Journal: Clinical and Translational Gastroenterology

Article Title: Cathepsin L and B as Potential Markers for Liver Fibrosis: Insights From Patients and Experimental Models

doi: 10.1038/ctg.2017.25

Figure Lengend Snippet: Upregulation of hepatic cathepsin L (CTSL) and cathepsin B (CTSB) expression in experimental liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl 4 ) or vehicle (olive oil) twice a week for 10 weeks. Hepatic CTSL and CTSB levels were evaluated by real-time PCR and immunohistochemistry. ( a ) Relative CTSL mRNA expression was significantly increased in the liver of CCl 4 -treated mice while CTSB levels were comparable. ( b , c ) Representative pictures of CTSL and CTSB immunostaining in control and CCl 4 -treated livers. Liver section from control exhibits granular CTSL immunostaining pattern while in CCl 4 -treated mice diffuse and strong cytoplasmic staining is evident in the hepatocytes, bile duct epithelium, fibroblasts, and Kupffer cells as indicated by arrows (Original magnification, × 200). ( d ) Quantification of immunostained CTSL and CTSB revealed overexpression of CTSL in the liver of CCl 4 -treated mice (values are mean±s.e.m., n =6), * P ≤0.05 compared with controls. ( e , f ) Temporal expression of hepatic CTSL and CTSB transcript levels were quantified in Abcb4−/− mice and their wild-type littermates. Compared with wild-type controls, CTSL and CTSB expression were significantly higher in Abcb4−/− at 4 weeks but gradually decreased to the basal levels by 16 weeks (mean±s.e.m., n =at least 7 mice in each group). * P ≤0.05 compared with wild-type controls.

Article Snippet: Plasma levels of CTSL and CTSB were measured using the ELISA Kit (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, respectively) as per the manufacturer’s instructions.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Immunohistochemistry, Immunostaining, Control, Staining, Over Expression

Journal: Clinical and Translational Gastroenterology

Article Title: Cathepsin L and B as Potential Markers for Liver Fibrosis: Insights From Patients and Experimental Models

doi: 10.1038/ctg.2017.25

Figure Lengend Snippet: Elevation in hepatic cathepsin L (CTSL) and cathepsin B (CTSB) expression in patients with liver cirrhosis. ( a , b ) Immunohistochemical localization of CTSL and CTSB in the human liver biopsies. In control liver tissue ( n =13), punctuate pericanalicular cytoplasmic CTSL and CTSB expression was observed in hepatocytes (arrow), along with mild focal positivity in bile duct epithelial cells, and kupffer cells. Similar to controls, mild CTSL and CTSB immunoreactivity is seen in patients with chronic hepatitis (CVH, n =10). Whereas strong and diffuse immunostaining for both these proteases was evident in hepatocytes, ductal cells, infiltrating histiocytes, and fibrotic septae (indicated by arrows) in the liver of cirrhotic subjects ( n =28) (Original magnification, × 200). ( c , d ) Total immunoscores of ( c ) CTSL and ( d ) CTSB were significantly higher in patients with liver cirrhosis compared with controls and CVH patients (mean±s.e.m., ** P ≤0.001 and *** P ≤0.0001, scores of fibrotic patients vs. the corresponding non-fibrotic controls, Kruskal–Wallis H test was used to compare the means of all the groups).

Article Snippet: Plasma levels of CTSL and CTSB were measured using the ELISA Kit (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, respectively) as per the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemical staining, Control, Immunostaining

Journal: Clinical and Translational Gastroenterology

Article Title: Cathepsin L and B as Potential Markers for Liver Fibrosis: Insights From Patients and Experimental Models

doi: 10.1038/ctg.2017.25

Figure Lengend Snippet: Liver collagen content correlate with hepatic cathepsin L (CTSL) and cathepsin B (CTSB) expression. ( a – f ) Morphometrically measured Sirius red–stained fibrotic area in the livers of chronic hepatitis and cirrhotic patients exhibited a strong positive correlation with the score of CTSL ( a – c ) and CTSB ( d – f ) immunopositivity in whole liver tissue, fibrotic septae, and bile ducts, respectively. Correlation analysis was performed using Spearman-rank test and the individual data points represent samples from individual patients.

