Specific DNAzymes cleave the 300-618 nt of 5'UTR to inhibit DHAV-1 translation and replication.

DNAzymes effectively inhibit the expression of viral genes. Duck hepatitis A virus type-1 (DHAV-1) genomic RNA carries an internal ribosome entry site (IRES). The IRES initiates the translation of DHAV-1 via a mechanism that differs from that of cap-dependent translation. Therefore, it is an attractive target for the treatment of DHAV-1. In this study, we designed 6 DNAzymes (Dzs) specifically targeting 300-618 nt sequence in the DHAV-1 5'untranslated region (UTR; a predicted IRES-like element). In the presence of divalent metal ions, three designed DNAzymes (DZ369, DZ454, and DZ514) efficiently cleaved the 300-618 nt sequence of the DHAV-1 5'UTR RNA. The activity of the Dzs was particularly dependent on Mg2+ ions. Subsequently, the translation inhibitory activity of these Dzs was determined by western blotting experiments. The Dzs effectively inhibited the translation mediated by the 300-618 nt of DHAV-1 5'UTR in duck embryo fibroblasts (DEFs). Importantly, DZ454 showed the strongest inhibitory effect, and its inhibition was time and dose dependent. However, none of the Dzs showed significant inhibition of cap-dependent translation. These results suggest that these Dzs show specificity for target RNA. Moreover, DZ454 inhibited the replication of DHAV-1. In conclusion, the designed DNAzymes can be used as inhibitors of DHAV-1 RNA translation and replication, providing new insights useful for the development of anti-DHAV-1 drugs.

Duck hepatitis A virus type 1 mediates cell cycle arrest in the S phase.

BACKGROUND: Duck hepatitis A virus type 1 (DHAV-1) is one of the most serious pathogens endangering the duck industry. However, there are few studies on the regulation of the cell cycle by DHAV-1. METHODS: In this study, flow cytometry was applied to analyze the effect of DHAV-1 infection on the cell cycle of duck embryo fibroblasts (DEFs). Subsequently, we analyzed the effects of cell cycle phases on DHAV-1 replication by real-time reverse transcriptase quantitative PCR (real-time RT-qPCR). RESULTS: Flow cytometry data analysis found that DEFs in the S phase increased by 25.85% and 54.21% at 24 h and 48 h after DHAV-1 infection, respectively. The levels of viral RNA detected by real-time RT-qPCR were higher in the DEFs with synchronization in the S phase or G0/G1 phase than in the control group. However, there was no difference in viral copy number between the G2/M phase arrest and control groups. In addition, non-structural protein 3D of DHAV-1 significantly increased cells in the S phase, indicating that 3D protein is one of the reasons for the cell cycle arrest in the S phase. CONCLUSIONS: In summary, DHAV-1 infection induces the cell cycle arrest of DEFs in the S phase. Both S phase and G0/G1 phase synchronization facilitate the replication of DHAV-1, and 3D protein is one of the reasons for the S phase arrest.

Evaluation of the properties of daughter bubbles generated by inertial cavitation of preformed microbubbles.

Inertial cavitation (IC) of the preformed microbubbles is being investigated for ultrasound imaging and therapeutic applications. However, microbubbles rupture during IC, creating smaller daughter bubbles (DBs), which may cause undesired bioeffects in the target region. Thus, it is important to determine the properties of DBs to achieve controllable cavitation activity for applications. In this study, we theoretically calculated the dissolution dynamics of sulfur hexafluoride bubbles. Then, we applied a 1-MHz single tone burst with different peak negative pressures (PNPs) and pulse lengths (PLs), and multiple 5-MHz tone bursts with fixed acoustic conditions to elicit IC of the preformed SonoVue microbubbles and scattering of DBs, respectively. After the IC and scattering signals were received by a 7.5-MHz transducer, time- and frequency-domain analysis was performed to obtain the IC dose and scattering intensity curve. The theoretical dissolution curves and measured scattering intensity curves were combined to determine the effect of the incident pulse parameters on the lifetime, mean radius and distribution range of DBs. Increased PNP reduced the lifetime and mean size of the DBs population and narrowed the size distribution. The proportion of small DBs (less than resonance size) increased from 36.83% to 85.98% with an increase in the PNP from 0.6 to 1.6 MPa. Moreover, increased PL caused a shift of the DB population to the smaller bubbles with shorter lifetime and narrower distribution. The proportion of small bubbles increased from 25.74% to 95.08% as the PL was increased from 5 to 100 µs. Finally, increased IC dose caused a smaller mean size, shorter lifetime and narrower distribution in the DB population. These results provide new insight into the relationship between the incident acoustic parameters and the properties of DBs, and a feasible strategy for achieving controllable cavitation activity in applications.

