Notes

Organized recognition involving m6A-modified records in single-molecule and single-cell solution.
Women are born with an abundant but finite pool of ovarian follicles, which naturally and progressively decreased during their reproductive years until menstrual periods stop permanently (menopause). Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioral changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, around 45-50 years old, women are more likely to express mood disorders, anxiety, irritability and vasomotor symptoms. The current animal models of reproductive aging do not successfully replicate human perimenopause and the gradual changes that occur in this phase. While the traditional rat model of menopause involves ovariectomy or surgical menopause consisting of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones, natural or transitional menopause is achieved by the selective loss of ovarian folliclrimordial and primary follicles, but retains residual ovarian tissue before brain alterations that occurs in women in perimenopause. Low doses of VCD cause the selective destruction of the small preantral follicles of the ovary without affecting other peripheral tissues.Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. selleck inhibitor The exogenously added, neutral charged-FRET reagent can diffuse across the membrane and stay in the cytosol only once it is charged in the cytosol. When bacteria translocate from subcellular organelles (e.g., phagosomes) to the cytosol, the bacteria-associated β-lactamase cleaves the β-lactam ring, resulting in loss of FRET signal. Here we describe the fluorometer-based approach optimized for direct measurement of cytosolic translocation as a result of the EsxAB complex of Mycobacterium marinum in RAW264.7 cells.Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer's Disease (AD). We have recently developed a novel optically-responsive tool (the 'CofActor' system) that couples cof ilin and act in (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a light-activated, genetically encoded redox sensor that can be activated with precise spatial and temporal control. Here we describe a protocol for the expression and activation of the CofActor system in dissociated hippocampal neuron cultures prepared from newborn mice. Cultures were transfected with Lipofectamine on the fifth day in vitro (DIV5), then exposed to cellular stress inducing stimuli, leading to the formation of actin-cofilin rods that can be observed using live cell imaging techniques. The protocol described here allows for studies of stress-related cytoskeletal dysregulation in live neurons exposed to neurodegenerative stimuli, such as toxic Aβ42 oligomers. Moreover, expression of the sensor in neurons isolated from transgenic mouse models of AD and/or mice KO for proteins involved in AD can advance our understanding of the molecular basis of early cytoskeletal dysfunctions associated with neurodegeneration.Mammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases. Developing new tools to monitor mTOR spatiotemporal activation is crucial to better understand its roles in physiological and pathological conditions. However, the most widely used method to report mTOR activity relies on the quantification of specific mTOR-phosphorylated substrates by western blot. This approach requires cellular lysate preparation, which restricts the quantification to a single time point. Here, we present a simple protocol to study mTOR activity in living cells in real time using AIMTOR, an intramolecular BRET-based (bioluminescence resonance energy transfer) biosensor that we recently designed ( Bouquier et al., 2020 ). We describe transfection of AIMTOR in the C2C12 cell line and procedures to monitor BRET in a cell population using a plate reader and in single cells by microscopy. Importantly, this protocol is transposable to any cell line and primary cells. In addition, several subcellular compartment-specific versions of AIMTOR have been developed, enabling compartmentalized assessment of mTOR activity. This protocol describes how to use the sensitive AIMTOR biosensor to investigate mTOR signaling dynamics in living cells. Graphic abstract AIMTOR protocol overview from seeding cells to live BRET recording.
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