Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 1. The Recombinant Soluble CD89 Receptor Interacts Directly with Bacteria (A and B) Dose-dependent binding of soluble recombinant CD89 (sCD89) to fixed S.p (A) and E. coli (B). Binding to albumin (Alb) was used as a control. (C) Comparison of sCD89 binding to various types of fixed bacteria. (D) Interaction of sCD89 with live (green bar) versus fixed (black bars) 106 E. coli or 106 S. p. (E and F) S.p (E) and E. coli (F) binding to BMMs grown from CD89 transgenic mice (CD89Tg) or from littermates, visualized by confocal laser-scanning mi- croscopy. Right: quantification of binding (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (G and H) S.p (G) and E. coli (H) binding to BMMs isolated from CD89Tg mice or from littermates in the presence or absence of the anti-CD89 blocking antibody MIP8a F(ab’)2 (10 mg/mL) or of sCD89 (500 mg/mL), analyzed by flow cytometry. MFI, mean fluorescence intensity. Data are presented as mean ± SEM; n = 5. *p < 0.05, **p < 0.01; t test. See also Figure S1.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Recombinant, Bacteria, Binding Assay, Control, Comparison, Transgenic Assay, Isolation, Blocking Assay, Cytometry

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 2. Bacterium-CD89 Interaction on Mouse Cells Induces Activating ITAM Signaling, Leading to Inflammatory Cytokine Production, Bacterial Phagocytosis, and Killing (A and B) IL-6, TNF-a, and IL-1 production in the supernatant of BMMs obtained from CD89Tg and CD89R209L transgenic mice and littermate controls. Cells were incubated for 16 h in the presence of S.p (A) and E. coli (B) and cytokines in the supernatants were measured by ELISA. All data are presented as mean ± SEM; n = 3. *p < 0.05, **p < 0.01; t test. (C) Confocal analysis of E. coli-pHrodo phagocytosis by BMMs obtained from CD89Tg mice compared with littermates in the presence or absence of MIP8a F(ab)’2 or sCD89 in a dose-dependent manner (100–800 mg/mL). Left: representative images. Right: quantification. Data are presented as mean ± SEM; n = 3. ***p < 0.001, t test. (D) ROS production over 30 min by littermate, CD89Tg, and CD89R209L transgenic BMMs stimulated by live S.p (left) or E. coli (right), measured by confocal microscopy. All data are presented as mean ± SEM; n = 15. **p < 0.01, t test. (E) Quantification of bacterial survival after 2 h of incubation with BMMs from CD89Tg, CD89R209L Tg, and littermate mice. Data are presented as mean ± SEM; n = 3. ***p < 0.001, t test.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Transgenic Assay, Incubation, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 3. IgA-Deficient CVID Phagocytes Mediate Phagocytosis, ROS Production, and Bacterial Killing through CD89 Interaction (A) Representative plots of CD89 expression on blood monocytes isolated from healthy donors (HDs) (left) and CVID patients (right) using a phycoerythrin (PE)-conjugated anti-CD89 antibody and its isotype control. (B) Binding of S.p or E. coli to blood monocytes from HDs (purple symbols) or from CVID patients (red symbols) in the presence of monomeric IgA (500 mg/mL) or of MIP8a F(ab’)2 (10 mg/mL). All data are presented as mean ± SEM; n = 4. ***p < 0.001, t test. (C) Phagocytosis of E. coli-pHrodo by human blood monocytes and/or macrophages isolated from HDs or from CVID patients. Left: representative images. Scale bars, 200 mm. Right: quantification (n = 3). All data are presented as mean ± SEM. ns, not significant. (D) IL-6, TNF-a, and IL-1 production in the supernatant of monocytes obtained from CVID patients. Cells were incubated for 16 h in the presence of E. coli or S.p and in the presence or absence of MIP8a F(ab)’2 (500 mg/mL), and cytokines in the supernatants were measured by ELISA. All data are presented as mean ± SEM; n = 3. *p < 0.05, **p < 0.01, ****p < 0.0001; t test.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Expressing, Isolation, Control, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 4. Role of CD89-Bacterium Interaction under Physiological Conditions (A) Competitive ELISA assays between sCD89 and S.p (blue line) or E. coli (red line) and ns-IgA. (B) Competitive ELISA assays between sCD89 and S.p (blue line) or E. coli (red line) and pd-IgA. (C) Bacterial phagocytosis by BMMs obtained from CD89Tg mice (left) compared with littermates (right). Bacteria were allowed to be phagocytosed by BMMs from the indicated mice in the presence or absence of ns-IgA at physiological concentration (2 mg/mL) or MIP8a F(ab)’2 (500 mg/mL). Cells were washed and analyzed by flow cytometry. Data are presented as mean ± SEM; n = 3. *p < 0.05, ***p < 0.001; t test. (D) S.p (left) or E. coli (right) phagocytosis by BMDCs obtained from CD89Tg mice compared with littermates. Bacteria were allowed to be phagocytosed by BMDCs from the indicated mice in the presence or absence of ns-IgA at physiological concentration (2 mg/mL). Cells were washed and analyzed by flow cytometry. Data are presented as mean ± SEM; n = 3. *p < 0.05, ***p < 0.0001; t test. (E) Representative images of E. coli (blue) and CD11c (red) staining by BMDCs derived from CD89Tg or wild-type (WT) mice captured by imaging flow cytometry (scale bars, 5 mm) and the percentages of the bacterial phagocytosis score. See also Figures S1C and S5C.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Competitive ELISA, Bacteria, Concentration Assay, Cytometry, Staining, Derivative Assay, Imaging

