Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Bioinformatics approach and experimental validation reveal the hepatoprotective effect of pachyman against acetaminophen-associated liver injury.

Pachyman, known as Poria cocos polysaccharides, refers to the bioactive compounds isolated from Poria cocos. Pachyman is thought to exert cytoprotective action. However, the detailed mechanisms of pachyman action for hepatoprotection remain unknown. In this study, we aimed to assess the therapeutic actions, molecular mechanisms, and key target proteins of pachyman in the treatment of liver injury through network pharmacology and molecular docking assays. Furthermore, these bioinformatic findings were validated by an acetaminophen (APAP)-induced liver injury in vivo. Primarily using bioinformatic analysis, we screened and characterized 12 genes that act as potential therapeutic targets of pachyman against APAP-induced liver injury, in which all core targets were obtained. By using enrichment analysis, these core target genes of pachyman were characterized to reveal the pharmacological functions and molecular mechanisms of anti-liver injury induced by APAP. A molecular docking simulation was further performed to certain anti-liver injury target proteins of pachyman, including cytochrome P450 3A4 enzyme (CYP3A4) and inducible nitric oxide synthase (NOS2). In animal experiments, pachyman exerted potent hepatoprotective activities in prenatal APAP-exposed offspring livers, characterized by activated hepatocellular CYP3A4 and NOS2 expressions. These current findings have thus indicated that pachyman exerts hepatoprotective effects and may be the promising nutraceuticals for the treatment of APAP-induced liver injury.

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Stable Transgenic Mouse Strain with Enhanced Photoactivatable Cre Recombinase for Spatiotemporal Genome Manipulation.

Optogenetic genome engineering is a powerful technology for high-resolution spatiotemporal genetic manipulation, especially for in vivo studies. It is difficult to generate stable transgenic animals carrying a tightly regulated optogenetic system, as its long-term expression induces high background activity. Here, the generation of an enhanced photoactivatable Cre recombinase (ePA-Cre) transgenic mouse strain with stringent light responsiveness and high recombination efficiency is reported. Through serial optimization, ePA-Cre is developed to generate a transgenic mouse line that exhibits 175-fold induction upon illumination. Efficient light-dependent recombination is detected in embryos and various adult tissues of ePA-Cre mice crossed with the Ai14 tdTomato reporter. Importantly, no significant background Cre activity is detected in the tested tissues except the skin. Moreover, efficient light-inducible cell ablation is achieved in ePA-Cre mice crossed with Rosa26-LSL-DTA mice. In conclusion, ePA-Cre mice offer a tightly inducible, highly efficient, and spatiotemporal-specific genome engineering tool for multiple applications.

Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice.

Microglia are important immune cells in the central nervous system (CNS) that undergo turnover throughout the lifespan. If microglial debris is not removed in a timely manner, accumulated debris may influence CNS function. Clearance of microglial debris is crucial for CNS homeostasis. However, underlying mechanisms remain obscure. We here investigate how dead microglia are removed. We find that although microglia can phagocytose microglial debris in vitro, the territory-dependent competition hinders the microglia-to-microglial debris engulfment in vivo. In contrast, microglial debris is mainly phagocytosed by astrocytes in the brain, facilitated by C4b opsonization. The engulfed microglial fragments are then degraded in astrocytes via RUBICON-dependent LC3-associated phagocytosis (LAP), a form of noncanonical autophagy. Interference with C4b-mediated engulfment and subsequent LAP disrupt the removal and degradation of microglial debris, respectively. Together, we elucidate the cellular and molecular mechanisms of microglial debris removal in mice, extending the knowledge on the maintenance of CNS homeostasis.

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration.

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.

Innovatively treat rural food waste through producing organic grains.

