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Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible

Purpose: This paper evaluates a theory-driven, interactive hand hygiene (HH) intervention, the Safe Hands project, based on theories of organizational learning and culture including leadership support, dialogue and co-creation., Design: This prospective quasi-experimental study used unobtrusive overt observations to evaluate adherence to HH recommendations after implementing an infection-prevention intervention., Methods: The primary outcome was differences in HH practices "Before aseptic/clean procedure" (WHO moment 2), "After body fluid exposure risk" (WHO moment 3) and performance of aseptic techniques. One operating room (OR) department served as the study hospital and the other as the control hospital, both at Swedish university hospitals. Adherence to HH guidelines was measured 4 times during 2015 to 2017., Findings: The intervention site displayed a significant improvement in adherence to HH guidelines and aseptic techniques. WHO 2; from 23.8% to 36.2%, (P = .014), WHO 3; from 22.2% to 42.3%, (P = .002), and aseptic techniques; from 17.5% to 31.6%, (P = .003). No changes in adherence were identified at the control site. The use of contaminated gloves decreased post intervention at the study operating department., Conclusions: This study shows that implementing tailored interventions that are underpinned by theories from organizational learning and culture can improve adherence to hand hygiene in a complex setting as the OR up to 6 months post-intervention. The interprofessional co-creation of standards operating procedures addressing specific care procedures and emphasizing the importance of aseptic techniques can be an acceptable and feasible way to reduce the risks of contaminating medical devices and patients during perioperative care., (Copyright © 2022 American Society of PeriAnesthesia Nurses. Published by Elsevier Inc. All rights reserved.)

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism., (© 2023. The Author(s).)

Determining the functional role of thousands of genetic sequence variants (mutations) associated with genetic diseases is a major challenge. Here we present clustered regularly interspaced short palindromic repeat (CRISPR)-Select TIME , CRISPR-Select SPACE and CRISPR-Select STATE , a set of flexible knock-in assays that introduce a genetic variant in a cell population and track its absolute frequencies relative to an internal, neutral control mutation as a function of time, space or a cell state measurable by flow cytometry. Phenotypically, CRISPR-Select can thereby determine, for example, pathogenicity, drug responsiveness/resistance or in vivo tumor promotion by a specific variant. Mechanistically, CRISPR-Select can dissect how the variant elicits the phenotype by causally linking the variant to motility/invasiveness or any cell state or biochemical process with a flow cytometry marker. The method is applicable to organoids, nontransformed or cancer cell lines. It is accurate, quantitative, fast and simple and works in single-well or 96-well higher throughput format. CRISPR-Select provides a versatile functional variant assay for research, diagnostics and drug development for genetic disorders., (© 2022. The Author(s).)

Background: Urinary retention is common in elderly patients undergoing acute hip fracture surgery. Avoiding overfilling the urinary bladder is important to avoid detrusor muscle damage and associated motility problems. The aim of this study was to analyse associations between the co-creation of a nurse-driven urinary catheterisation protocol and the incidence of bladder distension in patients undergoing hip fracture surgery., Methods: This is a single-centre implementation intervention with a retrospective longitudinal observation design, using five measures points, spanning from June 2015 to March 2020. The intervention was theory driven and the participants, together with the facilitators and researcher, co-created a nurse-driven urinary catheterisation protocol. Data were retrieved from the hip fracture register. Uni- and multivariable logistic regressions were used for analyses of changes in bladder distension and urinary volume of ≥500 ml over the years., Results: A total of 3078 patients were included over a five-year period. The implementation intervention was associated with a reduction in the proportion of patients with bladder distension of 31.5% (95% confidence interval 26.0-37.0), from year 1 to year 5. The multivariable analysis indicated a 39% yearly reduction in bladder distension, OR 0.61 (95% confidence interval 0.57-0.64, p <  0001). There was a reduction in the proportion of patients with a bladder volume of ≥500 ml of 42.8% (95% confidence interval 36.2-49.4), from year 1 to year 5. The multivariable analysis found a 41% yearly reduction in patients with a bladder volume of ≥500 ml, OR 0.59 (95% confidence interval 0.55-0.64, p <  0.0001). The intervention was associated with improved documentation of both catheter indications and removal plans., Conclusion: The use of predefined catheter indications and a tighter bladder scanning schedule were associated with a reduction in the incidence of both bladder distension and urine volume ≥ 500 ml in hip fracture patients. Registered nurses can play an active role in the facilitation of timely and appropriate catheter treatment in patients with hip fractures., Trial Registration: Clinical Trial Registry ISRCTN 17022695 registered retrospectively on 23 December 2021, in the end of the study, after data collection., (© 2022. The Author(s).)

