The role of SWI/SNF chromatin remodelers in the repair of DNA double strand breaks and cancer therapy.

Switch/Sucrose non-fermenting (SWI/SNF) chromatin remodelers hydrolyze ATP to push and slide nucleosomes along the DNA thus modulating access to various genomic loci. These complexes are the most frequently mutated epigenetic regulators in human cancers. SWI/SNF complexes are well known for their function in transcription regulation, but more recent work has uncovered a role for these complexes in the repair of DNA double strand breaks (DSBs). As radiotherapy and most chemotherapeutic agents kill cancer cells by inducing double strand breaks, by identifying a role for these complexes in double strand break repair we are also identifying a DNA repair vulnerability that can be exploited therapeutically in the treatment of SWI/SNF-mutated cancers. In this review we summarize work describing the function of various SWI/SNF subunits in the repair of double strand breaks with a focus on homologous recombination repair and discuss the implication for the treatment of cancers with SWI/SNF mutations.

Association Between Urinary Epidermal Growth Factor and Renal Prognosis in Lupus Nephritis.

OBJECTIVE: To evaluate the role of urinary epidermal growth factor (EGF) as a biomarker of chronic kidney damage in lupus nephritis (LN). METHODS: A proteomics approach was used to identify urinary EGF as a biomarker of interest in a discovery cohort of patients with LN. The expression of urinary EGF was characterized in 2 large multiethnic LN cohorts, and the association between urinary EGF levels at the time of flare and kidney outcomes was evaluated in a subset of 120 patients with long-term follow-up data. For longitudinal studies, the expression of urinary EGF over time was determined in 2 longitudinal cohorts of patients with LN from whom serial urine samples were collected. RESULTS: Discovery analysis showed the urinary EGF levels as being low in patients with active LN (median peptide count 8.4, interquartile range [IQR] 2.8-12.3 in patients with active LN versus median 48.0, IQR 45.3-64.6 in healthy controls). The peptide sequence was consistent with that of proEGF, and this was confirmed by immunoblotting. The discovery findings were verified by enzyme-linked immunosorbent assay. Patients with active LN had a significantly lower level of urinary EGF compared to that in patients with active nonrenal systemic lupus erythematosus (SLE), patients with inactive SLE, and healthy kidney donors (each P < 0.05). The urinary EGF level was inversely correlated with the chronicity index of histologic features assessed in kidney biopsy tissue (Spearman's r = -0.67, P < 0.001). Multivariate survival analysis showed that the urinary EGF level was associated with time to doubling of the serum creatinine level (DSCr), a marker of future end-stage kidney disease (ESKD) (hazard ratio 0.88, 95% confidence interval 0.77-0.99, P = 0.045). Patients whose LN symptoms progressed to DSCr and those who experienced progression to ESKD had a lower urinary EGF level at the time of flare, and urinary EGF levels decreased over the 12 months following flare. All patients who experienced progression to ESKD were identified based on a urinary EGF cutoff level of <5.3 ng/mg. CONCLUSION: Urinary EGF levels are correlated with histologic kidney damage in patients with LN. Low urinary EGF levels at the time of flare and decreasing urinary EGF levels over time are associated with adverse long-term kidney outcomes.

Factors associated with biosafety level-2 research workers' laboratory exit handwashing behaviors and glove removal compliance.