Food waste (FW) in a whole country contains a large amount of nitrogen which could be used to replace chemical fertilizers to produce organic grains, thus mitigating environmental pollution from the source. A 2-year field experiment was carried out using rural FW to grow organic grains in Shandong Province, China. Different proportions of FW and cattle manure were designed: FM0, 100% cattle manure compost (CMC); FM1, 75% CMC + 25% FW; FM2, 50% CMC + 50% FW; FM3, 25% CMC + 75% FW; FM4, 100% FW; CF, 100% chemical fertilizer; CK, without any fertilizers. Compared with CK and FM0, the application of FW significantly increased the total nitrogen, total phosphorus, and total potassium content of the soil. Simultaneously, all the three indicators increased with the increase of the proportion of FW. FW did not cause increase of contents of heavy metals such as cuprum, zinc, and chromium in the soils, nor did it increase the heavy metals in the grains. Using FW to replace all cattle manure, the total organic yield of grains reached to an average of 18,163 kg ha-1. We found that 1 kg dry FW could produce 1.64 kg organic grains under organic conditions, with the average net income being 5.42 times that of chemical mode. Our findings may provide an innovative solution for treating rural food wastes, ensuring food safety, and conservating the agriculture ecosystem.

Rescue of mis-splicing of a common SLC26A4 mutant associated with sensorineural hearing loss by antisense oligonucleotides.

A wide spectrum of SLC26A4 mutations causes Pendred syndrome and enlarged vestibular aqueduct, both associated with sensorineural hearing loss (SNHL). A splice-site mutation, c.919-2A>G (A-2G), which is common in Asian populations, impairs the 3' splice site of intron 7, resulting in exon 8 skipping during pre-mRNA splicing and a subsequent frameshift that creates a premature termination codon in the following exon. Currently, there is no effective drug treatment for SHNL. For A-2G-triggered SNHL, molecules that correct mis-splicing of the mutant hold promise to treat the disease. Antisense oligonucleotides (ASOs) can promote exon inclusion when targeting specific splicing silencers. Here, we systematically screened a large number of ASOs in a minigene system and identified a few that markedly repressed exon 8 skipping. A lead ASO, which targets a heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 intronic splicing silencer (ISS) in intron 8, promoted efficient exon 8 inclusion in cultured peripheral blood mononuclear cells derived from two homozygous patients. In a partially humanized Slc26a4 A-2G mouse model, two subcutaneous injections of the ASO at 160 mg/kg significantly rescued exon 8 splicing in the liver. Our results demonstrate that the ISS-targeting ASO has therapeutic potential to treat genetic hearing loss caused by the A-2G mutation in SLC26A4.

Differences in renal cortex transcriptional profiling of wild-type and novel type B cystinuria model rats.

Cystinuria is a genetic disorder of cystine transport that accounts for 1-2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b0,+AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated.

Immunogenicity of meningococcal polysaccharide conjugate vaccine as a replacement for meningococcal polysaccharide vaccine in children in Guangzhou, China.

To assess the durability of antibody persistence after substitution of the MPCV vaccine for the MPSV-A vaccine in children, an observational study was conducted in children who voluntarily received two doses of MPCV-AC instead of MPSV-A between March 2017 and March 2018 in Guangzhou, China. In total, 131 and 47 participants were enrolled in the 3-year-old and 6-year-old groups, respectively. In the 3-year-old group, the seroprotection rate and GMT values for Men A and Men C were raised significantly after 1-month post- dose 1 MPSV booster vaccination. All immune indicators were significantly lower in pre- dose 1 MPSV booster vaccination in the 3-year-old group than after pre- dose 2 MPSV booster vaccination in the 6-year-old group. While no significant differences were found in most immune indicators between the 1-month post- dose 1 MPSV booster vaccination in the 3-year-old group and pre- dose 2 MPSV booster vaccination in 6-year-old group. The substitute meningococcal immunization schedule showed a good immunogenicity in young children, and good sequential immunogenicity with MPSV booster immunization.

Comprehensive influence of topological location and neighbor information on identifying influential nodes in complex networks.

Identifying the influential nodes of complex networks is now seen as essential for optimizing the network structure or efficiently disseminating information through networks. Most of the available methods determine the spreading capability of nodes based on their topological locations or the neighbor information, the degree of node is usually used to denote the neighbor information, and the k-shell is used to denote the locations of nodes, However, k-shell does not provide enough information about the topological connections and position information of the nodes. In this work, a new hybrid method is proposed to identify highly influential spreaders by not only considering the topological location of the node but also the neighbor information. The percentage of triangle structures is employed to measure both the connections among the neighbor nodes and the location of nodes, the contact distance is also taken into consideration to distinguish the interaction influence by different step neighbors. The comparison between our proposed method and some well-known centralities indicates that the proposed measure is more highly correlated with the real spreading process, Furthermore, another comprehensive experiment shows that the top nodes removed according to the proposed method are relatively quick to destroy the network than other compared semi-local measures. Our results may provide further insights into identifying influential individuals according to the structure of the networks.

