Journal: Foods

Article Title: Boosting Recombinant Bovine Chymosin in Komagataella phaffii via Fusion Protein and Constitutive Promoter Expression

doi: 10.3390/foods15040731

Figure Lengend Snippet: Purification and deglycosylation analysis of recombinant bovine chymosin. ( a ) SDS-PAGE analysis of samples from the purification process. Proteins were separated on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue. Approximately 10 µg of protein was loaded per lane. Lane 1: Prestained protein molecular weight markers (sizes indicated on the left). Lane 2: The purified recombinant chymosin obtained after acidification activation (pH 2.5, 2 h) and subsequent concentration using a 5 kDa molecular weight cut-off (MWCO) ultrafiltration membrane. Lane 3: The culture supernatant containing the acid-activated recombinant chymosin prior to ultrafiltration. ( b ) SDS-PAGE analysis of the recombinant chymosin deglycosylation. Analysis was performed under the same conditions as in ( a ). Lane 1: Prestained protein molecular weight markers. Lane 2: Endo-β-N-acetylglucosaminidase H (Endo H) enzyme alone. Lane 3: The purified recombinant chymosin after treatment with Endo H for 2 h at 37 °C, showing the complete deglycosylated form. Lane 4: The purified recombinant chymosin without Endo H treatment, showing the glycosylated form.

Article Snippet: Endo-β-N-acetylglucosaminidase H (Endo H) was obtained from New England Biolabs (Ipswich, MA, USA).

Techniques: Purification, Recombinant, SDS Page, Staining, Molecular Weight, Activation Assay, Concentration Assay, Membrane

Journal: Nature cell biology

Article Title: Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver

doi: 10.1038/ncb3169

Figure Lengend Snippet: (a) Representative images and quantification of pre-metastatic niche markers in livers educated with PAN02 exosomes (Exo), PAN02shCTL exosomes (shCTLexo), PAN02shMIF exosomes (shMIFexo and shMIF(2)exo) or PBS control (CTL). Immunofluorescence analysis shows frequency of TGFβ-expressing F4/80+ cells, αSMA and FN expression as well as F4/80+ cell frequency. Inset shows TGFβ+/F4/80+ cell; n = 3 (CTL F4/80), n = 4 (CTL TGFβ, αSMA, FN; Exo TGFβ; all shCTL; shMIF TGFβ; all shMIF(2), n = 6 (Exo F4/80; shMIF F4/80), n = 7 (Exo αSMA, FN; shMIF αSMA, FN) mice pooled from two experiments. ***P < 0.001, **P < 0.001 by ANOVA. Scale bars, 100μm. (b) Evaluation of liver metastasis by liver weight (grams) in tumor-free mice (CTL), mice injected intra-splenically with PAN02 cells either pre-educated with PBS (TU), with PAN02 exosomes (Exo+TU), with PAN02 exosomes in combination with A83-01 (Exo+A83-01+TU), or with PAN02shMIF exosomes (shMIFexo+TU); n = 4 (Exo+A83-01+TU), n = 5 (CTL, TU, Exo+TU, and shMIFexo+TU) mice pooled from two experiments. **P < 0.01, *P < 0.05 by ANOVA. Scale bar, 1cm. (c) Evaluation of liver metastasis in mice injected intra-splenically with PAN02 cells either pre-educated with PBS (TU), with exosomes isolated from PAN02 cells infected with control shRNA (shCTLexo+TU) or shMIF(2) (shMIF(2)exo+TU) lentiviral vectors; n = 4 (shCTLexo+TU), n = 5 (shMIF(2)exo+TU) and n = 6 (TU) mice from one experiment. ***P < 0.01 by ANOVA. Scale bar, 1cm. (d) Enzyme-linked immune assay (ELISA) reveals increased levels of MIF (picogram per 108 exosomes) in exosomes isolated from patients with pancreatic ductal adenocarcinoma (PDAC) with progression of disease post-diagnosis (POD) compared to PDAC patients with no evidence of disease 5 years post-diagnosis (NED) and to healthy controls (CTL), but not PDAC patients with liver metastasis (LM); n = 10 (NED), n = 12 (POD), n = 15 (CTL) and n = 18 (LM) patients. All patient samples were analyzed once as part of three independent ELISA assays. **P < 0.01 by ANOVA. All data are represented as mean±s.e.m

Article Snippet: MIF knockdown PAN02 cells were generated by using MIF shRNA lentiviral particle infection, generating the PAN02 variants shMIF (sc-37138-V, Santa Cruz) and shMIF(2) (iV042354, abmGood).

Techniques: Control, Immunofluorescence, Expressing, Injection, Isolation, Infection, shRNA, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung

doi: 10.1152/ajplung.00324.2017

Figure Lengend Snippet: Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) defines genome-wide transcriptional regulation by pSmad1 protein upon growth differentiation factor 15 (GDF15) in human airway epithelial cells. Normal human tracheobronchial epithelial cells in submerged culture were treated with 0.1% BSA-HCl (control) or recombinant human GDF15 (25 ng/ml) for 2 h to perform the phosphorylated-Smad1 (pSmad1) ChIP-seq analysis.

