Rothbondesen4729

Partial differential equation (PDE) systems describing the nonlinear spatiotemporal behavior are derived by coupling fixed point dynamics with species diffusion.Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect on downstream target genes and cellular physiology. Biochemical assays, including reporter assays, AGO2 pulldown, ribonucleoprotein pulldown, and biotin-labeled RNA pulldown, are used to capture the association of miRNAs and RBPs with circRNAs. Only a few studies have used circRNA pulldown assays to capture the associated miRNAs and RBPs under physiological conditions. In this detailed protocol, the circRNA of interest (e.g., circHipk2) was captured using a biotin-labeled antisense oligo (ASO) targeting the circHipk2 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads. The specific enrichment of circRNA was analyzed using reverse transcription quantitative PCR (RT-qPCR). Furthermore, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA. In summary, the specific pulldown of circRNA using this quick and easy method makes it a useful tool for identifying and validating circRNA interaction with specific miRNAs and RBPs.The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. selleck In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract Flowchart for the analysis of the effects of HK2 detachment from MAMs.Single-cell technologies have allowed high-resolution profiling of tissues and thus a deeper understanding of tissue homeostasis and disease heterogeneity. Understanding this heterogeneity can be especially important for tailoring treatments in a patient-specific manner. Here, we detail methods for preparing human cartilage tissue for profiling via cytometry by time-of-flight (cyTOF). We have previously utilized this method to characterize several rare cell populations in cartilage, including cartilage-progenitor cells, inflammation-amplifying cells (Inf-A), and inflammation-dampening cells (Inf-D). Previous bio-protocols have focused on cyTOF staining of PBMCs. Therefore, here we detail the steps unique to the processing of human cartilage and chondrocytes. Briefly, cartilage tissue is digested to release individual chondrocytes, which can be expanded and manipulated in culture. These cells are then collected and fixed in preparation for cyTOF, followed by standard staining and analysis protocols.The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This article presents an optimized protocol for pipette cleaning, which enables it to be performed quickly ( 90%) for over 100 reuses of a single pipette. For most patch-clamp experiments ( less then 30 whole-cell recordings per day), this method enables a single pipette to be used for an entire day of experiments. In addition, we describe easily implementable hardware and software as well as troubleshooting tips to help other labs implement this method in their own experiments. Pipette cleaning enables patch-clamp experiments to be performed with higher throughput, whether manually or in an automated fashion, by eliminating the tedious and skillful task of replacing pipettes. From our experience with numerous electrophysiology laboratories, pipette cleaning can be integrated into existing patch-clamp setups in approximately one day using the hardware and software described in this article. Graphic abstract Rapid enzymatic cleaning for reuse of patch-clamp pipettes.Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports of their biological functions continue to expand; however, attaining quantitative estimates with high spatiotemporal resolution in single cells poses a major research challenge. Here, we detail the use of the genetically encoded pH-sensitive pHluorin reporter expressed in Arabidopsis thaliana pollen tubes to assess cytosolic measurements with calibration to provide actual pH values. This technique enabled us to identify critical phenotypes and establish the importance of tip-focused pH gradient for pollen tube growth, although it can be adapted to other experimental systems.Clonal hematopoiesis is a state in which substantial fraction of hematopoietic stem cells acquire mutations in specific driver genes and expand in the absence of an overt hematological malignancy. Recent clinical studies have shown that clonal hematopoiesis increases likelihood of hematological malignancy and cardiovascular disease. While clinical studies have identified countless candidate driver genes associated with clonal hematopoiesis, experimental studies are required to evaluate causal and mechanistic relationships with disease processes. This task is technically difficult and expensive to achieve with traditional genetically engineered mice. The versatility and programmability of CRISPR-Cas system enables investigators to evaluate the pathogenesis of each mutation in experimental systems. Technical refinements have enabled gene editing in a cell type specific manner and at a single base pair resolution. Here, we summarize strategies to apply CRISPR-Cas system to experimental studies of clonal hematopoiesis and concerns that should be addressed.We study the optical-pump induced ultrafast transient change of x-ray absorption at L 3 absorption resonances of the transition metals Ni and Fe in the Fe0.5Ni0.5 alloy. We find the effect for both elements to occur simultaneously on a femtosecond timescale. This effect may hence be used as a handy cross correlation scheme, providing a time-zero reference for ultrafast optical-pump soft x-ray-probe measurement. The method benefits from a relatively simple experimental setup as the sample itself acts as time-reference tool. In particular, this technique works with low flux ultrafast soft x-ray sources. The measurements are compared to the cross correlation method introduced in an earlier publication.Purpose We provide a review of the key computed tomography (CT) technologies developed since the late 1980s and offer an overview of one of the future technologies under development. The focus of this review is mainly on the hardware and system development. The topics on the historical event linked to the early days of CT development and other innovations that contributed to the CT development, such as advanced image reconstruction techniques, are covered by companion papers in this special issue. Approach The review is divided into five major sections, each linked to a key innovation in CT helical spiral data acquisition, multi-slice CT, wide-cone CT, dual-source CT, and spectral CT. Given the limited scope of this review, only one of the future technologies, photon-counting CT, is discussed in detail. Whenever possible, both theory of operation and clinical examples are provided. Results Theoretical analyses, phantom results, and clinical examples clearly demonstrate the efficacy and clinical relevancy of five historical technology developments and one future technology in CT. These technologies have improved and will continue to improve CT performance in terms of isotropic volume coverage, improved temporal resolution, and material differentiation and characterization capabilities. Conclusions Over the past 30 years, technological developments of CT have contributed to the success of CT in many clinical applications such as trauma, oncology, cardiac imaging, and stroke. Advanced clinical applications have and will continue to demand more advanced technology development.Significance Time domain diffuse correlation spectroscopy (TD-DCS) can offer increased sensitivity to cerebral hemodynamics and reduced contamination from extracerebral layers by differentiating photons based on their travel time in tissue. We have developed rigorous simulation and evaluation procedures to determine the optimal time gate parameters for monitoring cerebral perfusion considering instrumentation characteristics and realistic measurement noise. Aim We simulate TD-DCS cerebral perfusion monitoring performance for different instrument response functions (IRFs) in the presence of realistic experimental noise and evaluate metrics of sensitivity to brain blood flow, signal-to-noise ratio (SNR), and ability to reject the influence of extracerebral blood flow across a variety of time gates to determine optimal operating parameters. Approach Light propagation was modeled on an MRI-derived human head geometry using Monte Carlo simulations for 765- and 1064-nm excitation wavelengths. We use a virtual probe with a source-detector separation of 1 cm placed in the pre-frontal region. Performance metrics described above were evaluated to determine optimal time gate(s) for different IRFs. Validation of simulation noise estimates was done with experiments conducted on an intralipid-based liquid phantom. Results We find that TD-DCS performance strongly depends on the system IRF. Among Gaussian pulse shapes, ∼ 300    ps pulse length appears to offer the best performance, at wide gates (500 ps and larger) with start times 400 and 600 ps after the peak of the TPSF at 765 and 1064 nm, respectively, for a 1-s integration time at photon detection rates seen experimentally (600 kcps at 765 nm and 4 Mcps at 1064 nm). Conclusions Our work shows that optimal time gates satisfy competing requirements for sufficient sensitivity and sufficient SNR. The achievable performance is further impacted by system IRF with ∼ 300    ps quasi-Gaussian pulse obtained using electro-optic laser shaping providing the best results.