Granthamday5624

To help understand the mechanism of action of LBL1, we recently took an unbiased chemical proteomics approach to identify its direct binding targets from the entire human cellular proteome. In this chapter, we describe our detailed methods to identify and validate lamins as the direct targets of LBL1. In this approach, we developed a clickable photoaffinity probe called LBL1-P that contains acylpyrroloquinazoline, trifluoromethyldiazirine and alkyne groups. Furthermore, we described a fluorescence microscopic method to validate that LBL1 directly targets lamin A in living cells. When properly designed, this approach should be broadly applicable to other bioactive small molecules. © 2020 Elsevier Inc. All rights reserved.The cyclic-AMP response element binding protein (CREB) is an important nuclear transcription factor and has been shown to be overexpressed and/or over-activated in many different cancer types, suggesting that targeting CREB is a novel approach for developing cancer therapies. Our lab discovered the first cell-permeable small molecule inhibitor of CREB, from which we further developed a potent CREB inhibitor with in vivo anti-cancer activity. In this article, we detailed our biochemical and cell-based bioassays to assess different small molecule CREB inhibitors. © 2020 Elsevier Inc. All rights reserved.Cellular functions are controlled by sophisticated signal transduction pathways triggered by receptors responding to myriad environmental stimuli. With the rise of synthetic biology, we can now engineer artificial receptors enabling real-time interrogation and manipulation of cellular signaling, and providing new clues about the design principles of natural sensing systems. In this review, we describe the main classes of synthetic receptors engineered to date, their applications, and highlight recent developments that might improve synthetic receptor design in the future. © 2020 Elsevier Inc. All rights reserved.Nuclease-mediated DNA cleavage and subsequent repair lie at the heart of genome editing, and the RNA-guided endonuclease Cas9 has emerged as the most widely-used tool for facilitating this process. Extensive biochemical and biophysical efforts have revealed much regarding the structure, mechanism, and cellular properties of Cas9. This has enabled engineering of Cas9 variants with enhanced activity, specificity, and other features. However, we lack a detailed understanding of the kinetics of Cas9-mediated DNA cleavage and repair in vivo. To study in vivo Cas9 cleavage kinetics and activity dose-dependence, we have engineered a chemically-inducible, single-component Cas9, ciCas9. ciCas9 allows for temporal and rheostatic control of Cas9 activity using a small molecule activator, A115. We have also developed a droplet-digital PCR-based assay (DSB-ddPCR) to directly quantify Cas9-mediated double-stranded breaks (DSBs). The methods in this chapter describe the application of ciCas9 and DSB-ddPCR to study the kinetics and dose-dependence of Cas9 editing in vivo. © 2020 Elsevier Inc. All rights reserved.RAS GTPases are involved in a number of dynamic signaling processes and have been a major focus of research due to the prevalence of activating RAS mutations in cancer. However, despite decades of research, some fundamental aspects of RAS biology are still not well understood. Difficulty in fully defining RAS-driven signaling stems from the overall complexity of downstream pathways and a lack of tools for specifically perturbing RAS function. To better characterize RAS-driven signaling, we recently developed a chemical genetic system for activating endogenous RAS with a small molecule. In this chapter, we describe the use of chemically inducible activator of RAS (CIAR), a single-protein, chemical genetic system that allows the rapid and dose-dependent activation of endogenous RAS. Methods in this chapter also describe the validation of RAS activation with CIAR through the analysis of downstream signaling. © 2020 Elsevier Inc. All rights reserved.Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the B4F. The assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (±10%) was compensated by the 100-fold higher sensitivity and the deviations from the ANS assay were ≤5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n≤5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness. © 2020 Elsevier Inc. All rights reserved.PURPOSE The purpose of this study was to compare primary outcomes following insertion of balloon and nonballoon gastrostomy tubes (G-tubes). METHODS A retrospective chart review over a 5-year period comparing the need for emergency, radiologic, or operative interventions between balloon and nonballoon G-tube devices was performed. RESULTS 145 patient charts were reviewed (46.8% female, 53.1% male). The indication for G-tube insertion was failure to thrive in 83.4%. Average age at insertion was 4.3 years (0-17.9 years). 