Article Snippet: Lentiviruses encoding human Smad1 shRNA (sc-29483-v, shSmad1) or control shRNA (sc-108080, shControl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Chromatin Immunoprecipitation, Next-Generation Sequencing, ChIP-sequencing, Genome Wide, Control, Recombinant

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung

doi: 10.1152/ajplung.00324.2017

Figure Lengend Snippet: Overexpressing human growth differentiation factor 15 (GDF15) enhances activation of Smad1 and interferon regulatory factor 7 (IRF7) in mouse airway epithelial cells. Primary tracheal epithelial cells from naïve human GDF15 transgenic (hGDF15 Tg+) mice or wild-type (WT) littermates were grown at air-liquid interface and infected with HRV-1B [multiplicity of infection (MOI) 1] or PBS (control) for 24 h. A, left: quantitative Western blot densitometry data of phosphorylated-Smad1 (pSmad1) and total Smad1 protein. A, right: representative Western blot pictures; B, left: quantitative Western blot densitometry data of IRF7 and β-actin protein (loading control). B, right: representative Western blot pictures. Data are presented as means ± SE from three independent experiments. *P < 0.05, **P < 0.01, compared with PBS controls or HRV-infected WT cells.

Article Snippet: Lentiviruses encoding human Smad1 shRNA (sc-29483-v, shSmad1) or control shRNA (sc-108080, shControl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Transgenic Assay, Infection, Control, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung

doi: 10.1152/ajplung.00324.2017

Figure Lengend Snippet: Smad1 cooperates with interferon regulatory factor 7 (IRF7) in response to HRV infection via growth differentiation factor 15 (GDF15) signaling in human airway epithelial cells. A: normal human bronchial epithelial cells transduced with lentiviruses encoding human GDF15 shRNA (shGDF15) or control shRNA (shControl) were grown at air-liquid interface and infected with HRV-16 [multiplicity of infection (MOI) 1] or PBS (control) for 24 h. A, top: quantitative Western blot densitometry data of IRF7 and β-actin protein (loading control). A, bottom: representative Western blot pictures. B, top: normal human tracheobronchial epithelial cells transduced with lentiviruses encoding human Smad1 shRNA (shSmad1) or control shRNA (shControl) were grown at air-liquid interface for 28 days to examine Smad1 protein expression. B, bottom: quantitative Western blot densitometry data of Smad1 and GAPDH protein (loading control). C: normal human tracheobronchial epithelial cells transduced with lentiviruses encoding human Smad1 shRNA (shSmad1) or control shRNA (shControl) were grown at air-liquid interface, pretreated with 0.1% BSA-HCl (control) or recombinant human GDF15 (25 ng/ml) for 72 h, and infected with HRV-16 (MOI 1) or PBS for 24 h. C, top: quantitative Western blot densitometry data of IRF7 and β-actin protein (loading control). C, bottom: representative Western blot pictures. Data are presented as means ± SE from 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with PBS controls or shControl cells; NS, not significant.

Article Snippet: Lentiviruses encoding human Smad1 shRNA (sc-29483-v, shSmad1) or control shRNA (sc-108080, shControl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Infection, Transduction, shRNA, Control, Western Blot, Expressing, Recombinant

Journal: PLOS Neglected Tropical Diseases

Article Title: Effect of a gut commensal Lactobacillus strain Limosilactobacillus caviae JL20 on leptospiral whole-cell inactivated vaccine in hamsters

doi: 10.1371/journal.pntd.0013951

Figure Lengend Snippet: (A) Experimental schema of blood collection during the immune process. (B) Total IgG antibody titres in the serum at 3, 14, 17, and 28 days after the first immunization (n = 6/group). (C) Serum IgG1 and IgG2/3 titres on day 28 after immunization (n = 6/group). (D) The serum samples collected after the first and second immunizations were subjected to a microneutralization assay (MAT) using 15 standard Leptospira strains, with the data limited to those strains that exhibited agglutination (n = 6/group). (B) - (D) : PBS + VAC, orally gavaged with PBS, and vaccinated; JL20 + VAC, orally gavaged with JL20, and vaccinated. Results represent mean ± SD of values. Statistical significance was evaluated using the Wilcoxon rank-sum test, with *p < 0.05, **p < 0.01, ***p < 0.001. , created in BioRender. 603, C. (2025) https://BioRender.com/4jus9df .

Article Snippet: Serum samples (1:100) were added to the antigen-coated wells and incubated at 37°C for 1 h, followed by HRP conjugate secondary anti-hamster-IgG, IgG1 and IgG2/3 (1:10000) (Southernbiotech 6060-05, 1940-05, 1935-05), which was incubated for 30 minutes.

Techniques: Microneutralization Assay, Agglutination