Structure-guided rational design of the Geobacillus thermoglucosidasius feruloyl esterase GthFAE to improve its thermostability.

Feruloyl esterases are indispensable biocatalysts catalyzing the cleavage of ester bonds between polysaccharides and their hydroxycinnamoyl cross-links. GthFAE from Geobacillus thermoglucosidasius was identified as a thermophilic alkaline feruloyl esterase with potential applications in paper manufacturing. To improve the enzymatic properties rationally and efficiently, the structure of GthFAE was solved at 1.9 Å, revealing a core domain of classical α/ß hydrolase fold and an inserted α/ß cap domain. In silico analysis based on it helped us to investigate whether the residues at the active center have positive effects on the stability, and how. Several site-directed mutations were conducted, of which substitutions at residues T41 and T150 apparently improved the thermostability. The combination mutant T41N/T150R exhibited an optimal temperature of 65 °C, a 6.4 °C higher Tm compared to wild type by 80 °C, and a 35-fold longer in half-life (201 min) at 70 °C. Molecular dynamics simulations further illustrated that the structure of T41N/T150R was more stable than the wild type and T150R stabilized the cap domain by introducing salt bridges to the region with E154 and D164. This study not only highlighted residues within the active center on their thermostability improving effects, but also contributed to the prospective industrial application of GthFAE.

Structural insights into the CP312R protein of the African swine fever virus.

African swine fever (ASF) is a lethal hemorrhagic disease that affects domestic pigs and wild boars. There is no medication available for ASF to date. The ability to mount antigen-specific responses to viral vectored CP312R makes it a crucial potential target for designing vaccines or drugs. This study determined the crystal structure of ASFV CP312R at 2.32 Å and found it to be a monomer with a single-stranded DNA binding core domain with a clear five-strands ß-barrel OB-fold architecture. Electrophoretic mobility shift assay and size-exclusion chromatography characterization assay further confirmed the single-stranded DNA (ssDNA)-binding property of ASFV CP312R. This study revealed the structure and preliminary ssDNA interaction mechanisms of ASFV CP312R, providing new clues for developing new antiviral strategies.

Intrarenal resistance index for the assessment of acute renal injury in a rat liver transplantation model.

BACKGROUND: Acute kidney injury (AKI) is a common complication after liver transplantation (LT) and associated with a high mortality. The renal resistive index (RI) is used to assess early renal function impairment in critical care patients. However, limited data are available concerning changes of renal RI and the development of AKI early after reperfusion. We approached to investigate the changes of renal RI and AKI after reperfusion in a rat liver transplantation model. METHODS: Rats were randomly divided into sham group or LT group. Ten rats in each group were used for the hemodynamic study and twenty for Doppler measurements during the procedure. Ten rats were sacrificed 30 min or 2 h after the reperfusion. We harvested kidneys, serum and urine for further analysis of the renal function. RESULTS: The intrarenal RI increased significantly in the anhepatic stage and decreased significantly after the reperfusion in the LT group compared with sham group (P < 0.05). AKI was seen after the reperfusion in the LT group. No correlation was noted between the RI and renal function parameters 30 min after reperfusion. CONCLUSIONS: The intrarenal RI increased significantly during the anhepatic stage, and decreased significantly early after the reperfusion. Intrarenal RI was unable to assess renal function in a rat liver transplantation model.

Structural insight into African swine fever virus I73R protein reveals it as a Z-DNA binding protein.

African Swine Fever (ASF) is a highly contagious viral haemorrhagic disease of swine, leading to enormous economic losses in the swine industry. However, vaccines and drugs to treat ASF have yet to be developed. African swine fever virus (ASFV) encodes more than 150 proteins, but 50% of them have unknown functions. Here, we present the crystal structure of the ASFV I73R protein at a resolution of 2.0 Å. Similar search tools based solely on amino acid sequence shows that it has no relationships to any proteins of known function. Interestingly, the overall structure of the I73R protein shares a winged helix-turn-helix fold, structural similarity with the Z-DNA binding domain (Zα). In accordance with this result, the I73R is capable of binding to a CpG repeats DNA duplex, which has a high propensity for forming Z-DNA during the DNA binding assays. In addition, the I73R protein was shown to be expressed at both early and late stages of ASFV post-infection in PAM cells as an 8.9 kDa protein. Immunofluorescence studies revealed that the I73R protein is expressed in the nucleus at early times post-infection and gradually translocated from the nucleus to the cytoplasm. Taken together, these data indicate that the I73R could be a member of Zα family that is important in host-pathogen interaction, which paves the way for the design of inhibitors to target this severe pathogen. Further exploring the biological role of I73R during ASFV infection in vitro and in vivo will provide new clues for development of new antiviral strategies.

Lin28 mediates paclitaxel resistance by modulating p21, Rb and Let-7a miRNA in breast cancer cells.

Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemoresistance remained unknown. In this study, we investigated the association of Lin28 with paclitaxel resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines and tumor tissues. We found that the expression level of Lin28 was closely associated with the resistance to paclitaxel treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to paclitaxel than the MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which had low-level expression of Lin28. Knocking down of Lin28 in Lin28 high expression T47D cells increased the sensitivity to paclitaxel treatment, while stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to paclitaxel treatment, resulting in a significant increase of IC50 values of paclitaxel. Transfection with Lin28 also significantly inhibited paclitaxel-induced apoptosis. We also found that Lin28 expression was dramatically increased in tumor tissues after neoadjuvant chemotherapy or in local relapse or metastatic breast cancer tissues. Moreover, further studies showed that p21, Rb and Let-7 miRNA were the molecular targets of Lin28. Overexpression of Lin28 in breast cancer cells considerably induced p21 and Rb expression and inhibited Let-7 miRNA levels. Our results indicate that Lin28 expression might be one mechanism underlying paclitaxel resistance in breast cancer, and Lin28 could be a potential target for overcoming paclitaxel resistance in breast cancer.