Comparative molecular dynamics simulations provided insights into the mechanisms of cold-adaption of alginate lyases from the PL7 family.

Alginate is an important polysaccharide that is abundant in the marine environments, including the Polar Regions, and bacterial alginate lyases play key roles in its degradation. Many reported alginate lyases show characteristics of cold-adapted enzymes, including relatively low temperature optimum of activities (Topt) and low thermal stabilities. However, the cold-adaption mechanisms of alginate lyases remain unclear. Here, we studied the cold-adaptation mechanisms of alginate lyases by comparing four members of the PL7 family from different environments: AlyC3 from the Arctic ocean (Psychromonas sp. C-3), AlyA1 from the temperate ocean (Zobellia galactanivorans), PA1167 from the human pathogen (Pseudomonas aeruginosa PAO1), and AlyQ from the tropic ocean (Persicobacter sp. CCB-QB2). Sequence comparison and comparative molecular dynamics (MD) simulations revealed two main strategies of cold adaptation. First, the Arctic AlyC3 and temperate AlyA1 increased the flexibility of the loops close to the catalytic center by introducing insertions at these loops. Second, the Arctic AlyC3 increased the electrostatic attractions with the negatively charged substrate by introducing a high portion of positively charged lysine at three of the insertions mentioned above. Furthermore, our study also revealed that the root mean square fluctuation (RMSF) increased greatly when the temperature was increased to Topt or higher, suggesting the RMSF increase temperature as a potential indicator of the cold adaptation level of the PL7 family. This study provided new insights into the cold-adaptation mechanisms of bacterial alginate lyases and the marine carbon cycling at low temperatures.

The relationship between social support and academic engagement among university students: the chain mediating effects of life satisfaction and academic motivation.

BACKGROUND: University students' academic engagement has a significant impact on their academic performance and career development. METHODS: In order to explore the influential mechanisms of social support on university students' academic engagement and the mediating role of academic motivation and life satisfaction, this study used the Adolescent Social Support Scale, University Students' Academic Engagement Scale Questionnaire, Adolescent Student Life Satisfaction Scale and University Students' Academic Motivation Questionnaire, to conduct a questionnaire survey and empirical analysis on 2106 Chinese university students. RESULTS: (1) social support significantly and positively predicts academic engagement; (2) social support influences academic engagement through the mediating effect of life satisfaction; (3) social support influences academic engagement through the mediating effect of academic motivation; (4) life satisfaction and academic motivation play a chain mediating role in the effect of social support on academic engagement. CONCLUSIONS: This study contributes to understanding the underlying mechanisms of the relationship between social support and academic engagement, which in turn provides insights for universities and the departments concerned to make measures to improve the level of university students' academic engagement.

Dynamic remodeling of Escherichia coli interactome in response to environmental perturbations.

Proteins play an essential role in the vital biological processes governing cellular functions. Most proteins function as members of macromolecular machines, with the network of interacting proteins revealing the molecular mechanisms driving the formation of these complexes. Profiling the physiology-driven remodeling of these interactions within different contexts constitutes a crucial component to achieving a comprehensive systems-level understanding of interactome dynamics. Here, we apply co-fractionation mass spectrometry and computational modeling to quantify and profile the interactions of ∼2000 proteins in the bacterium Escherichia coli cultured under 10 distinct culture conditions. The resulting quantitative co-elution patterns revealed large-scale condition-dependent interaction remodeling among protein complexes involved in diverse biochemical pathways in response to the unique environmental challenges. The network-level analysis highlighted interactome-wide biophysical properties and structural patterns governing interaction remodeling. Our results provide evidence of the local and global plasticity of the E. coli interactome along with a rigorous generalizable framework to define protein interaction specificity. We provide an accompanying interactive web application to facilitate the exploration of these rewired networks.

Activation of the Epac/Rap1 signaling pathway alleviates blood-brain barrier disruption and brain damage following cerebral ischemia/reperfusion injury.

