A novel PCR strategy for high-efficiency, automated site-directed mutagenesis

We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (T(ra)), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T(m)) are used together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high T(m) mutagenic primer at a high annealing temperature, which prevents the priming of the low T(m) primers (i.e. the two flanking primers). A megaprimer is then produced using this smDNA as the template at a denaturing temperature that prevents wild-type template DNA activity. The desired mutant DNA is then obtained by cycling again through these first two steps, resulting in a mutagenic efficiency of 100% in all tested cases. This highly automated method not only eliminates the necessity of any intermediate manipulation and accomplishes the mutagenesis process in a single round of PCR but, most notably, enables complete success of mutagenesis. This novel method is also both cost and time efficient and fully automated

Enhanced gas barrier properties of graphene oxide/rubber composites with strong interfaces constructed by graphene oxide and sulfur

Constructing strong interfacial interactions and complex filler networks is crucial to establishing high gas barrier properties in rubber composites. In this research, sulfur-graphene oxide (S-GO) hybrids were prepared by in situ growth of sulfur on the surfaces of GO sheets. The S-GO hybrids were also introduced into butadiene styrene rubber (SBR) using a green method of latex compounding. Results showed that sulfur could melt and spread on the surface of the GO during the crosslinking process at high temperatures. This process prevented the aggregation of GO and resulted in a fine dispersion of GO and complex filler networks in S-GO/SBR composites. More importantly, the sulfur particles on the GO surface not only aided the crosslinking of rubber molecules, but also chemically reacted with the GO radicals generated at high temperatures. This occurred by the homolytic cleavage of oxygen-containing groups, which thereby constructed covalent interfaces between the GO and SBR molecules. Due to these strong interfaces and complex filler networks, the tensile and tear strength of S-GO/SBR composites increased by 66.2% and 26.6%, respectively, when compared with conventional GO/SBR composites. The gas permeability coefficient of S-GO/SBR composites was decreased dramatically by 50.7% and 23.3% by comparison with that of pure SBR and GO/SBR composites, respectively. The apparent improvement demonstrated that the facile and effective method used in this research may open up new opportunities for the development of multifunctional rubber crosslinking agent as well as the fabrication of rubber composites with high performance

Insight into the effect of hospital-based prehabilitation on postoperative outcomes in patients with total knee arthroplasty: A retrospective comparative study

Background: Osteoarthritis (OA) has become one of the most prevalent joint diseases worldwide, leading to a growing burden of pain and disability as populations age. Although there is consistent evidence to support postoperative rehabilitation and high-intensity prehabilitation for total knee arthroplasty (TKA), the clinical outcomes of hospital-based prehabilitation remain unclear. We aimed to evaluate the effect of a hospital-based prehabilitation program on knee score (KS), function score (FS), and length of stay (LOS) among patients with knee OA after TKA. Methods: A retrospective comparative study was conducted at Renmin Hospital of Wuhan University among patients with primary knee OA. Seventy-two postopearative patients who did not undergo the prehabilitation program were included as the control group, while 68 postoperative patients who underwent the prehabilitation program were assigned to the intervention group. All patients went through the same care after TKA. The KS, FS, and pain levels were measured 5 days before surgery, immediately preceding surgery, immediately after the surgery, and at 1 week and 1 month postoperatively. LOS for each patient was recorded. Results: The new prehabilitation training program significantly improved the KS over time in the intervention group. However, no significant between-group difference was identified in the change of FS. The prehabilitation program also provided shorter LOS. Conclusions: The hospital-based prehabilitation program leads to improved recovery, as indicated by higher KS postoperatively, which may result in improved clinical outcomes of TKA

Enhanced covalent interface, crosslinked network and gas barrier property of functionalized graphene oxide/styrene-butadiene rubber composites triggered by thiol-ene click reaction

The high gas barrier property of a rubber composite is of great significance for reducing the exhaust gas emissions due to tire rolling resistance and hence the contribution this factor makes to environmental protection. Enhanced covalent interfaces and crosslinked networks are crucial to the gas barrier property of rubber composites. In this research, γ-mercaptopropyltriethoxysilane (MPS) modified GO (MGO)/styrene-butadiene rubber (SBR) composites were prepared by a synergetic strategy of latex compounding method and thiol-ene click reaction. It was found that the mercapto groups in MGO reacted with the vinyl groups in SBR molecules through thiol-ene click reaction during the crosslinking process at 150 °C, thus forming strong chemical interactions at the interface in the form of GO-MPS-rubber and enhanced crosslinked networks. Meanwhile, the strong interface promoted the dispersion of MGO in SBR. The uniform dispersion of MGO, strong interface between MGO and SBR molecules and enhanced crosslinked networks resulted in improved mechanical and gas barrier properties. When filling 5 phr fillers, the tensile strength and gas barrier properties of an MGO/SBR composite improved by 19.0% and 37.5%, respectively, relative to the comparing GO/SBR composite

Enhanced Fatigue and Durability of Carbon Black/natural rubber Composites Reinforced with Graphene Oxide and Carbon Nanotubes

Graphene oxide (GO) sheets and carbon nanotubes (CNTs) are of nanometer size and offer large shape factors which are beneficial in reducing crack propagation rates of composites when used in carbon black (CB) reinforced natural rubber (NR), thereby prolonging the service lives of rubber composites. In this research, CNT-CB/NR and GO-CB/NR composites were prepared by partially replacing CB with one-dimensional CNTs and two-dimensional flaky graphene oxide GO, respectively. The results showed that the complex filler dispersion in NR matrices was improved due to the isolation effect between the different fillers. The strain-induced crystallization (SIC) ability of CB/NR was effectively enhanced by the addition of both GO and CNT. The modulus at 100% strain and tear strength of the composites were also improved. More branching and deflections were observed at the crack tips of the composites and both effectively hindered crack propagation in the materials. Under uniaxial and multi-axial cyclic loading, the fatigue lives of CNT-CB/NR and GO-CB/NR composites greatly increased when compared with the fatigue lives of CB/NR composites. The GO-CB/NR composites exhibited evident advantages in respect of fatigue resistance and durability among the three composites

