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: eLife

Article Title: The rosetteless gene controls development in the choanoflagellate S. rosetta

doi: 10.7554/eLife.04070

Figure Lengend Snippet: S. rosetta cells are polarized, each having a single apical flagellum encircled by a collar of microvilli (bracket), shown in cross-sectional diagram ( A ) and through DIC imaging of a live cell ( B ). In rosette colonies ( C ), each cell is oriented around a central point, with the flagella facing outward. Bacterial prey (∼1 µm rods) attach transiently to the collars of some cells prior to ingestion by phagocytosis. Scale bar = 10 µm. ( D ) S. rosetta transitions between several morphologically differentiated cell types during its life history: rosette colonies (RC), chain colonies (CC), slow swimmers (SS), fast swimmers (FS), and thecate cells (TC). The transition from slow swimmers to rosette colonies (star) is induced by lipids from the bacterium Algoriphagus machipongonensis and can be regulated in the laboratory. ( E ) S. rosetta undergoes a sexual cycle in the laboratory. When starved, haploid cultures produce anisogamous gametes that are capable of mating to produce diploids. Diploids undergo meiosis and thereby produce haploids when grown in nutrient-rich media. Haploids and diploids can also reproduce asexually through mitosis. DOI: http://dx.doi.org/10.7554/eLife.04070.003

Article Snippet: A. machipongonensis conditioned media (ACM) was made from the sterile supernatant of the liquid A. machipongonensis culture (ATCC BAA-2233 [ ]) grown shaking for 48 hr in HN media at 30°C to an OD 600 of 0.30–0.39 and filtered through a 0.2 μm filter to remove bacterial cells and detritus.

Techniques: Imaging

Journal: eLife

Article Title: The rosetteless gene controls development in the choanoflagellate S. rosetta

doi: 10.7554/eLife.04070

Figure Lengend Snippet: ( A ) Vertical scatter plot showing the effect of mutagenesis on cell number, shown as the number of mutagenized cells divided by the number of cells in a paired, unmutagenized culture at 24 hr post-mutagenesis. Each dot represents one mutagenesis experiment and the mutagenesis from which the Rosetteless mutant was isolated is highlighted (red). The dotted line at 1.0 represents no effect. The mutagen doses used in the screen were 0.3% EMS (vol/vol) and 6300 rem X-rays, which each resulted, on an average, in a decrease in cell number (p < 0.05, Wilcoxon signed rank test). ( B ) Growth curve of wild-type (open circles, dotted line) and Rosetteless mutant (filled circles, solid line) cells shows that the Rosetteless phenotype is not due to a growth defect. Error bars show standard deviation. ( C ) Quantification of rosette induction in the presence of live A. machipongonensis , shown as mean ± SEM. Ø represents cultures in which no rosettes were observed (limit of detection = 0.03%). Compare to to see rosette induction from A. machipongonensis conditioned media (ACM) rather than live bacteria. Notably, the insensate mutant shows a low level of rosette induction when exposed to live bacteria but not when exposed to ACM. ( D ) Single confocal slices through rosettes stained with FM 1–43X dye showed differences in cell packing within wild-type and uptight mutant rosettes. Bottom: inverted images were false colored to mark the space between cells in the center of the rosette. Scale bar = 5 µm. ( E ) Phenotypes of rosette defect mutants after vigorous pipetting. Rosettes (red arrowheads) form in the presence of ACM and are robust to pipetting, whereas chain colonies break up into single cells. The uptight mutant occasionally formed rosettes, but none were visible in this field of view. Scale bar = 20 µm. DOI: http://dx.doi.org/10.7554/eLife.04070.007

Article Snippet: A. machipongonensis conditioned media (ACM) was made from the sterile supernatant of the liquid A. machipongonensis culture (ATCC BAA-2233 [ ]) grown shaking for 48 hr in HN media at 30°C to an OD 600 of 0.30–0.39 and filtered through a 0.2 μm filter to remove bacterial cells and detritus.

Techniques: Mutagenesis, Isolation, Standard Deviation, Bacteria, Staining

Journal: eLife

Article Title: The rosetteless gene controls development in the choanoflagellate S. rosetta

doi: 10.7554/eLife.04070

Figure Lengend Snippet: S. rosetta chain colonies from wild-type and mutant cultures grown without exposure to A. machipongonensis signals are shown at low magnification to document the morphologies of numerous chain colonies. Each phase-bright circle is a choanoflagellate cell, while the dark specks are bacteria. To the right of each image, the cells of each in focus chain colony have been false colored blue to identify chains and emphasize chain morphology. Three mutants (Rosetteless, Insensate, and Slacker) exhibit essentially wild-type chains. Solo is largely single-celled and rarely forms chains. The five remaining mutants all form chains that are more branched or highly clustered than wild-type chains. Scale bar = 50 µm. DOI: http://dx.doi.org/10.7554/eLife.04070.008

Article Snippet: A. machipongonensis conditioned media (ACM) was made from the sterile supernatant of the liquid A. machipongonensis culture (ATCC BAA-2233 [ ]) grown shaking for 48 hr in HN media at 30°C to an OD 600 of 0.30–0.39 and filtered through a 0.2 μm filter to remove bacterial cells and detritus.