Blood brain barrier (BBB) destruction plays a key role in ischemia stroke, including promoting BBB leakage and brain edema, and leads to unfavorable patient prognosis. Epac/Rap1 signaling pathway is important in mediating endothelial cell barrier function. This study will investigate the regulatory role of Epac/Rap1 signaling pathway in BBB disruption after cerebral ischemia/reperfusion (CI/R) injury. CI/R model was induced by 90 min of transient middle cerebral artery occlusion (MCAO) in male C57BL/6J mice. Injection of Epac/Rap1 signaling pathway agonist was performed half an hour before the MCAO operation. The results showed that CI/R injured the tight connection of BBB and evoked the suppression of the Epac/Rap1 signaling pathway. Based on Epac activation with a cAMP analogue, 8-CPT could improve BBB disfunction by increasing the expression of tight junction protein and reducing the formation of stress fibers. In addition, 8-CPT could ameliorate neurobehavioral disorders, cerebral edema, and cerebral infarction volume in MCAO mice. Moreover, inhibition of Epac pathway with Rap1 inhibitor GGTI298 and Rac1 inhibitor NSC23766 could aggravate the damage of BBB and cerebral injury accordingly. Our results indicate that, the activation of Epac/Rap1 signaling pathway has neuroprotective effects on CI/R damaged brain, through the recovery of BBB.

Novel Antioxidant Peptides Identified from Arthrospira platensis Hydrolysates Prepared by a Marine Bacterium Pseudoalteromonas sp. JS4-1 Extracellular Protease.

Crude enzymes produced by a marine bacterium Pseudoalteromonas sp. JS4-1 were used to hydrolyze phycobiliprotein. Enzymatic productions showed good performance on DPPH radical and hydroxyl radical scavenging activities (45.14 ± 0.43% and 65.11 ± 2.64%, respectively), especially small peptides with MWCO <3 kDa. Small peptides were fractioned to four fractions using size-exclusion chromatography and the second fraction (F2) had the highest activity in hydroxyl radical scavenging ability (62.61 ± 5.80%). The fraction F1 and F2 both exhibited good antioxidant activities in oxidative stress models in HUVECs and HaCaT cells. Among them, F2 could upregulate the activities of SOD and GSH-Px and reduce the lipid peroxidation degree to scavenge the ROS to protect Caenorhabditis elegans under adversity. Then, 25 peptides total were identified from F2 by LC-MS/MS, and the peptide with the new sequence of INSSDVQGKY as the most significant component was synthetized and the ORAC assay and cellular ROS scavenging assay both illustrated its excellent antioxidant property.

Epstein-Barr virus DNA change level combined with tumor volume reduction ratio after inductive chemotherapy as a better prognostic predictor in locally advanced nasopharyngeal carcinoma.

BACKGROUND: To explore the prognosis predicting ability of the combined factors, Epstein-Barr virus DNA change level (EBVCL) and tumor volume reduction ratio (TVRR) after inductive chemotherapy (IC), in locally advanced nasopharyngeal carcinoma (LANPC). METHODS: From 2010 to 2018, 299 LANPC patients were included in this retrospective study. Receiver operating characteristic (ROC) curve analysis was performed to acquire the best critical values. According to the best critical values of EBVCL and TVRR, patients were stratified into low- and high-risk groups. Kaplan-Meier and ROC curve analyses were utilized to verify the prognostic ability of the new predictor (EBVCL+TVRR). The prognostic values among EBVCL+TVRR, EBVCL, TVRR, TNM stage, and the RECIST 1.1 criteria were compared by ROC curve. The primary end points were overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS), and locoregional failure-free survival (LRFFS). RESULTS: ROC curve analyses of TVRR on three-year survival showed the best critical values of TVRR was 32.72% for OS, 30.21% for PFS and LRFFS, 29.87% for DMFS. The best critical value of EBVCL was 127 copies/ml for OS, and 87.7 copies/ml for PFS, DMFS, and LRFFS. The three-year OS, PFS, DMFS, and LRFFS for low- and high-risk groups were 97.7% versus 78.3% (hazard ratio [HR] = 0.2398; 95% confidence interval [CI]: 0.1277-0.4502; p < 0.0001), 91.1% versus 60.9% (HR = 0.3294; 95% CI: 0.2050-0.5292; p < 0.0001), 94.2% versus 68.7% (HR = 0.2413; 95% CI: 0.1284-0.4535; p < 0.0001) and 97.8% versus 77.9% (HR = 0.3078; 95% CI: 0.1700-0.5573; p = 0.0001), respectively. The maximal area under ROC curve of EBVCL+TVRR, EBVCL, TVRR, TNM stage, and RECIST 1.1 criteria for three-year OS was 0.829, 0.750, 0.711, 0.555, and 0.605, respectively. CONCLUSION: The new-developed indicator (EBVCL+TVRR) could better predict the LANPC patient's survival after IC compared with TNM stage system or RECIST 1.1 criteria.

