Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

LARP7 overexpression alleviates aortic senescence and atherosclerosis.

Atherosclerosis, characterized by the accumulation of lipid plaques on the inner walls of arteries, is the leading cause of heart attack, stroke and severe ischemic injuries. Senescent cells have been found to accumulate within atherosclerotic lesions and contribute to the progression of atherosclerosis. In our previous study, we discovered that suppressing Larp7 accelerates senescence by inhibiting Sirt1 activity, resulting in increased atherosclerosis in high-fat diet (HFD) fed and ApoE deficient (ApoEKO) mice. However, there has been no direct evidence demonstrating Larp7 per se could attenuate atherosclerosis. To this end, we generated a tetO-controlled and Cre-activated Larp7 gain-of-function mouse. Through RT-PCR and western blotting, we confirmed Larp7 overexpression in the aortas of HFD-fed ApoEKO; Larp7tetO mice. Larp7 overexpression led to increased Sirt1 activity and decreased cellular senescence signals mediated by p53/p65 in the aortas. Additionally, Larp7 overexpression reduced the presence of p16-positive senescent cells in the aortic lesions. Furthermore, Larp7 overexpression resulted in a decrease in pro-inflammatory macrophages and SASP factors. Consequently, Larp7 overexpression led to a reduction in the area of atherosclerotic lesions in HFD-fed ApoEKO; Larp7tetO mice. In summary, our study provides evidence that Larp7 overexpression holds promise as an approach to inhibit cellular senescence and prevent atherosclerosis.

LARP7 Suppresses Endothelial-to-Mesenchymal Transition by Coupling With TRIM28.

[Figure: see text].

LARP7 Protects Against Heart Failure by Enhancing Mitochondrial Biogenesis.

BACKGROUND: Heart failure (HF) is among the leading causes of morbidity and mortality, and its prevalence continues to rise. LARP7 (La ribonucleoprotein domain family member 7) is a master regulator that governs the DNA damage response and RNAPII (RNA polymerase II) pausing pathway, but its role in HF pathogenesis is incompletely understood. METHODS: We assessed LARP7 expression in human HF and in nonhuman primate and mouse HF models. To study the function of LARP7 in heart, we generated global and cardiac-specific LARP7 knockout mice. We acutely abolished LARP7 in mature cardiomyocytes by Cas9-mediated LARP7 somatic knockout. We overexpressed LARP7 in cardiomyocytes using adeno-associated virus serotype 9 and ATM (ataxia telangiectasia mutated protein) inhibitor. The therapeutic potential of LARP7-regulated pathways in HF was tested in a mouse myocardial infarction model. RESULTS: LARP7 was profoundly downregulated in failing human hearts and in nonhuman primate and murine hearts after myocardial infarction. Low LARP7 levels in failing hearts were linked to elevated reactive oxygen species, which activated the ATM-mediated DNA damage response pathway and promoted LARP7 ubiquitination and degradation. Constitutive LARP7 knockout in mouse resulted in impaired mitochondrial biogenesis, myocardial hypoplasia, and midgestational lethality. Cardiac-specific inactivation resulted in defective mitochondrial biogenesis, impaired oxidative phosphorylation, elevated oxidative stress, and HF by 4 months of age. These abnormalities were accompanied by reduced SIRT1 (silent mating type information regulation 2 homolog 1) stability and deacetylase activity that impaired SIRT1-mediated transcription of genes for oxidative phosphorylation and energy metabolism and dampened cardiac function. Restoring LARP7 expression after myocardial infarction by either adeno-associated virus-mediated LARP7 expression or small molecule ATM inhibitor substantially improved the function of injured heart. CONCLUSIONS: LARP7 is essential for mitochondrial biogenesis, energy production, and cardiac function by modulating SIRT1 homeostasis and activity. Reduction of LARP7 in diseased hearts owing to activation of the ATM pathway contributes to HF pathogenesis and restoring LARP7 in the injured heart confers myocardial protection. These results identify the ATM-LARP7-SIRT1 pathway as a target for therapeutic intervention in HF.

Mitochondrial Pyruvate Carriers Prevent Cadmium Toxicity by Sustaining the TCA Cycle and Glutathione Synthesis.

