Activation of Sirtuin3 by honokiol ameliorates alveolar epithelial cell senescence in experimental silicosis via the cGAS-STING pathway.

BACKGROUND: Silicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair and structural integrity maintenance. Inhibiting ATII cell senescence has shown promise in silicosis treatment. However, the mechanism behind silica-induced senescence remains elusive. METHODS: The study employed male C57BL/6 N mice and A549 human alveolar epithelial cells to investigate silicosis and its potential treatment. Silicosis was induced in mice via intratracheal instillation of crystalline silica particles, with honokiol administered intraperitoneally for 14 days. Silica-induced senescence in A549 cells was confirmed, and SIRT3 knockout and overexpression cell lines were generated. Various analyses were conducted, including immunoblotting, qRT-PCR, histology, and transmission electron microscopy. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. RESULTS: This study elucidates how silica induces ATII cell senescence, emphasizing mtDNA damage. Notably, honokiol (HKL) emerges as a promising anti-senescence and anti-fibrosis agent, acting through sirt3. honokiol effectively attenuated senescence in ATII cells, dependent on sirt3 expression, while mitigating mtDNA damage. Sirt3, a class III histone deacetylase, regulates senescence and mitochondrial stress. HKL activates sirt3, protecting against pulmonary fibrosis and mitochondrial damage. Additionally, HKL downregulated cGAS expression in senescent ATII cells induced by silica, suggesting sirt3's role as an upstream regulator of the cGAS/STING signaling pathway. Moreover, honokiol treatment inhibited the activation of the NF-κB signaling pathway, associated with reduced oxidative stress and mtDNA damage. Notably, HKL enhanced the activity of SOD2, crucial for mitochondrial function, through sirt3-mediated deacetylation. Additionally, HKL promoted the deacetylation activity of sirt3, further safeguarding mtDNA integrity. CONCLUSIONS: This study uncovers a natural compound, HKL, with significant anti-fibrotic properties through activating sirt3, shedding light on silicosis pathogenesis and treatment avenues.

Enrichment of prime-edited mammalian cells with surrogate PuroR reporters.

Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.

Low Expression of Lipoic Acid Synthase Aggravates Silica-Induced Pulmonary Fibrosis by Inhibiting the Differentiation of Tregs in Mice.

Aims: In addition to reducing the respiratory function, crystalline silica (SiO2) disturbs the immune response by affecting immune cells during the progression of silicosis. Regulatory T cell (Treg) differentiation may play a key role in the abnormal polarization of T helper cell (Th)1 and Th2 cells in the development of silicosis-induced fibrosis. Alpha-lipoic acid (ALA) has immunomodulatory effects by promoting Tregs differentiation. Thus, ALA may have a therapeutic potential for treating autoimmune disorders in patients with silicosis. However, little is known regarding whether ALA regulates the immune system during silicosis development. Results: We found that the expression levels of collagen increased, and the antioxidant capacity was lower in the Lias-/-+SiO2 group than in the Lias+/++SiO2 group. The proportion of Tregs decreased in the peripheral blood and spleen tissue in mice exposed to SiO2. The proportion of Tregs in the Lias-/-+SiO2 group was significantly lower than that in the Lias+/++SiO2 group. Supplementary exogenous ALA attenuates the accumulation of inflammatory cells and extracellular matrix in lung tissues. ALA promotes the immunological balance between Th17 and Treg responses during the development of silicosis-induced fibrosis. Innovation and Conclusion: Our findings confirmed that low expression of lipoic acid synthase aggravates SiO2-induced silicosis, and that supplementary exogenous ALA has therapeutic potential by improving Tregs in silicosis fibrosis.

Oral VV116 versus placebo in patients with mild-to-moderate COVID-19 in China: a multicentre, double-blind, phase 3, randomised controlled study.