Effect of Solvents on the Color Recovery Responses of Swollen Structural-Color Epoxy Films Based on Inverse Opal Photonic Crystals.

Photonic crystal (PC) films have been widely applied in color displays and the anticounterfeiting field due to their facile fabrication process and easily tunable properties. However, the method for improving the reusability of the color-changed swollen PC films is still a challenge. In this paper, we report the color recovery behavior of epoxy resin inverse opal photonic crystal (EP-IOPC) films, which show different responses after being infiltrated with ethanol, acetone, and dimethyl sulfoxide (DMSO) based on the swelling and deswelling process. DMSO achieved the best effect on the color recovery of the swollen EP-IOPC films compared to ethanol and acetone, and the reflection spectrum blue-shifted in a small range and finally stabilized at a 60 nm deviation from the original spectrum after 10 times recovery. This strategy of color recovery not only solved the problem that the swollen EP-IOPC film's color changes to a certain extent but also showed promising potential in the color display and anticounterfeiting field.

A Method for Capture and Detection of Crop Airborne Disease Spores Based on Microfluidic Chips and Micro Raman Spectroscopy.

Airborne crop diseases cause great losses to agricultural production and can affect people's physical health. Timely monitoring of the situation of airborne disease spores and effective prevention and control measures are particularly important. In this study, a two-stage separation and enrichment microfluidic chip with arcuate pretreatment channel was designed for the separation and enrichment of crop disease spores, which was combined with micro Raman for Raman fingerprinting of disease conidia and quasi identification. The chip was mainly composed of arc preprocessing and two separated enriched structures, and the designed chip was numerically simulated using COMSOL multiphysics5.5, with the best enrichment effect at W2/W1 = 1.6 and W4/W3 = 1.1. The spectra were preprocessed with standard normal variables (SNVs) to improve the signal-to-noise ratio, which was baseline corrected using an iterative polynomial fitting method to further improve spectral features. Raman spectra were dimensionally reduced using principal component analysis (PCA) and stability competitive adaptive weighting (SCARS), support vector machine (SVM) and back-propagation artificial neural network (BPANN) were employed to identify fungal spore species, and the best discrimination effect was achieved using the SCARS-SVM model with 94.31% discrimination accuracy. Thus, the microfluidic-chip- and micro-Raman-based methods for spore capture and identification of crop diseases have the potential to be precise, convenient, and low-cost methods for fungal spore detection.

A potential diagnostic pitfall in ST elevation: Acute pulmonary embolism or ST-segment elevation myocardial infarction.

The diagnosis of acute pulmonary embolism (APE) is a great challenge for physicians due to its nonspecific symptoms, and often missed or misdiagnosed as acute coronary syndrome. Electrocardiographic (ECG) abnormalities are seen in majority of patients with APE. Recently, APE with ST-segment elevation (STE) in leads V1 -V3 /V4 , mimicking ST-segment elevation myocardial infarction (STEMI), has been described. However, coronary angiography showed that the patient's coronary arteries were mostly normal. Herein, we describe a case of APE presenting with STE in V1 -V4 , along with severe stenosis of the left anterior descending (LAD) artery.