Author Correction: Determination of critical cooling rates in metallic glass forming alloy libraries through laser spike annealing.

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How Many Bulk Metallic Glasses Are There?

Quantitative prediction of glass forming ability using a priori known parameters is highly desired in metallic glass development; however proven to be challenging because of the complexity of glass formation. Here, we estimate the number of potential metallic glasses (MGs) and bulk metallic glasses (BMGs) forming systems and alloys, from empirically determined alloy design rules based on a priori known parameters. Specifically, we take into account atomic size ratio, heat of mixing, and liquidus temperature, which we quantify on binary glasses and centimeter-sized BMGs. When expanding into higher order systems that can be formed among 32 practical elements, we reduce the composition space for BMG formation using developed criteria by 106 times and estimate ∼3 million binary, ternary, quaternary, and quinary BMGs alloys.

Determination of critical cooling rates in metallic glass forming alloy libraries through laser spike annealing.

The glass forming ability (GFA) of metallic glasses (MGs) is quantified by the critical cooling rate (R C). Despite its key role in MG research, experimental challenges have limited measured R C to a minute fraction of known glass formers. We present a combinatorial approach to directly measure R C for large compositional ranges. This is realized through the use of compositionally-graded alloy libraries, which were photo-thermally heated by scanning laser spike annealing of an absorbing layer, then melted and cooled at various rates. Coupled with X-ray diffraction mapping, GFA is determined from direct R C measurements. We exemplify this technique for the Au-Cu-Si system, where we identify Au56Cu27Si17 as the alloy with the highest GFA. In general, this method enables measurements of R C over large compositional areas, which is powerful for materials discovery and, when correlating with chemistry and other properties, for a deeper understanding of MG formation.

Combinatorial Strategies for Synthesis and Characterization of Alloy Microstructures over Large Compositional Ranges.

The exploration of new alloys with desirable properties has been a long-standing challenge in materials science because of the complex relationship between composition and microstructure. In this Research Article, we demonstrate a combinatorial strategy for the exploration of composition dependence of microstructure. This strategy is comprised of alloy library synthesis followed by high-throughput microstructure characterization. As an example, we synthesized a ternary Au-Cu-Si composition library containing over 1000 individual alloys using combinatorial sputtering. We subsequently melted and resolidified the entire library at controlled cooling rates. We used scanning optical microscopy and X-ray diffraction mapping to explore trends in phase formation and microstructural length scale with composition across the library. The integration of combinatorial synthesis with parallelizable analysis methods provides a efficient method for examining vast compositional ranges. The availability of microstructures from this vast composition space not only facilitates design of new alloys by controlling effects of composition on phase selection, phase sequence, length scale, and overall morphology, but also will be instrumental in understanding the complex process of microstructure formation in alloys.

Spectral descriptors for bulk metallic glasses based on the thermodynamics of competing crystalline phases.

Metallic glasses attract considerable interest due to their unique combination of superb properties and processability. Predicting their formation from known alloy parameters remains the major hindrance to the discovery of new systems. Here, we propose a descriptor based on the heuristics that structural and energetic 'confusion' obstructs crystalline growth, and demonstrate its validity by experiments on two well-known glass-forming alloy systems. We then develop a robust model for predicting glass formation ability based on the geometrical and energetic features of crystalline phases calculated ab initio in the AFLOW framework. Our findings indicate that the formation of metallic glass phases could be much more common than currently thought, with more than 17% of binary alloy systems potential glass formers. Our approach pinpoints favourable compositions and demonstrates that smart descriptors, based solely on alloy properties available in online repositories, offer the sought-after key for accelerated discovery of metallic glasses.

