Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA (crRNA) sequence. Our analysis further revealed robust non-specific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.S-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function, and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, while blocking S-palmitoylation with 2-Br-palmitate reduces Nav1.6 current and shifts the steady state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (C1169, C1170, C1978) were identified. These differentially modulate distinct Nav1.6 properties. Interestingly, C1978 is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. C1978 S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.In animals, miRNAs are the most prevalent small non-coding RNA molecules controlling posttranscriptional gene regulation. The Argonaute proteins (AGO) mediate miRNA-guided gene silencing by recruiting multiple factors involved in translational repression, deadenylation, and decapping. Here, we report that CSDE1, an RNA-binding protein linked to stem cell maintenance and metastasis in cancer, interacts with AGO2 within miRNA-induced silencing complex and mediates gene silencing through its N-terminal domains. We show that CSDE1 interacts with LSM14A, a constituent of P-body assembly and further associates to the DCP1-DCP2 decapping complex, suggesting that CSDE1 could promote the decay of miRNA-induced silencing complex-targeted mRNAs. Together, our findings uncover a hitherto unknown mechanism used by CSDE1 in the control of gene expression mediated by the miRNA pathway. © 2020 Kakumani et al.OBJECTIVES Infantile hemangiomas (IHs) are common; some cases require timely referral and treatment to prevent complications. We developed and validated a reliable instrument for timely and adequate referral of patients with IH to experts by nonexpert primary physicians. METHODS In this multicenter, cross-sectional, observational study, we used a 3-stage process (1) development of the Infantile Hemangioma Referral Score (IHReS) tool by IH experts who selected a representative set of 42 IH cases comprising images and a short clinical history, (2) definition of the gold standard for the 42 cases by a second independent committee of IH experts, and (3) IHReS validation by nonexpert primary physicians using the 42 gold standard cases. RESULTS A total of 60 primary physicians from 7 different countries evaluated the 42 gold standard cases (without reference to the IHReS tool); 45 primary physicians evaluated these cases using the IHReS questionnaire, and 44 completed retesting using the instrument. IHReS had a sensitivity of 96.9% (95% confidence interval 96.1%-97.8%) and a specificity of 55.0% (95% confidence interval 51.0%-59.0%). The positive predictive value and negative predictive value were 40.5% and 98.3%, respectively. Validation by experts and primary physicians revealed substantial agreement for interrater reliability and intrarater repeatability. CONCLUSIONS IHReS, a 2-part algorithm with a total of 12 questions, is an easy-to-use tool for primary physicians for the purpose of facilitating correct and timely referral of patients with IH. IHReS may help practitioners in their decision to refer patients to expert centers. Copyright © 2020 by the American Academy of Pediatrics.The nutritional and immunologic properties of human milk, along with clear evidence of dose-dependent optimal health outcomes for both mothers and infants, provide a compelling rationale to support exclusive breastfeeding. https://www.selleckchem.com/products/rvx-208.html US women increasingly intend to breastfeed exclusively for 6 months. Because establishing lactation can be challenging, exclusivity is often compromised in hopes of preventing feeding-related neonatal complications, potentially affecting the continuation and duration of breastfeeding. Risk factors for impaired lactogenesis are identifiable and common. Clinicians must be able to recognize normative patterns of exclusive breastfeeding in the first week while proactively identifying potential challenges. In this review, we provide new evidence from the past 10 years on the following topics relevant to exclusive breastfeeding milk production and transfer, neonatal weight and output assessment, management of glucose and bilirubin, immune development and the microbiome, supplementation, and health system factors.