BACKGROUND: Spread of SARS-CoV-2 led to a global pandemic, and there remains unmet medical needs in the treatment of Omicron infections. VV116, an oral antiviral agent that has potent activity against SARS-CoV-2, was compared with a placebo in this phase 3 study to investigate its efficacy and safety in patients with mild-to-moderate COVID-19. METHODS: This multicentre, double-blind, phase 3, randomised controlled study enrolled adults in hospitals for infectious diseases and tertiary general hospitals in China. Eligible patients were randomly assigned in a 1:1 ratio using permuted block randomisation to receive oral VV116 (0·6 g every 12 h on day 1 and 0·3 g every 12 h on days 2-5) or oral placebo (on the same schedule as VV116) for 5 days. Randomisation stratification factors included SARS-CoV-2 vaccination status and the presence of high-risk factors for progression to severe COVID-19. Inclusion criteria were a positive SARS-CoV-2 test, an initial onset of COVID-19 symptoms 3 days or less before the first study dose, and a score of 2 or more for any target COVID-19-related symptoms in the 24 h before the first dose. Patients who had severe or critical COVID-19 or who had taken any antiviral drugs were excluded from the study. The primary endpoint was the time to clinical symptom resolution for 2 consecutive days. Efficacy analyses were performed on a modified intention-to-treat population, comprising all patients who received at least one dose of VV116 or placebo, tested positive for SARS-CoV-2 nucleic acid, and did not test positive for influenza virus before the first dose. Safety analyses were done on all participants who received at least one dose of VV116 or placebo. This study was registered with ClinicalTrials.gov, NCT05582629, and has been completed. FINDINGS: A total of 1369 patients were randomly assigned to treatment groups and 1347 received either VV116 (n=674) or placebo (n=673). At the interim analysis, VV116 was superior to placebo in reducing the time to sustained clinical symptom resolution among 1229 patients (hazard ratio [HR] 1·21, 95% CI 1·04-1·40; p=0·0023). At the final analysis, a substantial reduction in time to sustained clinical symptom resolution was observed for VV116 compared with placebo among 1296 patients (HR 1·17, 95% CI 1·04-1·33; p=0·0009), consistent with the interim analysis. The incidence of adverse events was similar between groups (242 [35·9%] of 674 patients vs 283 [42·1%] of 673 patients). INTERPRETATION: Among patients with mild-to-moderate COVID-19, VV116 significantly reduced the time to sustained clinical symptom resolution compared with placebo, with no observed safety concerns. FUNDING: Shanghai Vinnerna Biosciences, Shanghai Science and Technology Commission, and the National Key Research and Development Program of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Curcumin alleviates traumatic brain injury induced by gas explosion through modulating gut microbiota and suppressing the LPS/TLR4/MyD88/NF-κB pathway.

Gas explosions (GE) are a prevalent and widespread cause of traumatic brain injury (TBI) in coal miners. However, the impact and mechanism of curcumin on GE-induced TBI in rats remain unclear. In this study, we simulated GE-induced TBI in rats and administered curcumin orally at a dose of 100 mg/kg every other day for 7 days to modulate the gut microbiota in TBI rats. We employed 16S rRNA sequencing and LC-MS/MS metabolomic analysis to investigate changes in the intestinal flora and its metabolic profile. Additionally, we utilized ELISA, protein assays, and immunohistochemistry to assess neuroinflammatory signaling molecules for validation. In a rat TBI model, GE resulted in weight loss, pathological abnormalities, and cortical hemorrhage. Treatment with curcumin significantly mitigated histological abnormalities and microscopic mitochondrial structural changes in brain tissue. Furthermore, curcumin treatment markedly ameliorated GE-induced brain dysfunction by reducing the levels of several neuroinflammatory signaling molecules, including neuron-specific enolase, interleukin (IL)-1ß, IL-6, and cryptothermic protein 3. Notably, curcumin reshaped the gut microbiome by enhancing evenness, richness, and composition. Prevotella_9, Alloprevotella, Bacilli, Lactobacillales, Proteobacteria, and Gammaproteobacteria were identified as prominent members of the gut microbiota, increasing the linear discriminant analysis scores and specifically enhancing the abundance of bacteria involved in the nuclear factor (NF)-κB signaling pathway, such as Lachnospiraceae and Roseburia. Additionally, there were substantial alterations in serum metabolites associated with metabolic NF-κB signaling pathways in the model group. Curcumin administration reduced serum lipopolysaccharide levels and downregulated downstream Toll-like receptor (TLR)4/myeloid differentiation primary response 88 (MyD88)/NF-κB signaling. Furthermore, curcumin alleviated GE-induced TBI in rats by modulating the gut microbiota and its metabolites. Based on these protective effects, curcumin may exert its influence on the gut microbiota and the TLR4/MyD88/NF-κB signaling pathways to ameliorate GE-induced TBI.

Time series analysis-based seasonal autoregressive fractionally integrated moving average to estimate hepatitis B and C epidemics in China.

