Engineering of cytosine base editors with DNA damage minimization and editing scope diversification.

Cytosine base editors (CBEs), which enable precise C-to-T substitutions, have been restricted by potential safety risks, including DNA off-target edits, RNA off-target edits and additional genotoxicity such as DNA damages induced by double-strand breaks (DSBs). Though DNA and RNA off-target edits have been ameliorated via various strategies, evaluation and minimization of DSB-associated DNA damage risks for most CBEs remain to be resolved. Here we demonstrate that YE1, an engineered CBE variant with minimized DNA and RNA off-target edits, could induce prominent DSB-associated DNA damage risks, manifested as γH2AX accumulation in human cells. We then perform deaminase engineering for two deaminases lamprey LjCDA1 and human APOBEC3A, and generate divergent CBE variants with eliminated DSB-associated DNA damage risks, in addition to minimized DNA/RNA off-target edits. Furthermore, the editing scopes and sequence preferences of APOBEC3A-derived CBEs could be further diversified by internal fusion strategy. Taken together, this study provides updated evaluation platform for DSB-associated DNA damage risks of CBEs and further generates a series of safer toolkits with diversified editing signatures to expand their applications.

Chemoselective 1,2-Reduction and Regiodivergent Deuteration of Chromium-Bound Arenes.

Herein, a general strategy for chemo- and regioselective 1,2-reduction of chromium-bound arenes was developed, thus providing rapid access to 1,3-cyclohexadienes. Selective arene activation via π-complexation along with the use of mild hydride Ph3 SiH can overcome the inherently low reactivity of arene π-bonds while tolerating various reduction-sensitive functional groups. Its versatility further enables a regiodivergent deuteration. Using different sequences of (non)deuterated hydride and acid reagents, the deuterated positions as well as the degrees of deuterium incorporation can be controlled precisely, which leads to a large and previously inaccessible chemical space for 1,3-cyclohexadiene isotopologues. A reasonable mechanism was proposed based on intermediate capture and control experiments. The synthetic value of this selective 1,2-reduction was demonstrated in the formal total synthesis of (±)-galanthamine and (±)-lycoramine.

Changes in metabolomics and lipidomics in brain tissue and their correlations with the gut microbiome after chronic food-derived arsenic exposure in mice.

Arsenic can cause neurodegenerative diseases of the brain, but the definite mechanism is still unknown. In this study, to discuss the disturbances on brain metabolome and lipidome under subchronic arsenic exposure, we treated mice with the arsenic-containing feed (concentration of total arsenic = 30 mg/kg) prepared in accordance with the proportion of rice arsenicals for 16 weeks and performed metabolomics and lipidomics studies respectively using UHPLC-Triple-TOF-MS/MS and UHPLC-Q Exactive Focus MS/MS on mice brain. In addition, the distributions of arsenical metabolites along the feed-gut-blood-brain chain were analyzed by ICP-MS and HPLC-ICP-MS, and fecal microbial variations were investigated by 16 s sequencing. The data showed that although only a tiny amount of arsenic (DMA=0.101 mg/kg, uAs=0.071 mg/kg) enters the brain through the blood-brain barrier, there were significant changes in brain metabolism, including 118 metabolites and 17 lipids. These different metabolites were involved in 30 distinct pathways, including glycometabolism, and metabolisms of lipid, nucleic acid, and amino acid were previously reported to be correlated with neurodegenerative diseases. Additionally, these different metabolites were significantly correlated with 12 gut bacterial OTUs, among which Lachnospiraceae, Muribaculaceae, Ruminococcaceae, and Erysipelotrichaceae were also previously reported to be related to the distortion of metabolism, indicating that the disturbance of metabolism in the brain may be associated with the disturbance of gut microbes induced by arsenic. Thus, the current study demonstrated that the brain metabolome and lipidome were significantly disturbed under subchronic arsenic exposure, and the disturbances also significantly correlated with some gut microbiome and may be associated with neurodegenerative diseases. Although preliminary, the results shed some light on the pathophysiology of arsenic-caused neurodegenerative diseases.

