Molecular epidemiological and genetic characterization of pseudorabies virus in Guangxi, China.

Pseudorabies virus (PRV) is an important pathogen that can cause harm to the pig population. Since 2011, there have been a number of large-scale outbreaks of pseudorabies on Chinese farms where animals had been vaccinated with the Bartha-K61 vaccine. In order to understand the epidemiological trend and genetic variations of PRV in Guangxi province, China, 819 tissue samples were collected from swine farms where PRV infection was suspected from 2013 to 2019, and these were tested for infectious wild strains of PRV. The results showed a positive rate of PRV in Guangxi province of 28.21% (231/819). Thirty-six wild-type PRV strains were successfully isolated from PRV-positive tissue samples, and a genetic evolutionary analysis was performed based on the gB, gC, gD, gE, and TK genes. Thirty of the PRV strains were found to be closely related to the Chinese variant strains HeN1-China-2012 and HLJ8-China-2013. In addition, five PRV strains were genetically related to Chinese classical strains, and one isolate was a recombinant of the PRV variant and the vaccine strain Bartha-K61. Amino acid sequence analysis showed that all 36 PRV strains had characteristic variant sites in the amino acid sequences of the gB, gC, gD, and gE proteins. Pathogenicity analysis showed that, compared to classical PRV strains, the PRV variant strains were more pathogenic in mice and had a lower LD50. Taken together, our results show that wild-type PRV infections are common on pig farms in Guangxi province of China and that the dominant prevalent strains were those of the PRV variants. The PRV variant strains also had increased pathogenicity in mice. Our data will provide a useful reference for understanding the prevalence and genetic evolution of PRV in China.

Isolation and identification of two novel pseudorabies viruses with natural recombination or TK gene deletion in China.

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has gained increased attention in China in recent years due to outbreaks of emergent pseudorabies. However, there is limited information about the evolution and pathogenicity of emergent PRV field strains in China. In this study, two PRV field strains were isolated from an intensive pig farm with suspected PRV infection. These were named the GXLB-2015 and GXGG-2016 strains and their growth characteristics together with their genome sequences and pathogenicity were determined. Nucleotide homology and phylogenetic analysis revealed the GXLB-2015 stain was relatively close to the foreign PRV isolated strains with respect to the whole genome sequence. However, it formed an independent branch between the foreign PRV isolates and the previous PRV variants isolated in China. Further recombination and genetic evolution analysis showed that the GXLB-2015 strain was a natural recombinant between the Bartha strain and PRV variants. The GXGG-2016 strain was highly homologous with the Chinese classical strains, but it has a natural deletion of 69 aa in the thymidine kinase (TK) gene. Pathogenicity analysis showed that, the GXLB-2015 strain had the strongest pathogenicity to mice with an LD50 of 103.5, while the GXGG-2016 strain with the TK gene deletion was not pathogenic to mice. Taken together, our data provide direct evidence for the genomic recombination and natural TK gene deletion of PRVs, which may provide a reference for a better understanding of PRV evolution in China and contribute to the clinical control of PRV infection in pig farms.

The antihypertensive effect of MK on spontaneously hypertensive rats through the AMPK/Akt/eNOS/NO and ERK1/2/Cx43 signaling pathways.

We investigated the antihypertensive effects of maximakinin (MK) on spontaneously hypertensive rats (SHRs). The effects of MK on arterial blood pressure in SHRs were observed, and flow cytometry and 4,5-diaminofluorescein-2 staining were used to examine MK-induced nitric oxide (NO) release in human umbilical vein endothelial cells (HUVECs). Western blotting was used to analyze the effects of MK on the expression of AMP-activated protein kinase (AMPK), Akt, Connexin 43, ERK1/2, p38, and p-eNOS in HUVECs. The results showed that MK induced a more significant antihypertensive effect on SHRs than bradykinin (BK). MK induced significant increases in endothelial nitric oxide synthase (eNOS) phosphorylation and NO release in HUVECs. MK also significantly increased the phosphorylation of Akt and AMPK in HUVECs. The AMPK inhibitor compound C blocked the effect of MK on the generation of NO. MK induced the phosphorylation of ERK1/2, p38, and Connexin 43. The expression of p-Connexin 43 was significantly decreased in the presence of the ERK1/2 inhibitor U0126 but not the p38 inhibitor SB203580. The effects of MK on the phosphorylation of AMPK and ERK1/2 were significantly decreased by the BK B2 receptor inhibitor HOE-140. In summary, MK can significantly reduce blood pressure in SHRs. The antihypertensive effect might be mediated through the activation of the BK B2 receptor, while the downstream AMPK/PI3K/Akt/eNOS/NO and ERK1/2/Connexin 43 signaling pathways play additional roles.

Algorithm-based coevolution network identification reveals key functional residues of the α/ß hydrolase subfamilies.

Covariant residues identified by computational algorithms have provided new insights into enzyme evolutionary routes. However, the reliability and accuracy of routine statistical coupling analysis (SCA) are unable to satisfy the needs of protein engineering because SCA depends only on sequence information. Here, we set up a new SCA algorithm, SCA.SIM, by integrating structure information and MD simulation data. The more reliable covariant residues with high-quality scores are obtained from sequence alignment weighted by residual movement for eight related subfamilies, belonging to α/ß hydrolase family, with Candida antarctica lipase B (CALB). The 38 predicted covariant residues are tested for function by high-throughput quantitative evaluation in combination with activity and thermostability assays of a mutant library and deep sequencing. Based on the landscapes of both activity and thermostability, most mutants play key roles in catalysis, and some mutants gain 2.4- to 6-fold increase in half-life at 50°C and 9- to 12-fold improvement in catalytic efficiency. The activity of double mutants for A225F/T103A is higher than those of A225F and T103A which means that SCA.SIM method might be useful for identifying the allosteric coupling. The SCA.SIM algorithm can be used for protein coevolution and enzyme engineering research.