Biosafety level-2 laboratories are designated for work with human-derived samples or moderate-risk microorganisms that transmit primarily by direct contact exposures. Many laboratory procedures generate unseen droplets that contaminate workers' hands, equipment, and work surfaces. Workers' strict adherence to glove removal and handwashing is required prior to laboratory exit to prevent inadvertent transmission of pathogens to self or others. However, little is known about biosafety level-2 workers' compliance with these behaviors. In this article, glove removal and handwashing compliance upon laboratory exit were measured by direct observation of 93 biosafety level-2 research workers from 21 university laboratories. Participants completed a 41-item survey measuring social cognitive theory-based variables related to handwashing, self-reported compliance, and demographic factors. Survey items, observed exit frequency, and laboratory characteristics were evaluated for associations with handwashing compliance. Overall, observed glove removal and handwashing compliance upon laboratory exit were 43.0% (Standard Error [SE] = 2.3%), and 8.2% (SE = 1.2%), respectively, while workers' self-reported glove removal and handwashing compliance were 73.7% (SE = 3.6%) and 35.5% (SE = 4.1%), respectively. The average number of observed laboratory exits per hour was 2.8 for workers with any handwashing compliance vs. 5.4 for workers with no handwashing compliance (p = 0.0013). Among the cognitive variables, behavioral modeling by supervisors and coworkers had the strongest association with workers' compliance (slope = 3.5, SE = 1.3, p = 0.0113). Workers in laboratories with a written handwashing policy had higher compliance (Mean = 14.1%, SE = 5.9%) than workers in laboratories with no written policy (Mean = 1.1%, SE = 1.0%; p = 0.0488). Multi-faceted interventions that encourage modeling of the behavior by supervisors and coworkers, implementation of written handwashing policies, and efforts to reduce exit frequency by furnishing laboratories with necessary equipment and supplies may help improve compliance.

Aberrant Notch Signaling in the Bone Marrow Microenvironment of Acute Lymphoid Leukemia Suppresses Osteoblast-Mediated Support of Hematopoietic Niche Function.

More than half of T-cell acute lymphoblastic leukemia (T-ALL) patients harbor gain-of-function mutations in the intracellular domain of Notch1. Diffuse infiltration of the bone marrow commonly occurs in T-ALL and relapsed B-cell acute lymphoblastic leukemia patients, and is associated with worse prognosis. However, the mechanism of leukemia outgrowth in the marrow and the resulting biologic impact on hematopoiesis are poorly understood. Here, we investigated targetable cellular and molecular abnormalities in leukemia marrow stroma responsible for the suppression of normal hematopoiesis using a T-ALL mouse model and human T-ALL xenografts. We found that actively proliferating leukemia cells inhibited normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. In addition, leukemia development was accompanied by the suppression of the endosteum-lining osteoblast population. We further demonstrated that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. Notch blockade reversed attenuated HSPC cycling, leukemia-associated abnormal blood lineage distribution, and thrombocytopenia as well as recovered osteoblast and HSPC abundance and improved the hematopoietic-supportive functions of osteoblasts. Finally, we confirmed that reduced osteoblast frequency and enhanced Notch signaling were also features of the marrow stroma of human ALL tissues. Collectively, our findings suggest that therapeutically targeting the leukemia-infiltrated hematopoietic niche may restore HSPC homeostasis and improve the outcome of ALL patients.

Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention.

Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.

Detecting respiratory syncytial virus using nanoparticle-amplified immuno-PCR.

Early-stage detection is essential for effective treatment of pediatric virus infections. In traditional -immuno-PCR, a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. In this protocol, we describe a nanoparticle-amplified immuno-PCR assay that combines antibody recognition of traditional ELISA with a 50-fold nanoparticle valence amplification step followed by amplification by traditional PCR. The assay detects a respiratory syncytial virus (RSV) surface fusion protein using a Synagis antibody bound to a 15 nm gold nanoparticle co-functionalized with thiolated DNA complementary to a hybridized 76-base Tag DNA. The Tag DNA to Synagis ratio is 50 to 1. The presence of virus particles triggers the formation of a "sandwich" complex comprised of the gold nanoparticle construct, virus, and a 1 µm antibody-functionalized magnetic particle used for extraction. Virus-containing complexes are isolated using a magnet, DNA tags released by heating to 95 °C, and detected via real-time PCR. The limit of detection of the nanoparticle-amplified immuno-PCR assay was compared to traditional ELISA and traditional RT-PCR using RSV-infected HEp-2 cell extracts. Nanoparticle-amplified immuno-PCR showed a ∼4,000-fold improvement in the limit of detection compared to ELISA and a fourfold improvement in the limit of detection compared to traditional RT-PCR. Nanoparticle-amplified immuno-PCR offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection.