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Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant—YE1-BE3-FNLS—that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits. Structural and biochemical insights help engineer a cytosine base editor variant that possesses improved on-target activity with minimal DNA and RNA off-target editing.

Phycobiliproteins have gained increasing attention for their antidiabetic potential, yet the specific bioactive peptides and their targets and molecular mechanisms have remained unclear. In this study, four peptides with potential hypoglycemic activity were identified through virtual screening. Network pharmacology was employed to elucidate their hypoglycemic mechanism in the treatment of T2DM. A subsequent in vitro assay confirmed that the synthesized peptides, GR-5, SA-6, VF-6, and IR-7, exhibited significant inhibitory activity against α-glucosidase and DPP-IV. In insulin-resistant HepG2 models, all four peptides exhibited no cytotoxicity. Among them, GR-5 demonstrated the most promising therapeutic potential by remarkably enhancing cellular glucose consumption capacity. Furthermore, GR-5 administration substantially increased glycogen synthesis and enzymatic activities of hexokinase and pyruvate kinase with statistically significant improvements compared to the control groups. This study provides novel peptide candidates for T2DM treatment and validates an integrative strategy for targeted bioactive peptide discovery, advancing the development of algal protein-based therapeutics.

Background The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown. Results Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells. Conclusions CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.

Background: Rutin is a natural flavonoid extracted primarily from plants with anti-inflammatory and antioxidant properties, and it is highly valuable in the cosmetics industry. However, the poor transdermal permeability of rutin limits its application via transdermal administration. Previous studies have predominantly focused on chemical methods for enhancing penetration. This study investigated the potential of ultrasound as a physical method by which to augment the transdermal absorption and anti-inflammatory effects of rutin. Method: Through in vitro diffusion experiments, we analyzed the effects of the ultrasonic frequency and intensity on percutaneous absorption. The optimal ultrasound parameters were determined based on the intradermal retention rate, which is defined as the proportion of intradermal retention to the total penetration. Parameters with higher retention rates were considered optimal. To validate the anti-inflammatory efficacy of rutin delivered using the ultrasound-assisted method, we employed a tape-stripping technique to induce inflammation in BALB/c nude mice. Eight mice were assigned to each treatment group: (A) self-repair (control group), (B) regular rutin treatment, and (C) ultrasound-assisted treatment. Results: The research findings indicate that ultrasound frequency and intensity of 1 MHz and 0.2 W/cm2, as well as 3 MHz and 0.2 W/cm2, result in the maximum proportion of rutin intradermal retention, exhibiting values 1.8 times (using porcine skin) and 2.63 times (using nude mouse skin) higher than those achieved without ultrasound, respectively. Group C showed the shortest recovery time and displayed complete skin barrier function restoration by the fourth day (p<0.05), whereas group A exhibited the slowest recovery. Conclusions: This study offers an innovative approach for the transdermal delivery of rutin to facilitate skin barrier function repair.

Due to the non-smooth characteristics of stick-slip friction, analytical solutions for the Dry Friction Constrained System (DFCS) are generally unavailable. Consequently, numerical simulation has become the most widely used approach for analyzing the DFCS. However, the accuracy and efficiency of the numerical algorithm considering the Coulomb stick-slip motion and determining whether stick-slip motion is considered in engineering design to further improve the computational efficiency remain a critical area of study. In this paper, a single-degree-of-freedom DFCS is introduced to address these issues. The Runge-Kutta method, combined with the dichotomy, is employed to accurately capture the stick-slip transition point. The normal load and dry friction are both symmetrically and evenly distributed at contact surfaces. Firstly, stick-slip motion analyses are performed, and response characteristics of the DFCS are discussed. Then, the convergence characteristics of the numerical algorithm are analyzed, and the optimal iteration step size and the zero-velocity interval are determined. Finally, whether stick-slip motion is considered in numerical simulation in the design of the DFCS in engineering practice is analyzed based on the dimensionless external force and frequency ratio. The criteria for determining whether stick-slip motion is considered in engineering design are established, which can improve both computational accuracy and efficiency.

Chatter stability analysis is an effective way to optimize the cutting parameters and achieve chatter-free machining. This paper proposes a milling chatter stability analysis method based on the localized differential quadrature method (LDQM), which has the advantages of simple principle, easy application, and high computational efficiency. The milling process, considering the regeneration effect, is modeled using linear periodic delay differential equations (DDE), then the state transition matrix during the adjacent cutting period is constructed based on the LDQM, and finally, the stability of the milling process is obtained based on the Floquet theory. The accuracy and computation efficiency of the proposed method are verified through two benchmark milling models. The simulation results demonstrate that the proposed method in this paper can accurately and quickly obtain the chatter stability lobe diagram (SLD) of the milling process, which will provide guidance for optimizing the process parameters.

On the basis of linear multistep method, the stability of milling process is investigated in this paper. The dynamics of milling process considering the regeneration effect is modeled as a delay differential equation (DDE) with periodic coefficient. The Floquet theory is adopted to predict the stability of milling by calculating the spectral radius of the transition matrix over one principal period. Two high-order starting methods for the Milne-Simpson method are introduced firstly. The effects of different starting methods on the convergence rate of the algorithm are studied. Subsequently, a Milne-Simpson-based predictor-corrector method (SSM) is proposed to further improve the numerical stability and convergence rate. The accuracy and computational efficiency of SSM are verified through two benchmark milling models. The simulation results demonstrate that the proposed method has excellent numerical stability and higher convergence rate compared with the Simpson-based method (SBM) and Adams-Simpson-based method (ASM).

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh ; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions. Gao et al. developed a CRISPR–Cas9-based system in which sgRNA production is controlled by the endogenous promoter to monitor the expression of weakly expressed genes and long non-coding RNAs in mammalian cells.

The yellow stem borer, Scripophaga incertulas (Crambidae, Lepidoptera), is a highly destructive pest damaging rice crops. Here, we combined Illumina, PacBio sequencing and Hi-C scaffolding to generate a high-quality chromosome-level genome assembly of S. incertulas . We initially obtained an 839.78 Mb assembly with scoffold N50 of 1.13 Mb. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis demonstrated that this genome assembly has a high-level completeness of 97.5% gene coverage. The assembly contained 45.67% repetitive sequences (383.50 Mb), and has been annotated with 19,874 protein-coding genes. Next, 3,369 scaffolds were anchored to 26 chromosomes, resulting in an increase of the N50 to 32.16 Mb. Our research provides insights into the evolution and ecology of S. incertulas , offering valuable resources for future pest management.