Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.

BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

Injectable and conductive cardiac patches repair infarcted myocardium in rats and minipigs.

Cardiac patches can help to restore the electrophysiological properties of the heart after myocardial infarction. However, scaffolds for the repair of heart muscle typically require surgical implantation or, if they are injectable, they are not electrically conductive or do not maintain their shape or function. Here, we report the performance, as demonstrated for the repair of infarcted heart muscle in rats and minipigs, of injectable and conductive scaffolds consisting of methacrylated elastin and gelatin, and carbon nanotubes that display shape-memory behaviour, a hierarchical porous structure and a negligible Poisson's ratio. In rats, the implantation of cell-free patches or patches seeded with rat cardiomyocytes onto the myocardium after ligation of the left anterior descending coronary artery led to functional repair after 4 weeks, as indicated by increases in fractional shortening and the ejection fraction, and by a decrease in the infarcted area. We also observed measures of functional recovery in minipigs with infarcted hearts after the delivery of cell-free patches or patches incorporating cardiomyocytes differentiated from human pluripotent stem cells.

Desymmetrization of Cyclic 1,3-Diketones under N-Heterocyclic Carbene Organocatalysis: Access to Organofluorines with Multiple Stereogenic Centers.

Symmetric 1,3-diketones with fluorine or fluorinated substituents on the prochiral carbon remain to be established. Herein, we have developed a novel prochiral fluorinated oxindanyl 1,3-diketone and successfully applied these substrates in carbene-catalyzed asymmetric desymmetrization. Accordingly, a versatile strategy for asymmetric generation of organofluorines with fluorine or fluorinated methyl groups has been developed. Multiple stereogenic centers were selectively constructed with satisfactory outcomes. Structurally diverse enantioenriched organofluorines were generated with excellent results in terms of yields, diastereoselectivities, and enantioselectivities. Notably, exchanging fluorinated methyl groups to fluorine for this prochiral 1,3-diketones leads to switchable stereoselectivity. Mechanistic aspects and origin of stereoselectivity were studied by DFT calculations. Notably, some of the prepared organofluorines demonstrated competitive antibacterial activities.

Immunosuppressive effects of hydroxychloroquine and artemisinin combination therapy via the nuclear factor-κB signaling pathway in lupus nephritis mice.

Lupus nephritis (LN) is one of the most common and severe manifestations of systemic lupus erythematosus, leading to permanent renal damage and chronic kidney disease. Hydroxychloroquine (HCQ) serves a protective role against lupus-associated clinical manifestations and medical complications; however, it results in numerous adverse reactions, limiting its long-term use. The aim of the present study was to investigate the combined effect of HCQ and artemisinin (ART) on LN, and to elucidate the underlying mechanisms. An in vivo LN mouse model was prepared, and the animals were administered prednisone (PDS; serving as a positive control), high-dose HCQ (H-HCQ) or low-dose HCQ combined with ART (L-HCQ + ART) once daily for 8 weeks. The body weight, serum biochemical parameters, immune and inflammatory indicators, renal and spleen histological alterations, and mRNA expression levels of Kruppel-like factor 15 (KLF15) and nuclear factor-κB (NF-κB) were analyzed. It was observed that L-HCQ + ART and H-HCQ ameliorated the LN-induced body weight decrease, and significantly decreased the levels of anti-double stranded DNA, antinuclear antibodies, immunoglobulin G, interferon γ, tumor necrosis factor-α and transforming growth factor-ß1, as well as improved the kidney and spleen pathology, when compared with the model group. In addition, L-HCQ + ART and H-HCQ treatments induced KLF15 upregulation and NF-κB downregulation. These results indicated that treatment with L-HCQ + ART exerted renoprotective effects by regulating the expression levels of cytokines, KLF15 and NF-κB. This combination treatment may have a similar immunosuppressive effect as PDS and H-HCQ, and may be a promising alternative for LN treatment.

A novel dihydroxylated derivative of artemisinin from microbial transformation.

Microbial transformation of artemisinin (1) by Cunninghamella elegans was investigated. Four isolated products were identified as 6ß-hydroxyartemisinin (2), 7α-hydroxyartemisinin (3), 7ß-hydroxyartemisinin (4), and 6ß,7α-dihydroxyartemisinin (5). The structures were elucidated by spectroscopic and X-ray crystallographic analysis. Product 5 is a novel compound and being reported here for the first time. It features two hydroxyl groups in its structure, and this is the first report on dihydroxylation of the artemisinin skeleton. Quantitative structure-activity relationship and molecular modeling studies indicate the modification of artemisinin skeleton will increase antimalarial activity and water solubility. The chemical syntheses of artemisinin derivatives at C6 or C7 position are impossible due to the lack of functional groups. 6ß,7α-Dihydroxyartemisinin is hydroxylated at both 6ß- and 7α-positions of artemisinin skeleton at the same time. Therefore, this new compound would be a good scaffold for further structural modification in the search for more potent antimalarial drugs.

Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine.