Background: Urinary catheter (UC)-associated infections are one of the most common preventable healthcare-associated infections (HAIs) and they frequently occur in older, frail populations., Aim: The study aim was to describe the incidence of UC-associated infection in elderly patients undergoing hip fracture surgery after implementing a preventive care bundle., Methods: A longitudinal prospective study using a before-and-after design. The bundle was theory driven and involved the co-creation of a standard operational procedure, education and practical training sessions. Prospectively collected registry data were analysed. Univariable statistics and multivariable logistic regressions were used for analyses., Results: 2,408 patients with an acute hip fracture were included into the study. There was an overall reduction in UC catheter associated-associated urinary tract infections, from 18.5% ( n = 75/406) over time to 4.2% ( n = 27/647). When adjusting for all identified confounders, patients in phase 4 were 74% less likely to contract an UC-associated infection (OR, 0.26; 95% CI, 0.15-0.45, p < 0.0001)., Discussion: Bundled interventions can reduce UC-associated infections substantially, even in elderly frail patients. Partnership and co-creation as implementation strategies appear to be promising in the fight against HAI., Competing Interests: Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Renal cell carcinoma (RCC) is the most common type of kidney cancer and has the highest mortality rate among genitourinary cancers. Despite the advances in molecular targeted therapies to treat RCC, the inevitable emergence of resistance has delineated the need to uncover biomarkers to prospectively identify patient response to treatment and more accurately predict patient prognosis. Fringe is a fucose specific β1, 3N-acetylglucosaminyltransferase that modifies the Notch receptors. Given the link between its function and aberrant Notch activation in RCC, Fringe may be implicated in this disease. The Fringe homologs comprise of Lunatic fringe (LFng), Manic fringe (MFng) and Radical fringe (RFng). MFng has been reported to play a role in cancer. MFng is also essential in the development of B cells. However, the expression profile and clinical significance of MFng, and its association with B cells in RCC are unknown. CD20 is a clinically employed biomarker for B cells. This pilot study aimed to determine if MFng protein expression can be utilized as a prospective biomarker for therapeutics and prognosis in RCC, as well as to determine its association with CD20+ B cells. Analysis of publicly available MFng gene expression datasets on The Cancer Genome Atlas Netlwork (TCGA) identified MFng gene expression to be up-regulated in Kidney Clear Cell Renal Carcinoma (KIRC) patients. However there was no significant association between the patient survival probability and the level of MFng expression in this cohort. Immunohistochemistry performed on a tissue microarray containing cores from 64 patients revealed an elevated MFng protein expression in the epithelial and stromal tissues of RCC compared to the normal kidney, suggesting a possible role in tumorigenesis. Our study describes for the first time to our knowledge, the protein expression of MFng in the nuclear compartment of normal kidney and RCC, implicating a prospective involvement in gene transcription. At the cellular level, cytoplasmic MFng was also abundant in the normal kidney and RCC. However, MFng protein expression in the malignant epithelial and stromal tissue of RCC had no positive correlation with the patients' overall survival, progression-free survival and time to metastasis, as well as the gender, age, tumor stage and RCC subtype, indicating that MFng may not be an appropriate prognostic marker. The association between CD20+ B cells and epithelial MFng was found to approach borderline insignificance. Nonetheless, these preliminary findings may provide valuable information on the suitability of MFng as a potential therapeutic molecular marker for RCC, thus warrants further investigation using a larger cohort., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient., Results: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis., Conclusion: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ARHGAP35 gene encoding p190A RhoGAP (p190A) is significantly altered by both mutation and allelic deletion in human cancer, but the functional implications of such alterations are not known. Here, we demonstrate for the first time that p190A is a tumor suppressor using a xenograft mouse model with carcinoma cells harboring defined ARHGAP35 alterations. In vitro, restoration of p190A expression in carcinoma cells promotes contact inhibition of proliferation (CIP) through activation of LATS kinases and phosphorylation of the proto-oncogenic transcriptional co-activator YAP. In contrast, p190A forms harboring recurrent cancer mutations exhibit loss of function in modulating the Hippo pathway, inducing CIP, as well as attenuated suppression of tumor growth in mice. We determine that p190A promotes mesenchymal to epithelial transition (MET) and elicits expression of a cassette of epithelial adherens junction-associated genes in a cell density-dependent manner. This cassette includes CDH1 encoding E-cadherin, which amplifies p190A-mediated LATS activation and is necessary for CIP. Oppositely, we establish that p190A is obligatory for E-cadherin to activate LATS kinases and induce CIP. Collectively, this work defines a novel mechanism by which p190A and E-cadherin cooperate in modulating Hippo signaling to suppress tumor cell growth.

