CDCA8's function as an oncogene, promoting HCC cell proliferation through cell cycle regulation, was observed in our study, suggesting its utility in HCC diagnostics and treatment.
For the synthesis of pharmaceuticals and high-value fine chemicals, chiral trifluoromethyl alcohols are highly valuable intermediates. This research focused on the initial biocatalytic application of the novel isolate Kosakonia radicincitans ZJPH202011 to synthesize (R)-1-(4-bromophenyl)-2,2,2-trifluoroethanol ((R)-BPFL) with promising enantioselectivity. Through refined fermentation procedures and bioreduction adjustments in an aqueous buffer environment, the substrate concentration of 1-(4-bromophenyl)-22,2-trifluoroethanone (BPFO) was doubled, rising from 10 mM to 20 mM, and the enantiomeric excess (ee) of (R)-BPFL correspondingly enhanced from 888% to 964%. In order to amplify the effectiveness of biocatalytic reactions, natural deep eutectic solvents, surfactants, and cyclodextrins (CDs) were introduced individually as co-solvents to the reaction mixture, thereby augmenting mass transfer. L-carnitine lysine (C Lys, with a molar ratio of 12), Tween 20, and -CD exhibited a higher (R)-BPFL yield compared to other similar co-solvents. Subsequently, due to the outstanding performance of both Tween 20 and C Lys (12) in elevating BPFO solubility and enhancing cellular permeability, a combined reaction system utilizing Tween 20/C Lys (12) was implemented for the effective bioproduction of (R)-BPFL. By optimizing the crucial components within the synergistic BPFO bioreduction reaction system, BPFO loading reached a maximum of 45 mM, resulting in a 900% yield after only 9 hours. In contrast, a neat aqueous buffer yielded only 376% under similar conditions. The first report on K. radicincitans cells introduces them as a novel biocatalyst applied to the preparation of (R)-BPFL. The developed synergistic reaction system, integrating Tween 20 and C Lys, has substantial promise for the production of various chiral alcohols.
Planarians have demonstrated a potent influence on both stem cell research and the study of regeneration. Apocynin The mechanistic investigation toolkit has seen notable expansion over the last ten years; however, the necessary genetic tools for transgene expression remain inadequate. We detail here methodologies for in vivo and in vitro mRNA transfection within the Schmidtea mediterranea planarian species. The commercially available TransIT-mRNA transfection reagent is crucial in these methods for efficiently transporting mRNA encoding a synthetic nanoluciferase reporter. Employing a luminescent reporter effectively eliminates the substantial autofluorescent background within planarian tissues, enabling precise quantitative measurements of protein expression levels. Collectively, our approaches allow for the expression of heterologous reporters in planarian cells, establishing a basis for future transgenic method development in this area.
The brown coloring of freshwater planarians is attributable to the ommochrome and porphyrin body pigments, manufactured by specialized dendritic cells, which are located immediately beneath the epidermis. genetic profiling During embryonic development and regeneration, the emergence of new pigment cells contributes to the progressive darkening of newly formed tissue. On the other hand, significant exposure to light triggers the demise of pigment cells through a porphyrin-based process, reminiscent of the light sensitivity mechanisms seen in rare human disorders, porphyrias. We present a novel program for quantifying the relative levels of pigments in living creatures via image-processing algorithms. This program is then used to examine the modifications of bodily pigmentation due to light exposure. This tool aids in the further characterization of genetic pathways that govern pigment cell differentiation, ommochrome and porphyrin production, and the photosensitivity stemming from porphyrins.
Planarians, an exemplary model organism, are utilized in the study of regeneration and homeostasis. Knowledge of planarian cellular homeostasis is crucial to understanding their capacity for change. Whole mount planarians allow for the quantification of both apoptotic and mitotic rates. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a technique used to detect cell death via apoptosis, specifically by identifying fragmented DNA. This chapter describes a protocol for scrutinizing apoptotic cells in planarian paraffin sections, providing enhanced cellular visualization and quantification capabilities compared with the whole-mount approach.
