Error correction of microchip synthesized genes using Surveyor nuclease

The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'→5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by >16-fold, yielding a final error rate of ～1 in 8700 bp.     

Identification and proteome analysis of the two-component VirR/VirS system in epidemic Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects pigs and sporadically causes serious infections in humans. Two recent large-scale outbreaks of human streptococcal toxic-shock-like syndrome with high mortality occurred in China, posing new challenges for global public health. However, the global regulation of the virulence of epidemic SS2 isolates lacks a systematic understanding. In this study, we performed a mutational and functional analysis of an SS2 two-component system that is orthologous to the VirR/VirS regulatory system of Clostridium perfringens. An isogenic knockout mutant of VirR/VirS (ΔvirRS) was found to exhibit marked phenotypic changes, including the formation of shorter chains and thinner capsular walls, more easily cleared in whole blood, and decreased oxidative stress tolerance. Furthermore, the ΔvirRS mutant was greatly attenuated in a mouse model. Comparative proteome analysis of the expression profiles of the wild-type strain with the ΔvirRS mutant allowed us to identify 72 proteins that are differentially expressed in the absence of the VirR/VirS system and that are directly responsible for the pleiotropic phenotype of the ΔvirRS mutant.     

Colocalization and identification of interaction sites between IGFBP-3 and GalNAc-T14

GalNAc-T14 was identified as a novel IGFBP-3 binding partner in previous studies. Here, we furtherly confirmed the interaction between them by confocal microscopy, and identified the binding domain and probable interaction sites of GalNAc-T14 with IGFBP-3. The result of subcellular localization indicated that GalNAc-T14 was distributed in the cytosol, whereas IGFBP-3 existed in the cytosol and nucleolus. Confocal analyses demonstrated that IGFBP-3 and GalNAc-T14 colocalized in the cytosol. The result from yeast two hybrid assay showed that the C terminus of GalNAc-T14 (408-552aa) was essential for the interaction between GalNAc-T14 and IGFBP-3, especially Tyr(408), Pro(409), and Glu(410) of GalNAc-T14 may play key roles in the interaction with IGFBP-3. In conclusion, these studies demonstrated that IGFBP-3 and GalNAc-T14 are colocalized in MCF-7 cells and confirmed the interaction between IGFBP-3 and GalNAc-T14. This interaction may play an important role in the functional regulation of IGFBP-3.     

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.     

Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

Apoptotic regulators promote cytokinetic midbody degradation in C. elegans

Cell death genes are essential for apoptosis and other cellular events, but their nonapoptotic functions are not well understood. The midbody is an important cytokinetic structure required for daughter cell abscission, but its fate after cell division remains elusive in metazoans. In this paper, we show through live-imaging analysis that midbodies generated by Q cell divisions in Caenorhabditis elegans were released to the extracellular space after abscission and subsequently internalized and degraded by the phagocyte that digests apoptotic Q cell corpses. We further show that midbody degradation is defective in apoptotic cell engulfment mutants. Externalized phosphatidylserine (PS), an engulfment signal for corpse phagocytosis, exists on the outer surface of the midbody, and inhibiting PS signaling delayed midbody clearance. Thus, our findings uncover a novel function of cell death genes in midbody internalization and degradation after cell division.     

PPARα activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4

Peroxisome proliferator-activated receptor alpha (PPARα) has been implicated in the pathogenesis of cardiac hypertrophy, although its mechanism of action remains largely unknown. To determine the effect of PPARα activation on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore its molecular mechanisms, we evaluated the interaction of PPARα with nuclear factor of activated T-cells c4 (NFATc4) in nuclei of cardiomyocytes from neonatal rats in primary culture. In ET-1-stimulated cardiomyocytes, data from electrophoretic mobility-shift assays (EMSA) and co-immunoprecipitation (co-IP) revealed that fenofibrate (Fen), a PPARα activator, in a concentration-dependent manner, enhanced the association of NFATc4 with PPARα and decreased its interaction with GATA-4, in promoter complexes involved in activation of the rat brain natriuretic peptide (rBNP) gene. Effects of PPARα overexpression were similar to those of its activation by Fen. PPARα depletion by small interfering RNA abolished inhibitory effects of Fen on NFATc4 binding to GATA-4 and the rBNP DNA. Quantitative RT-PCR and confocal microscopy confirmed inhibitory effects of PPARα activation on elevation of rBNP mRNA levels and ET-1-induced cardiomyocyte hypertrophy. Our results suggest that activated PPARα can compete with GATA-4 binding to NFATc4, thereby decreasing transactivation of NFATc4, and interfering with ET-1 induced cardiomyocyte hypertrophy.