BACKGROUND: Hepatitis B (HB) and hepatitis C (HC) place the largest burden in China, and a goal of eliminating them as a major public health threat by 2030 has been set. Making more informed and accurate forecasts of their spread is essential for developing effective strategies, heightening the requirement for early warning to deal with such a major public health threat. AIM: To monitor HB and HC epidemics by the design of a paradigmatic seasonal autoregressive fractionally integrated moving average (SARFIMA) for projections into 2030, and to compare the effectiveness with the seasonal autoregressive integrated moving average (SARIMA). METHODS: Monthly HB and HC incidence cases in China were obtained from January 2004 to June 2023. Descriptive analysis and the Hodrick-Prescott method were employed to identify trends and seasonality. Two periods (from January 2004 to June 2022 and from January 2004 to December 2015, respectively) were used as the training sets to develop both models, while the remaining periods served as the test sets to evaluate the forecasting accuracy. RESULTS: There were incidents of 23400874 HB cases and 3590867 HC cases from January 2004 to June 2023. Overall, HB remained steady [average annual percentage change (AAPC) = 0.44, 95% confidence interval (95%CI): -0.94-1.84] while HC was increasing (AAPC = 8.91, 95%CI: 6.98-10.88), and both had a peak in March and a trough in February. In the 12-step-ahead HB forecast, the mean absolute deviation (15211.94), root mean square error (18762.94), mean absolute percentage error (0.17), mean error rate (0.15), and root mean square percentage error (0.25) under the best SARFIMA (3, 0, 0) (0, 0.449, 2)12 were smaller than those under the best SARIMA (3, 0, 0) (0, 1, 2)12 (16867.71, 20775.12, 0.19, 0.17, and 0.27, respectively). Similar results were also observed for the 90-step-ahead HB, 12-step-ahead HC, and 90-step-ahead HC forecasts. The predicted HB incidents totaled 9865400 (95%CI: 7508093-12222709) cases and HC totaled 1659485 (95%CI: 856681-2462290) cases during 2023-2030. CONCLUSION: Under current interventions, China faces enormous challenges to eliminate HB and HC epidemics by 2030, and effective strategies must be reinforced. The integration of SARFIMA into public health for the management of HB and HC epidemics can potentially result in more informed and efficient interventions, surpassing the capabilities of SARIMA.

Facile fabrication of 3D hollow porous aminopyridine rings decorated polymeric carbon nitride for enhanced photocatalytic hydrogen evolution and dye elimination.

In consideration of energy shortages and environmental pollution, there is a critical need to develop a photocatalyst with high catalytic performance for rapid hydrogen production and efficient pollutant degradation. We synthesized a photocatalytic composite catalyst with three-dimensional (3D) porous aminopyridine rings grafted on the edge of g-C3N4 (APCN) using melamine, cyanuric acid and 4-aminopyridine as raw materials. The composite catalyst exhibited excellent photocatalytic performance for H2 production (2.44 mmol g-1h-1) and RhB degradation (97.08%) under visible light. Subsequently, a possible enhanced mechanism of the catalyst was proposed on the basis of a series of characterization and photocatalytic experiments. The 3D porous structure not only enhanced the structural stability but also increased the surface area of the APCN catalysts, which generated more exposed active sites. Moreover, the aminopyridine ring embellishment was beneficial for achieving a narrowed bandgap and charge migration and separation, which decreased the occurrence of photogenerated carrier recombination. In summary, these two structural features showed a synergistic effect to enhance the photocatalytic performance of the APCN catalyst. Finally, an integrated feasible enhanced mechanism of photocatalytic activity was elucidated according to the results of active substance capture tests, showing that O2•- played an important role during RhB degradation.

Protective effects of curcumin against thyroid hormone imbalance after gas explosion-induced traumatic brain injury via activation of the hypothalamic-pituitary-thyroid axis in male rats.