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 5. CD89-Bacterium Interaction Protects against Infection-Related Mortality in Mice (A) Survival of CD89Tg mice (red line) and littermates (black line) after intranasal inoculation (at time 0) with S. pneumonia (n = 25). Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. *p < 0.05. (B) Decreased lung contents of S.p in CD89 transgenic compared with littermate mice. All data are presented as mean ± SEM; n = 8. ***p < 0.001, t test. (C) H&E staining of lung sections from representative CD89Tg and littermate animals after intranasal infection. Scale bars, 200 mm. (D) Alveolitis invasion score of monomorphic inflammatory cells. All data are presented as mean ± SEM; n = 6. ***p < 0.001, t test. (E) mRNA expression of cytokines (IL-1, TNF-a, and IL-6) was assessed by qRT-PCR of 5 independent lung tissue RNA samples collected 6 and 48 h after intranasal infection. mRNA levels were normalized to b-actin mRNA levels. All data are presented as mean ± SEM; n = 6. *p < 0.05, t test. (F) Increased survival of CD89Tg mice (red line, n = 26) compared with littermates (black line, n = 22) after CLP. Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. **p < 0.01. (G–I) 48 h after CLP, peritoneal fluid was evaluated for total bacteria (G), E. coli (H), and Enterococcus (I) in CD89Tg mice and littermates. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; t test. (J) IL-1, TNF-a, and IL-6 levels in peritoneal lavage, assessed by ELISA 6 and 48 h after CLP. All data are presented as mean ± SEM; n = 3. *p < 0.05, t test. See also Figures S6A–S6C.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Infection, Transgenic Assay, Staining, Expressing, Quantitative RT-PCR, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 7. CD89 Protection against Sepsis Is Independent of CRP and IgA Antibodies during the Early Phase of Infection (A) Increased survival of CD89TgCRP-KO animals after intranasal infection with S.p compared with CRP-KO mice (n = 12 per group). CD89Tg mice and their littermates were used as controls. Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. (B) Decreased lung counts of S.p in CD89TgCRP-KO mice at 48 h compared with CRP-KO mice (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (C) Expression of cytokine mRNA (IL-1, TNF-a, and IL-6) was assessed by qPCR of independent lung tissue RNA samples collected 6 and 24 h after intranasal infection. Cytokine mRNA levels were normalized to b-actin mRNA levels, as indicated in Figure 5E (n = 4). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (D) Increased survival of CD89TgCRP-KO (n = 10) compared with CRP-KO mice (n = 10) after CLP. Kaplan-Meier curves and log rank test were used to compare mortality rates. CD89Tg mice and their littermates were used as controls. All data are presented as mean ± SEM. (E) Peritoneal fluid counts of bacteria 48 h after CLP (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (F) IL-1, TNF-a, and IL-6 levels in peritoneal lavage, assessed by ELISA 6 and 48 h after CLP (n = 4). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (G) Phagocytosis of S.p (left) and E. coli (right) after incubation with BMMs isolated from CD89TgCRP-KO or CRP-KO mice. (H) Phagocytosis of S.p (left) and E. coli (right) after incubation with BMMs isolated from CD89TgCRP-KO or CRP-KO mice in the presence of MIP8a anti-CD89 F(ab’)2. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (I) Measurement of mouse IgA antibodies against the indicated bacteria 48 or 168 h after S.p infection (left) or CLP (right) in CD89Tg or CD89TgCRP-KO mice. Data are presented as mean ± SEM. See also Figure S7.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Infection, Expressing, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Isolation

