Anti-hsa-miR-59 alleviates premature senescence associated with Hutchinson-Gilford progeria syndrome in mice.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature aging disorder without an effective therapeutic regimen. Because of their targetability and influence on gene expression, microRNAs (miRNAs) are attractive therapeutic tools to treat diseases. Here we identified that hsa-miR-59 (miR-59) was markedly upregulated in HGPS patient cells and in multiple tissues of an HGPS mouse model (LmnaG609G/G609G ), which disturbed the interaction between RNAPII and TFIIH, resulting in abnormal expression of cell cycle genes by targeting high-mobility group A family HMGA1 and HMGA2. Functional inhibition of miR-59 alleviated the cellular senescence phenotype of HGPS cells. Treatment with AAV9-mediated anti-miR-59 reduced fibrosis in the quadriceps muscle, heart, and aorta, suppressed epidermal thinning and dermal fat loss, and yielded a 25.5% increase in longevity of LmnaG609G/G609G mice. These results identify a new strategy for the treatment of HGPS and provide insight into the etiology of HGPS disease.

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.

Targeted mutagenesis in mice via an engineered AsCas12f1 system.

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

No apparent p53 activation in CRISPR-engineered gene-edited rabbits.

Clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) and base editors (BEs) are revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR-Cas9-mediated genome editing can induce a p53-mediated stress response and cell cycle arrest in human cells, while not illustrated in gene-editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene-edited rabbits using CRISPR-Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE-edited, suggesting that p53 may not need to consider for application in gene-edited animals.

Genetic deletion of a short fragment of glucokinase in rabbit by CRISPR/Cas9 leading to hyperglycemia and other typical features seen in MODY-2.

Glucokinase (GCK) is a key enzyme in glucose sensing and glycemic regulation. In humans, mutations in the GCK gene cause maturity-onset diabetes of the young 2 (MODY-2), a disease that is characterized by an early-onset and persistent hyperglycemia. It is known that Gck knockout (KO) is lethal in mice with Gck KO mice dying within 2 weeks after birth. Therefore, Gck KO mice are not suitable for preclinical study and have limited suitability to study the pathophysiological role of glucokinase in vivo. Here, we report the generation of a novel rabbit with a non-frameshift mutation of GCK gene (GCK-NFS) by cytoplasm microinjection of Cas9 mRNA and gRNA. These GCK-NFS rabbits showed typical features of MODY-2 including hyperglycemia and glucose intolerance with similar survival rate and weight compared to wild-type (WT) rabbits. The diabetic phenotype including pancreatic and renal dysfunction was also found in the F1-generation rabbits, indicating that the genetic modification is germline transmissible. Treatment of GCK-NFS rabbit with glimepiride successfully reduced the fasting blood glucose drastically and improved its islet function. In conclusion, this novel GCK mutant rabbit generated with the CRISPR/Cas9 system mimics most, if not all, histopathological and functional defects seen in MODY-2 patients such as hyperglycemia and will be a valuable rabbit model for preclinical studies and drug screening for diabetes as well as for studying the pathophysiological role of glucokinase.

Study on defect-induced damage behaviors of ADP crystals by 355†nm pulsed laser.

High-quality ammonium dihydrogen phosphate (NH4H2PO4, ADP) crystals were grown in Z direction and in defined crystallographic direction (θ=90°, φ=45°) by the rapid growth method, respectively. Defect-induced damage behavior in 355 nm of three types of ADP samples cutting in type-II matching and third harmonic generation direction from the as-grown crystals were investigated, including the initial laser induced damage (LID) characteristics and the physical and chemical properties of defects which serve as the damage precursors. The evaluations of damage behaviors include the "sampling" laser induced damage threshold (LIDT) by 1-on-1 and R-on-1 methods, bulk damage growth and bulk damage morphology. UV-visible transmittance spectrum, ultraviolet absorption spectrum, fluorescence spectrum, positron annihilation spectrum and the online light scattering measurements were carried out to investigate the defect-induced damage behavior in ADP crystals. The study will provide a reference for the investigations on laser induced damage properties of ADP crystals in short wavelength.

The disrupted balance between hair follicles and sebaceous glands in Hoxc13-ablated rabbits.

