Determination of Caspase Activation by Western Blot.

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.

Subcellular Fractionation to Demonstrate Activation of Intrinsic Apoptotic Pathway.

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.

Transcription Factor-Binding Site Identification and Enrichment Analysis.

Transcription factors orchestrate complex regulatory networks of gene expression. A better understanding of the common transcription factors, and their shared interactions, among a set of coregulated or differentially expressed genes can provide powerful insights into the key pathways governing such expression patterns. Critically, such information must also be considered in the context of the frequency in which a transcription factor is present in a properly selected background, and in the context of existing evidence of gene and transcription factor interaction. Given the vast amount of publicly available gene expression data that can be further scrutinized by the user-friendly analysis tools described here, many useful insights are assuredly to be revealed. The proceeding methods for application of the analysis tool CiiiDER for transcription factor-binding site identification, enrichment analysis, and coregulatory factor identification should be applicable to any dataset comparing differential gene expression in response to various stimuli and gene coexpression datasets. These methods should assist the researcher in identifying the most relevant regulators within a gene set, and refining the list of targets for future study to those which may share biologically important regulatory networks.

Covid-19 pandemic and pregnancy.

At the end of 2019, a new coronavirus disease, COVID-19, emerged and quickly spread around the world. Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), the causative virus of this disease, belongs to the ß-coronavirus family, together with SARS and middle east respiratory syndrome, and has similar biological characteristics to these viruses. For obstetricians, the susceptibility and prognoses of pregnant women and the effects of the infection on the fetus have been the focus of attention; however, at present, the seriousness of the disease in pregnant women is not apparent, and COVID-19 does not increase the rate of miscarriage, stillbirth, preterm labor or teratogenicity. Even so, carriers might transmit SARS-CoV-2 to pregnant women. Thus, we must keep in mind that all medical personnel must understand and maintain standard precautions in their clinical and laboratory practices.

Marijuana-derived cannabinoids inhibit uterine endometrial stromal cell decidualization and compromise trophoblast-endometrium cross-talk.

Marijuana (cannabis) use by pregnant women in the United States is increasing and there is a dire need to understand the beneficial or harmful effects of cannabis during pregnancy. Uterine endometrial stromal cells are fibroblast-like cells that differentiate into secretory cells, a process called decidualization, to create a microenvironment conducive for placenta formation and early embryonic growth. In this study, using model human cell lines, we for the first time demonstrate that Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) inhibit endometrial stromal cell decidualization and have adverse effects on trophoblast-endometrium cross-talk.