Notes

Repeat-swap homology custom modeling rendering associated with extra active transporters: updated standard protocol and idea associated with elevator-type mechanisms.
Intracellular delivery of therapeutic proteins remains a challenge for the success of protein-mediated disease treatment. We herein develop a robust nanoplatform made with a TME-pH responsive Meo-PEG-b-PPMEMA polymer and a cationic lipid-like compound G0-C14 for in vivo delivery of cytotoxic saporin and breast cancer therapy. This nanoplatform could respond to a TME pH to rapidly release saporin/G0-C14 complexes, which could significantly improve the uptake of cytosolic saporin by tumor cells and subsequent endosomal escape, thereby leading to an effective inhibition of tumor growth.The increasing incidence of hepatitis C viral (HCV) infection worldwide is a major concern for causing liver cirrhosis and hepatocellular carcinoma, leading to increased morbidity and mortality. Currently, the prevalence of HCV infection is estimated to be in the range of ∼3%. According to the World Health Organization, antiviral drugs can cure more than 95% of the HCV infected cases, if timely diagnosis and treatment are provided. The gold standard RT-qPCR assay is expensive and requires a minimum turnaround time of 4 h. Hence, a rapid and cost-effective detection assay that can be used even in resource-limited settings would be highly beneficial for mass level screening. Herein, we present an Au NP based facile strategy for rapid, early-stage, and sensitive detection of HCV RNA in clinical samples which avoids thiol tagging to the antisense oligonucleotide and expensive infrastructure. This technique utilizes the hybridization of a short-chain antisense oligonucleotide from the 5' untranslated region (UTR) of the viral genome with the isolated HCV RNA samples. Using a specific sequence universal to all HCV genotypes-obtained through the NCBI BLASTn tool-the HCV positive samples have stabilized the citrate capped Au NPs against salt-induced aggregation, retaining their red color. On the other hand, negative controls, including HBV and HIV positive samples, do not stabilize the Au NPs, which results in purple coloration. Besides, the assay is successfully tested with a RNase A enzyme-treated HCV positive sample, which does not stabilize the Au NPs, thus confirming the role of the viral HCV RNA in this strategy. This Au NP based assay takes about 30 min using the viral RNA isolate and has high specificity with a detection limit of 100 IU mL-1, which is ∼10 fold lower than the state-of-the-art Au NP based strategy.The direct quantification of programmed death-ligand 1 (PD-L1) as a biomarker for cancer diagnosis, prognosis and treatment efficacy is an unmet clinical need. BAY-1895344 Herein, we demonstrate the first report of rapid, ultrasensitive and selective electrochemical detection of PD-L1 directly in undiluted whole blood using modified gold-coated magnetic nanoparticles as "dispersible electrodes" with an ultralow detection limit of 15 attomolar and a response time of only 15 minutes.The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN2) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 °C achieved with the use of LN2 protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN2 to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN2 in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.
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