Article Snippet: Plasma levels of CTSL and CTSB were measured using the ELISA Kit (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, respectively) as per the manufacturer’s instructions.

Techniques: Expressing, Staining

Journal: Clinical and Translational Gastroenterology

Article Title: Cathepsin L and B as Potential Markers for Liver Fibrosis: Insights From Patients and Experimental Models

doi: 10.1038/ctg.2017.25

Figure Lengend Snippet: Plasma levels of cathepsin L (CTSL) and cathepsin B (CTSB) increase with Child–Pugh stage of liver cirrhosis. ( a , b ) Comparison of plasma ( a ) CTSL and ( b ) CTSB levels in normal controls ( n =15) and patients with liver cirrhosis (Child–Pugh stage A, n =20 and B+C, n =31). Plasma levels of CTSL and CTSB were significantly higher in patients with liver cirrhosis compared with healthy controls. CTSB levels were significantly increased in subjects with Child–Pugh stages B and C compared with patients with Child–Pugh class A liver cirrhosis (values are mean±s.e.m., * P ≤0.05, ** P ≤0.001 and *** P ≤0.0001, Kruskal–Wallis H test was used to compare the means of all the groups). ( c – f ) The diagnostic accuracy of plasma CTSL, CTSB, and their combination in chronic liver disease with cirrhosis. Receiver–operating characteristic (ROC) curves was performed to determine the area under the curve (AUC) using plasma CTSL, CTSB, or their combination to predict hepatic cirrhosis ( c ) control vs. patients with liver cirrhosis (Child–Pugh stage A+B+C); ( d ) control vs. patients with Child class A liver cirrhosis; ( e ) control vs. patients with Child class B+C liver cirrhosis; ( f ) patients with Child class A vs. B+C liver cirrhosis.

Article Snippet: Plasma levels of CTSL and CTSB were measured using the ELISA Kit (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, respectively) as per the manufacturer’s instructions.

Techniques: Clinical Proteomics, Comparison, Diagnostic Assay, Control

Journal: Clinical and Translational Gastroenterology

Article Title: Cathepsin L and B as Potential Markers for Liver Fibrosis: Insights From Patients and Experimental Models

doi: 10.1038/ctg.2017.25

Figure Lengend Snippet: CTSL and CTSB in relation to clinical parameters in patients with liver cirrhosis

Article Snippet: Plasma levels of CTSL and CTSB were measured using the ELISA Kit (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, respectively) as per the manufacturer’s instructions.

Techniques:

Journal: Clinical and Translational Gastroenterology

Article Title: Cathepsin L and B as Potential Markers for Liver Fibrosis: Insights From Patients and Experimental Models

doi: 10.1038/ctg.2017.25

Figure Lengend Snippet: Diagnostic accuracy of plasma CTSL, CTSB, or both for the assessment of liver cirrhosis

Article Snippet: Plasma levels of CTSL and CTSB were measured using the ELISA Kit (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, respectively) as per the manufacturer’s instructions.

Techniques: Diagnostic Assay, Clinical Proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Stress-resistant Translation of Cathepsin L mRNA in Breast Cancer Progression *

doi: 10.1074/jbc.M114.624353

Figure Lengend Snippet: CTSL expression in human breast cancer cells upon cell stress. A, immunoblot of hypoxia inducible factor-1α (HIF-1α) in cell lysates of MDA-MB 231 cultivated under hypoxic conditions. B, levels of phosphorylated eukaryotic initiation factor 2 (eIF2) in hypoxia and Torin-1-treated cells. Immunoblot for total and phosphorylated eIF2 in cell lysates of stress-treated MDA-MB 231. C, CTSL protein levels in stress-treated MDA-MB 231. D, abundance of total CTSL mRNA in stress-treated MDA-MB 231 (n = 3, mean ± S.E.).

