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Application of extended non-invasive prenatal analyze inside pre-natal diagnosing fetuses with an increase of nuchal translucency.
Here, we present a protocol for assessing virus-infected cells using electron cryo-tomography (cryoET). It includes the basic workflows of seeding cells, plunge-freezing, clipping, cryo-focused ion beam milling (cryoFIB-milling), and cryoET, as well as two optional modules micropatterning and live-cell fluorescence microscopy. We use an A549 human cell line and the virus HAdV5-pIX-mcherry in this protocol, but the comprehensive workflow can be easily transferred to other cell types and different types of virus infection or treatment. For complete details on the use and execution of this protocol, please refer to Pfitzner et al. (2021).Here, we describe a biosensor to assess meiotic cohesin subunit Rec8 cleavage in mouse oocytes. We detail oocyte collection and microinjection of the mRNA expressing the biosensor. The biosensor is targeted to chromosomes and consists of two fluorophores flanking a Rec8 fragment containing separase cleavage sites. Cleavage leads to dissociation of one fluorophore from chromosomes, and the efficiency can be estimated by live imaging. We detail the use of this biosensor in mouse oocytes with or without Aurora B/C inhibitor. For complete details on the use and execution of this protocol, please refer to Nikalayevich et al. (2022).Due to the unique structure of circular RNAs, it is challenging to use traditional pulldown approaches. Here, we describe the design and use of a probe that spans the back splicing junction (BSJ), enabling interaction with circular RNAs. The probe repeats four times, allowing efficient and specific pulldown of circular RNAs and their binding partners. This protocol describes the steps for mouse cardiac fibroblast (MCF) cells; we have also verified the protocol in other cell types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).Rho family GTPases are central regulators of cytoskeletal dynamics controlled by guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). This protocol presents a workflow for a robust high-throughput compatible biosensor assay to analyze changes in Rho GTPase activity by these proteins in the native cellular environment. The procedure can be used for semi-quantitative comparison of GEF/GAP function and extended for analysis of additional modulators. The experimental design is applicable also to other monomolecular ratiometric FRET sensors. For complete details on the use and execution of this protocol, please refer to Müller et al. (2020).We describe a pipeline for optimized and streamlined multiplexed immunofluorescence-guided laser capture microdissection allowing the harvest of individual cells based on their phenotype and tissue localization for transcriptomic analysis with next-generation RNA sequencing. Here, we analyze transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages, and melanoma cells in non-dissociated metastatic melanoma tissue. While this protocol is described for melanoma tissues, we successfully applied it to human tonsil, skin, and breast cancer tissues as well as mouse lung tissues. For complete details on the use and execution of this protocol, please refer to Martinek et al. (2022).Phase-field simulation is a powerful tool for understanding lithium metal electrodeposition. This protocol outlines the process of numerically solving the phase-field equations using the MOOSE framework. Here, we describe steps to obtain the spatiotemporal distribution of major physical characteristics such as phase-field, ion concentration, overpotential, and driving force. Such an approach may help to reveal the underlying physics and kinetics of dendrite growth, while also providing design principles for suppressing lithium dendrites. For complete details on the use and execution of this protocol, please refer to Hong and Viswanathan (2018).Infectious clone technology is universally applied for biological characterization and engineering of viruses. This protocol describes procedures that implement synthetic biology advances for streamlined assembly of virus infectious clones. Here, I detail homology-based cloning using biological material, as well as SynViP assembly using type IIS restriction enzymes and chemically synthesized DNA fragments. The assembled virus clones are based on compact T-DNA binary vectors of the pLX series and are delivered to host plants by Agrobacterium-mediated inoculation. For complete details on the use and execution of this protocol, please refer to Pasin et al. Lorlatinib concentration (2017, 2018) and Pasin (2021).Kinases are indispensable signaling components. Radioactive-based phosphorylation assays are widely used but require specific protective equipment and safety trainings. Here, we present a Phos-tag-based non-radioactive kinase assay to study Arabidopsis kinase activities. We expressed and purified both kinase and substrate proteins from E. coli cells and then used the Phos-tag technology to detect the kinase activities under either different temperatures or chemical treatments. This non-radioactive approach is environmentally friendly and applicable to other kinases and organisms. For complete details on the use