Journal: Nature Communications

Article Title: TRIB3 promotes MYC-associated lymphoma development through suppression of UBE3B-mediated MYC degradation

doi: 10.1038/s41467-020-20107-1

Figure Lengend Snippet: a , b The mRNA level of TRIB3 in lymphoma patients and the corresponding control groups. The center line indicates the median, bounds of box = 25th and 75th percentiles; bars = 10th and 90th percentiles; whiskers = min to max, show all points. Statistical significance was determined by two-tailed Student’s t test. P values: 8.52 × 10 −8 a ; 0.018 b . c TRIB3 gene copy numbers in the lymphoma set (GSE24881) were analyzed at the Oncomine website. The center line indicates the median, bounds of box = 25th and 75th percentiles; bars = 10th and 90th percentiles; whiskers = min to max, show all points. Statistical significance was determined by two-tailed Student’s t test. P value: 2.80 × 10 −7 . d Pie chart showing all the types and statistics of TRIB3 gene mutation in 89 lymphoma patients. e Schematic strategy for studying the development of B cell lymphoma (BCL) in Myc Eμ mice with myeloid cell-specific deletion of Trib3 ( Myc Eμ Cre Lysm Trib3 F/+ ), thymocyte-specific deletion of Trib3 ( Myc Eμ Cre L ck Trib3 F/+ ), or B lymphocyte-specific deletion of Trib3 ( Myc Eμ Cre CD19 Trib3 F/+ ). f Representative spleens and lymph nodes (LNs) obtained from WT, Myc Eμ , Myc Eμ Cre Lysm Trib3 F/+ , Myc Eμ Cre Lck Trib3 F/+ , Myc Eμ Cre CD19 Trib3 F/+ , or Myc Eμ Cre CD19 Trib3 F/F mice (4.5 months old). Scale bar, 1 cm. g , h The data indicate the spleen and LN weights of WT ( n = 10), Myc Eμ ( n = 13), Myc Eμ Cre Lysm Trib3 F/+ ( n = 8), Myc Eμ Cre L ck Trib3 F/+ ( n = 10), Myc Eμ Cre CD19 Trib3 F/+ ( n = 10), or Myc Eμ Cre CD19 Trib3 F/F mice ( n = 7) (4.5 months old). Data are represented as means ± SEM. Statistical significance was determined by two-tailed Student’s t test. P values: 7.34 × 10 −10 (WT vs. Myc Eμ ), 1.44 × 10 −5 (WT vs. Myc Eμ Cre L ck Trib3 F/+ ), 8.89 × 10 −6 (WT vs. Myc Eμ Cre Lysm Trib3 F/+ ), 2.94 × 10 −8 ( Myc Eμ vs. Myc Eμ Cre CD19 Trib3 F/+ ), 3.01 × 10 −5 ( Myc Eμ vs. Myc Eμ Cre CD19 Trib3 F/F ) g ; 4.56×10 −4 (WT vs. Myc Eμ ), 1.64 × 10 −5 (WT vs. Myc Eμ Cre L ck Trib3 F/+ ), 6.47 × 10 −5 (WT vs. Myc Eμ Cre Lysm Trib3 F/+ ), 5.14× 10 −4 ( Myc Eμ vs. Myc Eμ Cre CD19 Trib3 F/+ ), and 0.0011 ( Myc Eμ vs. Myc Eμ Cre CD19 Trib3 F/F ) h . i Kaplan–Meier survival curves for Myc Eμ ( n = 15) and Myc Eμ Cre CD19 Trib3 F/+ mice ( n = 12). Statistical difference was determined by two-sided log-rank test. P value: 0.0045. j Schematic representation of generating the inducible deletion of Trib3 in Myc Eμ mice ( Myc Eμ Cre ERT2 Trib3 F/+ ). k Kaplan–Meier survival curves for Myc Eμ Cre ERT2 Trib3 F/+ mice treated with vehicle ( n = 13) or tamoxifen ( n = 12 per group, 250 mg/kg). Statistical difference was determined by two-sided log-rank test. P value: 0.0012. l Representative H&E staining of the spleens and LNs of 5-month-old Myc Eμ Cre ERT2 Trib3 F/+ mice treated with or without tamoxifen. Scale bar, 50 μm. The data are presented as presentative from three independent experiments. The Myc Eμ Cre Lysm Trib3 F/+ , Myc Eμ Cre L ck Trib3 F/+ , Myc Eμ Cre CD19 Trib3 F/+ , Myc Eμ Cre CD19 Trib3 F/F , and Myc Eμ Cre ERT2 mice are heterozygous for the Myc Eμ allele. Source data are provided as a Source Data file.