Techniques: Mutagenesis, Bacteria

Journal: eLife

Article Title: The rosetteless gene controls development in the choanoflagellate S. rosetta

doi: 10.7554/eLife.04070

Figure Lengend Snippet: ( A ) The rtls gene (top) contains 12 exons (numbered) and encodes a protein (bottom) with an amino-terminal signal peptide (green), two C-type lectin-like domains (CTLDs), extended stretches of serines and threonines (wavy lines), and two internal repeats of unknown function (RP1 and RP2). The rtls l1 SNV interrupts a splice donor in intron 7 (GT → GC). The epitope used to generate the anti-Rtls antibody is shown (orange bracket). ( B ) An alignment of Rtls CTLDs with CTLDs from rat surfactant protein A (rat SP-A, 1R13_A) and rat mannose-binding protein (rat MBP, 2MSB_A) revealed that residues used in disulfide bonds (blue), mannose-type sugar binding (red), and calcium ion binding (*) are conserved. Other conserved or similar residues are highlighted in gray. ( C ) Alignment of the RP1 and RP2 regions. ( D ) RT-PCR of rtls with primers to the exon 5/6 junction and exon 12 showed that wild-type cells produce a single isoform while Rosetteless cells produce diverse splice isoforms. ( E ) Wild-type cDNA yielded the expected splice isoform (i) while Rosetteless mutant cDNA yielded isoforms with: (ii) intron 7 retention or (iii–iv) variants of exon 7 that were longer (*) or shorter (**) than wild type. Isoforms ii and iv contained early stop codons (arrows). ( F ) Semi-quantitative analysis of the fluorescent signal observed in Rtls dot blots, normalized to the intensity of the wild-type culture (WT). Rosetteless mutant cells ( rtls l1 ) showed reduced Rtls signal both with and without A. machipongonensis ( Alg ) relative to WT . Error bars show standard deviation. DOI: http://dx.doi.org/10.7554/eLife.04070.014

Article Snippet: A. machipongonensis conditioned media (ACM) was made from the sterile supernatant of the liquid A. machipongonensis culture (ATCC BAA-2233 [ ]) grown shaking for 48 hr in HN media at 30°C to an OD 600 of 0.30–0.39 and filtered through a 0.2 μm filter to remove bacterial cells and detritus.

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Standard Deviation

Journal: eLife

Article Title: The rosetteless gene controls development in the choanoflagellate S. rosetta

doi: 10.7554/eLife.04070

Figure Lengend Snippet: ( A ) RT-PCR with a primer bridging the exon 7/8 boundary of rtls paired with a primer in exon 12, amplified the wild-type rtls splice isoform from wild-type and rtls l1 cDNA. This was in contrast with the diverse alternative rtls splice isoforms amplified from Rosetteless cells when RT-PCR was performed with a primer bridging the exon 5/6 junction and a primer in exon 12 . ( B ) Purified, recombinant protein corresponding to the anti-Rtls epitope has a predicted size of approximately 38 kDa (arrowhead). The purity of 100 ng of recombinant protein was analyzed by silver stain (left) and by western blot with anti-Rtls (right) on two separate 4–12% gradient gels. ( C ) Raw data showing the validation of anti-Rtls on dot blots of wild-type cell lysates. Pre-incubation of anti-Rtls with the recombinant Rtls epitope competes away the staining, demonstrating that the majority of the signal is specific to the Rtls protein. Three replicate samples are shown. ( D ) Raw dot blot data showing levels of Rtls in wild-type (WT) or Rosetteless mutant ( rtls l1 ) cultures with or without inoculation with A. machipongonensis ( Alg .). Each spot is normalized for total S. rosetta cell number. DOI: http://dx.doi.org/10.7554/eLife.04070.015

Article Snippet: A. machipongonensis conditioned media (ACM) was made from the sterile supernatant of the liquid A. machipongonensis culture (ATCC BAA-2233 [ ]) grown shaking for 48 hr in HN media at 30°C to an OD 600 of 0.30–0.39 and filtered through a 0.2 μm filter to remove bacterial cells and detritus.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Purification, Recombinant, Silver Staining, Western Blot, Biomarker Discovery, Incubation, Staining, Dot Blot, Mutagenesis