MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo.

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.

TFAM is an autophagy receptor that limits inflammation by binding to cytoplasmic mitochondrial DNA.

When cells are stressed, DNA from energy-producing mitochondria can leak out and drive inflammatory immune responses if not cleared. Cells employ a quality control system called autophagy to specifically degrade damaged components. We discovered that mitochondrial transcription factor A (TFAM)-a protein that binds mitochondrial DNA (mtDNA)-helps to eliminate leaked mtDNA by interacting with the autophagy protein LC3 through an autolysosomal pathway (we term this nucleoid-phagy). TFAM contains a molecular zip code called the LC3 interacting region (LIR) motif that enables this binding. Although mutating TFAM's LIR motif did not affect its normal mitochondrial functions, more mtDNA accumulated in the cell cytoplasm, activating inflammatory signalling pathways. Thus, TFAM mediates autophagic removal of leaked mtDNA to restrict inflammation. Identifying this mechanism advances understanding of how cells exploit autophagy machinery to selectively target and degrade inflammatory mtDNA. These findings could inform research on diseases involving mitochondrial damage and inflammation.

Lycium barbarum glycopetide prolong lifespan and alleviate Parkinson's disease in Caenorhabditis elegans.

Introduction: Lycium barbarum glycopeptide (LbGp) is the main bioactive compound extracted from the traditional Chinese medicine. L. barbarum berries and has been proven to have numerous health benefits, including antioxidative, anti-inflammatory, anticancer, and cytoprotective activities. However, the antiaging effect of LbGp remains unknown. Methods: The lifespan and body movement of C. elegans were used to evaluate the effect of LbGp on lifespan and health span. The thrashing assay was used to determine the role of LbGp in Parkinson's disease. To investigate the mechanisms of LbGp-induced antiaging effects, we analyzed changes in lifespan, movement, and the expression of longevity-related genes in a series of worm mutants after LbGp treatment. Results: We found that LbGp treatment prolonged the lifespan and health span of C. elegans. Mechanistically, we found that LbGp could activate the transcription factors DAF-16/FOXO, SKN-1/Nrf2, and HSF-1, as well as the nuclear receptor DAF-12, thereby upregulating longevity-related genes to achieve lifespan extension. In addition, we found that the lifespan extension induced by LbGp partially depends on mitochondrial function. Intriguingly, LbGp also ameliorated neurodegenerative diseases such as Parkinson's disease in a DAF-16-, SKN-1-, and HSF-1-dependent manner. Conclusion: Our work suggests that LbGp might be a viable candidate for the treatment and prevention of aging and age-related diseases.

Influence of Government Price Regulation on the Price, Volume and Spending of Antibiotics in China: A Controlled Interrupted Time Series Study.

BACKGROUND: Chinese government established maximum retail prices for antibiotics listed in China's National Reimbursement List in February 2013. This study aimed to analyze the impact of pharmaceutical price regulation on the price, volume and spending of antibiotics in China. METHODS: An interrupted time series design with comparison series was used to examine impacts of the policy changes on average daily cost, monthly hospital purchase volume and spending of the 11 price-regulated antibiotics and 40 priceunregulated antibiotics in 699 hospitals. One intervention point was applied to assess the impact of policy. RESULTS: After government price regulation, compared to price-unregulated antibiotics, the average daily cost of the price-regulated group declined rapidly (ß=-5.68, P<.001). The average hospital monthly purchase spending of priceregulated antibiotics also decreased rapidly (ß=-0.49, P<.010) and a positive trend change (ß=0.04, P<.001) in average hospital spending of price-unregulated antibiotics was found. CONCLUSION: Government regulation can reduce the prices and spending of price-regulated antibiotics. To control increasing expenditure, besides price caps regulation, factors determining drug utilization also need to be considered in policy designing.

Gene activation in Caenorhabditis elegans using the Campylobacter jejuni CRISPR-Cas9 feeding system.

Clustered regularly interspaced palindromic repeats-based activation system, a powerful genetic manipulation technology, can modulate endogenous gene transcription in various organisms through fusing nuclease-deficient Cas9 to transcriptional regulatory domains. At present, this clustered regularly interspaced palindromic repeats-based activation system has been applied to activate gene expression by microinjection manner in Caenorhabditis elegans. However, this complicated and time-consuming injection manner is not suitable for efficient and high-throughput gene regulation with clustered regularly interspaced palindromic repeats-Cas9 system. Here, we engineered a Campylobacter jejun clustered regularly interspaced palindromic repeats-Cas9-based gene activation system through bacteria feeding technique to delivering gene-specific sgRNA in C. elegans. It enables to activate various endogenous genes efficiently, as well as induce the corresponding phenotypes with a more efficient and labor-saving manner. Collectively, our results demonstrated that our novel dCjCas9-based activation feeding system holds great promise and potential in C. elegans.