Prevalence, risk factors, and clinical correlations of underweight in Chinese newly diagnosed and drug-naïve patients with Parkinson's disease.

OBJECTIVE: Studies have shown that patients with Parkinson's disease (PD) will experience weight loss during the progression of the illness, which suggests an increased rate of underweight. However, few studies have addressed underweight in early de novo population. This study aimed to examine the prevalence, risk factors, and clinical correlations of underweight in Chinese newly diagnosed and drug-naïve patients with PD. METHODS: A total of 245 inpatients with newly diagnosed PD and 213 age-, sex-, and education-matched healthy controls were enrolled in Ningbo. BMI, demographics, supine and upright blood pressure, Montreal Cognitive Assessment (MoCA), Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD) together with fasting blood glucose, low-density lipoprotein, total cholesterol, uric acid (UA), and homocysteine were collected in all subjects. Hoehn and Yahr (HY) rating and Unified Parkinson's Disease Rating Scale (UPDRS) motor scores were also measured in patients. RESULTS: Overall, 35 (14.3%) patients were underweight and 14 (6.6%) controls were underweight. Binary regression analyses showed that low MoCA (p = 0.035), ΔSBP and ΔDBP values (both p < 0.001) were risk factors for underweight. Furthermore, correlation analysis indicated that BMI was associated with HY grade, UPDRS motor, HAMA, HAMD, MoCA, ΔSBP, ΔDBP, and UA values, stepwise multiple regression revealed significant correlations between BMI and ΔSBP (p < 0.001), ΔDBP (p = 0.001), MoCA (p = 0.002), UPDRS motor (p = 0.005), and HAMD scores (p = 0.014). CONCLUSIONS: Our study showed that the prevalence of underweight was significantly higher in Chinese newly diagnosed and drug-naïve patients with PD than in the healthy population, and several clinical variables were risk factors for underweight.

Sex differences in underweight and body mass index in Chinese early de novo patients with Parkinson's disease.

OBJECTIVE: There have been studies investigating sex differences in clinical manifestation of Parkinson's disease (PD). However, sex differences in underweight and body mass index (BMI) in de novo PD patients lacked systematic study. We aimed to compare sex differences in clinical features and related factors of underweight and BMI in Chinese de novo PD patients. MATERIALS AND METHODS: A total of 253 untreated PD inpatients and 218 controls were recruited from Ningbo. BMI, demographics, Montreal Cognitive Assessment (MoCA), supine and upright blood pressure, Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD), homocysteine (HCY), uric acid, glycated hemoglobin, and lipid parameters were examined. Patients were assessed using the Unified Parkinson's Disease Rating Scale (UPDRS) motor scores and Hoehn and Yahr (HY) Rating Scale. RESULTS: Female patients had a significantly lower incidence of underweight and higher BMI than male patients, and there were sex differences in serum lipids, HCY levels, and depression severity. Binary regression analysis showed that only in male patients was underweight associated with the UPDRS motor score and lower ΔSBP and ΔDBP values (all p < .05). Further multiple regression analysis indicated, in addition to the correlations between BMI and ΔSBP and ΔDBP values in both sexes (all p < .001), BMI was also associated with MoCA and lower UPDRS motor scores in male patients and lower HAMD scores in female patients. CONCLUSION: Our study suggests that there are significant sex differences in the prevalence of underweight, BMI, and factors associated with underweight and BMI among de novo PD patients.

Using organic fertilizers to increase crop yield, economic growth, and soil quality in a temperate farmland.

