Real-Time Detection of Multiple Intracellular MicroRNAs using an Ultrasound-Propelled Nanomotor-Based Dynamic Fluorescent Probe.

Multiple intracellular microRNA (miRNA) detection is essential for disease diagnosis and management. Nonetheless, the real-time detection of multiple intracellular miRNAs has remained challenging. Herein, we have developed an ultrasound (US)-powered nanomotor-based dynamic fluorescent probe for the real-time OFF-ON fluorescent determination of multiple intracellular miRNAs. The new probe relies on the utilization of multicolored quantum dot (QD)-labeled single-stranded DNA (ssDNA)/graphene oxide (GO)-coated US-powered gold nanowire (AuNW) nanomotors. The fluorescence of QDs is quenched due to π-π interactions with the GO. Upon binding to target miRNAs, the QDs-ssDNA is now distant from the AuNWs, resulting in effective OFF-ON QD fluorescence switching. Compared with conventional passive probes, the dynamic fluorescent probe enhances probe-target interactions by using the US-propelled nanomotor, resulting in exceptionally efficient and prompt hybridization. Simultaneous quantitative analysis of miR-10b and miR-21 in vitro can be achieved within 15 min with high sensitivity and specificity. Additionally, multicolor QDs provide strong signal intensity and multiplexed detection, enabling one-step real-time discrimination between cancer cells (A549) and normal cells (L02). The obtained results are in good agreement with those from qRT-PCR. This dynamic fluorescent probe based on a nanomotor and QDs enables rapid "on the move" specific detection of multiple intracellular miRNAs in intact cells, facilitating real-time monitoring of diverse intracellular miRNA expression, and it could pave the way for novel applications of nanomotors in biodetection.

Study on the extraction, purification, stability, free radical scavenging kinetics, polymerization degree and characterization of proanthocyanidins from Pinus koraiensis seed scales.

To efficiently harness resources from Pinus koraiensis seed scales, a type of forestry waste, rigorous studies on the extraction, purification, stability, and free radical scavenging capacity of the proanthocyanidins derived from these seed scales were conducted. Kinetic models showed that under ultrasonic conditions, the proanthocyanidins content reached 2.66 mg/g within 0.5 h. The optimal storage parameters include darkness, 4 °C, and pH 4. The degrees of polymerization of the mixture and the high- and low-polymer components were 4.89, 7.42 and 3.07, respectively, with the low-polymer component exhibiting the highest radical scavenging activity. Through HPLC-QE-MS/MS, 1H NMR, and FT-IR analyses, we identified proanthocyanidin B1, proanthocyanidin B2, (-)-epicatechin, and polymeric trimer esters. The Pinus koraiensis proanthocyanidins exhibited a high molecular weight, a complex internal molecular structure, and commendable stability, with crystallization requiring elevated temperatures. Therefore, the proanthocyanidins from Pinus koraiensis seed scales have emerged as highly promising novel natural antioxidants.

Room-Temperature Cascade Electrophilic Addition/Cyclization/Oxidation Reactions: Divergent Selective Synthesis of Brominated 2H-Chromenes, 2H-Chromen-2-ols and 2H-Chromen-2-ones.

The room temperature metal-free cascade electrophilic addition/cyclization/oxidation reactions of (3-phenoxyprop-1-yn-1-yl)benzenes to divergently synthesize various brominated benzopyran derivatives (3-bromo-2H-chromenes, 3-bromo-2H-chromen-2-ols and 3-bromo coumarins) by tuning the amount of Br2 and H2O have been developed. The method exhibited high selectivity, mild reaction conditions, broad substrate scope, high efficiency, and the applicability for derivatization of the brominated products. The importance of the strategies provides a great advantage for selective synthesis of brominated benzopyran derivatives.

Potential drug targets for tumors identified through Mendelian randomization analysis.