Cadmium (Cd) is a major heavy metal pollutant, and Cd toxicity is a serious cause of abiotic stress in the environment. Plants protect themselves against Cd stress through a variety of pathways. In a recent study, we found that mitochondrial pyruvate carriers (MPCs) are involved in Cd tolerance in Arabidopsis (Arabidopsis thaliana). Following the identification of MPCs in yeast (Saccharomyces cerevisiae) in 2012, most studies have focused on the function of MPCs in animals, as a possible approach to reduce the risk of cancer developing. The results of this study show that AtMPC protein complexes are required for Cd tolerance and prevention of Cd accumulation in Arabidopsis. AtMPC complexes are composed of two elements, AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). When the formation of AtMPCs was interrupted by the loss of AtMPC1, glutamate could supplement the synthesis of acetyl-coenzyme A and sustain the TCA cycle. With the up-regulation of glutathione synthesis following exposure to Cd stress, the supplementary pathway could not efficiently drive the tricarboxylic acid cycle without AtMPC. The ATP content decreased concomitantly with the deletion of tricarboxylic acid activity, which led to Cd accumulation in Arabidopsis. More importantly, ScMPCs were also required for Cd tolerance in yeast. Our results suggest that the mechanism of Cd tolerance may be similar in other species.

A novel injectable hydrogel prepared from phenylboronic acid modified gelatin and oxidized-dextran for bone tissue engineering.

Complicated fractures have always been challenging in orthopaedics. Designing a multifunctional biomaterial that can contribute to the treatment of fractures using a simple operation remains challenging. Here, we developed a trinity hydrogel system consisting of hydrogel prepared from phenylboronic acid modified gelatin and oxidized-dextran, lithium and cobalt co-doped mesoporous bioactive glass nanoparticles (MBGNs), and irisin. This hydrogel material exhibits considerable injectability, fat-to-shape, and self-healing characteristics. In addition, compared to hydrogel prepared from gelatin and oxidized-dextran, the hydrogel material presented a noticeable enhancement in compression stress and adhesion strength towards porcine bone fragments, which enables it more effectively splice bone fragments during surgery. Based on the various interactions between irisin and the hydrogel network, the system exhibited a clear sustained release of irisin. Based on the results of in vitro cell tests, the hydrogel material showed good cytocompatibility. And it also considerably enhanced the in vitro pro-osteogenic and pro-angiogenic capacities of bone marrow mesenchymal stromal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs). In vivo experimental results indicated that this hydrogel considerably improved the repair of cranial defects in rats. The current study provides a feasible strategy for the treatment of bone fractures and stimulation of fracture healing.

Inhibition of Heat Shock-Induced H3K9ac Reduction Sensitizes Cancer Cells to Hyperthermia.

Heat stress, clinically known as hyperthermia, is a promising adjunctive modality in cancer treatment. However, the efficacy of hyperthermia as a monotherapy is limited and the underlying mechanism remains poorly understood. Targeting histone modifications is an emerging strategy for cancer therapy, but little is known regarding the role of heat stress in altering these modifications. Here, we report that heat shock inhibits H3K9 acetylation (H3K9ac) via histone deacetylase 6 (HDAC6) regulation. Heat shock inhibits the interaction between HDAC6 and heat shock protein 90 (HSP90), enhances nuclear localization of HDAC6, and promotes HDAC6 phosphorylation, which is regulated by protein phosphatase 2A (PP2A). Combining hyperthermia with HDAC inhibitors vorinostat or panobinostat leads to better anti-cancer effects compared to monotherapy. KEAP1 and DPP7 as genes affected by heat-induced inhibition of H3K9ac, and combining them with hyperthermia can better induce apoptosis in tumor cells. This study reveals previously unknown mechanisms of H3K9ac decreased by heat shock in cancer cells and highlights a potential combinational therapy involving hyperthermia and targeting of these new mechanisms.

A lithium-scandium sulfate with second-harmonic generation response and deep-ultraviolet transparency.

A new beryllium-free deep-UV transparent NLO crystal Li(H2O)2Sc(SO4)2 features a two-dimensional [Sc(SO4)2] framework consisting of twisted [Sc3S4O9] units decorated by [LiO2(H2O)2] groups into a unique layer. Remarkably, Li(H2O)2Sc(SO4)2 exhibits a phase-matching SHG response of 0.7 × KDP and a deep-UV cutoff edge below 190 nm.

Lithium and cobalt co-doped mesoporous bioactive glass nanoparticles promote osteogenesis and angiogenesis in bone regeneration.