Public Awareness, Individual Prevention Practice, and Psychological Effect at the Beginning of the COVID-19 Outbreak in China.

BACKGROUND: The COVID-19 has spread to more than 200 countries and territories. But less is known about the knowledge, protection behavior and anxiety regarding the outbreak among the general population. METHODS: A cross-sectional, population-based online survey was conducted in China and abroad from January 28 to February 1, 2020. Socio-demographic information was collected and knowledge scores, practice scores, anxiety scores and perceived risk were calculated. General linear model and binary logistic regression were used to identify possible associations. RESULTS: We included 9,764 individuals in this study, and 156 (1.6%) were from Hubei Province. The average knowledge score was 4.7 (standard deviation, 1.0) (scored on a 6-point scale); 96.1% maintained hand hygiene, and 90.3% of participants had varying levels of anxiety. People in Hubei Province were the most anxious, followed by those in Beijing and Shanghai. People who had experienced risk behaviors did not pay more attention to wearing masks and hand hygiene. CONCLUSIONS: The public had high awareness on knowledge of COVID-19 outbreak, and a high proportion of people practiced good hand hygiene behavior. Many people claimed anxiety, especially in heavily affected areas during pandemic, suggesting the importance of closing the gap between risk awareness and good practice and conduct psychological counseling to public and patients.

Myasthenia gravis: Molecular mechanisms and promising therapeutic strategies.

Myasthenia gravis (MG) is a type of autoimmune disease caused by the blockage of neuromuscular junction transmission owing to the attack of autoantibodies on transmission-related proteins. Related antibodies, such as anti-AChR, anti-MuSK and anti-LRP4 antibodies, can be detected in most patients with MG. Although traditional therapies can control most symptoms, several challenges remain to be addressed, necessitating the development of more effective and safe treatment strategies for MG. With the in-depth exploration on the mechanism and immune targets of MG, effective therapies, especially therapies using biologicals, have been reported recently. Given the important roles of immune cells, cytokines and intercellular interactions in the pathological process of MG, B-cell targeted therapy, T-cell targeted therapy, proteasome inhibitors targeting plasma cell, complement inhibitors, FcRn inhibitors have been developed for the treatment of MG. Although these novel therapies exert good therapeutic effects, they may weaken the immunity and increase the risk of infection in MG patients. This review elaborates on the pathogenesis of MG and discusses the advantages and disadvantages of the strategies of traditional treatment and biologicals. In addition, this review emphasises that combined therapy may have better therapeutic effects and reducing the risk of side effects of treatments, which has great prospects for the treatment of MG. With the deepening of research on immunotherapy targets in MG, novel opportunities and challenges in the treatment of MG will be introduced.

TadA orthologs enable both cytosine and adenine editing of base editors.

Cytidine and adenosine deaminases are required for cytosine and adenine editing of base editors respectively, and no single deaminase could enable concurrent and comparable cytosine and adenine editing. Additionally, distinct properties of cytidine and adenosine deaminases lead to various types of off-target effects, including Cas9-indendepent DNA off-target effects for cytosine base editors (CBEs) and RNA off-target effects particularly severe for adenine base editors (ABEs). Here we demonstrate that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single or double mutations, which display minimized Cas9-independent DNA off-target effects and genotoxicity, with orthologs B5ZCW4, Q57LE3, E8WVH3, Q13XZ4 and B3PCY2 as promising candidates for further engineering. Furthermore, RNA off-target effects of TadA ortholog-derived base editors could be further reduced or even eliminated by additional single mutation. Taken together, our work expands the base editing toolkits, and also provides important clues for the potential evolutionary process of deaminases.

Injury-induced interleukin-1 alpha promotes Lgr5 hair follicle stem cells de novo regeneration and proliferation via regulating regenerative microenvironment in mice.