BACKGROUND: In Comoros, the widespread of chloroquine (CQ)-resistant Plasmodium falciparum populations was a major obstacle to malaria control, which led to the official withdrawal of CQ in 2004. Continuous monitoring of CQ-resistant markers of the P. falciparum CQ resistant transporter (pfcrt) and the P. falciparum multiple drug resistance 1 (pfmdr-1) is necessary inder to obtain first-hand information on CQ susceptibility of parasite populations in the field. The objective of this study is to assess the prevalence and evolution of CQ-resistance in the P. falciparum populations on the Comoros' Grande Comore island after withdrawal of CQ. METHODS: A total of 207 P. falciparum clinical isolates were collected from the island, including 118 samples from 2006 to 2007 and 89 samples from 2013 to 2014. Nucleotide substitutions in the pfcrt and pfmdr-1 genes linked to CQ response in parasite isolates were assessed using nested PCR and DNA sequencing. RESULTS: From the pfcrt gene segment sequenced, we detected C72S, M74I, N75E, and K76T substitutions in the parasite isolates collected from both 2006-2007 to 2013-2014 periods. Significant decline of pfcrt resistant alleles at C72S (42.6 to 6.9 %), M74I (39.1 to 14.9 %), N75E (63.5 to 18.3 %), and K76T (72.2 to 19.5 %) from 2006-2007 to 2013-2014 were observed, and the frequency of pfcrt wild type allele was significantly increased from 19.1 % in 2006-2007 to 75.8 % in 2013-2014. Sequence analysis of pfmdr-1 also detected point mutations at codons N86Y, Y184F, and D1246Y, but not S1034C and N1042D, in the isolates collected from both examined periods. An increasing trend in the prevalence of the pfmdr-1 wild type allele (NYD, 4.3 % in 2006-2007; and 28.7 % in 2013-2014), and a decreasing trend for pfmdr-1 N86Y mutation (87.0 % in 2006-2007; and 40.2 % in 2013-2014) were observed in our samples. CONCLUSIONS: The present data indicate that the prevalence and patterns of mutant pfcrt and pfmdr-1 dramatically decreased in the Grande Comore isolates from 2006 to 2014, suggesting that the CQ-sensitive P. falciparum strains have returned after the withdrawal of CQ. The data also suggests that the parasites with wild type pfcrt/pfdmr-1 genes may have growth and/or transmission advantages over the mutant parasites. The information obtained from this study will be useful for developing and updating anti-malarial treatment policy in Grande Comore island.

Polymorphisms of the artemisinin resistant marker (K13) in Plasmodium falciparum parasite populations of Grande Comore Island 10 years after artemisinin combination therapy.

BACKGROUND: Plasmodium falciparum malaria is a significant public health problem in Comoros, and artemisinin combination therapy (ACT) remains the first choice for treating acute uncomplicated P. falciparum. The emergence and spread of artemisinin-resistant P. falciparum in Southeast Asia, associated with mutations in K13-propeller gene, poses a potential threat to ACT efficacy. Detection of mutations in the P. falciparum K13-propeller gene may provide the first-hand information on changes in parasite susceptibility to artemisinin. The objective of this study is to determinate the prevalence of mutant K13-propeller gene among the P. falciparum isolates collected from Grande Comore Island, Union of Comoros, where ACT has been in use since 2004. METHODS: A total of 207 P. falciparum clinical isolates were collected from the island during March 2006 and October 2007 (n = 118) and March 2013 and December 2014 (n = 89). All isolates were analysed for single nucleotide polymorphisms (SNPs) and haplotypes in the K13-propeller gene using nested PCR and DNA sequencing. RESULTS: Only three 2006-2007 samples carried SNPs in the K13-propeller gene, one having a synonymous (G538G) and the other having two non-synonymous (S477Y and D584E) substitutions leading to two mutated haplotypes (2.2%, 2/95). Three synonymous mutations (R471R, Y500Y, and G538G) (5.9%, 5/85) and 7 non-synonymous substitutions (21.2%, 18/85) with nine mutated haplotypes (18.8%, 16/85) were found in isolates from 2013 to 2014. However, none of the polymorphisms associated with artemisinin-resistance in Southeast Asia was detected from any of the parasites examined. CONCLUSION: This study showed increased K13-propeller gene diversity among P. falciparum populations on the Island over the course of 8 years (2006-2014). Nevertheless, none of the polymorphisms known to be associated with artemisinin resistance in Asia was detected in the parasite populations examined. Our data suggest that P. falciparum populations in Grande Comore are still effectively susceptible to artemisinin. Our results provide insights into P. falciparum populations regarding mutations in the gene associated with artemisinin resistance and will be useful for developing and updating anti-malarial guidance in Comoros.

[Primary Pharmacodynamic Study on Chuanping Dropping Pills].

OBJECTIVE: To study the antitussive, antiasthmatic, and expectorant effects of Chuanoing dropping pills. METHODS: Histamine-acetylcholine induced asthma, ovalbumin sensitization, pulmonary overflow, spiral strip trachea of guinea pig, lung strip of guinea pig, ammonia water or citric acid induced cough of mice, and phenol red expectoration of mice were carried out. RESULTS: Chuanping dripping pills significantly prolonged the incubation induced by histamine-acetylcholine, reduced the reaction stage of ovalbumin sensitization of guinea pig with asthma, inhibited the increase of pulmonary overflow on histamine induced asthma, reduced the contraction of smooth muscle and lung strip by histamine induced asthma on guinea pig, reduced cough times on ammonia or citrate acid caused mice, and increased the output on mice tracheal phenol red. CONCLUSIONS: Chuanping dropping pills have significant effect on antitussive, antiasthmatic and expectorant actions.