Reconfigurable Inverse Opal Structure Film for a Rewritable and Double-Sided Photonic Crystal Paper.

A rewritable photonic crystal (PC) paper as an environmentally friendly and low-resource-consuming material for information storage and spreading has gradually become a research hotspot. In this work, a novel rewritable PC paper with inkless writing and double-sided rewritability properties was developed. A double-sided epoxy resin PC paper exhibiting an inverse opal structure and a bright structural color was fabricated using the sacrificial template method. Carbon black was doped into the material to increase color saturation and purity while preventing light transmission and protecting the double-sided structural color from interference. The force of sliding friction and deformation triggered by capillary pressure as well as swelling-triggered recovery of the inverse opal structure led to an easy rewriting of the PC paper. The PC paper exhibited excellent rewritability even after 50 runs of the rewriting process. Given the inkless and double-sided rewriting, this study provides a new method for the preparation of rewritable PC papers.

Combined therapy of adenovirus vector mediated IGF-1 gene with anti-CD20 mAbs exerts potential beneficial role on type 1 diabetes in nonobese diabetic mice.

AIMS: To assess the protective effects of combined treatment with anti-CD20 monoclonal antibody (mAb) and adenovirus mediated mouse insulin-like growth factor 1 (Adv-mIGF-1) gene on type 1 diabetes (T1D) in nonobese diabetic (NOD) mice at early stage. METHODS: To simultaneously restore the proportion of Th cells and block the interaction of B cells, NOD model mice were assigned to four groups which received PBS, Adv-mIGF-1 gene and anti-CD20 mAbs alone or combination, respectively. After 16 weeks of therapeutic intervention, blood samples and pancreatic tissues of mice were measured via the methods of ELISA, RT-PCR, western blotting, H&E staining, TUNEL and immunohistochemistry assays. KEY FINDINGS: Chronic combination intervention with Adv-mIGF-1 gene and anti-CD20 mAbs reduced the T1D-related morbidity, promoted the secretion of insulin, controlled the blood glucose levels (BGLs) and alleviated insulitis of experimental mice. In addition, current combination intervention also protected the pancreatic ß cells via suppressing the expression of Fas and TNF-α, inhibiting Caspase-3/8 related apoptotic pathway, and activating the Bcl-2-related antiapoptotic pathway. Furthermore, current combination therapy also increased the expression levels of PDX-1 and CK-19 genes, and finally accelerated the proliferation and differentiation of pancreatic ß-cells. In addition, combination therapy could also ameliorate the pathological characteristics of diabetic nephropathy in NOD mice. CONCLUSION: Combination treatment with Adv-mIGF-1 gene and anti-CD20 mAbs may exert a potential beneficial role on T1D in NOD mice.

Comparing the 7th and 8th editions of UICC/AJCC staging system for nasopharyngeal carcinoma in the IMRT era.

BACKGROUND: To compare the prognostic value of 7th and 8th editions of the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) staging system for patients with nonmetastatic nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy and simultaneous integrated boost- intensity-modulated radiation therapy (SIB-IMRT). METHODS: Patients with NPC (n = 300) who received SIB-IMRT were included. Survival by T-classification, N-classification, and stage group of each staging system was assessed. RESULTS: For T-classification, nonsignificant difference was observed between T1 and T3 and between T2 and T3 disease (P = 0.066 and 0.106, respectively) for overall survival (OS) in the 7th staging system, whereas all these differences were significant in the 8th staging system (all P < 0.05). The survival curves for disease-free survival (DFS) and locoregional recurrence-free survival (LRRFS) in both staging systems were similar, except for the comparison of T2 and T4 disease for LRRFS (P = 0.070 for 7th edition; P = 0.011 for 8th edition). For N-classification, significant differences were observed between N2 and N3 diseases after revision (P = 0.046 and P = 0.043 for OS and DFS, respectively). For staging system, no significant difference was observed between IVA and IVB of 7th edition. CONCLUSION: The 8th AJCC staging system appeared to have superior prognosis value in the SIB-IMRT era compared with the 7th edition.