According to the latest cancer research data, there are a significant number of new cancer cases and a substantial mortality rate each year. Although a substantial number of clinical patients are treated with existing cancer drugs each year, the efficacy is unsatisfactory. The incidence is still high and the effectiveness of most cancer drugs remains unsatisfactory. Therefore, we evaluated the human proteins for their causal relationship to for cancer risk and therefore also their potential as drug targets. We used summary tumors data from the FinnGen and cis protein quantitative trait loci (cis-pQTL) data from a genome-wide association study, and employed Mendelian randomization (MR) to explore the association between potential drug targets and nine tumors, including breast, colorectal, lung, liver, bladder, prostate, kidney, head and neck, pancreatic caners. Furthermore, we conducted MR analysis on external cohort. Moreover, Bidirectional MR, Steiger filtering, and colocalization were employed to validate the main results. The DrugBank database was used to discover potential drugs of tumors. Under the threshold of False discovery rate (FDR) < 0.05, results showed that S100A16 was protective protein and S100A14 was risk protein for human epidermal growth factor receptor 2-positive (HER-positive) breast cancer, phosphodiesterase 5A (PDE5A) was risk protein for colorectal cancer, and melanoma inhibitory activity (MIA) was protective protein for non-small cell lung carcinoma (NSCLC). And there was no reverse causal association between them. Colocalization analysis showed that S100A14 (PP.H4.abf = 0.920) and S100A16 (PP.H4.abf = 0.932) shared causal variation with HER-positive breast cancer, and PDE5A (PP.H4.abf = 0.857) shared causal variation with colorectal cancer (CRC). The MR results of all pQTL of PDE5A and MIA were consistent with main results. In addition, the MR results of MIA and external outcome cohort were consistent with main results. In this study, genetic predictions indicate that circulating S100 calcium binding protein A14 (S100A14) and S100 calcium binding protein A16 (S100A16) are associated with increase and decrease in the risk of HER-positive breast cancer, respectively. Circulating PDE5A is associated with increased risk of CRC, while circulating MIA is associated with decreased risk of NSCLC. These findings suggest that four proteins may serve as biomarkers for cancer prevention and as potential drug targets that could be expected for approval.

Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3.

Currently, the Cas9 and Cas12a systems are widely used for genome editing, but their ability to precisely generate large chromosome fragment deletions is limited. Type I-E CRISPR mediates broad and unidirectional DNA degradation, but controlling the size of Cas3-mediated DNA deletions has proven elusive thus far. Here, we demonstrate that the endonuclease deactivation of Cas9 (dCas9) can precisely control Cas3-mediated large-fragment deletions in mammalian cells. In addition, we report the elimination of the Y chromosome and precise retention of the Sry gene in mice using CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3, respectively. In conclusion, dCas9-controlled CRISPR/Cas3-mediated precise large-fragment deletion provides an approach for establishing animal models by chromosome elimination. This method also holds promise as a potential therapeutic strategy for treating fragment mutations or human aneuploidy diseases that involve additional chromosomes.

Unraveling the role of Xist in X chromosome inactivation: insights from rabbit model and deletion analysis of exons and repeat A.

X chromosome inactivation (XCI) is a process that equalizes the expression of X-linked genes between males and females. It relies on Xist, continuously expressed in somatic cells during XCI maintenance. However, how Xist impacts XCI maintenance and its functional motifs remain unclear. In this study, we conducted a comprehensive analysis of Xist, using rabbits as an ideal non-primate model. Homozygous knockout of exon 1, exon 6, and repeat A in female rabbits resulted in embryonic lethality. However, X∆ReAX females, with intact X chromosome expressing Xist, showed no abnormalities. Interestingly, there were no significant differences between females with homozygous knockout of exons 2-5 and wild-type rabbits, suggesting that exons 2, 3, 4, and 5 are less important for XCI. These findings provide evolutionary insights into Xist function.

La(OTf)3-Catalyzed [3+2] Cycloaddition Reactions for the Synthesis of Benzo[d]oxazoles/Benzofurans.

The La(OTf)3-catalyzed [3+2] cycloaddition reactions for the synthesis of benzo[d]oxazoles/benzofurans via quinones and 1,2-di-tert-butyl-3-(cyanimino)diaziridine (1,3-di-tert-butyl-2-cyanoguanidine)/vinyl azides have been explored. A series of 5-hydroxybenzofuran-4-carboxylic acid derivatives and 5-hydroxybenzo[d]oxazole-4-carboxylic acid derivatives were conveniently obtained with high yields and good stereoselectivities, which could be used for further transformations to valuable compounds.

Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.

5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

Genome-edited rabbits: Unleashing the potential of a promising experimental animal model across diverse diseases.

Animal models are extensively used in all aspects of biomedical research, with substantial contributions to our understanding of diseases, the development of pharmaceuticals, and the exploration of gene functions. The field of genome modification in rabbits has progressed slowly. However, recent advancements, particularly in CRISPR/Cas9-related technologies, have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases, including cardiovascular disorders, immunodeficiencies, aging-related ailments, neurological diseases, and ophthalmic pathologies. These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice. This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine, underscoring their impact and future potential in translational medicine.

Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope.

BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797-C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.

Development of a highly efficient prime editor system in mice and rabbits.