Mesoporous silica nanorods for improved oral drug absorption.

Mesoporous silica nanoparticles (MSNs) have been widely used in biomedical applications. However, most studies have been limited to spherical MSNs, while non-spherical MSNs have never been rigorously studied. In this study, we fabricated mesoporous silica nanospheres (MSNSs) and mesoporous silica nanorods (MSNRs), with different aspect ratios (ARs) but identical surface chemistries to explore the shape effects of MSNs on oral delivery. The results of cellular studies demonstrated that MSNRs exhibited a higher cellular uptake than MSNSs. Mechanistic studies showed that caveolae-mediated endocytosis was involved in the uptake of MSNRs, while the clathrin-dependent pathway contributed to the endocytosis of MSNSs. Meanwhile, the apparent permeability coefficient value (Papp) of doxorubicin hydrochloride (Dox)-loaded MSNRs was approximately 1.8-, 3.2- and 6.3-fold higher than that of Dox-loaded MSNS1, Dox-loaded MSNS2 and Dox solution, respectively. The in vivo pharmacokinetics study showed that the area under the plasma concentration-time curve (AUC) achieved by Dox-loaded MSNRs was 1.9-, 3.4- and 5.7-fold higher than the corresponding values for Dox-loaded MSNS1, MSNS2 and Dox solution, respectively. Taken together, our results demonstrated that tuning nanoparticle shape potentially determines the biological fate of nanoparticles with higher delivery efficiency, such as enhanced cellular uptake and oral bioavailability.

Fluid Retention Caused by Rosiglitazone Is Related to Increases in AQP2 and αENaC Membrane Expression.

Peroxisome proliferator activated receptor-γ (PPARγ) is a ligand-activated transcription factor of the nuclear hormone receptor superfamily. The decreased phosphorylation of PPARγ due to rosiglitazone (ROS) is the main reason for the increased insulin sensitivity caused by this antidiabetic drug. However, there is no clear evidence whether the nuclear translocation of p-PPARγ stimulated by ROS is related to fluid retention. It is also unclear whether the translocation of p-PPARγ is associated with the change of aquaporin-2 (AQP2) and epithelial sodium channel α subunit (αENaC) in membranes, cytoplasm, and nucleus. Our experiments indicate that ROS significantly downregulates nuclear p-PPARγ and increases membrane AQP2 and αENaC; however, SR1664 (a nonagonist PPARγ ligand) reduces p-PPARγ and has no effect on AQP2 and αENaC. Therefore, we conclude that in vitro the fluid retention caused by ROS is associated with the increases in membrane αENaC and AQP2 but has little relevance to the phosphorylation of PPARγ.

Modulation of the thermostability and substrate specificity of Candida rugosa lipase1 by altering the acyl-binding residue Gly414 at the α-helix-connecting bend.

Candida rugosa Lipase1 (LIP1) is widely used in industrial applications. Optimizing its catalytic performance is still a challenging goal for protein engineers. Mutagenesis of key residues in the active site of the enzyme may provide an effective strategy for enhancing stability and altering substrate specificity. In this study, multiple sequence alignment and structural analysis revealed that the acyl-binding residue, Gly414, of LIP1, which is located at a bend connecting α-helixes, was the non-conserved residue in five other isoenzymes. Using saturation mutagenesis, four mutants with improved stability (G414A, G414M, G414H and G414W) were obtained. Compared to the wild type, the best mutant (G414W) exhibited a remarkable 6.5-fold enhancement in half-life at 60 °C and a 14 °C higher T50(15). Its optimum temperature was increased by 15 °C. Simultaneously, G414W displayed a shift in substrate preference from medium-chain to short-chain pNP-ester. Modeling analysis showed that the multiple interactions formed by hydrophobic clusters and hydrogen bonds in the acyl-binding tunnel might lead to the observed thermostability improvement. Additionally, the bulky tryptophan substitution formed a strong steric hindrance to the accommodation of long-chain substrates in the tunnel. These results indicate that the key acyl-binding residues at the α-helix-connecting bend could mediate enzyme stability and catalytic substrate spectra.

Recognition mechanism between Lac repressor and DNA with correlation network analysis.

Lac repressor is a DNA-binding protein which inhibits the expression of a series of genes involved in lactose metabolism. Lac repressor can bind at a random DNA site via nonspecific interactions; then, it rapidly translocates through the double chain of DNA until it finds the specific binding site. Therefore, the site transform between these two modes is essential for the specific recognition between Lac repressor and DNA. Here, the recognition mechanism between Lac repressor and DNA was illustrated with molecular dynamics simulations and correlation network analyses. We have found that the correlation network of the specific system (2KEI) is more centralized and denser than that of the nonspecific system (1OSL). The significant difference in the networks between the nonspecific and specific systems is apparently due to the different binding modes. Then, different interaction modes were found where electrostatic and hydrogen bonding interactions in the nonspecific system are stronger than those in the specific system. Hydrophobic interactions were found only in specific complexes and mostly focused on the hinge helices. Furthermore, the hinge helix will induce the bending of DNA for the specific system. At the same time, a common specific sequence of DNA was revealed for three specific systems. Then, two design systems (positive and control) were used to evaluate the specific recognition between DNA and Lac repressor. These combined methods can be used to reveal the recognition mechanism between other transcription factors and DNA.