37.2% had a balloon type G-tube, and 62.8% had a nonballoon type. Patients with a nonballoon device had 1.14 (0-15) ER visits related to the G-tube vs. 0.48 (0-6) visits with a balloon device. Of the ER visits for patients with a nonballoon device, 26.9% were replaced in ER, 38.5% in radiology, and 34.6% required an operation for replacement. For patients with a balloon device, 47.8% were replaced in the ER, 52.2% were replaced in radiology (GJ), and none required operative replacement. The majority of patients who initially had a nonballoon G-tube placed required a second operation for device change (95.7%). Patients with nonballoon devices required significantly more operations (average 2.55, range 0-16) vs patients with balloon devices (average 0.40, range 0-3) (p  less then  .05). CONCLUSIONS Balloon-type G-tubes require less ER visits and operative interventions compared to nonballoon G-tubes. LEVEL OF EVIDENCE C. Various definitions and biomarkers have been developed in an unsuccessful attempt to obtain a "gold standard" for periprosthetic joint infection (PJI) diagnosis. The development of the 2011 Musculoskeletal Infection Society criteria facilitated further research and advances by allowing the use of a consistent PJI definition across studies. The newly proposed 2018 criteria do not rely at all on expert opinions/consensus. In this review, we describe the most relevant definitions developed throughout recent time, their rationale, characteristics, and supportive evidence for their clinical implementation. In the opinion of the authors, the orthopedic community should consider a probability and likelihood paradigm to create a PJI diagnostic definition. Probably not a single definition might be suited for all situations; the inclusion of serological findings could be the next step moving forward. BACKGROUND Prosthetic joint infection (PJI) is associated with significant morbidity, mortality, and costs. We developed a fast-track PJI care system using an infectious disease physician to work directly with the TJA service and coordinate in the treatment of PJI patients. We hypothesized that streamlined care of patients with hip and knee PJI decreases the length of the acute hospital stay without increasing the risk of complication or incorrect antibiotic selection. METHODS A single-center retrospective chart review was performed for all patients treated operatively for PJI. A cohort of 78 fast-track patients was compared to 68 control patients treated before the implementation of the program. Hospital length of stay (LOS) and cases of antibiotic mismatch were primary outcomes. Secondary outcomes, including 90-day readmissions, reoperations, mortality, rate of reimplantation, and 12-month reimplant survival, were compared. Cox regressions were analyzed to assess the effects on LOS of patient demographics and the type of surgery performed. RESULTS Average hospital LOS from infection surgery to discharge was significantly lower in the fast-track cohort (3.8 vs 5.7 days; P = .012). There were no episodes of antibiotic mismatch in the fast-track group vs 1 recorded episode in the control group. No significant differences were noted comparing 90-day complications, reimplantation rate, or 12-month reimplant survival rates. CONCLUSION Through the utilization of an orthopedic-specific infectious disease physician, a fast-track PJI protocol can significantly shorten hospital LOS while remaining safe. Streamlining care pathways may help decrease the overall healthcare costs associated with treating PJI. Periprosthetic joint infection represents a serious complication following total knee arthroplasty. ML264 KLF inhibitor In the setting of chronic or age-indeterminate total knee arthroplasty infection, a 2-staged approach has been traditionally the preferred method of treatment over single-stage debridement and reimplantation debridement or debridement, antibiotics and implant retention. Two-stage is the preferred treatment method in North America and has demonstrated better overall success than the single stage techniques. Additionally, the 2-stage method is the preferred treatment for difficult to treat pathogens as well as in patients who have already undergone a previous revision procedure. An articulating prefabricated antibiotic spacer has entered the armamentarium of 2-stage revision knee surgery, and has demonstrated comparable results to custom and static spacers in terms of the primary goal of infection control. Importantly, the potential for enhanced mobility and function hold promise by safely providing a more "livable" knee during the convalescent period prior to definitive reimplantation. BACKGROUND The purpose of this study was to compare patients who had chronic prosthetic joint infection treated using three methods of articulating polymethylmethacrylate spacers in two-stage reimplantation. METHODS We identified 77 patients who had chronic prosthetic joint infection with a minimum of one-year follow-up. Reinfection rates were determined using modified International Consensus group criteria. RESULTS The overall reinfection rate was 18% (14 of 77 patients). Despite a higher medical comorbidity in the second-generation spacer cohort, there were no statistical differences in reinfection rates between articulating spacer types. CONCLUSION This study suggests that there were no differences in efficacy between the traditional molded, first-generation premolded, and second-generation premolded articulating spacers, but more studies with high level of evidence are needed.