Proteins are important biomaterials and are generally produced in living cells. Here, we show a novel DNA hydrogel that is capable of producing functional proteins without any living cells. This protein-producing gel (termed 'the P-gel system' or 'P-gel') consists of genes as part of the gel scaffolding. This is the first time that a hydrogel has been used to produce proteins. The efficiency was about 300 times higher than current, solution-based systems. In terms of volumetric yield, the P-gel produced up to 5 mg ml(-1) of functional proteins. The mechanisms behind the high efficiency and yield include improved gene stability, higher local concentration and a faster enzyme turnover rate due to a closer proximity of genes. We have tested a total of 16 different P-gels and have successfully produced all 16 proteins including membrane and toxic proteins, demonstrating that the P-gel system can serve as a general protein production technology.

IMPORTANCE Data are limited regarding the association between CYP2C19 genetic variants and clinical outcomes of patients with minor stroke or transient ischemic attack treated with clopidogrel. OBJECTIVE To estimate the association between CYP2C19 genetic variants and clinical outcomes of clopidogrel-treated patients with minor stroke or transient ischemic attack. DESIGN, SETTING, AND PARTICIPANTS Three CYP2C19 major alleles (*2, *3, *17) were genotyped among 2933 Chinese patients from 73 sites who were enrolled in the Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events (CHANCE) randomized trial conducted from January 2, 2010, to March 20, 2012. INTERVENTIONS Patients with acute minor ischemic stroke or transient ischemic attack in the trial were randomized to treatment with clopidogrel combined with aspirin or to aspirin alone. MAIN OUTCOMES AND MEASURESThe primary efficacy outcome was new stroke. The secondary efficacy outcome was a composite of new composite vascular events (ischemic stroke, hemorrhagic stroke, myocardial infarction, or vascular death). Bleeding was the safety outcome. RESULTS Among 2933 patients, 1948 (66.4%) were men, with a mean age of 62.4 years. Overall, 1207 patients (41.2%) were noncarriers and 1726 patients (58.8%) were carriers of loss-of-function alleles (*2, *3). After day 90 follow-up, clopidogrel-aspirin reduced the rate of new stroke in the noncarriers but not in the carriers of the loss-of-function alleles (P = .02 for interaction; events among noncarriers, 41 [6.7%] with clopidogrel-aspirin vs 74 [12.4%] with aspirin; hazard ratio [HR], 0.51 [95% CI, 0.35-0.75]; events among carriers, 80 [9.4%] with clopidogrel-aspirin vs 94 [10.8%] with aspirin; HR, 0.93 [95% CI, 0.69 to 1.26]). Similar results were observed for the secondary composite efficacy outcome (noncarriers: 41 [6.7%] with clopidogrel-aspirin vs 75 [12.5%] with aspirin; HR, 0.50 [95% CI, 0.34-0.74]; carriers: 80 [9.4%] with clopidogrel-aspirin vs 95 [10.9%] with aspirin; HR, 0.92 [95% CI, 0.68-1.24]; P = .02 for interaction). The effect of treatment assignment on bleeding did not vary significantly between the carriers and the noncarriers of the loss-of-function alleles (2.3% for carriers and 2.5% for noncarriers in the clopidogrel-aspirin group vs 1.4% for carriers and 1.7% for noncarriers in the aspirin only group; P = .78 for interaction). CONCLUSIONS AND RELEVANCE Among patients with minor ischemic stroke or transient ischemic attack, the use of clopidogrel plus aspirin compared with aspirin alone reduced the risk of a new stroke only in the subgroup of patients who were not carriers of the CYP2C19 loss-of-function alleles. These findings support a role of CYP2C19 genotype in the efficacy of this treatment.

Bone marrow-derived mesenchymal stem cells (MSCs) can serve as a vehicle for gene therapy. Angiopoietin-1 (Ang1) is a critical factor for endothelial survival and vascular stabilization via the inhibition of endothelial permeability and leukocyte-endothelium interactions. We hypothesized that MSC-based Ang1 gene therapy might be a potential therapeutic approach for lipopolysaccharide (LPS)-induced lung injury. MSCs were isolated from 6 week-old inbred male mice and transduced with the Ang1 gene, using a lentivirus vector. The MSCs showed no significant phenotypic changes after transduction. In the in vivo mouse model, the LPS-induced lung injury was markedly alleviated in the group treated with MSCs carrying Ang1 (MSCs-Ang1), compared with groups treated with MSCs or Ang1 alone. The expression of Ang1 protein in the recipient lungs was increased after MSCs-Ang1 administration. The histopathological and biochemical indices of LPS-induced lung injury were improved after MSCs-based Ang1 gene treatment. MSCs-Ang1 administration also reduced pulmonary vascular endothelial permeability and the recruitment of inflammatory cells into the lung. Cells of MSC origin could be detected in the recipient lungs for 2 weeks after injection with MSCs. These results suggest that MSCs and Ang1 have a synergistic role in the treatment of LPS-induced lung injury. MSC-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of acute lung injury.

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BackgroundCell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process.Methodology/Principal FindingsUsing cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments.Conclusions/SignificanceThree macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE.

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Key Points Histamine and serotonin induce, but subsequently truncate, angiogenesis via a thrombspondin-1–mediated negative feedback loop.