Pain-related risk factors after arthroscopic minimally invasive treatment of meniscus injury of knee joints.

Pain-related risk factors after arthroscopic minimally invasive treatment of meniscus injury of knee joints were explored. Altogether 42 patients (conservative group), 40 patients (open group) and 46 patients (minimally invasive group) who received conservative treatment or arthroscopic knee surgery at the Quwo County People's Hospital were selected. The clinical effects of patients in the three groups at 24 weeks after treatment were observed. The knee joint activity, the knee injury and osteoarthritis outcome score (KOOS), Lysholm knee joint function score, VAS pain score and WOMAC score were recorded before treatment, at 24 weeks after treatment and at 2 years after treatment. Complications were also recorded. The related risk factors of postoperative pain were analyzed. There was no significant difference between the short-term efficacy of conservative treatment and that of surgical treatment (P>0.05); however, the long-term improvement effect of the surgical treatment on knee joint function and pain was better (P<0.05). The short-term and long-term effects of arthroscopic surgery were close to those of the open surgery. Arthroscopic surgery had a good long-term improvement effect on knee joint function and pain (P<0.05), and the incidence of postoperative pain was low (P<0.05). The results of logistic multivariate regression analysis manifested that WOMAC score, articular cartilage injury, time of postoperative weight bearing <1 week, no postoperative cold compress and open knee surgery were independent risk factors that affected postoperative pain (P<0.05). In conclusion, arthroscopic minimally invasive treatment has a good effect on patients with meniscus injury of knee joints who fail conservative treatment. Articular cartilage injury, postoperative weight bearing, cold compress and type of operation are independent risk factors that affect postoperative pain. Clinicians should bring patient attention to the prevention of meniscus injury and further improve the efficacy of treatment.

The comparison of prognostic value of tumour volumetric regression ratio and RECIST 1.1 criteria after induction chemotherapy in locoregionally advanced nasopharyngeal carcinoma.

PURPOSE: To compare the prognostic value of the sum volumetric regression ratio (SVRR) of the primary tumour and metastatic lymph nodes with treatment response based on RECIST 1.1 criteria after induction chemotherapy in locoregionally advanced nasopharyngeal carcinoma (NPC). METHODS AND MATERIALS: A total of 117 stage III-IVA NPC patients treated with induction chemotherapy followed by concurrent chemoradiotherapy (CCRT) were retrospectively reviewed. The SVRR and the treatment response based on RECIST 1.1 were measured using contrast-enhanced computed tomography (CT) localisations before and after induction chemotherapy. The receiver operating characteristic (ROC) curve analysis was used to identify the optimal cutoff point of the SVRR and compare the prognostic value of the SVRR and RECIST 1.1criteria. RESULTS: The optimal cutoff points of SVRR for progression-free survival (PFS), locoregional failure-free survival (LRFFS) and distant metastasis-free survival (DMFS) were all 25.15%, while for overall survival (OS) it was 16.63%. The area under the ROC curve (AUC) of optimal cutoff points of SVRR was superior than that of RECIST 1.1 for PFS (AUC: 0.716 vs. 0.578; P = 0.0022), LRFFS (AUC: 0.700 vs. 0.574; P = 0.0080) and DMFS (AUC: 0.736 vs. 0.606; P = 0.0053), respectively. The 3-year PFS, DMFS and OS rates for SVRR less than vs. greater than or equal to the cutoff points were 55.8% vs. 92.2% (P < 0.001, hazard ratio (HR): 0.209, 95% confidence interval (CI): 0.091-0.480), 59.7% vs. 96.7% (P < 0.001, HR: 0.120, 95% CI: 0.043-0.336) and 66.7% vs. 98.1% (P < 0.001, HR: 0.069, 95% CI: 0.014-0.342), while the responses [stable disease (SD), partial response (PR)] based on RECIST 1.1 were not significantly associated with 3-year survival rates. Multivariate analysis indicated that SVRR was an independent prognostic factor for PFS, DMFS and OS (all P < 0.05). CONCLUSIONS: The sum volumetric regression ratio and response based on RECIST 1.1 were related to the prognosis in locoregionally advanced NPC after induction chemotherapy. Sum volumetric regression ratio is an independent outcome predictor for survival in locoregionally advanced NPC, playing a better prognostic role than RECIST 1.1.

