Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches.

Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3\u27 of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment

Resminostat in EGFR-mutated lung cancer

Drug-tolerant cells are mediators of acquired resistance. BIM-intron2 deletion polymorphism (BIM-del) is one of the mechanisms underlying the resistance to epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI)-mediated apoptosis that induces drug tolerance. Here, we investigated whether resminostat, a histone deacetylase inhibitor, circumvents BIM-del-associated apoptosis resistance. The human EGFR-mutated non-small cell lung cancer (NSCLC) cell line PC-9 and its homozygous BIM-del-positive variant (PC-9 BIMi2-/-), established by editing with zinc finger nuclease, were used. In comparison with PC-9 cells, PC-9 BIMi2-/- cells were less sensitive to apoptosis mediated by EGFR-TKIs such as gefitinib and osimertinib. The combined use of resminostat and an EGFR-TKI preferentially induced the expression of the pro-apoptotic BIM transcript containing exon 4 rather than that containing exon 3, increased the level of pro-apoptotic BIM protein (BIMEL), and stimulated apoptosis in vitro. In a subcutaneous tumor model derived from PC-9 BIMi2-/- cells, gefitinib monotherapy decreased tumor size but retained residual lesions, indicative of the presence of tolerant cells in tumors. The combined use of resminostat and gefitinib increased BIMEL protein level and induced apoptosis, subsequently leading to the remarkable shrinkage of tumor. These findings suggest the potential of resminostat to circumvent tolerance to EGFR-TKIs associated with BIM deletion polymorphism

From basic mechanisms to clinical applications in heart protection, new players in cardiovascular diseases and cardiac theranostics: meeting report from the third international symposium on "New frontiers in cardiovascular research"

In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients' cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia-reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients' outcome

Long-term antithrombotic management patterns in Asian patients with acute coronary syndrome: 2-year observations from the EPICOR Asia study.

BACKGROUND: Despite guideline recommendations, dual antiplatelet therapy (DAPT) is frequently used for longer than 1 year after an acute coronary syndrome (ACS) event. In Asia, information on antithrombotic management patterns (AMPs), including DAPT post discharge, is sparse. This analysis evaluated real-world AMPs up to 2 years post discharge for ACS. HYPOTHESIS: There is wide variability in AMP use for ACS management in Asia. METHODS: EPICOR Asia (NCT01361386) is a prospective observational study of patients discharged after hospitalization for an ACS in eight countries/regions in Asia, followed up for 2 years. Here, we describe AMPs used and present an exploratory analysis of characteristics and outcomes in patients who received DAPT for ≤12 months post discharge compared with >12 months. RESULTS: Data were available for 12 922 patients; of 11 639 patients discharged on DAPT, 2364 (20.3%) received DAPT for ≤12 months and 9275 (79.7%) for >12 months, with approximately 60% still on DAPT at 2 years. Patients who received DAPT for >12 months were more likely to be younger, obese, lower Killip class, resident in India (vs China), and to have received invasive reperfusion. Clinical event rates during year 2 of follow-up were lower in patients with DAPT >12 vs ≤12 months, but no causal association can be implied in this non-randomized study. CONCLUSIONS: Most ACS patients remained on DAPT up to 1 year, in accordance with current guidelines, and over half remained on DAPT at 2 years post discharge. Patients not on DAPT at 12 months are a higher risk group requiring careful monitoring

Optical solitons: mathematical model and simulations

Optical solitons travel in nonlinear dispersive optical fiber that can mathematically modeled by forced nonlinear Schrödinger Equation (fNLS). A precise numerical simulation is employed to simulate optical solitons travel based on the mathematical model equation modeled in ideal lossless fiber and fiber loss. The outcomes from simulations further clarify the effects of fiber loss during transmission of signal which distorted the balanced effects between self-phase modulation (SPM) and group velocity dispersion (GVD) in nonlinear optical fiber with fiber. Furthermore, the outcomes have met the agreement with the simulation done by engineering software

Numerical solution of the gardner equation

The Gardner equation is commonly used to describe wave propagation in weakly nonlinear dispersive medium. The Gardner equation has a higher order nonlinear term, which could make the numerical calculation inaccurate. In this paper, the Gardner equation is solved using two numerical methods, i.e., the method of lines and pseudospectral method. The efficiency and accuracy of both methods were studied. Our results show that both methods are accurate and efficient methods to solve the Gardner equation. By comparing the accuracy of both the methods, the method of lines performs better than pseudospectral method most of the time

Identification of cis-acting elements and splicing factors involved in the regulation of BIM pre-mRNA splicing

Aberrant changes in the expression of the pro-apoptotic protein, BCL-2-like 11 (BIM), can result in either impaired or excessive apoptosis, which can contribute to tumorigenesis and degenerative disorders, respectively. Altering BIM pre-mRNA splicing is an attractive approach to modulate apoptosis because BIM activity is partly determined by the alternative splicing of exons 3 or 4, whereby exon 3-containing transcripts are not apoptotic. Here we identified several cis-acting elements and splicing factors involved in BIM alternative splicing, as a step to better understand the regulation of BIM expression. We analyzed a recently discovered 2,903-bp deletion polymorphism within BIM intron 2 that biased splicing towards exon 3, and which also impaired BIM-dependent apoptosis. We found that this region harbors multiple redundant cis-acting elements that repress exon 3 inclusion. Furthermore, we have isolated a 23-nt intronic splicing silencer at the 3′ end of the deletion that is important for excluding exon 3. We also show that PTBP1 and hnRNP C repress exon 3 inclusion, and that downregulation of PTBP1 inhibited BIM-mediated apoptosis. Collectively, these findings start building our understanding of the cis-acting elements and splicing factors that regulate BIM alternative splicing, and also suggest potential approaches to alter BIM splicing for therapeutic purposes.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

PTBP1 represses inclusion of <i>BIM</i> exon 3 independently of the 2,903-nt polymorphic fragment.

<p>(A) Schematic of the deletions made on the Δ10E minigene to remove the putative PTBP1 binding sites. +2,582 to +2,662 of the polymorphic fragment has been expanded to show the nucleotide sequence. The predicted PTBP1 binding sites on Δ10E are boxed, whereas the deletions are indicated by dashes. (B) Real-time RT-PCR analysis of RNA from K562 cells nucleofected with the minigene constructs described in (A) to assess the ratio of exon 3- to exon 4-containing minigene products. The Δ11 minigene serves as a positive control for enhanced exon 3 inclusion. Results are presented as an average of triplicates and the relative minigene E3: E4 ratio was determined by normalizing to the E3: E4 ratio of K562 cells nucleofected with the Δ10 minigene. Error bars represent ± SEM. (C) K562 cells were either nucleofected with control or PTBP1-specific siRNA duplexes. 24 hours later, these cells were nucleofected with either the WT, Δ10, Δ10E or Δ11 minigene. Real-time RT-PCR analysis of RNA from these cells was performed after another 24 hours to determine the ratio of exon 3- to exon 4-containing minigene products. Results are presented as an average of triplicates and the relative minigene E3: E4 ratio was determined by normalizing to the E3: E4 ratio of K562 cells nucleofected with the same minigene, but without any siRNA. Error bars represent ± SEM.</p