CHCHD2 and CHCHD10 regulate mitochondrial dynamics and integrated stress response.

Mitochondrial dysfunction is becoming one of the main pathology factors involved in the etiology of neurological disorders. Recently, mutations of the coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) which encode two homologous proteins that belong to the mitochondrial CHCH domain protein family, are linked to Parkinson's disease and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), respectively. However, the physiological and pathological roles of these twin proteins have not been well elaborated. Here, we show that, in physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 and suppress its enzyme activity, which not only restrains the initiation of the mitochondrial integrated response stress (mtISR), but also suppresses the processing of OPA1 for mitochondrial fusion. Further, during mitochondria stress-induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, CHCHD2 and CHCHD10 translocate to the cytosol and interacte with eIF2a, which attenuates mtISR overactivation by suppressing eIF2a phosphorylation and its downstream response. As such, knockdown of CHCHD2 and CHCHD10 triggers mitochondrial ISR, and such cellular response is enhanced by CCCP treatment. Therefore, our findings demonstrate the first "mtISR suppressor" localized in mitochondria for regulating stress responses in mammalian cells, which has a profound pathological impact on the CHCH2/CHCH10-linked neurodegenerative disorder.

Histone H3K4me3 modification is a transgenerational epigenetic signal for lipid metabolism in Caenorhabditis elegans.

As a major risk factor to human health, obesity presents a massive burden to people and society. Interestingly, the obese status of parents can cause progeny's lipid accumulation through epigenetic inheritance in multiple species. To date, many questions remain as to how lipid accumulation leads to signals that are transmitted across generations. In this study, we establish a nematode model of C. elegans raised on a high-fat diet (HFD) that leads to measurable lipid accumulation, which can transmit the lipid accumulation signal to their multigenerational progeny. Using this model, we find that transcription factors DAF-16/FOXO and SBP-1/SREBP, nuclear receptors NHR-49 and NHR-80, and delta-9 desaturases (fat-5, fat-6, and fat-7) are required for transgenerational lipid accumulation. Additionally, histone H3K4 trimethylation (H3K4me3) marks lipid metabolism genes and increases their transcription response to multigenerational obesogenic effects. In summary, this study establishes an interaction between a network of lipid metabolic genes and chromatin modifications, which work together to achieve transgenerational epigenetic inheritance of obesogenic effects.

Factors Associated with Free Medicine Use in Patients with Hypertension and Diabetes: A 4-Year Longitudinal Study on Full Coverage Policy for Essential Medicines in Taizhou, China.

Full coverage policies for medicines have been implemented worldwide to alleviate medicine cost burden and promote access to medicines. However, few studies have explored the factors associated with free medicine use in patients with chronic diseases. This study aimed to analyze the utilization of free medicines by patients with hypertension and diabetes after the implementation of the full coverage policy for essential medicines (FCPEM) in Taizhou, China, and to explore the factors associated with free medicine use. We conducted a descriptive analysis of characteristics of patients with and without free medicine use and performed a panel logit model to examine factors associated with free medicine use, based on an electronic health record database in Taizhou from the baseline year (12 months in priori) to three years after FCPEM implementation. After FCPEM implementation, the proportion of patients without any free medicine use decreased from 31.1% in the baseline year to 28.9% in the third year, while that of patients taking free medicines rose from 11.0% to 22.8%. Patients with lower income or education level, those with agricultural hukou, patients aged 65 and above, married patients, and patients in the Huangyan district were more likely to take free medicines. In conclusion, FCPEM contributed to improved medicine access, especially in vulnerable populations. Local policy makers should consider expanding the coverage of FCPEM to other types of medicines and cultivate the potential of social supports for patients to enhance the effectiveness of FCPEM policies.

Can locally developed me-too drugs aid price negotiation? An example of cancer therapies from China.