Effects of different probiotic combinations on the components and bioactivity of Spirulina.

Spirulina acts as a good dietary nutritional supplement. However, few research studies have been conducted on its fermentation. Three groups of probiotic combinations, lactic acid bacteria, Bacillus strains, and their mixture, were used to investigate Spirulina fermentation. The results showed that lactic acid bacteria significantly increased the content of amino acids and the ratio of essential amino acids to total amino acids in the fermented Spirulina, compared with the unfermented Spirulina, and this trend was enhanced by the strains' mixture. However, compared to unfermented Spirulina, the amino acid levels were significantly decreased after fermentation with Bacillus strains and so was the total free amino acid and essential amino acid content. Fermentation significantly reduced the contents of the offensive components of Spirulina, with significant differences among the three mixed bacterial treatments. Moreover, Bacillus strain fermentation increased the contents of flavonoids and polyphenols compared to the unfermented Spirulina, and significantly enhanced 1,1-diphenyl-2-trinitrophenylhydrazine free-radical scavenging ability and total antioxidant ability. On the contrary, treatments with lactic acid bacteria and the mixture of lactic acid bacteria and Bacillus strains endowed the fermented supernatants with good antibacterial ability. The results showed that probiotic fermentation has a good effect on Spirulina and can serve as a new procedure for developing new Spirulina-containing food items.

Classification and structural insight into vibriolysin-like proteases of Vibrio pathogenicity.

Vibriolysin-like proteases (VLPs) are important virulence agents in the arsenal of Vibrio causing instant cytotoxic effects during infection. Most of Vibrio secreted VLPs show serious pathogenicity, while some species of Vibrio with VLPs are non-pathogenic, like Vibrio tasmaniensis and Vibrio pacinii. To investigate the relation between VLPs and Vibrio pathogenicity, one phylogenetic tree of VLPs was constructed and compared consensus sequences at the N-terminus of VLPs. Based on these results, VLPs were defined into nine phylogenetic clades. Pathogenicity analysis of Vibrio showed that Vibrio species with VLPs III, VI, VII or VIII are serious pathogenic bacteria, while species with VLPs I, II, IV or IX are opportunistic pathogens. Multiple sequence alignment showed that the N-terminal 5-16 nucleotides of each clade are highly conservative. Topological analysis of VLPs exhibited the structural differences in N-terminal regions of each VLP clade. These results suggest that structure of N-terminus might play a key role in the pathogenicity of VLPs. Our findings give new insights into the classification of VLPs and the relationship between VLPs and Vibrio pathogenicity.

Variant Amino Acid Residues Alter the Enzyme Activity of Peanut Type 2 Diacylglycerol Acyltransferases.

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol (TAG) biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. This reaction is a major control point in the Kennedy pathway for biosynthesis of TAG, which is the most important form of stored metabolic energy in most oil-producing plants. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14.' Sequence analysis of 11 different peanut cultivars revealed a gene family of 8 peanut DGAT2 genes (designated AhDGAT2a-h). Sequence alignments revealed 21 nucleotide differences between the eight ORFs, but only six differences result in changes to the predicted amino acid (AA) sequences. A representative full-length cDNA clone (AhDGAT2a) was characterized in detail. The biochemical effects of altering the AhDGAT2a sequence to include single variable AA residues were tested by mutagenesis and functional complementation assays in transgenic yeast systems. All six mutant variants retained enzyme activity and produced lipid droplets in vivo. The N6D and A26P mutants also displayed increased enzyme activity and/or total cellular fatty acid (FA) content. N6D mutant mainly increased the content of palmitoleic acid, and A26P mutant mainly increased the content of palmitic acid. The A26P mutant grew well both in the presence of oleic and C18:2, but the other mutants grew better in the presence of C18:2. AhDGAT2 is expressed in all peanut organs analyzed, with high transcript levels in leaves and flowers. These levels are comparable to that found in immature seeds, where DGAT2 expression is most abundant in other plants. Over-expression of AhDGAT2a in tobacco substantially increased the FA content of transformed tobacco seeds. Expression of AhDGAT2a also altered transcription levels of endogenous tobacco lipid metabolic genes in transgenic tobacco, apparently creating a larger carbon 'sink' that supports increased FA levels.