CRISPR/Cas9 technology allows facile modification of the genome in virtually any desired way through the use of easily designed plasmid constructs that express a gRNA targeting a genomic site-of-interest and Cas9. Hydrodynamic tail vein injection, on the other hand, is a simple method to deliver "naked" plasmid DNA to 5-40% of the hepatocytes of the liver of adult mice. Here, we describe how these two techniques can be combined to create a workflow for fast, easy, and cost-efficient in vivo genome editing of the adult mouse liver. Using this method, large cohorts of mice with genetically modified livers can be established within 3 weeks to generate models for gene function in normal physiology and diseases of the liver.

Background: The role of B-lymphocytes in solid tumours is unclear. Tumour biology studies have implied both anti- and pro-tumoural effects and prognostic studies have mainly linked B-cells to increased survival. This study aimed to analyse the clinical relevance of B-lymphocytes in renal cell cancer (RCC), where information on the prognostic impact is lacking., Methods: Following immunohistochemistry (IHC) stainings with a CD20 antibody, density of CD20+ B-cells was quantified in an RCC discovery- and validation cohort. Associations of B-cell infiltration, determined by CD20 expression or a B-cell gene-signature, and survival was also analysed in 14 publicly available gene expression datasets of cancer, including the kidney clear cell carcinoma (KIRC) dataset., Results: IHC analyses of the discovery cohort identified a previously unrecognised subgroup of RCC patients with high infiltration of CD20+ B-cells. The B-cell-high subgroup displayed significantly shorter survival according to uni- and multi-variable analyses. The association between poor prognosis and high density of CD20+ B-cells was confirmed in the validation cohort. Analyses of the KIRC gene expression dataset using the B-cell signature confirmed findings from IHC analyses. Analyses of other gene expression datasets, representing 13 different tumour types, indicated that the poor survival-association of B-cells occurred selectively in RCC., Conclusion: This exploratory study identifies a previously unrecognised poor-prognosis subset of RCC with high density of CD20-defined B-cells.

ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35 ., (© 2018 Frank et al.)

Aberrant expression of O -glycans is a hallmark of epithelial cancers. Mucin-type O- glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N -acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6 GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell-cell adhesion, and formation of crypts in tissue cultures. O- Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6-specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Hand hygiene and aseptic techniques are essential preventives in combating hospital-acquired infections. However, implementation of these strategies in the operating room remains suboptimal. There is a paucity of intervention studies providing detailed information on effective methods for change. This study aimed to evaluate the process of implementing a theory-driven knowledge translation program for improved use of hand hygiene and aseptic techniques in the operating room., Methods: The study was set in an operating department of a university hospital. The intervention was underpinned by theories on organizational learning, culture and person centeredness. Qualitative process data were collected via participant observations and analyzed using a thematic approach., Results: Doubts that hand-hygiene practices are effective in preventing hospital acquired infections, strong boundaries and distrust between professional groups and a lack of psychological safety were identified as barriers towards change. Facilitated interprofessional dialogue and learning in "safe spaces" worked as mechanisms for motivation and engagement. Allowing for the free expression of different opinions, doubts and viewing resistance as a natural part of any change was effective in engaging all professional categories in co-creation of clinical relevant solutions to improve hand hygiene., Conclusion: Enabling nurses and physicians to think and talk differently about hospital acquired infections and hand hygiene requires a shift from the concept of one-way directed compliance towards change and learning as the result of a participatory and meaning-making process. The present study is a part of the Safe Hands project, and is registered with ClinicalTrials.gov (ID: NCT02983136 ). Date of registration 2016/11/28, retrospectively registered.