This protocol emphasizes the recently-developed planarian infection model, focusing on host-pathogen interactions during fungal infections. immune sensor The infection of Schmidtea mediterranea, the planarian, with the human fungal pathogen Candida albicans is meticulously detailed. This easily replicated model system provides a swift visual method to monitor tissue damage across different infection durations. This model system, while primarily designed for Candida albicans, is likely applicable to other infectious agents of interest.
Imaging living animals allows researchers to understand the relationship between metabolic processes and their underlying cellular structures, or associated larger functional units. Planarian in vivo imaging over extended timeframes was enabled by our combined and optimized adaptation of existing protocols, resulting in a cost-effective and easily reproducible approach. Immobilization using low-melting-point agarose circumvents the need for anesthesia, averting any influence on the animal's imaging-related function or physical state, and allows for the subsequent recovery of the organism. For the purpose of imaging the highly dynamic and rapidly altering reactive oxygen species (ROS) inside living creatures, we implemented the immobilization procedure. The in vivo study of reactive signaling molecules, including the mapping of their location and dynamics across diverse physiological states, is fundamental to comprehending their roles in developmental processes and regeneration. In this current protocol, we provide the details of the immobilization and ROS detection procedures. To confirm the signal's specificity, we used pharmacological inhibitors alongside signal intensity measurements, differentiating it from the planarian's intrinsic autofluorescence.
The long-established practice of employing flow cytometry and fluorescence-activated cell sorting to roughly isolate cell subpopulations in Schmidtea mediterranea is well-recognized. In this chapter, we illustrate a technique for immunostaining live planarian cells, utilizing either single or double staining protocols, using mouse monoclonal antibodies specific for S. mediterranea plasma membrane antigens. Employing this protocol, live cell populations can be categorized based on their membrane signatures, permitting a detailed analysis of S. mediterranea cells, and opening up possibilities for subsequent applications including transcriptomics and cell transplantation, all at a single-cell level.
There is an escalating need for highly viable cells derived from the Schmidtea mediterranea species. Within this chapter, a cell dissociation approach is detailed, relying on papain (papaya peptidase I). Cells with complex morphologies are effectively dissociated by this cysteine protease, which boasts broad specificity and leads to a notable improvement in both the yield and viability of the separated cell suspension. Cell dissociation, specifically with papain, is preceded by a treatment to remove mucus. This pre-treatment significantly improved cell dissociation yield, employing any dissociation technique. Among downstream applications, live immunostaining, flow cytometry, cell sorting, transcriptomics, and single-cell level cell transplantation are particularly well-suited to the use of papain-dissociated cells.
Enzymatic methods for dissociating planarian cells are a well-established and widely used technique in the field. Their application in transcriptomics, and particularly in single-cell studies, unfortunately, raises concerns about the dissociation of live cells, which can lead to stress responses within the cellular machinery. This protocol details planarian cell dissociation using ACME, a dissociation-fixation method reliant on acetic acid and methanol. Modern single-cell transcriptomic techniques are applicable to ACME-dissociated cells, which can be both fixed and cryopreserved.
Sorting specific populations of cells by fluorescence or physical properties is a long-standing and widely practiced method of flow cytometry. Flow cytometry has proven indispensable in the study of planarians, species resistant to transgenic methods, providing an alternative approach to investigate stem cell biology and lineage tracing during the regeneration process. In planarian research, flow cytometry applications have progressed significantly, from the initial use of broad Hoechst staining to isolate cycling stem cells to the more nuanced and functional methodologies involving vital dyes and surface antibody markers. We refine the classic DNA-labeling Hoechst staining by coupling it with pyronin Y staining to identify RNA within the same sample. Although Hoechst staining alone permits the isolation of stem cells situated within the S/G2/M phases of cellular division, the inherent diversity present amongst the stem cell population exhibiting a 2C DNA content remains unresolved. This protocol, through the assessment of RNA levels, enables the categorization of this stem cell population into two subgroups: G1 stem cells with a relatively high RNA level and a slow-cycling population with a lower RNA level, which we identify as RNAlow stem cells. We also describe the procedure for combining the RNA/DNA flow cytometry protocol with EdU labeling, including an optional step for immunostaining prior to sorting with the pluripotency marker TSPAN-1. A novel staining approach and instances of combinatorial flow cytometry applications are integrated into the existing flow cytometry toolkit for investigating planarian stem cells, as detailed in this protocol.