Having previously shown that the binding of neutrophil gelatinase-associated lipocalin (NGAL) to matrix metalloproteinase-9 (MMP-9) protects this extracellular matrix remodeling enzyme from ...autodegradation, we hypothesized that the addition of NGAL to breast cancer cells, which do not express this protein but do express MMP-9, might result in a more aggressive phenotype in vivo. Based on our previous reports that MMPs can be detected in the urine of cancer patients, we also asked whether MMP-9/NGAL could be detected in the urine of breast cancer patients and whether it might be predictive of disease status.
Clones of MCF-7 human breast cancer cells differentially expressing NGAL were generated by stable transfection with human NGAL expression constructs. The established clones were then implanted s.c. in immunodeficient mice and tumor growth was monitored. In addition, we analyzed the urine of individuals with breast cancer and age-matched, sex-matched controls using gelatin zymography for the presence of MMP-9/NGAL.
Increased NGAL expression resulted in significant stimulation of tumor growth. Immunohistochemical analysis of MCF-7 tumors revealed that the NGAL-overexpressing ones exhibited increased growth rates that were accompanied by increased levels of MMP-9, increased angiogenesis, and an increase in the tumor cell proliferative fraction. In addition, MMP-9/NGAL complex was detected in 86.36% of the urine samples from breast cancer patients but not in those from healthy age and sex-matched controls.
These findings suggest, for the first time, that NGAL may play an important role in breast cancer in vivo by protecting MMP-9 from degradation thereby enhancing its enzymatic activity and facilitating angiogenesis and tumor growth. Clinically, these data suggest that the urinary detection of MMP-9/NGAL may be useful in noninvasively predicting disease status of breast cancer patients.
Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, ..."AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.
RNA-guided endonucleases such as Cas9 provide efficient on-target genome editing in cells but may also cleave at off-target loci throughout the genome. Engineered variants of Streptococcus pyogenes ...Cas9 (SpCas9) have been developed to globally reduce off-target activity, but individual off-targets may remain, or on-target activity may be compromised. In order to evolve against activity at specific off-targets while maintaining strong on-target editing, we developed a novel M13 bacteriophage-mediated selection method. Using this method, sequential rounds of positive and negative selection are used to identify mutations to Cas9 that enhance or diminish editing activity at particular genomic sequences. We also introduce scanning mutagenesis of oligo-directed targets (SMOOT), a comprehensive mutagenesis method to create highly diverse libraries of Cas9 variants that can be challenged with phage-based selection. Our platform identifies novel SpCas9 mutants which mitigate cleavage against off-targets both in biochemical assays and in T-cells while maintaining higher on-target activity than previously described variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity Cas9 (SpartaCas), composed of the most enriched mutations, each of unknown function. This evolved Cas9 mutant reduces off-target cleavage while preserving efficient editing at multiple therapeutically relevant targets. Directed evolution of Cas9 using our system demonstrates an improved structure-independent methodology to effectively engineer nuclease activity.
Abstract
Understanding morphological changes of nanoparticles in solution is essential to tailor the functionality of devices used in energy generation and storage. However, we lack experimental ...methods that can visualize these processes in solution, or in electrolyte, and provide three-dimensional information. Here, we show how X-ray ptychography enables in situ nano-imaging of the formation and hollowing of nanoparticles in solution at 155 °C. We simultaneously image the growth of about 100 nanocubes with a spatial resolution of 66 nm. The quantitative phase images give access to the third dimension, allowing to additionally study particle thickness. We reveal that the substrate hinders their out-of-plane growth, thus the nanocubes are in fact nanocuboids. Moreover, we observe that the reduction of Cu
2
O to Cu triggers the hollowing of the nanocuboids. We critically assess the interaction of X-rays with the liquid sample. Our method enables detailed in-solution imaging for a wide range of reaction conditions.
•Super Skills for Life (SSL) is a transdiagnostic prevention program for children and adolescents with emotional problems. It is based on the principles of Cognitive Behavior Therapy (CBT), ...behavioral activation, and social skills training.•Young children with emotional problems who participated in SSL showed significant reduction in their anxiety and depressive symptoms 12 months after the intervention compared to children in the wait-list control group.•SSL also has a positive impact in children's behavioral difficulties and psychosocial impairment 12 months after the intervention.
The present study examined the long-term efficacy of a transdiagnostic prevention program, Super Skills for Life (SSL), among young children with emotional problems. SSL is based on the principles of cognitive behavioral therapy, behavioral activation, and social skills training.
One-hundred and twenty-three Spanish-speaking children, aged 6 to 8 years, and their parents participated in the study. They were recruited from ten schools. Schools were randomly allocated to either the intervention (IG) or waiting-list control (WLC) groups. The children's parent completed a set of questionnaires to measure their child's anxiety and depressive symptoms, general difficulties and positive attributes, and psychosocial impairment. Parent's assessment at baseline and at a 12-month follow-up was assessed using an intent-to-treat approach.
The IG, compared to WLC group, showed significant baseline-to-follow-up reductions in anxiety and depressive symptoms, as well as in behavioral difficulties and psychosocial impairment in various life domains.
The small sample size and the reliance on parental reports were the major limitations of this study.
This study provides initial evidence of the long-term effects of SSL, suggesting that it is a promising indicated preventive intervention for young children with emotional problems.
Abstract Background The Spence Children’s Anxiety Scale (SCAS) is a widely used instrument for assessing symptoms of anxiety disorders among children and adolescents. Previous studies have ...demonstrated its good reliability for children and adolescents from different backgrounds. However, remarkable variability in the reliability of the SCAS across studies and inconsistent results regarding its factor structure has been found. Methods The present study aims to examine the SCAS factor structure by means of a systematic review with narrative synthesis, the mean reliability of the SCAS by means of a meta-analysis, and the influence of the moderators on the SCAS reliability. Databases employed to collect the studies included Scholar Google, PsycARTICLES, PsycINFO, Web of Science, and Scopus since 1997. Results Twenty-nine and 32 studies, which examined the factor structure and the internal consistency of the SCAS, respectively, were included. The SCAS was found to have strong internal consistency, influenced by different moderators. The systematic review demonstrated that the original six-factor model was supported by most studies. Limitations Factorial invariance studies (across age, gender, country) and test–retest reliability of the SCAS were not examined in this study. Conclusions It is concluded that the SCAS is a reliable instrument for cross-cultural use, and it is suggested that the original six-factor model is appropriate for cross-cultural application.
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