BACKGROUND: The hair follicles (HFs) are barely regenerated after loss in injuries in mammals as well as in human beings. Recent studies have shown that the regenerative ability of HFs is age-related; however, the relationship between this phenomenon and the stem cell niche remains unclear. This study aimed to find a key secretory protein that promotes the HFs regeneration in the regenerative microenvironment. METHODS: To explore why age affects HFs de novo regeneration, we established an age-dependent HFs regeneration model in leucine-rich repeat G protein-coupled receptor 5 (Lgr5) + /mTmG mice. Proteins in tissue fluids were analyzed by high-throughput sequencing. The role and mechanism of candidate proteins in HFs de novo regeneration and hair follicle stem cells (HFSCs) activation were investigated through in vivo experiments. The effects of candidate proteins on skin cell populations were investigated by cellular experiments. RESULTS: Mice under 3-week-old (3W) could regenerate HFs and Lgr5 HFSCs, which were highly correlated with the immune cells, cytokines, IL-17 signaling pathway, and IL-1α level in the regeneration microenvironment. Additionally, IL-1α injection induced de novo regeneration of HFs and Lgr5 HFSCs in 3W mouse model with a 5 mm wound, as well as promoted activation and proliferation of Lgr5 HFSCs in 7-week-old (7W) mice without wound. Dexamethasone and TEMPOL inhibited the effects of IL-1α. Moreover, IL-1α increased skin thickness and promoted the proliferation of human epidermal keratinocyte line (HaCaT) and skin-derived precursors (SKPs) in vivo and in vitro, respectively. CONCLUSIONS: In conclusion, injury-induced IL-1α promotes HFs regeneration by modulating inflammatory cells and oxidative stress-induced Lgr5 HFSCs regeneration as well as promoting skin cell populations proliferation. This study uncovers the underlying molecular mechanisms enabling HFs de novo regeneration in an age-dependent model.

TadA reprogramming to generate potent miniature base editors with high precision.

Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.

Potential Therapeutic Strategies for Skeletal Muscle Atrophy.

The maintenance of muscle homeostasis is vital for life and health. Skeletal muscle atrophy not only seriously reduces people's quality of life and increases morbidity and mortality, but also causes a huge socioeconomic burden. To date, no effective treatment has been developed for skeletal muscle atrophy owing to an incomplete understanding of its molecular mechanisms. Exercise therapy is the most effective treatment for skeletal muscle atrophy. Unfortunately, it is not suitable for all patients, such as fractured patients and bedridden patients with nerve damage. Therefore, understanding the molecular mechanism of skeletal muscle atrophy is crucial for developing new therapies for skeletal muscle atrophy. In this review, PubMed was systematically screened for articles that appeared in the past 5 years about potential therapeutic strategies for skeletal muscle atrophy. Herein, we summarize the roles of inflammation, oxidative stress, ubiquitin-proteasome system, autophagic-lysosomal pathway, caspases, and calpains in skeletal muscle atrophy and systematically expound the potential drug targets and therapeutic progress against skeletal muscle atrophy. This review focuses on current treatments and strategies for skeletal muscle atrophy, including drug treatment (active substances of traditional Chinese medicine, chemical drugs, antioxidants, enzyme and enzyme inhibitors, hormone drugs, etc.), gene therapy, stem cell and exosome therapy (muscle-derived stem cells, non-myogenic stem cells, and exosomes), cytokine therapy, physical therapy (electroacupuncture, electrical stimulation, optogenetic technology, heat therapy, and low-level laser therapy), nutrition support (protein, essential amino acids, creatine, ß-hydroxy-ß-methylbutyrate, and vitamin D), and other therapies (biomaterial adjuvant therapy, intestinal microbial regulation, and oxygen supplementation). Considering many treatments have been developed for skeletal muscle atrophy, we propose a combination of proper treatments for individual needs, which may yield better treatment outcomes.

Inflammation: Roles in Skeletal Muscle Atrophy.