and execution of this protocol, please refer to Lin et al. (2022).People with chronic pain often fear and avoid movements and activities that were never paired with pain. Safe movements may be avoided if they share some semantic relationship with an actual pain-associated movement. This study investigated whether pain-associated operant responses (movements) can become categorically associated with perceptually dissimilar responses, thus motivating avoidance of new classes of safe movements-a phenomenon known as category-based avoidance generalization. Using a robotic arm, 2 groups were trained to categorize arm movements in different ways. Subsequently, the groups learned through operant conditioning that an arm movement from one of the categories was paired with a high probability of pain, whereas the others were paired with either a medium probability of pain or no pain (acquisition phase). Self-reported pain-related fear and pain expectancy were collected as indices of fear learning. During a final generalization test phase, the movements categorically related to those from the acquisition phase were made available but in the absence of pain. Results showed that the generalization of outcome measures depended on the categorical connections between arm movements, ie, the groups avoided and feared the novel generalization movement categorically related to the pain-associated acquisition movement, depending on how they had previously learned to categorize the movements. This suggests that operant pain-related avoidance can generalize to safe behaviors, which are not perceptually, but categorically, similar to a pain-associated behavior. This form of pain-related avoidance generalization is problematic because category-based relations can be extremely wide reaching and idiosyncratic. Thus, category-based generalization of operant pain-related avoidance merits further investigation.
Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance.

Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues.

Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were idy contribute to a better understanding of SCLC biology and the development of novel therapies.
We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.This article explores the reception of the contraceptive implant, Implanon, by healthcare workers and patients in family planning units in South Africa's public health sector. Based on observations conducted at public health facilities in the Eastern Cape Province, and on interviews with nurses and patients in the same province, the study explored real-world experiences of the implant. This article examines the strategies used by nurses to promote use of the device, and explores how patients themselves responded to a widescale, national rollout of the implant within government family planning services. The study examines the reception of Implanon in the context of the post-Apartheid era in South Africa, in which the vestiges of Apartheid-era healthcare provision, and lack thereof, continue to animate personal experiences of contraception.Poly(furfuryl alcohol) is a bio-based thermoset resin with a limited application portfolio due to its brittleness. Side ring-opening reactions that occur during polymerization lead to carbonyl moieties. Such unique self-generated functionality was exploited to generate tough and ductile materials via the creation of Schiff-based macromolecular architectures.The periaqueductal gray (PAG) represents a key target of projection neurons residing in the spinal dorsal horn. In comparison to lamina I spinoparabrachial neurons, little is known about the intrinsic and synaptic properties governing the firing of spino-PAG neurons, or whether such activity is modulated by neonatal injury. In this study, this issue was addressed using ex vivo whole-cell patch clamp recordings from lamina I spino-PAG neurons in adult male and female FVB mice after hindpaw incision at postnatal day (P)3. Spino-PAG neurons were classified as high output, medium output, or low output based on their action potential discharge after dorsal root stimulation. The high-output subgroup exhibited prevalent spontaneous burst firing and displayed initial burst or tonic patterns of intrinsic firing, whereas low-output neurons showed little spontaneous activity. Interestingly, the level of dorsal root-evoked firing significantly correlated with the resting potential and membrane resistance but not with the strength of primary afferent-mediated glutamatergic drive. Neonatal incision failed to alter the pattern of monosynaptic sensory input, with most spino-PAG neurons receiving direct connections from low-threshold C-fibers. Furthermore, primary afferent-evoked glutamatergic input and action potential discharge in adult spino-PAG neurons were unaltered by neonatal surgical injury. Finally, Hebbian long-term potentiation at sensory synapses, which significantly increased afferent-evoked firing, was similar between P3-incised and naive littermates. Collectively, these data suggest that the functional response of lamina I spino-PAG neurons to sensory input is largely governed by their intrinsic membrane properties and appears resistant to the persistent influence of neonatal tissue damage.
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