Metallic glass nanostructures of tunable shape and composition.

Metals of hybrid nano-/microstructures are of broad technological and fundamental interests. Manipulation of shape and composition on the nanoscale, however, is challenging, especially for multicomponent alloys such as metallic glasses. Although top-down approaches have demonstrated nanomoulding, they are limited to very few alloy systems. Here we report a facile method to synthesize metallic glass nanoarchitectures that can be applied to a broad range of glass-forming alloys. This strategy, using multitarget carousel oblique angle deposition, offers the opportunity to achieve control over size, shape and composition of complex alloys at the nanoscale. As a consequence, nanostructures of programmable three-dimensional shapes and tunable compositions are realized on wafer scale for metallic glasses including the marginal glass formers. Realizing nanostructures in a wide compositional range allows chemistry optimization for technological usage of metallic glass nanostructures, and also enables the fundamental study on size, composition and fabrication dependences of metallic glass properties.

Combinatorial development of bulk metallic glasses.

The identification of multicomponent alloys out of a vast compositional space is a daunting task, especially for bulk metallic glasses composed of three or more elements. Despite an increasing theoretical understanding of glass formation, bulk metallic glasses are predominantly developed through a sequential and time-consuming trial-and-error approach. Even for binary systems, accurate quantum mechanical approaches are still many orders of magnitude away from being able to simulate the relatively slow kinetics of glass formation. Here, we present a high-throughput strategy where ∼3,000 alloy compositions are fabricated simultaneously and characterized for thermoplastic formability through parallel blow forming. Using this approach, we identified the composition with the highest thermoplastic formability in the glass-forming system Mg-Cu-Y. The method provides a versatile toolbox for unveiling complex correlations of material properties and glass formation, and should facilitate a drastic increase in the discovery rate of metallic glasses.

Directed evolution of a cellodextrin transporter for improved biofuel production under anaerobic conditions in Saccharomyces cerevisiae.

Introduction of a cellobiose utilization pathway consisting of a cellodextrin transporter and a ß-glucosidase into Saccharomyces cerevisiae enables co-fermentation of cellobiose and xylose. Cellodextrin transporter 1 (CDT1) from Neurospora crassa has been established as an effective transporter for the engineered cellobiose utilization pathways. However, cellodextrin transporter 2 (CDT2) from the same species is a facilitator and has the potential to be more efficient than CDT1 under anaerobic conditions due to its energetic benefits. Currently, CDT2 has a very low activity and is considered rate-limiting in cellobiose fermentation. Here, we report the directed evolution of CDT2 with an increased cellobiose uptake activity, which results in improved cellobiose fermentation under anaerobic conditions. After three rounds of directed evolution, the cellobiose uptake activity of CDT2 was increased by 2.2-fold, which resulted from both increased specific activity and transporter expression level. Using high cell density fermentation under anaerobic conditions, the evolved mutant conferred 4.0- and 4.4-fold increase in the cellobiose consumption rate and ethanol productivity, respectively. In addition, although the cellobiose uptake activity was still lower than that of CDT1, the engineered CDT2 showed significantly improved cellobiose consumption and ethanol production under anaerobic conditions, representing the energetic benefits of a sugar facilitator for anaerobic cellobiose fermentation. This study demonstrated that anaerobic biofuel production could be significantly improved via directed evolution of a sugar transporter protein in yeast.

The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructs.