Phenotypic switching of vascular smooth muscle cells in the 'normal region' of aorta from atherosclerosis patients is regulated by miR-145.

Switching of vascular smooth muscle cells (VSMCs) from a contractile phenotype to an adverse proliferative phenotype is a hallmark of atherosclerosis or vascular restenosis. However, the genetic modulators responsible for this switch have not been fully elucidated in humans nor have they been correlated with clinical abnormalities. This study investigated genetic mechanisms involved in phenotypic switching of VSMCs at non-defect areas of the aorta in patients with atherosclerosis. Aortic wall samples were obtained from patients with (N = 53) and without (N = 27) atherosclerosis undergoing cardiovascular surgery. Vascular smooth muscle cell cultures were generated, and expression of microRNA-145 (miR-145), its target gene Kruppel-Like Factor 5 (KLF5) and Myocardin (MYOCD, a smooth muscle-specific transcriptional coactivator) were analysed using RT-qPCR, along with expression of relevant proteins. Vascular smooth muscle cells were transduced with miR-145 inhibitor and mimic to determine the effect of miR-145 expression on VSMC proliferation. miR-145 expression decreased while KLF5 expression increased in atherosclerotic aortas. Atherosclerotic samples and VSMCs had decreased expression of contractile markers calponin and alpha smooth muscle actin (α-SMA) and MYOCD. miR-145 inhibitor-transduced VSMCs from non-atherosclerotic patients showed decreased expression of calponin and α-SMA and increased proliferation compared with non-transduced controls, and these levels were close to those of atherosclerotic patients. miR-145 mimic-transduced VSMCs from atherosclerotic patients showed increased expression of calponin and α-SMA and decreased proliferation compared with non-transduced controls, and these levels were close to those found in non-atherosclerotic patients. These data demonstrate that miR-145 modulates the phenotypic switch of VSMCs from a contractile to a proliferative state via KLF5 and MYOCD in atherosclerosis.

A comprehensive alanine-scanning mutagenesis study reveals roles for salt bridges in the structure and activity of Pseudomonas aeruginosa elastase.

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (kcat/Km), half-lives (t1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.

[Development of transgenic maize with anti-rough dwarf virus artificial miRNA vector and their disease resistance].

Maize is one of the most important food crops. Rice black-streaked dwarf virus is a maize rough dwarf disease pathogen. The occurrence and transmission of maize rough dwarf disease brings great damage to maize production. The technology of using artificial miRNA to build antiviral plant has been proven effective in a variety of plants. However, such trials in maize have not been reported. We designed primers based on the sequence of maize zea-miR159a precursor and sequence of function protein genes and silencing RBSDV coding genes in RBSDV genome. We constructed amiRNA (artificial miRNA) gene for silencing RBSDV coding gene and gene silencing suppressor. We constructed pCAMBIA3301-121-amiRNA plant expression vector for transforming maize inbred lines Z31 by using agrobacterium mediated method. After molecular analysis of transgenic maize, homozygous lines with high miRNA expression were selected by molecular detection for a subsequent natural infection experiment. We studied the severity of maize rough dwarf disease according to a grading standard (grade 0 to 4). The experiment results showed that the disease resistance of transgenic homozygous maize with the anti-rough dwarf virus amiRNA vector was better than that of wild type. Among the transgenic maize, S6-miR159 transgenic maize had high disease resistance. It is feasible to create new maize variety by the use of artificial miRNA.