Various diseases can cause skeletal muscle atrophy, usually accompanied by inflammation, mitochondrial dysfunction, apoptosis, decreased protein synthesis, and enhanced proteolysis. The underlying mechanism of inflammation in skeletal muscle atrophy is extremely complex and has not been fully elucidated, thus hindering the development of effective therapeutic drugs and preventive measures for skeletal muscle atrophy. In this review, we elaborate on protein degradation pathways, including the ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), the calpain and caspase pathways, the insulin growth factor 1/Akt protein synthesis pathway, myostatin, and muscle satellite cells, in the process of muscle atrophy. Under an inflammatory environment, various pro-inflammatory cytokines directly act on nuclear factor-κB, p38MAPK, and JAK/STAT pathways through the corresponding receptors, and then are involved in muscle atrophy. Inflammation can also indirectly trigger skeletal muscle atrophy by changing the metabolic state of other tissues or cells. This paper explores the changes in the hypothalamic-pituitary-adrenal axis and fat metabolism under inflammatory conditions as well as their effects on skeletal muscle. Moreover, this paper also reviews various signaling pathways related to muscle atrophy under inflammatory conditions, such as cachexia, sepsis, type 2 diabetes mellitus, obesity, chronic obstructive pulmonary disease, chronic kidney disease, and nerve injury. Finally, this paper summarizes anti-amyotrophic drugs and their therapeutic targets for inflammation in recent years. Overall, inflammation is a key factor causing skeletal muscle atrophy, and anti-inflammation might be an effective strategy for the treatment of skeletal muscle atrophy. Various inflammatory factors and their downstream pathways are considered promising targets for the treatment and prevention of skeletal muscle atrophy.

Identification of potential biomarkers in ovarian carcinoma and an evaluation of their prognostic value.

BACKGROUND: Ovarian cancer is one of the most common malignant tumors in female genital organs, and its incidence rate is high. However, the pathogenesis and prognostic markers of ovarian cancer are unclear. This study sought to screen potential markers of ovarian cancer and to explore their prognostic value. METHODS: The Cancer Genome Atlas, Gene Expression Omnibus, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used in this study. The least absolute shrinkage and selection operator (LASSO), multivariate Cox regression and stepwise regression analysis were chosen to screen genes and construct risk model. Gene Set Enrichment Analysis (GSEA) and an immune-infiltration analysis were performed. RESULTS: One hundred thirty two co-expressed genes were found. They involved in metabolism, protein phosphorylation, mitochondria, and immune signaling pathways. Twelve genes significantly related to the survival of ovarian cancer were identified. Eight risk genes (i.e., CACNB1, FAM120B, HOXB2, MED19, PTPN2, SMU1, WAC.AS1, and BCL2L11) were further screened and used to construct the risk model. The risk status might be an independent prognostic factor of ovarian cancer, and most of the biological functions of genes expressed in high-risk ovarian cancer were related to synapse, adhesion, and immune-related functions. The clusters of CD4+ T cells and M2 macrophages were high in high-risk status samples. CONCLUSIONS: In ovarian cancer, the abnormal expression of 8 genes, including CACNB1, FAM120B, HOXB2, MED19, PTPN2, SMU1, WAC.AS1, and BCL2L11, is closely related to ovarian cancer progression, and these genes can serve as independent prognosis markers of ovarian cancer.

Dietary supplementation with N-3 polyunsaturated fatty acid-enriched fish oil promotes wound healing after ultraviolet B-induced sunburn in mice.

N-3 polyunsaturated fatty acids (n-3 PUFA) can alleviate ultraviolet B (UVB)-induced skin cancers, but their effects on sunburn and upcoming wound healing remain controversial. This study aimed to explore the impact of n-3 PUFA-enriched fish oil (n-3 PUFA-FO) on UVB-induced sunburns and subsequent healing. Sixty C57BL/6 female mice were divided into two groups. The treated group mice were fed n-3 PUFA-FO for the entire duration of the experiment. Mice in the control group were fed a standard diet. After two weeks of n-3 PUFA-FO feeding, mice were exposed to UVB for 20 min and sacrificed 20 d later. Skin photodamage and lesion area were recorded during wound healing. Epidermal lesion thickness was quantified in hematoxylin and eosin-stained skin sections. Inflammation and macrophage polarization were assessed by qRT-PCR. Oxidative stress and antioxidant enzyme activity were quantified using specific ELISA kits. N-3 PUFA-FO feeding decreased UVB photodamage and accelerated wound healing progression, both of which were coupled with less intense inflammation and increased macrophage M2 phenotype polarization. Furthermore, n-3 PUFA-FO brought about a decrease in malondialdehyde (MDA) levels but increased the activity of catalase (CAT) and glutathione peroxidase (GP), without changing superoxide dismutase (SOD) activity. N-3 PUFA-FO protects against UVB-induced skin photodamage and promotes wound healing by modulating macrophage phenotypic polarization and antioxidant enzyme activities. N-3 PUFA-FO could be a novel therapeutic approach for both the prevention and treatment of sunburns.