The recently developed prime-editing (PE) technique is more precise than previously available techniques and permits base-to-base conversion, replacement, and insertions and deletions in the genome. However, previous reports show that the efficiency of prime editing is insufficient to produce genome-edited animals. In fact, prime-guide RNA (pegRNA) designs have posed a challenge in achieving favorable editing efficiency. Here, we designed prime binding sites (PBS) with a melting temperature (Tm) of 42 °C, leading to optimal performance in cells, and we found that the optimal Tm was affected by the culture temperature. In addition, the ePE3max system was developed by updating the PE architecture to PEmax and expressing engineered pegRNA (epegRNA) based on the original PE3 system. The updated ePE3max system can efficiently induce gene editing in mouse and rabbit embryos. Furthermore, we successfully generated a Hoxd13 (c. 671 G > T) mutation in mice and a Tyr (c. 572 del) mutation in rabbits by ePE3max. Overall, the editing efficiency of modified ePE3max systems is superior to that of the original PE3 system in producing genome-edited animals, which can serve as an effective and versatile genome-editing tool for precise genome modification in animal models.

Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.

Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.

A strategy for Cas13 miniaturization based on the structure and AlphaFold.

The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.

A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.

BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.

Exercise-based cardiac rehabilitation programmers for patients after transcatheter aortic valve implantation: A systematic review and meta-analysis.

BACKGROUND: Currently, transcatheter aortic valve implantation (TAVI) is presently a recognized treatment modality for patients with severe aortic stenosis who are often old, disabled, frail, and have low exercise capacity (ExCap). It is further expected from this therapy to improve quality of life by improving of the cardio function performance. The aim of this study is to evaluate the effect of exercise-based cardiac rehabilitation (CR) on patients after TAVI. METHODS: PubMed, Embase, Cochrane Library, and Web of Science were searched from inception to December 10, 2022 for relevant studies that evaluated the effect of CR on patients after TAVI. The primary outcome was the improvement of 6-minute walked distance and Barthel index score after CR. The secondary outcomes included other parameters such as SF-12 scale, HADS score, Morse Fall Scale, Frailty-Index, ExCap, and FIM score. All statistical analyses were performed using the standard statistical procedures provided in Review Manager 5.2. RESULTS: A total of 12 observational studies were identified, with 2365 participants. Pooled data indicated that CR programmers significantly improved the 6-minute walked distance (SMD 0.65; 95% confidence intervals [CI] 0.51-0.79) and Barthel index score (SMD 0.83; 95% CI 0.61-1.06). In addition, compared with admission, patients experienced significant improvement in SF-12 scale at CR discharge, with a pooled mean differences (MD) of 2.74 (95% CI 0.86-4.61) in physical component score and 2.76 (95% CI 0.59-4.93) in mental component score. Similar results were also observed in ExCap (MD 8.10 W; 95% CI 1.57 W-14.63 W) and FIM score (MD 11.0; 95% CI 6.22-15.78). CONCLUSIONS: Our analysis indicated that exercise-based CR programmers had significant effect on patients after TAVI in improving exercise tolerance and functional independence.

A high-performance metal halide perovskite-based laser-driven display.

Laser-driven liquid crystal displays (LCDs) comprising metal halide perovskites (MHPs) as the blue-to-green/red color converters are at the forefront of ongoing intense research on the development and improvement of display devices. However, the inferior high photoluminescence quantum yield (PLQY) of MHPs under the excitation of high-power blue light and photoluminescence deterioration at high temperatures remain major concerns. Herein, we design a kind of octylamine-modified MHP via binding energy engineering, and the synthesized materials show PLQY of 97.6% under the excitation of a blue laser at 450 nm. Meanwhile, this design endows a structural self-healing ability to achieve a high PLQY and luminescence stability under high temperature (90 °C) and high flux excitation (386 mW cm-2). The blue light-excitable MHPs with a near unity PLQY, strong stability, and low PLQY deterioration are further encapsulated into a laser-driven LCD device. This prototype demonstrates excellent color gamut (132% NTSC, 98% Rec. 2020), illuminance intensity (>10 000 lux), and energy consumption (47.5% of commercial consumption), and hence is expected to be beneficial for the reduction of energy consumption in backlight display devices, particularly in large-screen outdoor displays.

YIPF5 (p.W218R) mutation induced primary microcephaly in rabbits.