Gas explosion (GE)-induced traumatic brain injury (TBI) can affect thyroid hormone (TH) homeostasis in miners. This study evaluated the effects of hepatic transthyretin and hypothalamic-pituitary-thyroid (HPT) axis on thyroids and explored the protective effect and mechanism of curcumin on GE-induced TBI. Thirty rats were randomly divided into three groups (10 per group): first group (control group)-rats received GE treatment once; second group (GE group)-rats received GE treatment (200 m from the source of the explosion once); third group (GE + Cur group)-rats received curcumin (Cur) by lavage at a dose of 100 mg/kg/day once every other day for 7 days after receiving GE. After GE, the pathological changes were analyzed by hemotoxylin and eosin staining, and the levels of serum reactive oxygen species (ROS), urine iodine (UI), THs, nuclear factor-kappa B (NF-κB), superoxide dismutase (SOD), glutathione peroxidase (Gpx), and malondialdehyde (MDA) were analyzed using ELISA. Expression of proteins in the HPT axis of rats was examined by immunohistochemistry and Western blotting. We found that GE could induce pathologic changes in rat thyroid and liver. Serum levels of THs, NF-κB and serum redox state became unbalanced in rats after GE. GE could inhibit the biosynthesis and biotransformation of THs by affecting key HPT axis proteins. Additionally, GE reduced the level of hepatic transthyretin. Serum THs levels and thyroid sections were almost recovered to normal after curcumin treatment. The aforementioned key HPT axis proteins in the curcumin group showed opposite expression trends. In summary, GE affected THs balance while curcumin can protect against these injury effects by affecting TH biosynthesis, biotransformation, and transport, and inducing oxidative stress and inflammatory responses.

Estimating the Effects of the COVID-19 Outbreak on the Reductions in Tuberculosis Cases and the Epidemiological Trends in China: A Causal Impact Analysis.

OBJECTIVE: COVID-19 may have a demonstrable influence on disease patterns. However, it remained unknown how tuberculosis (TB) epidemics are impacted by the COVID-19 outbreak. The purposes of this study are to evaluate the impacts of the COVID-19 outbreak on the decreases in the TB case notifications and to forecast the epidemiological trends in China. METHODS: The monthly TB incidents from January 2005 to December 2020 were taken. Then, we investigated the causal impacts of the COVID-19 pandemic on the TB case reductions using intervention analysis under the Bayesian structural time series (BSTS) method. Next, we split the observed values into different training and testing horizons to validate the forecasting performance of the BSTS method. RESULTS: The TB incidence was falling during 2005-2020, with an average annual percentage change of -3.186 (95% confidence interval [CI] -4.083 to -2.281), and showed a peak in March-April and a trough in January-February per year. The BSTS method assessed a monthly average reduction of 14% (95% CI 3.8% to 24%) in the TB case notifications from January-December 2020 owing to COVID-19 (probability of causal effect=99.684%, P=0.003), and this method generated a highly accurate forecast for all the testing horizons considering the small forecasting error rates and estimated a continued downward trend from 2021 to 2035 (annual percentage change =-2.869, 95% CI -3.056 to -2.681). CONCLUSION: COVID-19 can cause medium- and longer-term consequences for the TB epidemics and the BSTS model has the potential to forecast the epidemiological trends of the TB incidence, which can be recommended as an automated application for public health policymaking in China. Considering the slow downward trend in the TB incidence, additional measures are required to accelerate the progress of the End TB Strategy.

Estimating the Long-Term Epidemiological Trends and Seasonality of Hemorrhagic Fever with Renal Syndrome in China.

OBJECTIVE: We aim to examine the adequacy of an innovation state-space modeling framework (called TBATS) in forecasting the long-term epidemic seasonality and trends of hemorrhagic fever with renal syndrome (HFRS). METHODS: The HFRS morbidity data from January 1995 to December 2020 were taken, and subsequently, the data were split into six different training and testing segments (including 12, 24, 36, 60, 84, and 108 holdout monthly data) to investigate its predictive ability of the TBATS method, and its forecasting performance was compared with the seasonal autoregressive integrated moving average (SARIMA). RESULTS: The TBATS (0.27, {0,0}, -, {<12,4>}) and SARIMA (0,1,(1,3))(0,1,1)12 were selected as the best TBATS and SARIMA methods, respectively, for the 12-step ahead prediction. The mean absolute deviation, root mean square error, mean absolute percentage error, mean error rate, and root mean square percentage error were 91.799, 14.772, 123.653, 0.129, and 0.193, respectively, for the preferred TBATS method and were 144.734, 25.049, 161.671, 0.203, and 0.296, respectively, for the preferred SARIMA method. Likewise, for the 24-, 36-, 60-, 84-, and 108-step ahead predictions, the preferred TBATS methods produced smaller forecasting errors over the best SARIMA methods. Further validations also suggested that the TBATS model outperformed the Error-Trend-Seasonal framework, with little exception. HFRS had dual seasonal behaviors, peaking in May-June and November-December. Overall a notable decrease in the HFRS morbidity was seen during the study period (average annual percentage change=-6.767, 95% confidence intervals: -10.592 to -2.778), and yet different stages had different variation trends. Besides, the TBATS model predicted a plateau in the HFRS morbidity in the next ten years. CONCLUSION: The TBATS approach outperforms the SARIMA approach in estimating the long-term epidemic seasonality and trends of HFRS, which is capable of being deemed as a promising alternative to help stakeholders to inform future preventive policy or practical solutions to tackle the evolving scenarios.

