Journal: Microorganisms

Article Title: Bacteriome Signature in SARS-CoV-2-Infected Patients Correlates with Increased Gut Permeability and Systemic Inflammatory Cytokines

doi: 10.3390/microorganisms13061407

Figure Lengend Snippet: Zonulin plasma concentrations ( A ), and fecal secretory IgA ( B ) in control subjects (CTL), acute COVID-19 patients, and post-COVID-19 condition (PCC) patients.

Article Snippet: Fecal secretory immunoglobulin A (sIgA) was quantified by the commercial Human IgA ELISA kit (Elabscience, Bethesda, MD, USA), according to the manufacturer’s protocol.

Techniques: Clinical Proteomics, Control

Journal: Microorganisms

Article Title: Bacteriome Signature in SARS-CoV-2-Infected Patients Correlates with Increased Gut Permeability and Systemic Inflammatory Cytokines

doi: 10.3390/microorganisms13061407

Figure Lengend Snippet: Correlations between taxonomic counts of the gut bacteriome and clinical and laboratory data in COVID-19 patients. ( A ) C-reactive protein with differentially increased genera. ( B , C ) Plasma zonulin levels with differentially increased families and genera. ( D , E ) Fecal sIgA levels with differentially increased Bacillota, Prevotellacea, and some genera. ( F – H ) Plasma concentrations of IL-6, IL-2 and IFN-γ with differentially increased taxa.

Article Snippet: Fecal secretory immunoglobulin A (sIgA) was quantified by the commercial Human IgA ELISA kit (Elabscience, Bethesda, MD, USA), according to the manufacturer’s protocol.

Techniques: Clinical Proteomics

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Representative image of leptin expression in lung adenocarcinoma. Leptin expression in tumors and paired normal lung tissues was detected by immunochemical staining analysis. (A) Leptin protein was overexpressed in a moderately differentiated lung adenocarcinoma samples with acinar predominant growth compared with normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma (epithelial cells) of lung cancer tissues. (B) There was no difference in the expression of leptin protein in a case of well-differentiated lung adenocarcinoma with lepidic predominant (LPA) growth compared with normal control lung tissue. Leptin was seen to be expressed at low levels in the parenchyma of lung cancer tissue and in normal lung tissue. (C) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma sample with papillary predominant growth (PPA) compared to normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma of lung cancer tissues. (D) Leptin protein was overexpressed in a poorly differentiated lung adenocarcinoma sample with micropapillary predominant growth (MPA) compared to normal control lung tissue. It can be seen that leptin protein was expressed in both parenchyma and mesenchyme of lung cancer tissues. (E) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma with solid predominant growth compared to normal control lung tissue. It can be seen that leptin protein was expressed in the parenchyma, mesenchyme, and normal control lung tissues of lung cancer, but the expression level was low in normal tissues. (F) Leptin protein was overexpressed in a lung minimally invasive adenocarcinoma (MIA) compared to normal control lung tissue from the same patient. All experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin-peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope.

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: The effect of endogenous leptin expression on the proliferation of pulmonary adenocarcinoma cells. (A) The clone formation test of H1299 and A549 cells on the effect of proliferation of pulmonary adenocarcinoma; 50 ng/mL artificial recombinant leptin protein was used to simulate the impact of exogenous leptin in circulating blood on tumor cells; (B) the flow cell cycle analysis of PI staining showing the effect of endogenous leptin expression on the cell cycle of lung adenocarcinoma in H1299 and A549 cells; (C) AV/PI double-staining flow cytometry apoptotic cell test showing the effect of endogenous leptin expression in the apoptosis of lung adenocarcinoma in H1299-sh and A549 cells. All the experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin–peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope (100×).

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Recombinant, Cell Cycle Assay, Staining, Double Staining, Flow Cytometry, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Molecular mechanisms of endogenous leptin expression effect on the proliferative ability of lung adenocarcinoma cells. (A) Effect of endogenous leptin expression on key signaling molecules of PI3K/AKT pathway and its downstream signaling pathway in H1299 and A549 cells detected by western blotting; (B) effect of endogenous leptin expression on the p53 signaling pathway in A549 cells by western blotting; (C) endogenous leptin expression level on the expression level of autophagy-related protein LC3-II in H1299 cell lines detected by protein immunofluorescence. (D) Effect of endogenous leptin expression on key signaling molecules of the mTOR pathway and its downstream signalings in H1299 and A549 cells detected by western blotting. All the experiments were repeated three times. Scale bar: 50 µm.

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal:

Article Title: Increased immunoglobulin A levels in milk by over-expressing the murine polymeric immunoglobulin receptor gene in the mammary gland epithelial cells of transgenic mice

doi: 10.1046/j.1365-2567.2000.00094.x

Figure Lengend Snippet: IgA and SC in the milk of transgenic mice. Western blot analysis of milk samples, collected from F2 female mice of all transgenic lines. Samples were diluted in PBS as indicated below and 3 µl was fractionated on a 7·5% SDS–PAGE gel under reducing conditions (a) or non-reducing conditions (b). (a) The blot was incubated with a rabbit anti-human SC antibody and a rabbit anti-mouse IgA (α) antibody. Milk samples used: -M, non-transgenic littermate (13 days lactation); line 3642 (mouse 5967, 12 days lactation); line 3643 (mouse 8068, 13 days lactation); line 3644 (mouse 9507, 13 days lactation); line 3646 (mouse 5971, 14 days lactation). Dilutions used: line 3642, 1 : 10; line 3643, 1 : 50; line 3644, 1 : 500; line 3646, 1 : 200; non-transgenic littermate, 1 : 10. C1, purified human sIgA from colostrum (hsIgA, 20 ng SC). The 80 000 MW SC protein of the human sIgA molecule is visible and used as a reference. C2, mouse myeloma protein IgA (MOPC 315, 30 ng). The IgA murine heavy chain is shown as a band of 60 000 MW. The numbers on the left indicate the molecular weight of the protein standards (in kDa). mSC, murine secretory component (MW 95 000–100 000); mIgA (α), murine IgA heavy chain (MW 60 000). (b) The blot was incubated with a rabbit anti-mouse IgA (α) antibody. Milk samples used were diluted 1 : 10 in PBS. -M, non-transgenic littermate (14 days lactation); line 3642 (mouse 8189, 12 days lactation); line 3643 (mouse 4779, 14 days lactation; mouse 8068, 13 days lactation); line 3644 (mouse 9507, 13 days lactation; mouse 9287, 12 days lactation); line 3646 (mouse 5971, 14 days lactation; mouse 10695, 12 days lactation). C1, purified human sIgA from colostrum (415 000); msIgA, murine secretory IgA (MW 435 000).

Article Snippet: Purified human secretory IgA (sIgA) from pooled human colostrum (Sigma–Aldrich Chemie BV, Zwyndrecht, the Netherlands) and mouse myeloma protein IgA (λ2) (ICN-Biomedicals BV, Veenendaal, the Netherlands) were used as controls.

Techniques: Transgenic Assay, Western Blot, SDS Page, Incubation, Purification, Molecular Weight