Weberlundsgaard3813

We propose that regular moderate intensity exercise should be considered as part of a combinatorial approach including widespread hygiene initiatives, properly planned and well-executed social distancing policies, and use of efficacious facial coverings like N95 respirators. Studies discerning COVID-19 pathogenesis mechanisms, transfer dynamics, and individual responses to pharmaceutical and adjunct treatments are needed to reduce viral transmission and bring an end to the COVID-19 pandemic.Selective identification of newly synthesized proteins is challenging because all proteins, both existing and nascent, have the same amino acid pool and are therefore chemically indistinguishable. L-homopropargylglycine is an amino acid analog of methionine containing an alkyne moiety that can undergo a classic click chemical reaction with azide containing Alexa Fluor. Here, we present an integrated tool based on immunofluorescence staining to accurately trace and localize the newly synthesized protein in isolated primary mouse hepatocytes. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021).Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with Nuclear Export Mediator Factor (NEMF), a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative Polymerase chain reaction (PCR). Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers. For complete details on the use and execution of this protocol, please refer to Udagawa et al. (2021).Lipid-filled adipocytes are incompatible with droplet-based single-cell methods, such as 10x Genomics-based technology, thus restricting droplet-based single-cell analyses of adipose tissues to the stromal vascular fraction. To overcome this limitation and obtain cellular and molecular insight into adipose tissue composition and plasticity, single-nucleus sequencing-based technologies can be applied. Here, we provide an optimized protocol for nuclei isolation from mouse adipose tissues suitable for single-nucleus RNA sequencing. This allows for transcriptomic profiling of the entire adipose tissue at single-cell resolution. For complete details on the use of this protocol, please refer to Sárvári et al., 2021.Genetic manipulation in mice allows the discovery of gene function and biological mechanisms in vivo. The widely used Cre/LoxP system usually takes months to years especially when starting with the production of floxed alleles of a new gene of interest (GOI). Here, we describe a protocol using the CRISPR-Cas9 system to acutely inactivate the GOI in adult mice. This protocol enables hepatocyte-specific gene editing within 4 weeks in adult mice and avoids compensatory effects of traditional gene inactivation initiated during various developmental stages. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).The use of germ-free mice is integral to the understanding of host-gut microbiome relationships. Such models rely on faithful replication of the donor microbiome to establish causal effects of the gut microbiota on host pathophysiology. This protocol describes the preparation and transfer of donor microbiota, focusing on strict anaerobic processing methods and multiple instillations by gavage for optimal gut microbiota recovery. For complete details on the generation and use of this protocol, please refer to Choo and Rogers (2021).Quantifying differential genome occupancy by chromatin immunoprecipitation (ChIP) remains challenging due to variation in chromatin fragmentation, immunoprecipitation efficiencies, and intertube variability. In this protocol, we add heterologous spike-ins from Drosophila chromatin as an internal control to the mice chromatin before immunoprecipitation to normalize for technical variation in ChIP-qPCR or ChIP-seq. The choice of spike-in depends on the evolutionary conservation of the protein of interest and the antibody used. For complete details on the use and execution of this protocol, please refer to Greulich et al. (2021).13C nuclear spin hyperpolarization can increase the sensitivity of detection in an MRI experiment by more than 10,000-fold. 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13C label exchange between injected [1-13C]pyruvate and the endogenous tumor lactate pool can be used clinically to assess tumor grade and response to treatment. We describe here an experimental protocol for using this technique in patient-derived and established cell line xenograft models of breast cancer in the mouse. For complete details on the use and execution of this protocol, please refer to Ros et al. (2020).The identification and validation of circulating small non-coding RNA (sncRNA) as biomarkers for disease diagnosis, staging, and response to novel therapies is still a compelling challenge. Pre-analytical variables, such as storage temperature or blood hemolysis, and different analytical approaches affect sncRNA stability, detection, and expression, resulting in discrepancies among studies. Here, we report a systematic standardized protocol to reproducibly analyze circulating sncRNAs, employing high-throughput sncRNA sequencing and qRT-PCR validation, from 200 μL of human plasma samples. For details on the use and execution of this protocol, please refer to Ventriglia et al. (2020), Sebastiani et al. (2017), and Dotta et al. (2018).Protein-peptide interactions are involved in many fundamental cellular functions and constitute promising drug targets. Selleckchem U0126 Here, we provide a detailed protocol for the cost-effective preparation of a cellulose-based solid support for synthesis of nanoscale to micromolar-scale peptide libraries. Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating in vitro assessment and development of high-affinity binders. For complete details on the use and execution of this protocol, please refer to Schulte et al., (2020).Lysine succinylation is a recently discovered posttranslational modification that plays critical roles in metabolism, epigenetic signaling, and human diseases. To investigate the effects of site-specific histone lysine succinylation on nucleosome dynamics requires the generation of homogeneously modified histones, which is a significant challenge. Here, we report a protocol for the rapid site-specific installation of a succinyl lysine analog onto histone. We then use a Förster resonance energy transfer approach to characterize the impact on nucleosome dynamics. For complete details on the use and execution of this protocol, please refer to Jing et al. (2018).Covalent DNA-protein crosslinks (DPCs) have emerged as pervasive sources of genome instability. DPCs are targeted for repair by DNA-dependent proteases of the Wss1/SPRTN family. However, understanding how these enzymes achieve specificity has been hampered by the lack of suitable in vitro model substrates. Here, we describe the generation of defined protein-oligonucleotide conjugates as DPC model substrates, which enable the analysis of DPC proteases in activity and binding assays. For complete details on the use and execution of this protocol, please refer to Reinking et al. (2020).We describe a genome-wide DNA double-strand break (DSB) mapping technique, Break-seq. In this protocol, we provide step-by-step instructions for cell embedment in agarose, in-gel DSB labeling and subsequent capture, followed by standard Illumina library construction and sequencing. We also provide the framework for sequence data processing and DSB peak identification. Finally, we present a custom-designed 3D-printed device for processing agarose-embedded DNA samples. The protocol is applicable to Saccharomyces cerevisiae, as well as mammalian suspension, adherent, and 3D organoid cell cultures. For complete details on the use and execution of this protocol, please refer to Hoffman et al. (2015) and Chakraborty et al. (2020).Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species. For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020).Maceration techniques which promote the extraction of color pigments and tannin from grapes are often sought in Pinot noir winemaking to optimise color stability; alternatively, exogenous grape tannins may be included during fermentation. To examine the effect of seed-derived tannins and the use of pectolytic enzymes on color development in wines, conventional must preparations of Vitis vinifera L. cv Pinot noir grapes were compared with wines made using a supplementary addition of either a commercial seed tannin product or previously fermented seeds, while in a complementary experiment, seeds were sequentially removed during fermentation. After 6 months bottle aging, wines supplemented with either a commercial seed tannin solution (0.4 g/L), or fermented seeds (20% w/w seeds) had from 60% to 95% higher tannin concentration than the untreated wine, and up to 60% more monomeric anthocyanins. Conversely, when a third of the seeds were removed from the fermenting wine, the concentration of both tannin and non-bleachable pigments was 20-30% lower than in untreated wines and the wine hue had more red-purple tones. Exploration of the use of pectolytic enzymes in conjunction with seed removal was also found to have a significant impact on wine color parameters. Further insights on the timing of egress of tannin precursors from seeds was obtained from histochemical examination of the seeds that had been removed during alcoholic fermentation.Mitochondrial medicine has experienced significant progress in recent years and is expected to grow significantly in the near future, yielding many opportunities to translate novel bench discoveries into clinical medicine. Multiple lines of evidence have linked mitochondrial dysfunction to a variety of metabolic diseases, including diabetic nephropathy (DN). Mitochondrial dysfunction presumably precedes the emergence of key histologic and biochemical features of DN, which provides the rationale to explore mitochondrial fitness as a novel therapeutic target in patients with DN. Ultimately, the success of mitochondrial medicine is dependent on a better understanding of the underlying biology of mitochondrial fitness and function. To this end, recent advances in mitochondrial biology have led to new understandings of the potential effect of mitochondrial dysfunction in a myriad of human pathologies. We have proposed that molecular mechanisms that modulate mitochondrial dynamics contribute to the alterations of mitochondrial fitness and progression of DN.