateral tail vein.

Techniques: Infection

Journal:

Article Title: Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

doi: 10.1128/IAI.71.5.2365-2372.2003

Figure Lengend Snippet: Bacterial species and strains used in this study and their invasion of HUVEC monolayers

Article Snippet: Streptococcus gordonii strain CH1 (Challis) was chosen as the paradigm for most of the experiments reported here because it is virulent in the rat model of infection ( 59 ), several adhesion-deficient mutants were available for comparison ( 14 , 39 , 40 ), and it produces large amounts of the alpha-hemolysin (hydrogen peroxide) ( 3 , 4 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Bacterium Description Source or reference HUVEC invasion a S. gordonii DL1 (Challis) Wild-type reference strain 52 17 ± 5 EM230 DL1 hsa :: ermAM 52 4 ± 2 OB219 DL1 sspA ′ sspB ′:: ermAM 14 20 ± 4 OB220 DL1 sspA :: ermAM 14 19 ± 6 OB235 DL1 cshA3 :: ermAM 40 2 ± 0.4 OB271 DL1 cshB2 :: ermAM 40 2 ± 1 OB277 DL1 cshA31 :: cat cshB2 :: ermAM 40 2 ± 0.6 CH1 (Challis) Wild-type reference strain 59 82 ± 15 AMS12 CH1 with 1.7-kb Hin d fragment of gtfG replaced with lacZ /Erm r 59 14 ± 0.5 M5 Wild type from human dental plaque 28 5 ± 2.5 S. sanguis L22 Wild type from human dental plaque 28 76 ± 11 L52 Wild type from human dental plaque 28 9 ± 2 L74 Wild type from human dental plaque 28 52 ± 8 L79 Wild type from human dental plaque 28 14 ± 5 133-79 Wild type from human endocarditis 28 21 ± 5 2017-78 Wild type from human endocarditis 28 25 ± 3 10556 Wild type from human endocarditis ATCC 98 ± 18 S. oralis KS32AR Wild type from dental plaque 8 9 ± 8 16532AR Wild type from dental plaque 8 3 ± 2 S. mitis NCTC 10712 Wild type from dental plaque 8 23 ± 6 OP51 Wild type from dental plaque 8 35 ± 9 S. mutans UA159 Wild type from dental plaque 60 26 ± 7 S. salivarius 13419 Wild type from human saliva ATCC 2 ± 1 E. coli HB101 Hybrid of strains K-12 and B, transformable strain 7 <0.01 Open in a separate window a Average number of intracellular bacteria (× 10 4 ) per HUVEC monolayer ± standard deviation of the mean ( n = 6). b ATCC, American Type Culture Collection.

Techniques:

Journal: Scientific reports

Article Title: Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections.

doi: 10.1038/s41598-022-15543-6

Figure Lengend Snippet: Figure 4. Diagnostic performance evaluation of the multiplex real-time RT-PCR. A, schematic representation of flow chart of the multiplex real-time PCR assay for detection of clinical specimens. After the patient’s nasopharyngeal/throat swab is taken, the nucleic acid extracted from the sample is amplified simultaneously in three tubes by the multiplex PCR assay. If the fluorescence channel of RSV in tube A shows an amplification curve and GAPDH is detected in all three tubes, a valid sample collection can be diagnostic for RSV infection in the sample. B, the number of each pathogen detected by three-tube nine-plex real-time PCR. The total number of pathogens detected in each type of infection were as follows: Single, 7; Dual, 7; Triple, 7; Quadruple, 4. LP: Legionella pneumophila, HI: Haemophilus influenzae, MP: Mycoplasma pneumoniae, CP: Chlamydia pneumoniae, SP: Streptococcus pneumoniae.

Article Snippet: Number Species Subtypes Strain ID Supplier Description 1 Influenza A virus H1N1 ATCC VR95 American Type Culture Collection 2 H3N2 GDV124 The State Key Laboratory of Virology, Wuhan University, CHINA3 H1N1 2009 GDV108 4 H5N1 The National Institute for Food and Drug Control, CHINA Supplied as virus cultures5 H7N9 6 Influenza B virus IVB Yamataga GDV105 The State Key Laboratory of Virology, Wuhan University, CHINA7 IVB Victoria GDV104 8 Respiratory syncytial virus RSV A ATCC VR1540 American Type Culture Collection 9 RSV B ATCC VR 955 10 Adenovirus Human adenovirus 1 ATCC VR-1 American Type Culture Collection 11 Human adenovirus 2 ATCC VR-846 12 Human adenovirus 3 ATCC VR-3 13 Human adenovirus 4 ATCC VR-1572 14 Human adenovirus 5 ATCC VR-5 15 Human adenovirus 46 ATCC VR-1308 16 Human adenovirus 48 ATCC VR-1406 17 Human mastadenovirus 7 ATCC VR-7 18 Human adenovirus 55 N/A The State Key Laboratory of Virology, Wuhan University, CHINA Supplied as clinical isolates 19 Chlamydia pneumoniae ATCC VR2282 American Type Culture Collection 20 Mycoplasma pneumoniae Mycoplasma pneumoniae Somerson et al ATCC 15531 21 Streptococcus pneumoniae Streptococcus pneumoniae Serotype 19F ATCC 49619 22 Streptococcus pneumoniae (Klein) Chester ATCC 700669 23 Legionella pneumoniae Legionella pneumophila subsp. fraseri Brenner et al ATCC 33156 24 Legionella pneumophila subsp. pneumophila Brenner et al ATCC 43290 25 Haemophilus influenzae Haemophilus influenzae (Lehmann and Neumann) Winslow et al ATCC 10211 12 Vol:. (1234567890) Scientific Reports | (2022) 12:13306 | https://doi.org/10.1038/s41598-022-15543-6 real-time RT-PCR assay followed the pattern of selecting conserved gene sequence regions.