Article Snippet: Cloning of Inducible Expression Vectors The different CTSL transcript variants (CTSL-A: catalog number SC124186; CTSL-A3: SC100267; Origene, Rockville, MD) were amplified by PCR with mutagenesis primers, generating two new restriction sites at the ends of the PCR product.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Stress-resistant Translation of Cathepsin L mRNA in Breast Cancer Progression *

doi: 10.1074/jbc.M114.624353

Figure Lengend Snippet: Stress resistance is mediated by the UTRs but is not dependent on the IRES element. A, inducible expression of single CTSL cDNA variants in murine PyMT cells. Overview of expressed variants. The variant CTSL-A contains the complete described IRES motif. CTSL-A3 corresponds to the shortest described splice variant. CTSL-CDS does not contain any UTR. B, treatment scheme to assess newly synthesized CTSL under stress conditions. C, production of CTSL protein under hypoxia upon expression of different cDNA variants. D, production of CTSL protein under Torin-1 upon expression of different cDNA variants. E, treatment scheme to assess newly synthesized CTSL under Torin-1 and nocodazole treatment. F, production of cathepsin L protein under Torin-1 and nocodazole upon expression of different cDNA variants. G, polyribosome association of CTSL-CDS mRNA under Torin-1 treatment with/without addition of nocodazole.

Article Snippet: Cloning of Inducible Expression Vectors The different CTSL transcript variants (CTSL-A: catalog number SC124186; CTSL-A3: SC100267; Origene, Rockville, MD) were amplified by PCR with mutagenesis primers, generating two new restriction sites at the ends of the PCR product.

Techniques: Expressing, Variant Assay, Synthesized

Journal: RSC Advances

Article Title: Design and synthesis of non-hydroxamate lipophilic inhibitors of 1-deoxy- d -xylulose 5-phosphate reductoisomerase (DXR): in silico , in vitro and antibacterial studies †

doi: 10.1039/d4ra05083e

Figure Lengend Snippet: The selected members of MCL displaying significant in vitro Ec DXR inhibition at 100 μM. The corresponding molecular docking scores and predicted binding energies are also provided. The metal chelating atoms are shown in bold red fonts

Article Snippet: The commercially available Ec DXR enzyme assay kits from Echelon Biosciences (Product number: K-2000C) were purchased and used to screen all compounds.

Techniques: In Vitro, Inhibition, Binding Assay

Journal: RSC Advances

Article Title: Design and synthesis of non-hydroxamate lipophilic inhibitors of 1-deoxy- d -xylulose 5-phosphate reductoisomerase (DXR): in silico , in vitro and antibacterial studies †

doi: 10.1039/d4ra05083e

Figure Lengend Snippet: The binding mode of 22b in the Ec DXR (PDB ID 3R0I ) active site. (A) The comparison of the predicted pose of 22b (pink ball and stick) with the docked pose of 1 (green-coloured ball and stick). The metal ion (purple-coloured ball) is chelated by the NA ring's O , O donor motif. As hypothesized, NA (F7) occupies the hydrophobic pocket B lined by Pro274 and Met276 (cyan coloured), whereas phosphonic acid of FSM and 22b occupy the hydrophilic region (B) a closer docked view of 22b overlaid with 1 and the cocrystallized ligand (moss green sticks) in the DXR active site (PDB code 3R0I ). The phosphonic groups and the 22b and 1 linker atoms are predicted to adopt a similar conformation.

Article Snippet: The commercially available Ec DXR enzyme assay kits from Echelon Biosciences (Product number: K-2000C) were purchased and used to screen all compounds.

Techniques: Binding Assay, Comparison