Influence of graphene oxide and carbon nanotubes on the fatigue properties of silica/styrene-butadiene rubber composites under uniaxial and multiaxial cyclic loading

The influence of equivalent replacement of silica (SiO2) by carbon nanotubes (CNT) or graphene oxide (GO) on the microstructure, mechanical and fatigue behaviors of SiO2/styrene-butadiene rubber (SBR) composites was investigated. Results showed that the synergistic effect between CNT (or GO) and SiO2 was beneficial for the filler network and improved the mechanical properties of SBR composites. Furthermore, the introduction of CNT (or GO) led to low crack growth rates and the crack propagation tips were easy to deflection. Under multiaxial fatigue conditions, the maximum engineering stress was determined to be a reliable fatigue life predictor for SBR composites

MicroRNA-Mediated Responses to Chromium Stress Provide Insight Into Tolerance Characteristics of Miscanthus sinensis

Chromium (Cr) is a heavy metal in nature, which poses a potential risk to toxicity to both animals and plants when releasing into the environment. However, the regulation of microRNA (miRNA)-mediated response to heavy metal Cr has not been studied in Miscanthus sinensis. In this study, based on high-throughput miRNA sequencing, a total of 104 conserved miRNAs and 158 nonconserved miRNAs were identified. Among them, there were 45 differentially expressed miRNAs in roots and 13 differentially expressed miRNAs in leaves. The hierarchical clustering analysis showed that these miRNAs were preferentially expressed in a certain tissue. There were 833 differentially expressed target genes of 45 miRNAs in roots and 280 differentially expressed target genes of 13 miRNA in leaves. After expression trend analysis, five significantly enriched modules were obtained in roots, and three significantly enriched trend blocks in leaves. Based on the candidate gene annotation and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) function analysis, miR167a, novel_miR15, and novel_miR22 and their targets were potentially involved in Cr transportation and chelation. Besides, miR156a, miR164, miR396d, and novel_miR155 were identified as participating in the physiological and biochemical metabolisms and the detoxification of Cr of plants. The results demonstrated the critical role of miRNA-mediated responses to Cr treatment in M. sinensis, which involves ion uptake, transport, accumulation, and tolerance characteristics

Radiomic signature of the FOWARC trial predicts pathological response to neoadjuvant treatment in rectal cancer

Background We aimed to develop a radiomic model based on pre-treatment computed tomography (CT) to predict the pathological complete response (pCR) in patients with rectal cancer after neoadjuvant treatment and tried to integrate our model with magnetic resonance imaging (MRI)-based radiomic signature. Methods This was a secondary analysis of the FOWARC randomized controlled trial. Radiomic features were extracted from pre-treatment portal venous-phase contrast-enhanced CT images of 177 patients with rectal cancer. Patients were randomly allocated to the primary and validation cohort. The least absolute shrinkage and selection operator regression was applied to select predictive features to build a radiomic signature for pCR prediction (rad-score). This CT-based rad-score was integrated with clinicopathological variables using gradient boosting machine (GBM) or MRI-based rad-score to construct comprehensive models for pCR prediction. The performance of CT-based model was evaluated and compared by receiver operator characteristic (ROC) curve analysis. The LR (likelihood ratio) test and AIC (Akaike information criterion) were applied to compare CT-based rad-score, MRI-based rad-score and the combined rad-score. Results We developed a CT-based rad-score for pCR prediction and a gradient boosting machine (GBM) model was built after clinicopathological variables were incorporated, with improved AUCs of 0.997 [95% CI 0.990–1.000] and 0.822 [95% CI 0.649–0.995] in the primary and validation cohort, respectively. Moreover, we constructed a combined model of CT- and MRI-based radiomic signatures that achieve better AIC (75.49 vs. 81.34 vs.82.39) than CT-based rad-score (P = 0.005) and MRI-based rad-score (P = 0.003) alone did. Conclusions The CT-based radiomic models we constructed may provide a useful and reliable tool to predict pCR after neoadjuvant treatment, identify patients that are appropriate for a 'watch and wait' approach, and thus avoid overtreatment. Moreover, the CT-based radiomic signature may add predictive value to the MRI-based models for clinical decision making

Folding by Numbers: Primary Sequence Statistics and Their Use in Studying Protein Folding

The exponential growth over the past several decades in the quantity of both primary sequence data available and the number of protein structures determined has provided a wealth of information describing the relationship between protein primary sequence and tertiary structure. This growing repository of data has served as a prime source for statistical analysis, where underlying relationships between patterns of amino acids and protein structure can be uncovered. Here, we survey the main statistical approaches that have been used for identifying patterns within protein sequences, and discuss sequence pattern research as it relates to both secondary and tertiary protein structure. Limitations to statistical analyses are discussed, and a context for their role within the field of protein folding is given. We conclude by describing a novel statistical study of residue patterning in β-strands, which finds that hydrophobic (i,i+2) pairing in β-strands occurs more often than expected at locations near strand termini. Interpretations involving β-sheet nucleation and growth are discussed

Free Cysteine Modulates the Conformation of Human C/EBP Homologous Protein

The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid l-cysteine. Addition of l-cysteine caused a significant dose-dependent decrease in the protein helicity – dropping from 69.1% to 23.8% in the presence of 1 mM of l-cysteine – and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 µM, an 8∶1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free l-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of l-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress