Development of a robust nitrilase by fragment swapping and semi-rational design for efficient biosynthesis of pregabalin precursor

Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi-rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi-rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4-fold increase in whole-cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes.     

MiR-20a-containing exosomes from umbilical cord mesenchymal stem cells alleviates liver ischemia/reperfusion injury

Mesenchymal stem cells (MSCs) have been proved to exert considerable therapeutic effects on ischemia-reperfusion (I/R)-induced injury, but the underlying mechanism remains unknown. In this study, we aimed to explore the potential molecular mechanism underlying the therapeutic effect of MSCs-derived exosome reinforced with miR-20a in reversing liver I/R injury. Quantitative real-time polymerase chain reaction, Western blot, and IHC were carried out to compare the differential expressions of miR-20a, Beclin-I, FAS, Caspase-3, mTOR and P62 in IR rats and normal rats. TUNEL was performed to assess IR-induced apoptosis in IR rats, and luciferase assay was used to confirm the inhibitory effect of miR-20a on Beclin-I and FAS expression. Among the 12 candidate microRNAs (miRNAs), miR-486, miR-25, miR-24, miR-20a,miR-466 and miR-433-3p were significantly downregulated in I/R. In particular, miR-20a, a miRNA highly expressed in umbilical cord-derived mesenchymal stem cells, was proved to bind to the 3ʹ UTR of Beclin-I and FAS to exert an inhibitory effect on their expressions. Since Beclin-I and FAS were aberrantly upregulated in IR, exosomes separated from UC-MSCs showed therapeutic efficacy in reversing I/R induced apoptosis. In addition, exosomes reinforced with miR-20a and separated from UC-MSCs almost fully alleviated I/R injury. Furthermore, our results showed that miR-20a could alleviate the abnormal expression of genes related to apoptosis and autophagy, such as active Caspase-3, mTOR, P62, and LC3II. This study presented detailed evidence to clarify the mechanism underlying the therapeutic efficacy of UC-MSCs in the treatment of I/R injury.     

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Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5–5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-P_thl-augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.     

High temporal and individual variation in the prevalence and intensity of chytrid infection in the southernmost Leaf Frog of the genus Pithecopus (Anura,Phyllomedusidae)

Hydrobiologia - Pithecopus rusticus is an endemic amphibian restricted to the type locality, in southern Brazil, and possibly endangered to extinction, due to habitat degradation. However, an...     

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I_Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on I_Na. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca²⁺ and Na⁺ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.     

Expression scoring of a small-nucleolar-RNA signature identified by machine learning serves as a prognostic predictor for head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy with high mortality and poor prognosis due to a lack of predictive markers. Increasing evidence has demonstrated small nucleolar RNAs (snoRNAs) play an important role in tumorigenesis. The aim of this study was to identify a prognostic snoRNA signature of HNSCC. Survival-related snoRNAs were screened by Cox regression analysis (univariate, least absolute shrinkage and selection operator, and multivariate). The predictive value was validated in different subgroups. The biological functions were explored by coexpression analysis and gene set enrichment analysis (GSEA). One hundred and thirteen survival-related snoRNAs were identified, and a five-snoRNA signature predicted prognosis with high sensitivity and specificity. Furthermore, the signature was applicable to patients of different sexes, ages, stages, grades, and anatomic subdivisions. Coexpression analysis and GSEA revealed the five-snoRNA are involved in regulating malignant phenotype and DNA/RNA editing. This five-snoRNA signature is not only a promising predictor of prognosis and survival but also a potential biomarker for patient stratification management.     

miR-222 inhibits cardiac fibrosis in diabetic mice heart via regulating Wnt/β-catenin-mediated endothelium to mesenchymal transition

miR-222 participates in many cardiovascular diseases, but its effect on cardiac remodeling induced by diabetes is unclear. This study evaluated the functional role of miR-222 in cardiac fibrosis in diabetic mice. Streptozotocin (STZ) was used to establish a type 1 diabetic mouse model. After 10 weeks of STZ injection, mice were intravenously injected with Ad-miR-222 to induce the overexpression of miR-222. miR-222 overexpression reduced cardiac fibrosis and improved cardiac function in diabetic mice. Mechanistically, miR-222 inhibited the endothelium to mesenchymal transition (EndMT) in diabetic mouse hearts. Mouse heart fibroblasts and endothelial cells were isolated and cultured with high glucose (HG). An miR-222 mimic did not affect HG-induced fibroblast activation and function but did suppress the HG-induced EndMT process. The antagonism of miR-222 by antagomir inhibited HG-induced EndMT. miR-222 regulated the promoter region of β-catenin, thus negatively regulating the Wnt/β-catenin pathway, which was confirmed by β-catenin siRNA. Taken together, our results indicated that miR-222 inhibited cardiac fibrosis in diabetic mice via negatively regulating Wnt/β-catenin-mediated EndMT.     

Dielectric Properties of Normal and Metastatic Lymph Nodes Ex Vivo From Lung Cancer Surgeries

The dielectric properties of normal and tumor human tissues have been widely reported in recent years. However, the dielectric properties of intrathoracic lymph nodes (LNs) have not been reported. In this communication, we measured the dielectric properties (i.e., permittivity and conductivity) of ex vivo intrathoracic LNs obtained from lung cancer surgeries. Results show that the permittivity and conductivity of metastatic LNs are higher than those of normal LNs over the frequency range of 1 MHz–4 GHz. Statistically significant differences are observed at single specific frequencies (64, 128, 298, 433, and 915 MHz and 2.45 GHz). Our study provides the basic data to support future-related research and fills the research gap on the dielectric properties of LNs in the lungs. Bioelectromagnetics. 2020;41:148–155. © 2020 Bioelectromagnetics Society.     

Integrated bioinformatics analysis revealed the regulation of angiogenesis by tumor cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, metastasis accounts for most of the cases. Angiogenesis plays an important role in cancer metastasis, but how tumor cells affect the function of endothelial cells by dictating their microRNA (miRNA) expression remains largely unknown.Differentially expressed miRNAs (DEMs) were identified through dataset downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. We then used online tools to obtain potential targets of candidate miRNAs and functional enrichment analysis, as well as the protein-protein interaction (PPI). Finally, the function of miR-302c-3p was validated through in vitro assay.In the current study, we found that HCC cells altered miRNA expression profiles of human umbilical vein endothelial cells (HUVECs) and miR-302c-3p was the most down-regulated miRNA in HUVECs when they were co-cultured with HCC-LM3 cells. Functional enrichment analysis of the candidate targets revealed that these genes were involved in epigenetic regulation of gene expression, in particular, cytosine methylation. In addition, PPI network demonstrated distinct roles of genes targeted by miR-302c-3p. Importantly, inhibition of angiogenesis, migration and permeability by the most down-regulated miR-302c-3p in HUVECs was confirmed in vitro. These findings brought us novel insight into the regulation of angiogenesis by HCC cells and provided potential targets for the development of therapeutic strategies.