Turn-on fluorescent assay for antioxidants based on their inhibiting polymerization of dopamine on graphene quantum dots.

We describe a sensitive turn-on fluorescent assay for antioxidants by using fluorescence-tunable graphene quantum dots (GQDs). GQDs exhibited strong fluorescence without dopamine (DA). DA could self-polymerize to a thin polydopamine (PDA) film on the surface of GQDs under alkaline environment, resulting in the fluorescence quenching of GQDs via fluorescence resonance energy transfer (FRET). However, the self-polymerization of DA could be effectively inhibited in the presence of antioxidants including glutathione (GSH), ascorbic acid (AA), cysteine (Cys), and homocysteine (Hcys). Thus, the fluorescence of GQDs restored. The "turn-on" sensing of antioxidants could be achieved with high sensitivity. The detection limit for GSH, AA, Cys, and Hcys could be achieved as low as 2.4 nM, 1.5 nM, 4.2 nM, and 4.4 nM, respectively. Finally, the GQDs@PDA system was applied for monitoring cerebral antioxidants in rat brain microdialysates. This work promises new opportunities to evaluate antioxidant capacity in physiological and pathological fields.

Isoquercitrin Delays Denervated Soleus Muscle Atrophy by Inhibiting Oxidative Stress and Inflammation.

Although denervated muscle atrophy is common, the underlying molecular mechanism remains unelucidated. We have previously found that oxidative stress and inflammatory response may be early events that trigger denervated muscle atrophy. Isoquercitrin is a biologically active flavonoid with antioxidative and anti-inflammatory properties. The present study investigated the effect of isoquercitrin on denervated soleus muscle atrophy and its possible molecular mechanisms. We found that isoquercitrin was effective in alleviating soleus muscle mass loss following denervation in a dose-dependent manner. Isoquercitrin demonstrated the optimal protective effect at 20 mg/kg/d, which was the dose used in subsequent experiments. To further explore the protective effect of isoquercitrin on denervated soleus muscle atrophy, we analyzed muscle proteolysis via the ubiquitin-proteasome pathway, mitophagy, and muscle fiber type conversion. Isoquercitrin significantly inhibited the denervation-induced overexpression of two muscle-specific ubiquitin ligases-muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), and reduced the degradation of myosin heavy chains (MyHCs) in the target muscle. Following isoquercitrin treatment, mitochondrial vacuolation and autophagy were inhibited, as evidenced by reduced level of autophagy-related proteins (ATG7, BNIP3, LC3B, and PINK1); slow-to-fast fiber type conversion in the target muscle was delayed via triggering expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α); and the production of reactive oxygen species (ROS) in the target muscle was reduced, which might be associated with the upregulation of antioxidant factors (SOD1, SOD2, NRF2, NQO1, and HO1) and the downregulation of ROS production-related factors (Nox2, Nox4, and DUOX1). Furthermore, isoquercitrin treatment reduced the levels of inflammatory factors-interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α)-in the target muscle and inactivated the JAK/STAT3 signaling pathway. Overall, isoquercitrin may alleviate soleus muscle atrophy and mitophagy and reverse the slow-to-fast fiber type conversion following denervation via inhibition of oxidative stress and inflammatory response. Our study findings enrich the knowledge regarding the molecular regulatory mechanisms of denervated muscle atrophy and provide a scientific basis for isoquercitrin as a protective drug for the prevention and treatment of denervated muscle atrophy.

Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination.

Base editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.