Pure hair and nail ectodermal dysplasia 9 (ECTD-9) is an autosomal recessive genetic disease caused by mutation of HOXC13 and is characterized by hypotrichosis and nail dystrophy in humans. Unlike patients with ECTD-9, Hoxc13-mutated mice and pigs do not faithfully recapitulate the phenotype of hypotrichosis, so there is a limited understanding of the molecular mechanism of Hoxc13-mediated hypotrichosis in animal models and clinically. Here, the homozygous Hoxc13-/- rabbits showed complete loss of hair on the head and dorsum, whereas hypotrichosis in the limbs and tail were determined in the Hoxc13-/- rabbits. In addition, reduced hair follicles (HFs) while the enlarged and increased number of sebaceous glands (SGs) were also found in the Hoxc13-/- rabbits, showing that the disrupted balance between HFs and SGs may respond to hypotrichosis of ECTD-9 in an animal model and clinically. Therefore, our findings demonstrate that Hoxc13-/- rabbits can be used as a model for human ECTD-9, especially to understand the pathologic mechanism of hypotrichosis. Moreover, the disrupted balance between HFs and SGs, especially in the Hoxc13-/- rabbits, can be used as an ideal animal model for dermatology ailments, such as acne and hypotrichosis, in preclinical studies.-Deng, J., Chen, M., Liu, Z., Song, Y., Sui, T., Lai, L., Li, Z. The disrupted balance between hair follicles and sebaceous glands in Hoxc13-ablated rabbits.

The minimal promoter (P1) of Xist is non-essential for X chromosome inactivation.

The previous report shows the minimal promoter (P1) contributes to the Xist RNA activation in cells, while the role of the Xist P1 has not yet been investigated in animal individuals. Here, female Xist P1 knockout rabbits (Xist P1-/-) were generated for the studies. The results showed that there is no significant difference in transmission ratio, Xist and X-linked genes expression, and Xist RNA localization between the female wild type (WT) and Xist P1-/- rabbits, suggesting that P1 is non-essential for Xist expression and XCI in rabbits. Our study has explored the function of Xist P1 in animal level for the first time, and the results provide new ideas for future studies of XCI mechanisms.

Comparison of hydrogen vacancies in KDP and ADP crystals: a combination of density functional theory calculations and experiment.

The hydrogen vacancy (VH) is the most common point defect that may lead to optical damage of potassium dihydrogen phosphate (KDP) and its analog ammonium dihydrogen phosphate (ADP), further limiting their practical application in high-power laser systems. In this work, we have grown KDP and ADP crystals by using a rapid growth method, and investigated the physical origin of the different stability of VH as well as the defect-induced electronic structure and optical absorption in KDP and ADP crystals. The inclusion of van der Waals correction to density functional theory calculations is found to have little influence on VH energetics of KDP whereas it largely reduces the charge transition level ε(+/-) of VH by >2 eV in ADP. It is found that hydrogen vacancies mainly contribute to the redshift of the measured absorption edges of both KDP and ADP crystals. Owing to the varied lattice environments and locations, the VH defects exhibit different stability, and electronic and optical properties in KDP and ADP crystals. Notably, the extra optical absorption caused by the positively-charged VH in KDP could be largely reduced by decreasing the defect concentration, whereas ADP exhibits defect-location dependence - the optical damage center of the VH in the NH4+ group could not be eliminated because of electron capture of its neighboring N atoms. The calculation results help us to better understand the origin of laser damage in KDP and ADP crystals.

CRISPR/Cas9-mediated mutation of PHEX in rabbit recapitulates human X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) is the most common cause of inheritable rickets, with an incidence of 1/20 000 in humans. Inactivation or mutation of the gene PHEX, a phosphate-regulating endopeptidase, leads to hypophosphatemia and defective bone mineralization in XLH patients. Presently, there is no adequate animal model for safety assessments of physiotherapies and drug screening for XLH rickets. In this study, an XLH model was generated via PHEX gene knockout (KO) through coinjection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)/sgRNA mRNA into rabbit zygotes. The typical phenotypes of growth retardation, hypophosphatemia, elevated serum FGF23 and bone mineralization were observed in the PHEX KO rabbits but not in normal controls. In summary, for the first time, we have successfully obtained PHEX KO rabbits and recapitulated human XLH using the CRISPR/Cas9 system. This novel XLH rabbit model could be utilized as a drug screening model for XLH prevention and preclinical therapy.

Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system.

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms.

Investigation of surface damage precursor evolutions and laser-induced damage threshold improvement mechanism during Ion beam etching of fused silica.

