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Prevention of bile duct injury and vasculo-biliary injury while performing laparoscopic cholecystectomy (LC) is an unsolved problem. Clarifying the surgical difficulty using intraoperative findings can greatly contribute to the pursuit of best practices for acute cholecystitis. In this study, multiple evaluators assessed surgical difficulty items in unedited videos and then constructed a proposed surgical difficulty grading.<br />We previously assembled a library of typical video clips of the intraoperative findings for all LC surgical difficulty items in acute cholecystitis. Fifty-one experts on LC assessed unedited surgical videos. Inter-rater agreement was assessed by Fleiss's κ and Gwet's agreement coefficient (AC).<br />Except for one item ("edematous change"), κ or AC exceeded 0.5, so the typical videos were judged to be applicable. The conceivable surgical difficulty gradings were analyzed. According to the assessment of difficulty factors, we created a surgical difficulty grading system (agreement probability=0.923, κ=0.712, 90% CI 0.587-0.837; AC<br />=0.870, 90% CI 0.768-0.972).<br />The previously published video clip library and our novel surgical difficulty grading system should serve as a universal objective tool to assess surgical difficulty in LC.<br />The previously published video clip library and our novel surgical difficulty grading system should serve as a universal objective tool to assess surgical difficulty in LC.<br />To assess the benefits and harms associated with biopsychosocial rehabilitation in patients with inflammatory arthritis (IA) and osteoarthritis (OA).<br />We performed a systematic review and meta-analysis. Data were collected through electronic searches of Cochrane CENTRAL, Medline, Embase, PsycINFO, and CINAHL databases up to March 2019. Trials examining the effect of biopsychosocial rehabilitation in adults with IA and/or OA were considered eligible, excluding rehabilitation adjunct to surgery. The primary outcome for benefit was pain, and total withdrawals for harm.<br />Of the 27 trials meeting the eligibility criteria, 22 trials (3,750 participants) reported sufficient data to be included in the quantitative synthesis. For patient reported outcome measures, biopsychosocial rehabilitation was slightly superior to control for pain relief (SMD -0.19 [95%CI, -0.31 to -0.07]), had a small effect on patient global (SMD -0.13 [95%CI, -0.26 to -0.00]), with no apparent effect on health-related quality of life, fatice in the estimates of effect.Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substmetry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 Preparation of N-(ε)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2 In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3 In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4 In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5 In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.<br />Salivary gland epithelial cells (SGECs) are key cellular drivers in the pathogenesis of primary Sjӧgren's Syndrome (pSS); however, the mechanisms sustaining SGECs activation in pSS remain undetermined. The aim of this study is to determine the role of autophagy in the survival and activation of SGECs in pSS.<br />Primary SGECs isolated from minor salivary glands (SG) of patients with pSS or sicca syndrome were evaluated by flow-cytometry, immunoblotting, and immunofluorescence to assess autophagy (autophagic-flux, LC3IIB, p62, LC3B+/LAMP1+ staining), apoptosis (annexin V/PI, Caspase-3) and activation (ICAM, VCAM). Focus score and germinal centers presence was assessed in SG from the same patients to correlate with histological severity. Human salivary gland (HSG) cells were stimulated in vitro with PBMCs and serum from pSS patients in the presence or absence of autophagy inhibitors to determine changes in autophagy and epithelial cell activation.<br />SGECs from pSS patients (n=24) exhibited increased autophagy (t.Amplification of genomic DNA fragments by PCR is necessary for plant molecular biology approaches such as genotyping. While this is a routine molecular technique in a modern laboratory, there are still significant hurdles when analyzing a large number of samples or collecting and storing samples while in the field. Because PCR amplification directly from plant tissue is often unsuccessful due to various inhibitors, genomic DNA purification is usually required, which involves laborious and time-consuming procedures or costly materials, particularly when using commercial kits. These undermine scalability and use in less-equipped settings. In addition, plant tissues and purified DNA need to be stored under proper conditions to avoid degradation. Here, we describe a low-cost, high-throughput PCR method to amplify genomic DNA fragments from plant tissue pounded to cellulose-based filter paper without the need for DNA purification or special equipment for sample storage. In this protocol, a small punch of plant tissue is pounded to a commercially available or homemade DNA storage card and directly placed into a PCR mixture containing Tween-20, a non-ionic detergent, directly followed by PCR. [https://www.selleckchem.com/products/5-n-ethyl-n-isopropyl-amiloride-eipa.html selleck chemicals llc] We also describe the steps to prepare a homemade DNA storage card, which is easy to make and can be stored with plant tissue at room temperature