Article Snippet: Purified His-tagged MYC (TP760019) was purchased from Origene Inc. GST-tagged TRIB3 (10731-H09B) and KDC domains, GST-tagged MAX (12885-H20B) and His-tagged MAX (12269-H08H) were purchased from Sinobiological Inc.

Techniques: Two Tailed Test, Mutagenesis, Staining

Journal: Nature Communications

Article Title: TRIB3 promotes MYC-associated lymphoma development through suppression of UBE3B-mediated MYC degradation

doi: 10.1038/s41467-020-20107-1

Figure Lengend Snippet: a In vitro ubiquitination assays show that the TRIB3 or TRIB3-KDC protein decreased polyubiquitination of MYC mediated by UBE3B and UBCH3. b The TRIB3 protein or the KDC domain of TRIB3 decreased the interaction of MYC and UBE3B in the purified system. c Neither the TRIB3 protein nor the KDC domain of TRIB3 affected the interaction of UHCH3 and UBE3B in the purified system. d The interaction of TRIB3 and MYC in Raji cells was evaluated by Co-IP assays. e Colocalization of TRIB3 and MYC in Raji cells was detected with immunostaining (PLA assay). Data are representative images from three independent experiments. Scale bar, 2 μm. f Mapping TRIB3 regions binding to MYC. Top: deletion mutants of TRIB3. Bottom: HEK 293T cells were cotransfected with the indicated constructs of TRIB3 (HA tag) and MYC (Flag tag). Cell extracts were IP with an anti-Flag Ab. g , h Mapping MYC regions binding to TRIB3 ( g ) or UBE3B ( h ). Top: deletion mutants of MYC. Bottom: HEK293T cells were cotransfected with the indicated constructs of MYC and TRIB3 or UBE3B. Cell extracts were IP with an anti-Flag Ab. i TRIB3 overexpression interfered with the interaction of UBE3B and MYC. HEK 293T cells were cotransfected with the indicated constructs. Cell extracts were IP with an anti-MYC Ab. j TRIB3 decreased the binding of UBE3B and MYC. Kinetic interactions of the UBE3B and MYC proteins were determined by SPR analyses with the BSA or TRIB3 proteins. k TRIB3 overexpression increased the interaction of MAX and MYC. HEK 293T cells were cotransfected with the indicated constructs. Cell extracts were IP with an anti-MYC Ab. l TRIB3 overexpression increased the binding of MAX and MYC. Kinetic interactions of the MAX and MYC proteins were determined by SPR analyses with the CTRL (GST) or TRIB3 (TRIB3-GST) protein. m The heterotrimers of MYC, TRIB3, and MAX in Raji cells were detected by Co-IP assays. Cellular extracts were IP with mouse IgG as a negative control or anti‐MYC, anti‐TRIB3, or anti-MAX antibodies. Western blots were performed with anti‐MYC, anti‐TRIB3, and anti-MAX antibodies. n Recovery of the TRIB3 -HA wild-type or KDC -HA mutant enhanced the decreased cell viabilities of TRIB3 Cas9 Jurkat cells for the indicated times. The colors represent different time points; the diameter indicates the relative cell viability. Source data are provided as a Source Data file.

Article Snippet: Purified His-tagged MYC (TP760019) was purchased from Origene Inc. GST-tagged TRIB3 (10731-H09B) and KDC domains, GST-tagged MAX (12885-H20B) and His-tagged MAX (12269-H08H) were purchased from Sinobiological Inc.

Techniques: In Vitro, Purification, Co-Immunoprecipitation Assay, Immunostaining, Binding Assay, Construct, FLAG-tag, Over Expression, Negative Control, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: TRIB3 promotes MYC-associated lymphoma development through suppression of UBE3B-mediated MYC degradation

doi: 10.1038/s41467-020-20107-1

Figure Lengend Snippet: TRIB3 expression is enhanced in human lymphoma cells due to either the increased copy number in response to various stresses and nonsynonymous (Q84R) mutation of TRIB3 . Whereas, UBE3B, an E3 ubiquitin ligase, and UBCH3, an E2 ubiquitin-conjugated enzyme, have been identified to target MYC for proteasomal degradation. The enhanced TRIB3 interacts with MYC to decrease the UBE3B-mediated ubiquitination of MYC K427 and increase MYC stability; this interaction also supports the formation of MYC and MAX heterodimer. These actions enhance MYC transcriptional activity, causing high proliferation and self-renewal of lymphoma cells. Interfering with the TRIB3/MYC interaction enhances MYC degradation and suppresses the development and progression of high TRIB3/MYC-associated lymphoma.