Rapid growth in pharmaceutical expenditures and high prices have greatly hampered access to medicines, especially targeted anticancer medicines. Confronted with such difficulties, the Chinese government has put more effort into supporting local research and development of cancer medicines, resulting in locally developed me-too drugs. Since 2016, the government has implemented a central reimbursement-linked drug price negotiation policy aimed at reducing the prices of expensive medicines. Locally developed me-too drugs marketed at lower prices may inject price competition and help negotiate reduced prices of similar internationally-developed products. As an example, we selected 3 tyrosine kinase inhibitors (TKIs) developed for the therapy of advanced non-small cell lung cancer harboring mutations in the epidermal growth factor receptor (EGFR). Descriptive analysis was applied to data from the Chinese Medical Economic Information database to describe the impact on the price and utilization of three TKIs after the introduction of icotinib, a locally developed me-too TKI and two national negotiations regarding the price of EGFR-TKIs in China. After two national negotiations, the daily costs of all three EGFR-TKIs were reduced to around $30. From the first quarter of 2013 to the second quarter of 2016, the market share of the purchasing volume of icotinib, China's locally developed TKI, increased from 13% to 40%, while the market shares of two internationally developed TKIs decreased from 35% to 15% and from 52% to 45%, for erlotinib and gefitinib, respectively. The prices of EGFR-TKIs decreased and China's locally developed TKI accounted for a considerable proportion of market share. Locally developed me-too drugs aid price negotiation by injecting price competition and helping negotiate reduced prices of similar internationally-developed products. Through efforts to develop me-too drugs, combined with national drug price negotiation and reimbursement policies, developing countries might improve access to more affordable targeted cancer therapies.

N6-methyldeoxyadenine and histone methylation mediate transgenerational survival advantages induced by hormetic heat stress.

Environmental stress can induce survival advantages that are passed down to multiple generations, representing an evolutionarily advantageous adaptation at the species level. Using the nematode worm Caenorhabditis elegans as a model, we found that heat shock experienced in either parent could increase the longevity of themselves and up to the fifth generation of descendants. Mechanistic analyses revealed that transcription factor DAF-16/FOXO, heat shock factor HSF-1, and nuclear receptor DAF-12/FXR functioned transgenerationally to implement the hormetic stress response. Histone H3K9me3 methyltransferases SET-25 and SET-32 and DNA N6-methyl methyltransferase DAMT-1 participated in transmitting high-temperature memory across generations. H3K9me3 and N6-methyladenine could mark heat stress response genes and promote their transcription in progeny to extend life span. We dissected the mechanisms responsible for implementing and transmitting environmental memories in descendants from heat-shocked parents and demonstrated that hormetic stress caused survival benefits could be transmitted to multiple generations through H3K9me3 and N6-mA modifications.

Health Care Utilization and Costs of Patients With Prostate Cancer in China Based on National Health Insurance Database From 2015 to 2017.

BACKGROUND: In terms of medical costs, prostate cancer is on the increase as one of the most costly cancers, posing a tremendous economic burden, but evidence on the health care utilization and medical expenditure of prostate cancer has been absent in China. OBJECTIVE: This study aimed to analyze health care utilization and direct medical costs of patients with prostate cancer in China. METHODS: Health care service data with a national representative sample of basic medical insurance beneficiaries between 2015 and 2017 were obtained from the China Health Insurance Association database. We conducted descriptive and statistical analyses of health care utilization, annual direct medical costs, and composition based on cancer-related medical records. Health care utilization was measured by the number of hospital visits and the length of stay. RESULTS: A total of 3,936 patients with prostate cancer and 24,686 cancer-related visits between 2015 and 2017 were identified in the database. The number of annual outpatient and inpatient visits per patient differed significantly from 2015 to 2017. There was no obvious change in length of stay and annual direct medical costs from 2015 to 2017. The number of annual visits per patient (outpatient: 3.0 vs. 4.0, P < 0.01; inpatient: 1.5 vs. 2.0, P < 0.001) and the annual medical direct costs per patient (US$2,300.1 vs. US$3,543.3, P < 0.001) of patients covered by the Urban Rural Resident Basic Medical Insurance (URRBMI) were both lower than those of patients covered by the Urban Employee Basic Medical Insurance (UEBMI), and the median out-of-pocket expense of URRBMI was higher than that of UEBMI (US$926.6 vs. US$594.0, P < 0.001). The annual direct medical costs of patients with prostate cancer in Western regions were significantly lower than those of patients in Eastern and Central regions (East: US$4011.9; Central: US$3458.6; West: US$2115.5) (P < 0.001). CONCLUSIONS: There was an imbalanced distribution of health care utilization among regions in China. The direct medical costs of Chinese patients with prostate cancer remained stable, but the gap in health care utilization and medical costs between two different insurance schemes and among regions still needed to be further addressed.