Surface damage precursor evolution has great influence on laser-induced damage threshold improvement of fused silica surface during Ion beam etching. In this work, a series of ion sputtering experiment are carried out to obtain the evolutions of damage precursors (dot-form microstructures, Polishing-Induced Contamination, Hertz scratches, and roughness). Based on ion sputtering theory, surface damage precursor evolutions are analyzed. The results show that the dot-form microstructures will appear during ion beam etching. But as the ion beam etching depth goes up, the dot-form microstructures can be mitigated. And ion-beam etching can broaden and passivate the Hertz scratches without increasing roughness value. A super-smooth surface (0.238nm RMS) can be obtained finally. The relative content of Fe and Ce impurities both significantly reduce after ion beam etching. The laser-induced damage threshold of fused silica is improved by 34% after ion beam etching for 800nm. Research results can be a reference on using ion beam etching process technology to improve laser-induced damage threshold of fused silica optics.

D-repeat in the XIST gene is required for X chromosome inactivation.

XIST is a long non-coding RNA, which expressed exclusively from the inactive X chromosome. Although it has been revealed that the A-repeat contributes to the X chromosome inactivation (X-inactivation), the role of the longest D-repeat has not yet been investigated. Here, a sgRNA directed CRISPR/Cas9 system which have multiple target sites within repeat D of XIST, were used to generate D-repeat deletion and studied its roles on X-inactivation. The results showed that the deletion of D-repeat caused a significantly decreased expression of XIST, and up regulated expression of X-linked genes, suggesting that the D-repeat may play an important role in the regulation of XIST expression and silencing of the X-linked genes, which could provide a new idea in the molecular mechanisms of X-inactivation.

Screening effective short interfering RNA/short hairpin RNA for inhibition of human astrovirus ORF2 gene expression in cultured cells.

In this study, we have evaluated four different 21-nt duplexes of small interfering RNA (siRNA-469, siRNA-852, siRNA-1802 and siRNA-1806) that specifically target the ORF2 gene of human astrovirus (HAstV) in inhibiting HAstV capsid protein expression in transfected BHK-21 cells. Furthermore, fluorescence analysis, real-time quantitative PCR (RT-qPCR) and western blot assays showed that pGPU6/GFP/Neo-shRNA inhibits ORF2 gene expression in Caco2 cells. The results indicate that siRNA/shRNA-469 and siRNA/shRNA-1802 can interfere with capsid protein expression in cell culture, and this provides a powerful tool for the study of HAstV gene functions and the biological properties of the capsid protein.

The Influence of B4C Film Density on Damage Threshold Based on Monte Carlo Method for X-ray Mirror.

The uniformity and consistency of X-ray mirror film materials prepared by experimental methods are difficult to guarantee completely. These factors directly affect the service life of free electron laser devices in addition to its own optical properties. Therefore, the quality of the film material, especially the density, has a critical effect on its application. Boron carbide film and monocrystalline silicon substrate were suitable examples to explore their influence of density on the damage threshold based on Monte Carlo and heat-conduction methods. Through simulation results, it was found that the change in film density could affect the energy deposition depth and damage threshold. When the film density was 2.48 g/cm3, it had relatively high damage threshold in all energy ranges. And then the specific incident parameter for practical application was investigated. It was found that the damage mechanism of the B4C/Si was the melting of the interface. And the damage threshold was also higher with the film density of 2.48 g/cm3. Therefore, it was recommended to maintain the density at this value as far as possible when preparing the film, and to ensure the uniformity and consistency of the film material.

Effect of Comprehensive Nursing on Pain Relief, Comfort and Burden of Family Care of Infantile Anal Fistula.

Objective: To explore the effect of comprehensive nursing on pain relief, comfort and burden of family care of infantile anal fistula. Methods: This was a randomized, double-blind, controlled clinical trial. A total of 106 cases of children with anal fistula from January 2021 to December 2021 were selected and divided into observation group and control group, there were 53 cases in each group. The control group were underwent with routine nursing intervention. The observation group were underwent with video health education, peer support, music relaxation training, physiotherapy and auricular point pressing on the basis of routine nursing therapy and other measures (comprehensive nursing). The wound healing time, comfort score, complication rate and family care burden of two groups were compared. Results: After intervention, the scores of pain degree in the observation group were significantly lower than those in the control group (4.02 ± 0.85 vs 5.89 ± 2.36, p < 0.05), and the scores of comfort degree in the observation group were higher than those in the control group (70.23 ± 5.98 vs 46.88 ± 5.23, p < 0.05). After intervention, the wound healing time of the observation group was shorter than that of the control group (3.98 ± 0.63 vs 5.77 ± 1.02, p < 0.05), and the crying times of the observation group were less than that of the control group (1.22 ± 0.26 vs 4.02 ± 0.48, p < 0.05). After intervention, the total scores of family members' care load in the observation group were significantly lower than those in the control group (37.26 ± 4.78 vs 55.99 ± 5.02, p < 0.05). Conclusion: Comprehensive nursing can effectively promote wound healing in infantile anal fistula, reduce pain, prompt children's comfort, reduce the number of children crying, and reduce the burden of care for children's families.