Primary microcephaly (PMCPH) is a rare autosomal recessive neurodevelopmental disorder with a global prevalence of PMCPH ranging from 0.0013% to 0.15%. Recently, a homozygous missense mutation in YIPF5 (p.W218R) was identified as a causative mutation of severe microcephaly. In this study, we constructed a rabbit PMCPH model harboring YIPF5 (p.W218R) mutation using SpRY-ABEmax mediated base substitution, which precisely recapitulated the typical symptoms of human PMCPH. Compared with wild-type controls, the mutant rabbits exhibited stunted growth, reduced head circumference, altered motor ability, and decreased survival rates. Further investigation based on model rabbit elucidated that altered YIPF5 function in cortical neurons could lead to endoplasmic reticulum stress and neurodevelopmental disorders, interference of the generation of apical progenitors (APs), the first generation of progenitors in the developing cortex. Furthermore, these YIPF5-mutant rabbits support a correlation between unfolded protein responses (UPR) induced by endoplasmic reticulum stress (ERS), and the development of PMCPH, thus providing a new perspective on the role of YIPF5 in human brain development and a theoretical basis for the differential diagnosis and clinical treatment of PMCPH. To our knowledge, this is the first gene-edited rabbit model of PMCPH. The model better mimics the clinical features of human microcephaly than the traditional mouse models. Hence, it provides great potential for understanding the pathogenesis and developing novel diagnostic and therapeutic approaches for PMCPH.

Unique progerin C-terminal peptide ameliorates Hutchinson-Gilford progeria syndrome phenotype by rescuing BUBR1.

An accumulating body of evidence indicates an association between mitotic defects and the aging process in Hutchinson-Gilford progeria syndrome (HGPS), which is a premature aging disease caused by progerin accumulation. Here, we found that BUBR1, a core component of the spindle assembly checkpoint, was downregulated during HGPS cellular senescence. The remaining BUBR1 was anchored to the nuclear membrane by binding with the C terminus of progerin, thus further limiting the function of BUBR1. Based on this, we established a unique progerin C-terminal peptide (UPCP) that effectively blocked the binding of progerin and BUBR1 and enhanced the expression of BUBR1 by interfering with the interaction between PTBP1 and progerin. Finally, UPCP significantly inhibited HGPS cellular senescence and ameliorated progeroid phenotypes, extending the lifespan of LmnaG609G/G609G mice. Our findings reveal an essential role for the progerin-PTBP1-BUBR1 axis in HGPS. Therapeutics designed around UPCP may be a beneficial strategy for HGPS treatment.

Lp-PLA2 (Lipoprotein-Associated Phospholipase A2) Deficiency Lowers Cholesterol Levels and Protects Against Atherosclerosis in Rabbits.

BACKGROUND: Elevated plasma Lp-PLA2 (lipoprotein-associated phospholipase A2) activity is closely associated with an increased risk of cardiovascular events. However, whether and how Lp-PLA2 is directly involved in the pathogenesis of atherosclerosis is still unclear. To examine the hypothesis that Lp-PLA2 could be a potential preventative target of atherosclerosis, we generated Lp-PLA2 knockout rabbits and investigated the pathophysiological functions of Lp-PLA2. METHODS: Lp-PLA2 knockout rabbits were generated using CRISPR/Cas9 system to assess the role of Lp-PLA2 in plasma lipids regulation and identify its underlying molecular mechanisms. Homozygous knockout rabbits along with wild-type rabbits were fed a cholesterol-rich diet for up to 14 weeks and their atherosclerotic lesions were compared. Moreover, the effects of Lp-PLA2 deficiency on the key cellular behaviors in atherosclerosis were assessed in vitro. RESULTS: When rabbits were fed a standard diet, Lp-PLA2 deficiency led to a significant reduction in plasma lipids. The decreased protein levels of SREBP2 (sterol regulatory element-binding protein 2) and HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase) in livers of homozygous knockout rabbits indicated that the cholesterol biosynthetic pathway was impaired with Lp-PLA2 deficiency. In vitro experiments further demonstrated that intracellular Lp-PLA2 efficiently enhanced SREBP2-related cholesterol biosynthesis signaling independently of INSIGs (insulin-induced genes). When fed a cholesterol-rich diet, homozygous knockout rabbits exhibited consistently lower level of hypercholesterolemia, and their aortic atherosclerosis lesions were significantly reduced by 60.2% compared with those of wild-type rabbits. The lesions of homozygous knockout rabbits were characterized by reduced macrophages and the expression of inflammatory cytokines. Macrophages of homozygous knockout rabbits were insensitive to M1 polarization and showed reduced DiI-labeled lipoprotein uptake capacity compared with wild-type macrophages. Lp-PLA2 deficiency also inhibited the adhesion between monocytes and endothelial cells. CONCLUSIONS: These results demonstrate that Lp-PLA2 plays a causal role in regulating blood lipid homeostasis and Lp-PLA2 deficiency protects against dietary cholesterol-induced atherosclerosis in rabbits. Lp-PLA2 could be a potential target for the prevention of atherosclerosis.