Poly(lactide-co-glycolide) (PLGA) sponge was filled with fibrin gel, bone marrow mesenchymal stem cells (BMSCs) and transforming growth factor-ß1 (TGF-ß1) to obtain a construct for cartilage restoration in vivo. The PLGA sponge lost its weight steadily in vitro, but degraded much faster in the construct of PLGA/fibrin gel/BMSCs implanted in the full-thickness cartilage defects. The in vivo degradation of the fibrin gel inside the construct was prolonged to 12 wk too. The CM-DiI labeled allogenic BMSCs were detectable after transplantation (implantation) into the defects for 12 wk by small animal in vivo fluorescence imaging and confocal laser scanning microscopy. In vivo repair experiments were firstly performed by implantation of the PLGA/fibrin gel/BMSCs and PLGA/BMSCs constructs into full-thickness cartilage defects (3 mm in diameter and 4 mm in depth) of New Zealand white rabbits for 12 wk. The defects implanted with the PLGA/fibrin gel/BMSCs constructs were filled with cartilage-like tissue containing collagen type II and glycosaminoglycans (GAGs), while those by the PLGA/BMSCs constructs were filled with fibrous-like tissues. To repair the defects of larger size (4 mm in diameter), addition of growth factors was mandatory as exemplified here by further loading of TGF-ß1. Implantation of the PLGA/fibrin gel/BMSCs/TGF-ß1 constructs into the full-thickness cartilage defects for 12 wk resulted in full restoration of the osteochondral tissue. The neo-cartilage integrated well with its surrounding cartilage and subchondral bone. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the cartilage special genes were significantly up-regulated compared with those of the TGF-ß1 absent constructs.

HO-1 modified mesenchymal stem cells modulate MMPs/TIMPs system and adverse remodeling in infarcted myocardium.

The present study was to determine the effects of the heme oxygenase-1 (HO-1) modified mesenchymal stem cells (MSCs) transplantation into acute MI hearts on normalizing the ratio of MMPs/TIMPs and remodeling in infarcted myocardium. HO-1 was transfected into cultured MSCs using an adenoviral vector. 1 x 10(6) Ad-HO-1-transfected MSCs (HO-1-MSCs) or Ad-Null-transfected MSCs (Null-MSCs) or PBS was respectively injected into rat hearts 1 h intramyocardially after myocardial infarction. The cardiac performance was significantly improved and left ventricular dilatation was significantly attenuated in HO-1-MSCs transplanted hearts. Moreover, a significant increase in microvessel density was observed in HO-1-MSCs transplanted hearts. TIMP2,3 expression in HO-1-MSCs transplanted hearts was significantly increased, and MMP2,9 expression in HO-1-MSCs transplanted hearts was significantly lower than Null-MSCs transplanted and PBS-treated hearts. TIMP1 expression did not vary significantly. Null-MSCs transplantation did not decrease the expression of MMP2,9 significantly compared with PBS-treated hearts. The ratio of TIMP2 to MMP2, and TIMP3 to MMP9 in cell-grafted hearts was increased significantly. HO-1-MSCs transplantation normalize the ratio of MMPs/TIMPs, contributing to the reversion of myocardial extracellular remodeling.

In vivo restoration of full-thickness cartilage defects by poly(lactide-co-glycolide) sponges filled with fibrin gel, bone marrow mesenchymal stem cells and DNA complexes.

A composite construct comprising of bone marrow mesenchymal stem cells (BMSCs), plasmid DNA encoding transforming growth factor-beta1 (pDNA-TGF-beta1), fibrin gel and poly (lactide-co-glycolide) (PLGA) sponge was designed and employed to repair articular cartilage defects. To improve the gene transfection efficiency, a cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 9% to BMSCs and showed heterogeneous TGF-beta1 expression in a 10-day culture period in vitro. In vivo culture of the composite constructs was performed by implantation into full-thickness cartilage defects of New Zealand white rabbit joints, using the constructs absence of pDNA-TGF-beta1 or BMSCs as controls. Heterogeneous expression of TGF-beta1 in vivo was detected at 4 weeks, but its level was decreased in comparison with that of 2 weeks. After implantation for 12 weeks, the cartilage defects were successfully repaired by the composite constructs of the experimental group, and the neo-cartilage integrated well with its surrounding tissue and subchondral bone. Immunohistochemical and glycosaminoglycans (GAGs) staining confirmed the similar amount and distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The cartilage special genes expressed in the neo-tissue were closer to those of the normal cartilage. An overall score of 2.83 was obtained according to Wakitani's standard. By contrast, only part of the defects was repaired by the pDNA-TGF-beta1 absence constructs, and no cartilage repair but fibrous tissue was found for the BMSCs absence constructs. Therefore, combination of the PLGA sponge/fibrin gel scaffold with BMSCs and gene therapy is an effective method to restore cartilage defects and may have a great potential for practical applications in the near future.