The Wnt-β-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a ...process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator β-catenin to displace it on TCF. Through a genome-scale screen in cultured
cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during
and
development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the β-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of β-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.
The number of anterior cruciate ligament (ACL) injuries reported in athletes younger than 18 years has increased over the past 2 decades. Reasons for the increasing ACL injury rate include the ...growing number of children and adolescents participating in organized sports, intensive sports training at an earlier age, and greater rate of diagnosis because of increased awareness and greater use of advanced medical imaging. ACL injury rates are low in young children and increase sharply during puberty, especially for girls, who have higher rates of noncontact ACL injuries than boys do in similar sports. Intrinsic risk factors for ACL injury include higher BMI, subtalar joint overpronation, generalized ligamentous laxity, and decreased neuromuscular control of knee motion. ACL injuries often require surgery and/or many months of rehabilitation and substantial time lost from school and sports participation. Unfortunately, regardless of treatment, athletes with ACL injuries are up to 10 times more likely to develop degenerative arthritis of the knee. Safe and effective surgical techniques for children and adolescents continue to evolve. Neuromuscular training can reduce risk of ACL injury in adolescent girls. This report outlines the current state of knowledge on epidemiology, diagnosis, treatment, and prevention of ACL injuries in children and adolescents.
Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we ...combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.
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•Genetic and functional analyses of myeloid preleukemia and leukemia in Down syndrome•Non-GATA1 preleukemic mutations are often not required for preleukemia•Previously undescribed transforming hotspot mutation in CSF2RB identified•Loss of function of 18 genes validated in transformation of preleukemia to leukemia
Myeloid leukemia in Down syndrome (ML-DS) evolves from transient abnormal myelopoiesis (TAM). Labuhn et al. show that trisomy 21 and GATA1 mutations are sufficient for the development of TAM. They further identify and functionally validate additional variants that drive TAM to ML-DS transformation.
Novel pyrazolopyrimidines displaying high potency and selectivity toward SRC family kinases have been developed by combining ligand-based design and phenotypic screening in an iterative manner. ...Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening against MCF7 mammary adenocarcinoma cells generated target-agnostic structure–activity relationships that biased subsequent designs toward breast cancer treatment rather than to a particular target. This strategy led to the discovery of two potent antiproliferative leads with phenotypically distinct anticancer mode of actions. Kinase profiling and further optimization resulted in eCF506, the first small molecule with subnanomolar IC50 for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, halts SRC-associated neuromast migration in zebrafish embryos without inducing life-threatening heart defects, and inhibits SRC phosphorylation in tumor xenografts in mice.
In September 2012, the New York City Department of Health and Mental Hygiene identified an outbreak of Neisseria meningitidis serogroup C invasive meningococcal disease among men who have sex with ...men (MSM). Twenty-two case-patients and 7 deaths were identified during August 2010-February 2013. During this period, 7 cases in non-MSM were diagnosed. The slow-moving outbreak was linked to the use of websites and mobile phone applications that connect men with male sexual partners, which complicated the epidemiologic investigation and prevention efforts. We describe the outbreak and steps taken to interrupt transmission, including an innovative and wide-ranging outreach campaign that involved direct, internet-based, and media-based communications; free vaccination events; and engagement of community and government partners. We conclude by discussing the challenges of managing an outbreak affecting a discrete community of MSM and the benefits of using social networking technology to reach this at-risk population.
Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, ...especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of
(cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of
variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a
variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca
sensitivity and Ca
dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca
sensitivity and decreases the Ca
off-rate constant (
). To investigate further, a heterozygous I79N
variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N
hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (
) and cytosolic Ca
transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca
transients due to the enhanced cytosolic Ca
buffering. These changes in Ca
handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both
and Ca
transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N
hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of
(atrial natriuretic peptide),
(brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N
vs. the isogenic control. This significant shift demonstrates that this missense in the
transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.
It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 ...(COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.
Genetic factors influence chronic obstructive pulmonary disease (COPD) risk, but the individual variants that have been identified have small effects. We hypothesised that a polygenic risk score ...using additional variants would predict COPD and associated phenotypes.
We constructed a polygenic risk score using a genome-wide association study of lung function (FEV1 and FEV1/forced vital capacity FVC) from the UK Biobank and SpiroMeta. We tested this polygenic risk score in nine cohorts of multiple ethnicities for an association with moderate-to-severe COPD (defined as FEV1/FVC <0·7 and FEV1 <80% of predicted). Associations were tested using logistic regression models, adjusting for age, sex, height, smoking pack-years, and principal components of genetic ancestry. We assessed predictive performance of models by area under the curve. In a subset of studies, we also studied quantitative and qualitative CT imaging phenotypes that reflect parenchymal and airway pathology, and patterns of reduced lung growth.
The polygenic risk score was associated with COPD in European (odds ratio OR per SD 1·81 95% CI 1·74–1·88 and non-European (1·42 1·34–1·51) populations. Compared with the first decile, the tenth decile of the polygenic risk score was associated with COPD, with an OR of 7·99 (6·56–9·72) in European ancestry and 4·83 (3·45–6·77) in non-European ancestry cohorts. The polygenic risk score was superior to previously described genetic risk scores and, when combined with clinical risk factors (ie, age, sex, and smoking pack-years), showed improved prediction for COPD compared with a model comprising clinical risk factors alone (AUC 0·80 0·79–0·81 vs 0·76 0·75–0·76). The polygenic risk score was associated with CT imaging phenotypes, including wall area percent, quantitative and qualitative measures of emphysema, local histogram emphysema patterns, and destructive emphysema subtypes. The polygenic risk score was associated with a reduced lung growth pattern.
A risk score comprised of genetic variants can identify a small subset of individuals at markedly increased risk for moderate-to-severe COPD, emphysema subtypes associated with cigarette smoking, and patterns of reduced lung growth.
US National Institutes of Health, Wellcome Trust.
Background
Randomized trials have established the safety of observation or axillary radiation (AxRT) as an alternative to axillary lymph node dissection (ALND) in patients with limited nodal disease ...who undergo upfront surgery. Variability remains in axillary management strategies in cN0 patients undergoing mastectomy found to have one to two positive sentinel lymph nodes (SLNs). We examined the impact of intraoperative pathology assessment in axillary management in a national cohort of AMAROS-eligible mastectomy patients.
Methods
The National Cancer Database was used to identify AMAROS-eligible cT1-2N0 breast cancer patients undergoing upfront mastectomy and SLN biopsy (SLNB) and found to have one to two positive SLNs, from 2018 to 2019. We constructed a variable defining intraoperative pathology as ‘not performed/not acted on’ if ALND was either not performed or performed at a later date than SLNB, or ‘performed/acted on’ if SLNB and ALND were completed on the same day. Adjusted multivariable analysis examined predictors of treatment with both ALND and AxRT.
Results
Overall, 8222 patients with cT1-2N0 disease underwent upfront mastectomy and had one to two positive SLNs. Intraoperative pathology was performed/acted on in 3057 (37.2%) patients. These patients were significantly more likely to have both ALND and AxRT than those without intraoperative pathology (41.0% vs. 4.9%;
p
< 0.001). On multivariate analysis, the strongest predictor of receiving both ALND and AxRT was use of intraoperative pathology (odds ratio 8.99, 95% confidence interval 7.70–10.5;
p
< 0.001).
Conclusions
We advocate that consideration should be made for omission of routine intraoperative pathology in mastectomy patients likely to be recommended postmastectomy radiation to minimize axillary overtreatment with both ALND and AxRT in appropriate patients.
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