This study aimed to develop and validate a predictive model based on S100 calcium-binding protein B (S100B) and neuron-specific enolase (NSE) as the core of epilepsy secondary to cerebral infarction. ...For this aim, 156 cases of cerebral infarction from June 2018 to December 2019 were selected. According to the ratio of 7:3, 109 cases were used for training and 47 cases were used for validation. The factors influencing cerebral infarction secondary to epilepsy were analyzed by a univariate analysis comparing the general data of the two groups and binary logistic regression, and the prediction model was established and validated. Results showed that there was no statistically significant comparison of general information between the training and validation groups (p>0.05). The comparison of NIHSS score, lesion location, lesion size, infarct staging, involved arterial system, large infarct, NSE, and S100B levels between the two groups was significant (P<0.05). The difference between the two groups will be secondary epilepsy= 1, non-epilepsy=0 as dependent variables and factors with significant differences in the univariate analysis as covariates for logistic regression analysis showed that NIHSS score > 15, cortical lesion, lesion size ≥ 5cm, carotid circulation involvement, large infarct, S100B, NSE were risk factors for secondary epilepsy in cerebral infarction. In conclusion, serum S100B and NSE levels were abnormally elevated in patients with epilepsy secondary to cerebral infarction, NIHSS score > 15, cortical lesions, lesion size ≥ 5 cm, carotid circulation involvement, large infarct, S100B and NSE are risk factors for epilepsy secondary to cerebral infarction, and the AUC area of S100B and NSE is large, based on S100B and NSE as The prediction model based on S100B and NSE has good predictive value.
Bacillus licheniformis
is an important industrial microorganism that can utilize a wide range of biomass. However, the lack of expression elements in
B. licheniformis
, especially regulated ...promoters, significantly restricts its applications. In this study, two promoters involved in the sugar alcohol uptake pathway, P
mtlA
and P
mtlR
, were characterized and developed as regulated promoters for expression. The results showed that mannitol, mannose, sorbitol, sorbose, and arabinose can act as inducers to activate expression from P
mtlA
at different levels. The induction by sorbitol was the strongest, and the optimal induction conditions were 15 g/L sorbitol during mid-logarithmic growth at 28 °C. In this work, the palindrome-like sequence ‘TTGTCA-cacggctcc-TGCCAA’ in P
mtlA
was identified as the binding site of the MtlR protein. This study helps to enrich the known inducible expression systems in
B. licheniformis
.
The xylose operon is an efficient biological element used for the regulation of gene expression in
. Although the mechanism underlying the xylose-mediated regulation of this operon has been ...elucidated, the transcriptional changes that occur under various fermentation conditions remain unclear. In this study, the effects of different conditions on xylose operon expression were investigated. Significant upregulation was observed during the transition from the logarithmic phase to the stationary phase (2.5-fold,
= 3,
< 0.01). Glucose suppressed transcription over 168-fold (
= 3,
< 0.01). Meanwhile, the inhibitory effect of glucose hardly strengthened at concentrations from 20 to 180 g/L. Furthermore, the transcription of the xylose operon increased at elevated temperatures (25-42 °C) and was optimal at a neutral pH (pH 6.5-7.0). Based on these findings, relevant fermentation strategies (delaying the induction time, using dextrin as a carbon source, increasing the fermentation temperature, and maintaining a neutral pH) were proposed. Subsequently, these strategies were validated through the use of maltogenic amylase as a reporter protein, as an 8-fold (
= 3,
< 0.01) increase in recombinant enzyme activity compared to that under unoptimized conditions was observed. This work contributes to the development of fermentation optimization and furthers the use of the xylose operon as an efficient expression element.
•Copper-phosphotriesterase (Cu-PTE) hybrid nanoflowers were successfully prepared.•The catalytic activity and stability of Cu-PTE hybrid nanoflowers were higher than that of free ...phosphotriesterase.•Cu-PTE hybrid nanoflowers could maintain 72.3 % relative activity after ten consecutive reactions.
Phosphotriesterase (PTE) is considered to be a good biodegradation agent for organophosphorus pesticides. However, the instability of the free PTE limits its application. In this study, the free PTE was hybridized with copper ions (Cu2+) to enhance its catalytic stability and activity. The acquired particles were freeze-dried after precipitation with PO43− at 4 °C for 72 h. Scanning electron microscopy showed that the Cu-PTE complexes formed flower-like nanoparticles after hybridization. The characteristic peaks of both the enzyme and metal material were revealed by Fourier transform–infrared spectroscopy. X-ray diffraction analysis indicated that PTE was encapsulated in the Cu3(PO4)2·3H2O based hybrid nanoflowers. Compared with free PTE, the catalytic activity of Cu-PTE hybrid nanoflowers was significantly increased about 2.2 fold. The catalytic efficiency (kcat/Vmax) of Cu-PTE hybrid nanoflowers was 1.76 fold than that of free PTE. The stability of the immobilized PTE under thermal and pH conditions was improved and the tolerance of it to organic solvents was also enhanced. Moreover, the Cu-PTE hybrid nanoflowers still exhibited 72.3 % relative activity after ten consecutive reactions. In general, this is the first time to use copper based hybrid nanoflowers to immobilize PTE, and the immobilized enzyme shows excellent performance on OPs degradation. The Cu-PTE hybrid nanoflowers may have great potential in the biodegradation of organophosphorus compounds in future.
Bacillus genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They ...can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 Bacillus constitutive promoters were screened for strength in Bacillus licheniformis. The motif in UP elements from a strong native promoter, PLan, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter–α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, Pacpp, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for Bacillus promoter engineering.