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.

Exploring moral algorithm preferences in autonomous vehicle dilemmas: an empirical study.

Introduction: This study delves into the ethical dimensions surrounding autonomous vehicles (AVs), with a specific focus on decision-making algorithms. Termed the "Trolley problem," an ethical quandary arises, necessitating the formulation of moral algorithms grounded in ethical principles. To address this issue, an online survey was conducted with 460 participants in China, comprising 237 females and 223 males, spanning ages 18 to 70. Methods: Adapted from Joshua Greene's trolley dilemma survey, our study employed Yes/No options to probe participants' choices and Likert scales to gauge moral acceptance. The primary objective was to assess participants' inclinations toward four distinct algorithmic strategies-Utilitarianism, Rawlsianism, Egoism, and a Hybrid approach-in scenarios involving AVs. Results: Our findings revealed a significant disparity between participants' preferences in scenarios related to AV design and those focused on purchase decisions. Notably, over half of the respondents expressed reluctance to purchase AVs equipped with an "egoism" algorithm, which prioritizes the car owner's safety. Intriguingly, the rejection rate for "egoism" was similar to that of "utilitarianism," which may necessitate self-sacrifice. Discussion: The hybrid approach, integrating "Utilitarianism" and "Egoism," garnered the highest endorsement. This highlights the importance of balancing self-sacrifice and harm minimization in AV moral algorithms. The study's insights are crucial for ethically and practically advancing AV technology in the continually evolving realm of autonomous vehicles.

Development of Woolly Hair and Hairlessness in a CRISPR-Engineered Mutant Mouse Model with KRT71 Mutations.

Hypotrichosis simplex (HS) and woolly hair (WH) are rare and monogenic disorders of hair loss. HS, characterized by a diffuse loss of hair, usually begins in early childhood and progresses into adulthood. WH displays strong coiled hair involving a localized area of the scalp or covering the entire side. Mutations in the keratin K71(KRT71) gene have been reported to underlie HS and WH. Here, we report the generation of a mouse model of HS and WH by the co-injection of Cas9 mRNA and sgRNA, targeting exon6 into mouse zygotes. The Krt71-knockout (KO) mice displayed the typical phenotypes, including Krt71 protein expression deletion and curly hair in their full body. Moreover, we found that mice in 3-5 weeks showed a new phenomenon of the complete shedding of hair, which was similar to nude mice. However, we discovered that the mice exhibited no immune deficiency, which was a typical feature of nude mice. To our knowledge, this novel mouse model generated by the CRISPR/Cas9 system mimicked woolly hair and could be valuable for hair disorder studies.

Constructing abundant interfaces by decorating MoP quantum dots on CoP nanowires to induce electronic structure modulation for enhanced hydrogen evolution reaction.

Interface engineering is a method of enhancing catalytic activity while maintaining a material's surface properties. Thus, we explored the interface effect mechanism via a hierarchical structure of MoP/CoP/Cu3P/CF. Remarkably, the heterostructure MoP/CoP/Cu3P/CF demonstrates an outstanding overpotential of 64.6 mV at 10 mA cm-2 with a Tafel slope of 68.2 mV dec-1 in 1 M KOH. DFT calculations indicate that the MoP/CoP interface in the catalyst exhibited the most favorable H* adsorption characteristics (-0.08 eV) compared to the pure phases of CoP (0.55 eV) and MoP (0.22 eV). This result can be attributed to the apparent modulation of electronic structures within the interface domains. Additionally, the CoCH/Cu(OH)2/CF‖MoP/CoP/Cu3P/CF electrolyzer demonstrates excellent overall water splitting performance, achieving 10 mA cm-2 in 1 M KOH solution with a modest voltage of only 1.53 V. This electronic structure adjustment via interface effects provides a new and efficient approach to prepare high-performance hydrogen production catalysts.