Article Snippet: Purified His-tagged MYC (TP760019) was purchased from Origene Inc. GST-tagged TRIB3 (10731-H09B) and KDC domains, GST-tagged MAX (12885-H20B) and His-tagged MAX (12269-H08H) were purchased from Sinobiological Inc.

Techniques: Expressing, Mutagenesis, Activity Assay

Journal: PLoS ONE

Article Title: Distinct Patterns of IgG and IgA against Food and Microbial Antigens in Serum and Feces of Patients with Inflammatory Bowel Diseases

doi: 10.1371/journal.pone.0106750

Figure Lengend Snippet: Serum IgG (A–F) and IgA (G–L) specific for ovalbumin (A/G), wheat (B/H), milk (C/I), mannan from S. cerevisiae = ASCA (D/J), and lysates from E. coli K12 (E/K) and B. fragilis ATCC 25285 (F/L) were quantified by ELISA in control patients/healthy controls (Con; n = 61) and patients suffering from CD (n = 52), UC (n = 29) and acute gastroenteritis/colitis (AGE; n = 12). Each dot represents one patient. Medians with interquartile ranges are indicated. P values (*<0.05; **<0.01; ***<0.001) were determined by Kruskal-Wallis test followed by a Dunn's post hoc test.

Article Snippet: All antigens were vigorously mixed for 1 h. E. coli K12 DH5α and B. fragilis ATCC 25285 were grown over night in LB or thioglycolate medium under aerobic or anaerobic culture conditions, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A-D) Human colonic EDMs were infected with Fn at moi 100 for 24 h. The RNA from EDMs was used for qRT-PCR to determine the expression of genes involved in base excision repair, mismatch repair and for non-homologous end joining (NHEJ). (A) Schematic showing the experimental design. (B) The level of BER transcripts, NEIL1, NEIL2, NTH1, OGG1, (C) The level of MMR transcripts, MLH1, MLH3, MSH2, MSH6, PMS2, (D) The transcript level of NHEJ marker Ku70 were determined by qRT-PCR. (E-F) Human colonic EDMs were infected with commensal E. coli -K12 strain (E), or pathogenic IBD-associated adherent invasive E. coli LF-82 (F) to determine the expression level of NEIL2 following infection. In (B-F), the expression level of the transcripts was normalized to the housekeeping gene (18srRNA), and the normalized expression value was compared with the respective uninfected control cells. Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, and ** indicates p≤0.01 as calculated by the unpaired two-tailed student’s t-test.

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Infection, Quantitative RT-PCR, Expressing, Non-Homologous End Joining, Marker, Control, Two Tailed Test

Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A) APC Min /+ EDMs derived from the uninvolved region of the colon were infected with different microbes; commensal E. coli K12, IBD-associated adherent-invasive E.coli LF82 and colon cancer-associated pathogens (NC101, H. pylori and Fn ). The supernatants were collected from the uninfected and infected EDMs done in the same experiments and assessed for oxidative DNA damage (right). Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, ** indicates p≤0.01 as assayed by student’s t-test. (B) The relative level of the oxidized bases produced by each microbe was compared with uninfected cells, which is considered as 1. The relative production of the oxidized base was compared between different microbes

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Derivative Assay, Infection, Produced

Journal: Scientific Reports

Article Title: Fluorescence in situ hybridisation in Carnoy’s fixed tonsil tissue

doi: 10.1038/s41598-022-16309-w

Figure Lengend Snippet: Bacterial cell cultures used in this study to test and optimise FISH probes.

Article Snippet: Figure 3 Fluorescence in situ hybridisation staining of Streptococcus pyogenes ATCC 700,294, Pseudomonas aeruginosa ATCC BAA-47 and Escherichia coli K-12 DH5 alpha, ATCC PTA-4079 mixed cells from broth culture. ( A ) Nucleic material/DAPI in blue, ( B ) Eubacterial probe (EUB) in green, ( C ) Streptococcus spp . specific probe (STRC493) in red, ( D ) Pseudomonas spp . specific probe (PSE227) in yellow, ( E ) composite image of ( A – D ).

Techniques: