Journal: Journal of Clinical Microbiology

Article Title: Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci

doi: 10.1128/JCM.00744-15

Figure Lengend Snippet: Reference PMEN ^a genomes and cps locus sequences used in this study

Article Snippet: PMEN17 , Maryland 6B -17 , ST384 , ATCC BAA342; ENA , 1997 , Invasive disease isolate, United States , 10608770.

Techniques: Isolation

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured ex vivo with 5 μg heat-killed S. pneumoniae (Sp); cell culture supernatants were collected after two, 18, and 24 hours had elapsed. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (2A) AUD subjects exhibited higher secretion of IFNγ, but this did not achieve statistical significance (p=0.10). IFNγ quantity increased significantly over time for both subject types (p<0.0001). (2B) No significant differences in secretion of IL-1β were observed between different subject types, however, secretion of IL-1β increased over time (p=0.0003). (2C) Secretion of IL-6 by AMs from subjects with AUDs tended to be higher (p=0.12). Secretion of IL-6 increased significantly over time in each subject group (p<0.0001). (2D) Secretion of TNFα did not differ between groups (p=0.26), but did increase in each group of subjects over time (p<0.0001).

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture, Ex Vivo

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured in the presence of heat-killed S. pneumoniae protein, at doses ranging from 0 μg to 10 μg. Cell culture supernatants were collected at 18 hours. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (3A) IFNγ secretion by AMs rose with exposure to increasing doses of pneumococcal protein (p=0.0002). IFNγ secretion was more elevated in the AUD group (p=0.008). (3B) IL-1β secretion by AMs rose with increasing doses of pneumococcal protein (p=0.02), but was not different between non-AUD and AUD groups. (3C) IL-6 secretion rose with increasing pneumococcal protein doses (p<0.0001), but was not different between non-AUD and AUD groups. (3D) TNFα secretion rose with increasing pneumococcal protein doses (p<0.0001), but was not different between non-AUD and AUD groups.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=15) and AUD subjects (n=15) were cultured up to 42 hours, with and without the addition of heat-killed S. pneumoniae (Sp, 10μg). In a subset of wells, N-acetylcysteine (NAC) was added after 18 hours of exposure to S. pneumoniae. Cell culture supernatants were collected at the 18 hour and 42 hour time points for analysis of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (4A) At the 18 hour time point, unstimulated AUD subjects’ AMs secreted more IFNγ (p=0.02) (denoted with asterisk). With pneumococcal protein stimulation, IFNγ secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.002). The addition of NAC at 18 hours during the 42-hour time course was associated with less IFNγ in cell culture supernatants compared to non-NAC treated AMs at 42 hours (p<0.0001 for both non-AUD and AUD subjects). (4B) After pneumococcal protein stimulation, IL-1β secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.0004). Compared to cell culture supernatants at 42 hours without NAC, the addition of NAC at 18 hours during the 42-hour time course was associated with a non-significant rise in supernatant IL-1β among non-AUD subjects (p=0.06), and significantly higher IL-1β in AUD subjects (p=0.009). Cell culture supernatants from pneumococcal protein-stimulated, NAC-treated AMs at 42 hours compared supernatants from pneumococcal protein-stimulated AMs at 18 hours contained IL-1β values that were higher both in non-AUD (p=0.003) and AUD subjects (p=0.01). (4C) At the 18 hour time point, unstimulated AUD subjects’ AMs secreted more IL-6 (p=0.03) (denoted with asterisk). With pneumococcal protein stimulation, IL-6 secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.009). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-6 secretion than that by non-NAC treated AMs at 42 hours (p=0.0001 for both non-AUD and AUD subjects). Supernatant IL-6 quantities at the 18 hour time point were significantly higher than those in supernatants from pneumococcal protein-stimulated, NAC-treated AMs in both subject types (p=0.05 for non-AUD, p=0.0001 for AUD). (4D) With pneumococcal protein stimulation, TNFα secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.004). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less TNFα in supernatants than what was measured in non-NAC treated AM culture supernatants at 42 hours (p=0.0001 for both non-AUD and AUD subjects), with values that were significantly less than AM culture supernatants at the 18 hour time point (p=0.02 for non-AUD, and p=0.0006 for AUD). * indicates p≤0.03 between non-AUD and AUD subjects, without pneumococcal stimulation, at 18 hours in culture. # indicates p<0.0001 between NAC and non-NAC treated conditions at 42 hours. & indicates p≤0.05 between non-NAC-treated 18-hour condition and NAC-treated, 42-hour condition.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=8) and AUD subjects (n=8) were cultured in the presence of 5 μg heat-killed S. pneumoniae (Sp); cell culture supernatants were collected at two, 18, and 24 hours. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (5A) For IFNγ, AUD subjects exhibited non-significantly higher secretion (p=0.10). Values increased significantly over time (p<0.0001). (5B) For IL-1β, no significant between-groups differences in secretion were observed (p=0.97), however, secretion increased over time (p=0.002). (5C) For IL-6, secretion values from AUD PBMCs did not differ between groups (p=0.35), but secretion increased over time (p=0.003). (5D) For TNFα, values did not differ between groups (p=0.54), and but did increase in subjects over time (p=0.007).

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=8) and AUD subjects (n=8) were cultured in the presence of heat-killed S. pneumoniae (Sp), at doses ranging from 0 μg to 10 μg. Cell culture supernatants were collected at 18 hours, and interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured. (6A) IFNγ secretion over the dose range increased with increasing doses of pneumococcal protein (p=0.04), but values between groups did not differ. (6B) IL-1β secretion increased with increasing pneumococcal protein doses (p=0.004), but were not different between non-AUD and AUD groups. (6C). IL-6 secretion increased with increasing pneumococcal protein doses (p=0.004), but were not different between non-AUD and AUD groups, except at the 0μg (media only) condition (p=0.04, asterisk). (6D) TNFα secretion increased with increasing pneumococcal protein doses (p=0.001), but were not different between non-AUD and AUD groups.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=10) and AUD subjects (n=10) were cultured up to 42 hours, with and without the addition of 10 μg heat-killed S. pneumoniae (Sp). In some wells, N-acetylcysteine (NAC) was added after 18 hours in culture. Culture media was collected at the 18 hour and 42 hour time points for analysis of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (7A) With pneumococcal protein stimulation, IFNγ secretion by PBMCs rose significantly between the 18 and 42 hour time points (p=0.04). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IFNγ secretion than that by non-NAC treated PBMCs (p=0.002 for both non-AUD and AUD subjects), with values that were significantly different than PBMCs at the 18 hour time point in non-AUD subjects only (p=0.02). (7B) With pneumococcal protein stimulation, IL-1β secretion by PBMCs did not rise significantly between the 18 and 42 hour time points (p=0.92). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-1β secretion than that by non-NAC treated PBMCs at the 42 hour time point (p=0.002 for non-AUD subjects; p=0.02 for AUD subjects). Values among NAC-treated cells at the 42 hour time point approximated those in untreated cells at the 18 hour time point (p=ns). (7C) With pneumococcal protein stimulation, IL-6 secretion by PBMCs tended to rise between the 18 hour and 42 hour time points, but not significantly (p=0.09). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-6 in cell culture supernatants than that found in supernatants from non-NAC treated PBMCs at the 42 hour time point (p=0.002 for both non-AUD and AUD subjects); values were not significantly different than PBMCs at the 18 hour time point for both subject types. (7D) With pneumococcal protein stimulation, TNFα secretion by PBMCs did not rise significantly between the 18 and 42 hour time points (p=0.44). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less TNFα in cell culture supernatants than in non-NAC treated PBMCs (p=0.002 for both non-AUD and AUD subjects); TNFα quantity in cell culture supernatant from pneumococcal protein -stimulated, NAC treated cells at 42 hours were significantly less than pneumococcal protein-stimulated PBMCs at the 18 hour time point among non-AUD subjects only (p=0.002). # indicates p≤0.02 between NAC and non-NAC treated conditions. & indicates p≤0.02 between 18-hour condition and NAC-treated, 42-hour condition.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Utility of MALDI-TOF MS as a new tool for Streptococcus pneumoniae serotyping

doi: 10.1371/journal.pone.0212022

Figure Lengend Snippet: Serotypes and sequence types of the isolates used in this study.

Article Snippet: Besides, 5 reference strains of serotype 6B (ATCC 700670, ATCC 700675 ATCC BAA-342, ATCC BAA-658, ATCC 700903), 3 of serotype 19A (ATCC 700678, ATCC 700904, ATCC 700674), and one each of serotypes 19F (ATCC 700905) and of serotype 35B (Statens Serum Institute serotype 35B) were also included [ ].

Techniques: Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter

doi: 10.1111/j.1582-4934.2008.00621.x

Figure Lengend Snippet: DNase-chip detects cell-type-specific DHS 10 kb into the first intron of CFTR . ( A ) Shown are DHS tracks in four different cell types: CFTR - primary human skin fibroblasts, CFTR + human bronchial epithelial cell line 16HBE14o- and CFTR + human colon carcinoma cell lines Caco2 and HT29. Significant peaks representing DHS are apparent in the 5′ promoter region of CFTR + cells, whereas Caco2 and HT29 cells display another significant DHS within the first intron. Peak height is a measurement of –log10 ( P -value) between 0 and 16, as determined by ACME (see Methods). The sequence associated with the identified intronic DHS is shown in panel B . Highlighted sequence corresponds to previously identified positive regulatory element 7/8 . Sequence is numbered based on GenBank reference sequence AC000111. (Sequence corresponds to bases 116916908–116918007 of human genome build 17.) ( C ) CFTR expression in cell types used in this study measured by RT-PCR analysis compared with a β-glucocerebrosidase housekeeping gene control.

Article Snippet: Primary skin fibroblasts (ATCC: GM08333) were grown in MEM (Gibco) supplemented with 15% FBS.

Techniques: Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter

doi: 10.1111/j.1582-4934.2008.00621.x

Figure Lengend Snippet: The 7/8 enhancer directly interacts with the CFTR promoter. Chromosome conformation analysis of CFTR + Caco2 ( A ), CFTR + 16HBE14o- ( B ) and CFTR - primary skin fibroblasts ( C ) show higher interaction of the first intron with the promoter in cells with the intron 1 DHS. Cross-linked chro-matin from each cell type was digested with Hind III (sites indicated by dashed lines) and re-ligated. The interaction frequency between a fixed Hind III fragment at the CFTR promoter and Hind III fragments 5′ and within the first, third and seventh introns was measured by Taqman® quantitative PCR. Each reaction was normalized to a control region in the ERCC3gene, as previously described . The amplification efficiencies of all primer sets were verified using Hind III-digested CFTR and ERCC3 BACs. Each experiment was repeated at least twice; a single representative experiment is shown with PCR reactions performed in triplicate. Error bars represent standard error of the mean ( n = 3).

Article Snippet: Primary skin fibroblasts (ATCC: GM08333) were grown in MEM (Gibco) supplemented with 15% FBS.

Techniques: Real-time Polymerase Chain Reaction, Control, Amplification

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