Provided are a method of screening an inhibitor of caspase activity by lipopolysaccharide and a method of screening a therapeutic agent for inflammatory diseases or sepsis using the same. Accordingly, it is possible to develop a caspase-4-specific inhibitor.

Disclosed are methods for diagnosing a myelodysplastic syndrome (MDS) in a subject. In some embodiments, the method involves assaying a sample from the subject to detect inflammasome activation, wherein an increase in inflammasome activation in the sample compared to a control is an indication of MDS in the subject. The disclosed methods can further involve treating the subject for MDS if an increase in inflammasome activation is detected.

Provided herein are methods and devices related to inducing a population of self-renewing or senescent stem cells, to produce one or more beneficial factors for the treatment of a disease or disorder in an individual. Also provided are compositions and methods for inducing senescence, useful for inducing senescence in a population of stem cells, in order to produce one or more beneficial factors for the treatment of a disease or disorder in an individual. Methods and devices to control and customize the production of the beneficial factors for the requirements of a disease or disorder being treated are described. Also provided are factor production units for the production of the beneficial factors, and devices for the delivery of the beneficial factors to an individual in need.

Methods and materials for specific imaging of active enzyme in a live or intact cell are disclosed. The enzyme of interest tagged to reporter protein (donor) is exogenously expressed in a cell. The conversion of proenzyme to active enzyme (containing reporter protein) is achieved upon applying an appropriate stimulus to the target cells. The activated enzyme is labelled with an activity-based probe carrying a fluorophore (acceptor). The covalent labelling of active enzyme by the activity-based probe creates a FRET pair which provides the opportunity to exquisitely image the function of an “active enzyme”. This method is used for specific imaging of the function of active caspase-3,-7,-8,-9 and cathepsin-B and also for profiling of inhibitors of caspases and cathepsin B.

An activity sensor sensitive to enzymes indicative of tissue condition are co-administered with control or normalizing activity sensors providing levels of the same enzyme in other tissues or levels of control enzymes indicative of assay success. Levels of control reporters can be used to normalize activity sensor data across samples such as in analyte velocity analyses. Control reporters can also be used to differentiate localized enzyme activity from systemic activity and to confirm activity sensor localization or to troubleshoot activity sensor uptake problems. Activity sensors and controls sensitive to immunological enzymes are particularly useful in assessing immuno-oncology treatments.

Herein is reported a method for determining the binding affinity of the binding sites of a bivalent full length antibody of the human IgG1 subclass to a homo-multimeric antigen comprising the steps of i) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis at a pH of from pH 7.5 to pH 8.5, in the presence of a reducing agent, at a temperature of from 30° C. to 42° C., for time of from 10 min. to 240 min. to cleave the antibody into Fabs and Fc-region, and ii) determining the binding affinity of the Fabs of the antibody for its antigen using a surface plasmon resonance method by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method and therewith determining the binding affinity of the binding sites of the bivalent full length antibody of the human IgG1 subclass.

Herein is reported a method for determining the binding interaction with a multimeric antigen of an antibody of the human IgG1 subclass that has at least two binding sites specifically binding to the antigen comprising the steps of 1) determining the binding affinity of the antibody for the multimeric antigen, and 2) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis under conditions and for a time sufficient to cleave the antibody into Fabs and Fc-region, and determining the binding affinity of the Fabs of the antibody for the multimeric, whereby the binding affinity of the antibody to the multimeric antigen to be affinity-driven if the binding affinity determined in both steps are comparable and to be avidity-driven if the binding affinity determined in both steps are different.

The present invention relates to the use as a biomarker of the active form of a human caspase protein, preferably the human caspase-4 or caspase-1, or of the active form of the protein encoded by an orthologue gene of the human caspase protein, preferably by an orthologue gene of the human caspase-4, for example the murine caspase-11 protein, in a method of diagnosis and/or prognosis and/or of monitoring the progression of a tumor, particularly lung cancer.

Provided herein are compounds, enzyme substrates, compositions, kits, uses, and methods for detecting the presence or absence of a caspase enzyme, measuring the activity of a caspase enzyme, or detecting the presence or absence of apoptosis. The detection or measurement can occur through intracellular cleavage of a compound or enzyme substrate, which can lead to an increase in fluorescence, e.g., in the violet or red channel, through liberation of a nucleic acid binding dye from a peptide, such as liberation of a DNA-binding dye from a negatively charged peptide comprising a sequence recognized and cleaved by a caspase.

Provided are improved methods for identifying the substrate recognition specificity or activity of a protease, convertase (sortase), or kinase. In some embodiments, methods are provided for identifying the endogenous protease or convertase cleaving patterns (e.g., cleaveOme) inside the secretory pathway of a living cell. Select embodiments involve aspects of yeast endoplasmic reticulum sequestration screening and next generation sequencing. Methods of producing polypeptides in Kex2 knockout yeast are also provided.