Healing of severe fractures and bone defects involves many complex biological processes, including angiogenesis and osteogenesis, presenting significant clinical challenges. Biomaterials used for bone tissue engineering often possess multiple functions to meet these challenges, including proangiogenic, proosteogenic, and antibacterial properties. We fabricated lithium and cobalt co-doped mesoporous bioactive glass nanoparticles (Li-Co-MBGNs) using a modified sol-gel method. Physicochemical analysis revealed that the nanoparticles had high specific surface areas (>600 m2/g) and a mesoporous structure suitable for hydroxyapatite (HA) formation and sustained release of therapeutic ions. In vitro experiments with Li-Co-MBGNs showed that these promoted angiogenic properties in HUVECs and pro-osteogenesis abilities in BMSCs by releasing Co2+ and Li+ ions. We observed their antibacterial activity against Staphylococcus aureus and Escherichia coli, indicating their potential applications in bone tissue engineering. Overall, our findings indicate the feasibility of its application in bone tissue engineering.

Effect of different bone cement distributions in percutaneous kyphoplasty on clinical outcomes for osteoporotic vertebral compression fractures: A retrospective study.

Osteoporotic fractures and their complications are becoming increasingly harmful to the elderly. This study aimed to evaluate the clinical results of connected or unconnected bilateral cement after bilateral percutaneous kyphoplasty (PKP) in patients with osteoporotic vertebral compression fractures (OVCF). The clinical data of 217 patients with single-segment OVCF were retrospectively collected. Patients were allocated into 2 groups according to the bilateral bone cement in the vertebrae was connected or unconnected after surgery. The surgery-related indexes of the 2 groups were compared, including operation time; bone cement injection volume; contact situation between bone cement and the upper and lower endplates of the vertebral body; visual analogue scale (VAS) scores before surgery, 1 week and 1 year after surgery; Oswestry disability index (ODI) before surgery, 1 week and 1 year after surgery; local kyphosis angle (LKA) before surgery, 1 week and 1 year after surgery; postoperative vertebral body height at 1 week and 1 year after surgery; vertebral body height restoration rate (HRR) at 1 week and 1 year after surgery. The follow-up results of all patients were recorded. The postoperative VAS, ODI, vertebral body height, LKA and other indexes of the 2 groups were significantly improved compared with those before the operation (P < .05), and there was no significant difference between the 2 groups (P > .05). At the same time, there were no significant difference in vertebral body HRR and bone cement leakage rate between the 2 groups (P > .05). X-ray examination showed that 21 of 217 patients (21/217, 9.8%) had a refracture of the injured vertebral body, including 16 cases (16/121, 13.2%) in the unconnected group and 5 cases (5/96, 5.2%) in the connected group (P < .05). Adjacent vertebrae fractures occurred in 25 cases (25/217, 11.5%), while 19 cases (19/121, 15.7%) were in the unconnected group and 6 cases (6/96, 6.3%) were in the connected group (P < .05). PKP has a good therapeutic effect on OVCF no matter whether the bilateral bone cement is connected or not. However, if the bilateral cement inside the vertebra was connected, the risk of recollapse of the injured vertebrae and the new fracture of adjacent vertebrae could be reduced.

Rare copy number variation analysis identifies disease-related variants in atrioventricular septal defect patients.

Atrioventricular septal defect (AVSD) is a deleterious subtype of congenital heart diseases (CHD) characterized by atrioventricular canal defect. The pathogenic genetic changes of AVSD remain elusive, particularly for copy number variation (CNV), a large segment variation of the genome, which is one of the major forms of genetic variants resulting in congenital heart diseases. In the present study, we recruited 150 AVSD cases and 100 healthy subjects as controls for whole exome sequencing (WES). We identified total 4255 rare CNVs using exon Hidden Markov model (XHMM) and screened rare CNVs by eliminating common CNVs based on controls and Database of Genomic Variants (DGV). Each patient contained at least 9 CNVs, and the CNV burden was prominently presented in chromosomes 19,22,21&16. Small CNVs (<500 kb) were frequently observed. By leveraging gene-based burden test, we further identified 20 candidate AVSD-risk genes. Among them, DYRK1A, OBSCN and TTN were presented in the core disease network of CHD and highly and dynamically expressed in the heart during the development, which indicated they possessed the high potency to be AVSD-susceptible genes. These findings not only provided a roadmap for finally unveiling the genetic cause of AVSD, but also provided more resources and proofs for clinical genetics.