Uric acid induces stress resistance and extends the life span through activating the stress response factor DAF-16/FOXO and SKN-1/NRF2.

Uric acid is a common metabolite found in mammals' serum. Recently, several metabolites have been identified that modulate aging, and uric acid levels are positively correlated with mammals' lifespan. However, the molecular mechanisms underlying this are largely undefined. Here we show that uric acid, an end product of purine metabolism, enhances the resistance of oxidative stress and extends the life span of Caenorhabditis elegans (C. elegans). We show that uric acid enhances a variety of pathways and leads to the upregulation of genes that are required for uric acid-mediated life span extension. We find that the transcription factors DAF-16/FOXO, SKN-1/NRF2 and HSF-1 contribute to the beneficial longevity conferred by uric acid. We also show that uric acid induced life span extension by regulating the reproductive signaling and insulin/IGF-1 signaling (IIS) pathways. In addition, we find that mitochondrial function plays an important role in uric acid-mediated life span extension. Taken together, these data suggest that uric acid prolongs the life span of C. elegans, in part, because of its antioxidative activity, which in turn regulates the IIS and the reproductive signaling pathways, thereby activating the function of the transcription factors DAF-16, HSF-1 and SKN-1.

Hypotaurine promotes longevity and stress tolerance via the stress response factors DAF-16/FOXO and SKN-1/NRF2 in Caenorhabditis elegans.

Hypotaurine, an important sulfur-containing and nonpeptidic amino acid, is a precursor of taurine and an antioxidant. Our previous study indicated that hypotaurine levels are associated with the ageing of Caenorhabditis elegans (C. elegans). However, whether hypotaurine plays a role in the lifespan regulation of C. elegans and the mechanism remains undetermined. Here, we found that hypotaurine enhances oxidative stress resistance and ameliorates ageing in C. elegans. Our results show that hypotaurine regulates a variety of pathways and leads to the upregulation of some age-related genes to extend lifespan. We also found that the stress response-related transcription factors DAF-16/FOXO and SKN-1/NRF2 contribute to the beneficial longevity conferred by hypotaurine. Moreover, our results demonstrate that hypotaurine induced lifespan extension by regulating the insulin/IGF-1 signaling (IIS) pathway, the reproductive signaling pathway and DR-like mechanisms. Additionally, our results also indicated that mitochondrial function also plays a crucial role in the lifespan extension induced by hypotaurine. Taken together, these data indicate that hypotaurine delays the ageing of C. elegans, due, at least in part, to its antioxidant activity, which in turn regulates IIS, and reproductive and DR-related pathways, thereby inducing the activity of the transcription factors DAF-16 and SKN-1.

Antiobesity Effect of Flaxseed Polysaccharide via Inducing Satiety due to Leptin Resistance Removal and Promoting Lipid Metabolism through the AMP-Activated Protein Kinase (AMPK) Signaling Pathway.

Obesity is a metabolic syndrome worldwide that causes many chronic diseases. Recently, we found an antiobesity effect of flaxseed polysaccharide (FP), but the mechanism remains to be elucidated. In this study, rats were first induced to develop obesity by being fed a high-fat diet. The obese rats were then fed a control diet, AIN-93M (group HFD), or a 10% FP diet (group FPD). The body weight, body fat, adipose tissue and liver sections, serous total triglycerides, levels of fasting blood glucose in serum, serous insulin, inflammatory cytokines in serum, and serous proteins within the leptin-neuropeptide Y (NPY) and AMP-activated protein kinase (AMPK) signaling pathway were determined and analyzed. FP intervention significantly reduced body weight and abdominal fat from 530 ± 16 g and 2.15% ± 0.30% in group HFD to 478 ± 10 g and 1.38% ± 0.48% in group FPD, respectively. This effect was achieved by removing leptin resistance possibly by inhibiting inflammation and recovering satiety through the significant downregulation of NPY and the upregulation of glucagon-like peptide 1. Adiponectin was then significantly upregulated probably via the gut-brain axis and further activated the AMPK signaling pathway to improve lipid metabolism including the improvement of lipolysis and fatty acid oxidation and the suppression of lipogenesis. This is the first report of the proposed antiobesity mechanism of FP, thereby providing a comprehensive understanding of nonstarch polysaccharides and obesity.