for a long time without any special equipment, allowing us to test the same sample multiple times. We have used this method in at least eleven plant species, including arabidopsis, tomato, soybean, potato, cotton, and rice. Altogether, our method decreases labor and cost, thereby increasing throughput and making plant DNA-based molecular diagnostic assays accessible to resource-limited settings, including classrooms, and facilitating sample collection in the field. © 2021 Wiley Periodicals LLC. link2 Basic Protocol 1 Making a homemade cellulose-based DNA storage card Basic Protocol 2 Pounding plant tissue on a DNA storage card Basic Protocol 3 DNA-purification free PCR.Genome editing of primary human cells with CRISPR-Cas9 is a powerful tool to study gene function. For many cell types, there are efficient protocols for editing with optimized plasmids for Cas9 and sgRNA expression. link3 Vascular cells, however, remain refractory to plasmid-based delivery of CRISPR machinery for in vitro genome editing due to low transfection efficiency, poor expression of the Cas9 machinery, and toxic effects of the selection antibiotics. Here, we describe a method for high-efficiency editing of primary human vascular cells in vitro using nucleofection for direct delivery of sgRNACas9-NLS ribonucleoprotein complexes. This method is more rapid and its high editing efficiency eliminates the need for additional selection steps. The edited cells can be employed in diverse applications, such as gene expression measurement or functional assays to assess various genetic perturbation effects in vitro. This method proves effective in vascular cells that are refractory to standard genome manipulation techniques using viral plasmid delivery. We anticipate that this technique will be applied to other non-vascular cell types that face similar barriers to efficient genome editing. © 2021 Wiley Periodicals LLC. Basic Protocol CRISPR-Cas9 genome editing of primary human vascular cells in vitro.3-Methylhistidine (3MH) is an indicator of muscle catabolism. Subclinical protein malnutrition is an independent predictor of aortic stiffness (AS). We aimed to study the relationship between serum 3MH level and AS among patients undergoing maintenance hemodialysis (MHD). Carotid-femoral pulse wave velocity was applied to measure AS of 110 MHD patients. Serum 3MH levels were analyzed using high-performance liquid chromatography and mass spectrometry. AS was defined as cfPWV &gt;10 m/s. Forty-five (40.9%) patients were categorized as having AS. Multivariable logistic (odds ratio 0.792, p  less then  0.001) and linear (β = -0.322, p  less then  0.001) regression analysis revealed that serum 3MH is an independent factor associated with AS among MHD patients. The diagnostic power of 3MH for AS in patients undergoing MHD was 0.691 (95% CI 0.595-0.775, p = 0.0002). Low serum 3MH levels could be a potential biomarker related to AS among MHD patients.Melanoma is highly heterogeneous with diverse genomic alterations and partial therapeutic responses. Emergence of drug-resistant tumor cell clones accompanied with high AXL expression level is one of the major challenges for anti-tumor clinical care. Recent studies have demonstrated that high AXL expression in melanoma cells mediated drug-resistance, epithelial-mesenchymal transition (EMT) and elevated survival of cancer stem cells (CSCs). Given that we have identified several non-steroidal anti-inflammatory drugs (NSAIDs) including Aspirin potently induce the degradation of AXL, we questioned whether NSAIDs could counteract the AXL-mediated neoplastic phenotypes. Here we found NSAIDs downregulate PKA activity via the PGE2 /EP2/cAMP/PKA signaling pathway and interrupt the PKA-dependent interaction between CDC37 and HSP90, resulting in an incorrect AXL protein folding and finally AXL degradation through the ubiquitination-proteasome system (UPS) pathway. Furthermore, NSAIDs not only sensitized the MEK inhibitor treatment, but also reduced EMT and relapse mediate by AXL in tumor tissue. Our findings suggest that the combination of inhibitors and NSAIDs, especially Aspirin, could be a simple but efficient modality to treat melanoma in which AXL is a key factor for drug-resistance, metastasis, and relapse.Systemic Lupus Erythematosus (SLE) is a complex and heterogenous autoimmune disease, where genetics, immunology, and environmental factors all play a role. Murine models have contributed critical information on mechanisms of disease and prospective therapeutics. The key features that have been used to study the disease include the development of anti-nuclear autoantibodies (ANAs), splenomegaly, and kidney disease. The loss of tolerance and subsequent autoimmune features, and the progression to severe disease, are all dependent on immune dysregulation. In this article, we will describe the methods used to evaluate the underlying immunological features of the disease, as a more sensitive strategy to understand the disease itself and the mechanisms of potential novel therapeutics. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 End study protocols for tissue harvesting Basic Protocol 2 End study protocols for tissue processing Basic Protocol 3 Immunophenotyping using flow cytometry protocols Support Protocol Tissue processing for cold storage Basic Protocol 4 Additional tissue processing for later analyses Basic Protocol 5 Analysis of serum auto-antibodies by ELISAs (ANAs, snRNP, and dsDNA).