Whole exome sequencing reveals genetic landscape associated with left ventricular outflow tract obstruction in Chinese Han population.

Left ventricular outflow tract obstruction (LVOTO), a major form of outflow tract malformation, accounts for a substantial portion of congenital heart defects (CHDs). Unlike its prevalence, the genetic architecture of LVOTO remains largely unknown. To unveil the genetic mutations and risk genes potentially associated with LVOTO, we enrolled a cohort of 106 LVOTO patients and 100 healthy controls and performed a whole-exome sequencing (WES). 71,430 rare deleterious mutations were found in LVOTO patients. By using gene-based burden testing, we further found 32 candidate genes enriched in LVOTO patient including known pathological genes such as GATA5 and GATA6. Most variants of 32 risk genes occur simultaneously rather exclusively suggesting polygenic inherence of LVOTO and 14 genes out of 32 risk genes interact with previously discovered CHD genes. Single cell RNA-seq further revealed dynamic expressions of GATA5, GATA6, FOXD3 and MYO6 in endocardium and neural crest lineage indicating the mutations of these genes lead to LVOTO possibly through different lineages. These findings uncover the genetic architecture of LVOTO which advances the current understanding of LVOTO genetics.

Exosomes from miRNA-378-modified adipose-derived stem cells prevent glucocorticoid-induced osteonecrosis of the femoral head by enhancing angiogenesis and osteogenesis via targeting miR-378 negatively regulated suppressor of fused (Sufu).

BACKGROUND: Local ischemia and defective osteogenesis are implicated in the progression of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Recent studies have revealed that exosomes released from adipose-derived stem cells (ASCs) play important roles in ONFH therapy. The present study aimed to investigate whether exosomes derived from miR-378-overexpressing ASCs (miR-378-ASCs-Exos) could promote angiogenesis and osteogenesis in GC-induced ONFH. METHODS: In vitro, we investigated the osteogenic potential of miR-378-ASCs-Exos on bone marrow stromal cells (BMSCs) by alkaline phosphatase staining and western blotting. The angiogenic effects of miR-378-ASCs-Exos on human umbilical vein endothelial cells (HUVECs) were examined by evaluating their proliferation, migration, and tube-forming analyses. We identified the underlying mechanisms of miR-378 in osteogenic and angiogenic regulation. In addition, an ONFH rat model was established to explore the effects of miR-378-ASCs-Exos through histological and immunohistochemical staining and micro-CT in vivo. RESULTS: Administration of miR-378-ASCs-Exos improved the osteogenic and angiogenic potentials of BMSCs and HUVECs. miR-378 negatively regulated the suppressor of fused (Sufu) and activated Sonic Hedgehog (Shh) signaling pathway, and recombinant Sufu protein reduced the effects triggered by miR-378-ASCs-Exos. In vivo experiments indicated that miR-378-ASCs-Exos markedly accelerated bone regeneration and angiogenesis, which inhibited the progression of ONFH. CONCLUSION: Our study indicated that miR-378-ASCs-Exos enhances osteogenesis and angiogenesis by targeting Sufu to upregulate the Shh signaling pathway, thereby attenuating GC-induced ONFH development.

LARP7 ameliorates cellular senescence and aging by allosterically enhancing SIRT1 deacetylase activity.

Cellular senescence is associated with pleiotropic physiopathological processes, including aging and age-related diseases. The persistent DNA damage is a major stress leading to senescence, but the underlying molecular link remains elusive. Here, we identify La Ribonucleoprotein 7 (LARP7), a 7SK RNA binding protein, as an aging antagonist. DNA damage-mediated Ataxia Telangiectasia Mutated (ATM) activation triggers the extracellular shuttling and downregulation of LARP7, which dampens SIRT1 deacetylase activity, enhances p53 and NF-κB (p65) transcriptional activity by augmenting their acetylation, and thereby accelerates cellular senescence. Deletion of LARP7 leads to senescent cell accumulation and premature aging in rodent model. Furthermore, we show this ATM-LARP7-SIRT1-p53/p65 senescence axis is active in vascular senescence and atherogenesis, and preventing its activation substantially alleviates senescence and atherogenesis. Together, this study identifies LARP7 as a gatekeeper of senescence, and the altered ATM-LARP7-SIRT1-p53/p65 pathway plays an important role in DNA damage response (DDR)-mediated cellular senescence and atherosclerosis.

Programming Bacteria With Light-Sensors and Applications in Synthetic Biology.

Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.