Techniques: Diagnostic Assay, Multiplex Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Amplification, Fluorescence, Infection

Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 Screens for Zika and Yellow Fever Viruses Identify TMEM41B and VMP1 as Required Host Factors (A) Bubble plot of genes significantly enriched in a genome-wide CRISPR KO screen in HAP1 cells challenged with ZIKV (top) and YFV (bottom). Colors indicate association with cellular pathways or protein complexes and domains. Red lines denote Z = ± 2. (B) Heatmap of Z scores for genes in the autophagy pathway ordered sequentially by functional role: L, lipid mobilization; 1, initiation; 2, nucleation; 3, elongation; 4, sequestration; 5, tethering/fusion. Rows represent replicate screens. (C) Scatterplot of gene-wise log 2 fold change (LFC) from this study (ZIKV) versus Moretti et al. (2018) autophagy screen. (D) HAP1 WT and (n = 3) individual KO clones for VTT domain-containing proteins infected with ZIKV. (E) WT and TMEM41B KO HAP1 cells overexpressing individual VTT domain proteins infected with ZIKV. (F) Same as (E) but in VMP1 KO HAP1 cells. (G) HAP1 WT and (n = 3–5) individual KO clones for autophagy genes infected with ZIKV. (H–K) Same as (D–G) but infected with YFV Asibi. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen-positive cells. Dots in (D), (G), (H), and (K) represent the average of n = 3 replicates from individual single-cell clones. Error bars in (E), (F), (I), and (J) depict a single KO clone with standard deviation (SD) of n = 3 replicates. See also B–S1I.

Article Snippet: CRISPR/Cas9 plasmids were generated from the pDDC6 vector, which encodes the human codon-optimized Streptococcus pyogenes Cas9 ( hSpCas9 ; a gift from Peter Duchek) (Addgene plasmid: #59985; http://n2t.net/addgene:59985 ; RRID: Addgene_59985).

Techniques: Genome Wide, CRISPR, Functional Assay, Clone Assay, Infection, Flow Cytometry, Standard Deviation

Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet:

Article Snippet: CRISPR/Cas9 plasmids were generated from the pDDC6 vector, which encodes the human codon-optimized Streptococcus pyogenes Cas9 ( hSpCas9 ; a gift from Peter Duchek) (Addgene plasmid: #59985; http://n2t.net/addgene:59985 ; RRID: Addgene_59985).

Techniques: Subcloning, Western Blot, Recombinant, Infection, Transfection, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, SYBR Green Assay, Sequencing, Derivative Assay, Plasmid Preparation, Software

Journal: Cancer Discovery

Article Title: Transcriptional Silencing of ALDH2 Confers a Dependency on Fanconi Anemia Proteins in Acute Myeloid Leukemia

doi: 10.1158/2159-8290.cd-20-1542

Figure Lengend Snippet: Figure 3. Inactivation of FA genes in AML leads to p53-induced cell-cycle arrest and apoptosis. A, Representative flow cytometry analysis of BrdU incorporation and DNA content to infer cell status following lentiviral transduction of MOLM-13 cells with the indicated sgRNAs (day 6). B, Quantifica- tion of different cell-cycle stages, average of three biological replicates. Paired Student t test was applied to calculate P values. C, Representative flow cytometry analysis of DAPI (indicating permeable dead cells) and Annexin-V staining (a preapoptotic cell marker) following lentiviral transduction of MOLM-13 cells (day 6). D, Quantification of live and apoptotic cells, average of three biological replicates. Paired Student t test was applied to calculate P values. E, Gene set enrichment analysis of RNA-seq data obtained from MOLM-13 cells lentivirally transduced with the indicated sgRNAs (70). Normalized enrichment score (NES) and family-wise error rate (FWER) P value are shown. F, Western blot analysis performed on lysates obtained from MOLM-13 cells on day 6 following sgRNA transduction. G, Competition-based proliferation assays in MOLM-13 cells following sequential sgRNA transduc- tion. Negative sgRNA or TP53 sgRNAs were infected first, selected with neomycin, followed by transduction with the sgRNAs indicated at the bottom of the graph (linked with GFP). n = 3. All bar graphs represent the mean ± SEM. All sgRNA experiments were performed in Cas9-expressing cell lines.

Article Snippet: Plasmid Construction: sgRNA and shRNA Cloning For CRISPR screening, the optimized sgRNA lentiviral expression vector (LRG2.1T) and the lentiviral human codon–optimized Streptococcus pyogenes Cas9 vector (LentiV_Cas9_Puro, Addgene: 108100) were used.

Techniques: Flow Cytometry, BrdU Incorporation Assay, Transduction, Staining, Marker, RNA Sequencing, Western Blot, Infection, Expressing