We used a constant total N application base rate to conduct a two-year field experiment comparing the effects of three organic fertilizers (rapeseed meal (RSM), soybean meal (SBM), and cattle manure (CM)) on the crop yield, economic growth, and soil quality of a winter wheat-summer maize rotation system. Winter wheat and summer maize in rapeseed meal treatment (RSMT), soybean meal treatment (SBMT), and cattle manure treatment (CMT) showed yield increases of 161%, 299%, and 256%, respectively, when compared to no organic fertilizer treatment (CK) (P < 0.05). The annual net incomes of SBMT and CMT were 1.46 and 1.42 times higher, respectively, than RSMT. Compared to the results of the CK group, RSM, SBM, and CM stimulated the soil physically, chemically, and biologically. We found the highest soil macroaggregate proportions, soil organic matter (SOM) levels, total N (TN) levels, and phospholipid fatty acid (PLFA) levels in SBMT. The highest soil pH, microbial biomass carbon (MBC) levels, and microbial biomass nitrogen (MBN) levels were observed in CMT. We used a soil quality index (SQI) to evaluate soil quality. After the two-year fertilization treatments, we calculated the SQI using a minimum data set (MDS). We used SOM levels and actinomycete quantity for the MDS properties. The SQI values were significantly different across the four treatments, with the highest values occurring in SBMT, then CMT and RSMT. In conclusion, SBM and CM were more effective than RSM at maintaining crop yield, economic growth, and soil quality.

Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing.

Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the start codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a start codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.

Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain.

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of ß-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.

Network analysis, and human and animal studies disclose the anticystitis glandularis effects of vitamin C.

BACKGROUND: Our present study aimed to unravel the therapeutic biotargets of vitamin C (VC) against cystitis glandularis (CG), and to elucidate the molecular mechanisms for VC treating CG. METHODS: Network pharmacology was used to predict therapeutic targets of VC against CG, and to identify molecular mechanisms. In addition, further human and animal studies were designed to validate the bioinformatic findings through biochemical tests, computerized tomography scans, and immunostaining assays. RESULTS: In bioinformatic analyses, pathogenic targets of CG and putative targets of VC were identified, respectively. An interaction network between biological target and functional protein was produced before screening and collecting the key therapeutic targets of VC against CG, biological processes, and signaling pathways. In addition, ingenuity pathway analysis with cloud platform indicated that anti-CG mechanisms of VC were achieved through modulating a cluster of molecular pathways, such as tumor necrosis factor (TNF) pathway. Meanwhile, 18 core targets of VC against CG were identified, and the most important TNF, interleukin-6 (IL6), and Jun biotargets were obtained, respectively. In further validation in human study, cellular TNF-α, IL6, and c-Jun expressions in patient's CG samples were elevated significantly, accompanied with detectable urinary tract infection. Beneficially, VC-dosed CG mice resulted in downregulated expressions of endogenous TNF-α, IL6, and c-Jun in blood and bladder samples. CONCLUSION: Collectively, these bioinformatic findings and experimentative data uncover the therapeutic targets and biological mechanisms of VC for treating CG, in which the key biomarkers of TNF-α, IL6, and c-Jun may be the potential molecules for treating CG in clinical application.

Clinical features of aflatoxin B1-exposed patients with liver cancer and the molecular mechanism of aflatoxin B1 on liver cancer cells.

Aflatoxin B1 (AFB1) induces hepatocellular carcinoma (HCC) through consumption of contaminated food in Southern China. Aldo-keto reductase-7A (AKR7A) functionally plays a potent role in the biodetoxification in the liver. In addition, hepatocellular lipid disorder has found to be closely linked to the development of HCC. This study was, therefore, designed to investigate the potent bioeffect of AKR7A on the lipid metabolism in AFB1-exposed hepatocellular carcinoma cells through assaying human cancerous samples and cell culture. In the baseline data, the HCC patients showed increased contents of AFB1 in sera and cancerous samples. In the clinical parameters, the HCC patients demonstrated changed lipid settings in sera. As revealed by immunostaining and immunoblotting, AFB1-elevated HCC sections showed marked down-regulation of AKR7A expression, accompanied with reduced ApoB expression and increased CD36, S6K1 expressions in the HCC. Studies in the human hepatocarcinoma line HepG2 also showed AFB1-exposure to increase ApoA1, LDL, TC, and TG contents; induce cell proliferation; and reduce hepatocellular AKR7A expression. Furthermore, AKR7A bioactivity was inactivated after treatment with perfluorooctane sulfonate (PFOS), an ApoB activator, in AFB1-dosed HepG2 cells. Collectively, our current findings suggest that hepatocellular AKR7A has a protective role against AFB1-induced cytotoxicity through the regulation of CD36, S6K1 and ApoB expression through the reduction of lipid utilization in malignant liver cells.