Time Series Analysis and Forecasting of the Hand-Foot-Mouth Disease Morbidity in China Using An Advanced Exponential Smoothing State Space TBATS Model.

OBJECTIVE: The high morbidity, complex seasonality, and recurring risk of hand-foot-and-mouth disease (HFMD) exert a major burden in China. Forecasting its epidemic trends is greatly instrumental in informing vaccine and targeted interventions. This study sets out to investigate the usefulness of an advanced exponential smoothing state space framework by combining Box-Cox transformations, Fourier representations with time-varying coefficients and autoregressive moving average (ARMA) error correction (TBATS) method to assess the temporal trends of HFMD in China. METHODS: Data from January 2009 to December 2019 were drawn, and then they were split into two segments comprising the in-sample training data and out-of-sample testing data to develop and validate the TBATS model, and its fitting and forecasting abilities were compared with the most frequently used seasonal autoregressive integrated moving average (SARIMA) method. RESULTS: Following the modelling procedures of the SARIMA and TBATS methods, the SARIMA (1,0,1)(0,1,1)12 and TBATS (0.024, {1,1}, 0.855, {<12,4>}) specifications were recognized as being the optimal models, respectively, for the 12-step ahead forecasting, along with the SARIMA (1,0,1)(0,1,1)12 and TBATS (0.062, {1,3}, 0.86, {<12,4>}) models as being the optimal models, respectively, for the 24-step ahead forecasting. Among them, the optimal TBATS models produced lower error rates in both 12-step and 24-step ahead forecasting aspects compared to the preferred SARIMA models. Descriptive analysis of the data showed a significantly high level and a marked dual seasonal pattern in the HFMD morbidity. CONCLUSION: The TBATS model has the capacity to outperform the most frequently used SARIMA model in forecasting the HFMD incidence in China, and it can be recommended as a flexible and useful tool in the decision-making process of HFMD prevention and control in China.

Preclinical and clinical studies of smoke-inhalation-induced acute lung injury: update on both pathogenesis and innovative therapy.

Smoke-inhalation-induced acute lung injury (SI-ALI) is a leading cause of morbidity and mortality in victims of fire tragedies. SI-ALI contributes to an estimated 30% of burn-caused patient deaths, and recently, more attention has been paid to the specific interventions for this devastating respiratory illness. In the last decade, much progress has been made in the understanding of SI-ALI patho-mechanisms and in the development of new therapeutic strategies in both preclinical and clinical studies. This article reviews the recent progress in the treatment of SI-ALI, based on pathophysiology, thermal damage, airway obstruction, the nuclear-factor kappa-B signaling pathway, and oxidative stress. Preclinical therapeutic strategies include use of mesenchymal stem cells, hydrogen sulfide, peroxynitrite decomposition catalysts, and proton-pump inhibitors. Clinical interventions include high-frequency percussive ventilation, perfluorohexane, inhaled anticoagulants, and nebulized epinephrine. The animal model, dose, clinical application, and pharmacology of these medications are summarized. Future directions and further needs for developing innovative therapies are discussed.

Efficient SSA-mediated precise genome editing using CRISPR/Cas9.

CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34-base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing. Here, we report a novel two-step precise gene-editing method by leveraging the SSA-mediated repair mechanism into the CRISPR/Cas9-mediated gene-editing system. An integrating cassette was developed with positive and negative selection markers, which was flanked by direct repeat sequences with desired mutations as SSA arms. After the targeted integration of the cassette mediated by CRISPR/Cas9-induced homologous-directed repair, cell clones were first selected through the positive selection. In the second round targeting, the selection cassette was removed by the SSA-mediated DNA double-strand break (DSB) repair without any scar left behind. The novel seamless genome editing technique was tested on CCR5 and APP loci, and finally demonstrated, respectively, up to 45.83% and 68% of precise genome editing efficiency. This study provides a new efficient approach for precise genome editing and gene correction.

Gene editing as a promising approach for respiratory diseases.

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52.

Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays. Moreover, the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different (plasmid, PCR and ssDNA) donor templates were confirmed. We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7. In addition, we also applied the fusion strategy for modifying the IGF2 gene of porcine PK15 cells, which further demonstrated a 2.2-fold increase in HDR frequency. In conclusion, our data suggests that Rad52-Cas9 fusion is a good option for enhancing CRISPR/Cas9-mediated HDR, which may be of use in future studies involving precise genome editing.

Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System.

In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the Puro(R) gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.

[Research progress in developing reporter systems for the enrichment of positive cells with targeted genome modification].

Targeted genome editing technology plays an important role in studies of gene function, gene therapy and transgenic breeding. Moreover, the efficiency of targeted genome editing is increased dramatically with the application of recently developed artificial nucleases such as ZFNs, TALENs and CRISPR/Cas9. However, obtaining positive cells with targeted genome modification is restricted to some extent by nucleases expression plasmid transfection efficiency, nucleases expression and activity, and repair efficiency after genome editing. Thus, the enrichment and screening of positive cells with targeted genome modification remains a problem that need to be solved. Surrogate reporter systems could be used to reflect the efficiency of nucleases indirectly and enrich genetically modified positive cells effectively, which may increase the efficiency of the enrichment and screening of positive cells with targeted genome modification. In this review, we mainly summarized principles and applications of reporter systems based on NHEJ and SSA repair mechanisms, which may provide references for related studies in future.

Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture.

Molecular cloning and SNP association analysis of chicken PMCH gene.

The pre-melanin-concentrating hormone (PMCH) gene is an important gene functionally concerning the regulations of body fat content, feeding behavior and energy balance. In this study, the full-length cDNA of chicken PMCH gene was amplified by SMART RACE method. The single nucleotide polymorphisms (SNPs) in the PMCH gene were screened by comparative sequence analysis. The obtained non-synonymous coding SNPs (ncSNPs) were designed for genotyping firstly. Its effects on growth, carcass characteristics and meat quality traits were investigated employing the F2 resource population of Gushi chicken crossed with Anak broiler by AluI CRS-PCR-RFLP. Our results indicated that the cDNA of chicken PMCH shared 67.25 and 66.47% homology with that of human and bovine PMCH, respectively. The deduced amino acid sequence of chicken PMCH (163 amino acids) were 52.07 and 50.89% identical to those of human and bovine PMCH, respectively. The PMCH protein sequence is predicted to have several functional domains, including pro-MCH, CSP, IL7, XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. gga-miR-18a, gga-miR-18b, gga-miR-499 microRNA targeting site was predicted in the 3' untranslated region of chicken PMCH mRNA. In addition, a total of seven SNPs including an ncSNP and a synonymous coding SNP, were identified in the PMCH gene. The ncSNP c.81 A>T was found to be in moderate polymorphic state (polymorphic index=0.365), and the frequencies for genotype AA, AB and BB were 0.3648, 0.4682 and 0.1670, respectively. Significant associations between the locus and shear force of breast and leg were observed. This polymorphic site may serve as a useful target for the marker assisted selection of the growth and meat quality traits in chicken.

Polymorphisms of the pro-opiomelanocortin and agouti-related protein genes and their association with chicken production traits.

Pro-opiomelanocortin (POMC) and agouti-related protein (AGRP) are hypothalamic neuropeptides that play a central role in the regulation of food intake and energy expenditure and for this reason the variations in the POMC and AGRP genes in chicken were examined by screening the DNA pools. Two silent cSNPs mutations in POMC gene and one silent cSNP mutation in AGRP gene were identified. PCR-restriction fragment length polymorphism (RFLP) was used to test the cSNPs c. C495T in the POMC and c. C9T in the AGRP gene in the F2 resource population of Gushi chicken crossed with Anak broiler. The association analysis on the polymorphisms of POMC, AGRP gene and production traits showed that the c. C495T mutation in the POMC gene was significantly linked to the pelvis breadth at 4 weeks of age (P = 0.035), body weight at 2 weeks of age (P = 0.013) and was highly significantly linked to the chest depth at 12 weeks of age (P = 0.006). The c. T9T genotype in the AGRP gene was associated with a low breast muscle water loss rate (P = 0.025), increased chest width at 12 weeks of age (P = 0.005) and body weight at 2 weeks of age (P = 0.036), a high slaughter rate (P = 0.049) and semi-evisceration weight (P = 0.019). These findings may have important implications for the molecular aspects of chicken breeding.