Background & Aims: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development., Methods: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing., Results: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC., Conclusions: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

A novel set of integrated procedures for quantification of fibroblast-rich stroma and vascular characteristics has recently been presented allowing discovery of novel perivascular and stromal biomarkers in colorectal, renal cell, and ovarian cancer. In the present study, data obtained through these procedures from clinically well-annotated collections of these three tumour types have been used to address two novel questions. First, data have been used to investigate if the three tumour types demonstrate significant differences regarding features such as vessel diameter, vessel density, and perivascular marker expression. Second, analyses of the cohorts have been used to explore the prognostic significance of a novel vascular metric, 'vessel distance inter-quartile range (IQR)' that describes intra-case heterogeneity regarding vessel distribution. The comparisons between the three tumour types demonstrated a set of significant differences. Vessel density of renal cell cancer was statistically significantly higher than in colorectal and ovarian cancer. Vessel diameter was statistically significantly higher in ovarian cancer. Concerning perivascular status, colorectal cancer displayed significantly higher levels of perivascular PDGFR-β expression than the other two tumour types. Intra-case heterogeneity of perivascular PDGFR-β expression was also higher in colorectal cancer. Notably, these fibroblast-dominated stroma phenotypes matched previously described experimental tumour stroma characteristics, which have been linked to differential sensitivity to anti-VEGF drugs. High 'vessel distance IQR' was significantly associated with poor survival in both renal cell cancer and colorectal cancer. In renal cell cancer, this characteristic also acted as an independent prognostic marker according to multivariate analyses including standard clinico-pathological characteristics. Explorative subset analyses indicated particularly strong prognostic significance of 'vessel distance IQR' in T stage 4 of this cancer type. Together, these analyses identified tumour-type-specific vascular-stroma phenotypes of possible functional significance, and suggest 'vessel distance IQR' as a novel prognostic biomarker.

This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

Background: Renal cell carcinoma (RCC) is a highly vascularised tumour, where anti-angiogenic treatment with multi-tyrosine-kinase-inhibitor, is used for first-line treatment of metastatic disease. Variations in vascular characteristics are likely to contribute to variations in intrinsic aggressiveness of the disease. Emerging studies are identifying perivascular status, including perivascular PDGFR-β, as a determinant of prognosis in other tumour types., Methods: This work explored the impact on prognosis of vascular characteristics in RCC through analyses of a population-based collection of tumours from surgery-alone-treated patients. The quantitative data from a panel of vascular metrics were obtained through computerised image analysis of sections double-stained for expression of the endothelial cell marker CD34 together with perivascular markers α-SMA or PDGFR-β., Results: Perivascular expression of PDGFR-β and α-SMA were positively correlated to each other, and negatively correlated to vessel density. High expression of PDGFR-β and α-SMA as well as low vessel density was significantly associated with short survival in uni- and multivariate analyses. Subgroup analyses demonstrated that the prognostic impact of the perivascular markers was particularly prominent in the T4-subgroup. A novel metric, related to PDGFR-β perivascular heterogeneity, was also associated with prognosis in uni-and multi-variate analyses. This novel metric also acted as a prognosis marker in ovarian cancer., Conclusions: The study demonstrates previously unrecognised associations between RCC survival and the absolute levels, and variability, of perivascular PDGFR-β. This marker should be further explored in other RCC cohorts. Findings also suggest mechanistic analyses and studies on the relationship between perivascular status and efficacy of multi-tyrosine-kinase-inhibitors.

Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and ∼ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9., (© The Author(s) 2014. Published by Oxford University Press.)

Background: During epithelial morphogenesis, a complex comprising the βPIX (PAK-interacting exchange factor β) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds βPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. Although a role for the Scrib-βPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown., Results: Here, we establish that Scrib targets βPIX and PAK2 to adherens junctions where a βPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, we show that this signaling pathway regulates cell survival in response to osmotic stress. Finally, we determine that in suspension cultures, the Scrib-βPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the βPIX-PAK2 complex suppressing anoikis, respectively., Conclusions: Our findings demonstrate that the Scrib-βPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-βPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.

The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of a potent promotile/invasive gene program by FRA1-dependent and -independent mechanisms. The program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties.

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.