Aerobic denitrification is considered as a promising biological method to eliminate the nitrate contaminants in waterbodies. However, the molecular mechanism of this process varies in different ...functional bacteria. In this study, the nitrogen removal characteristics for a newly isolated aerobic denitrifier Bacillus subtilis JD-014 were investigated, and the potential functional genes involved in the aerobic denitrification process were further screened through transcriptome analysis. JD-014 exhibited efficient denitrification performance when having sodium succinate as the carbon source with the range of nitrate concentration between 50 and 300 mg/L. Following the transcriptome data, most of the up-regulated differentially expressed genes (DEGs) were associated with cell motility, carbohydrate metabolism, and energy metabolism. Moreover, gene nirsir annotated as sulfite reductase was screened out and further identified as a regulator participating in the nitrogen removal process within JD-014. The findings in present study provide meaningful information in terms of a comprehensive understanding of genetic regulation of nitrogen metabolism, especially for Bacillus strains.
Engineering the yeast Yarrowia lipolytica as an efficient host to produce recombinant proteins remains a longstanding goal for applied biocatalysis. During the protein overproduction, the ...accumulation of unfolded and misfolded proteins causes ER stress and cell dysfunction in Y. lipolytica. In this study, we evaluated the effects of several potential ER chaperones and translocation components on relieving ER stress by debottlenecking the protein synthetic machinery during the production of the endogenous lipase 2 and the E. coli β-galactosidase. Our results showed that improving the activities of the non-dominant translocation pathway (SRP-independent) boosted the production of the two proteins. While the impact of ER chaperones is protein dependent, the nucleotide exchange factor Sls1p for protein folding catalyst Kar2p is recognized as a common contributor enhancing the secretion of the two enzymes. With the identified protein translocation components and ER chaperones, we then exemplified how these components can act synergistically with Hac1p to enhance recombinant protein production and relieve the ER stress on cell growth. Specifically, the yeast overexpressing Sls1p and cytosolic heat shock protein Ssa8p and Ssb1p yielded a two-fold increase in Lip2p secretion compared with the control, while co-overexpressing Ssa6p, Ssb1p, Sls1p and Hac1p resulted in a 90% increase in extracellular β-galp activity. More importantly, the cells sustained a maximum specific growth rate (μmax) of 0.38 h−1 and a biomass yield of 0.95 g-DCW/g-glucose, only slightly lower than that was obtained by the wild type strain. This work demonstrated engineering ER chaperones and translocation as useful strategies to facilitate the development of Y. lipolytica as an efficient protein-manufacturing platform.
Angiogenetic inhibitors are crucial in tumor therapy, and endogenous angiogenesis inhibitors have attracted considerable attention due to their effectiveness, safety, and multi-targeting ability. ...Arresten and canstatin, which have anti-angiogenesis effects, are the c-terminal fragments of the α1 and α2 chains of type IV collagen, respectively. In this study, human arresten and canstatin were recombinantly expressed in Escherichia coli (E. coli), and their effects on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated. Regarding the cell cycle distribution test and 5-ethynyl-2′-deoxyuridine (EdU) assays, arresten and canstatin could repress the proliferation of HUVECs at a range of concentrations. Transwell assay indicated that the migration of HUVECs was significantly decreased in the presence of arresten and canstatin, while tube formation assays suggested that the total tube length and junction number of HUVECs were significantly inhibited by these two proteins; moreover, they could also reduce the expression of vascular endothelial growth factor (VEGF) and the phosphorylation levels of PI3K and Akt, which indicated that the activation of the 3-kinase/serine/threonine-kinase (PI3K/Akt) signaling pathway was inhibited. These findings may have important implications for the soluble recombinant expression of human arresten and canstatin, and for the related therapy of cancer.
As a biologically active peptide, L-carnosine has been widely used in the pharmaceutical, cosmetic and health care industries due to its various physiological properties. However, relatively little ...research is available regarding L-carnosine's enzymatic synthesis function. In this study, a potential enzyme sequence with the function of carnosine synthesizing was screened out using the ancestral sequence reconstruction (ASR) technique. Identified with L-carnosine synthesis activity, this enzyme was further confirmed using autoproteolytic phenomenon via Western blot and N-terminal sequencing. After purification, the enzymatic properties of LUCA-DmpA were characterized. The melting temperature (
) and denaturation enthalpy (Δ
) of LUCA-DmpA were 60.27 ± 1.24 °C and 1306.00 ± 26.73 kJ·mol
, respectively. Circular dichroism (CD) spectroscopy results showed that this ancestral enzyme was composed of α-helix (35.23 ± 0.06%), β-sheet (11.06 ± 0.06%), β-turn (23.67 ± 0.06%) and random coil (32.03 ± 0.06%). The enzyme was characterized with the optimal temperature and pH of 45 °C and 9.0, respectively. Notably, LUCA-DmpA was also characterized with remarkable pH tolerance based on the observation of more than 85% remaining enzymatic activity after incubation at different pH buffers (pH = 6-11) for 12 h. Additionally, rather than being improved or inhibited by metal ions, its enzymatic activity was found to be promoted by introducing organic solvent with a larger log
value. Based on these homology modeling results, the screened LUCA-DmpA is suggested to have further optimization potential, and thereafter to be offered as a promising candidate for real industrial applications.
TrLipE is a thermophilic lipase that has potential commercial applications because of its catalytic ability under extreme conditions. Consistent with most lipases, the lid of TrLipE is located over ...the catalytic pocket, controls the substrate channel to the active center, and regulates the substrate specificity, activity, and stability of the enzyme through conformational changes. TrLipE from
has potential industrial applications, which is hindered by its weak enzymatic activity. Here, 18 chimeras (TrL1-TrL18) were reconstructed by N-terminal lid swapping between TrLipE and structurally similar enzymes. The results showed that the chimeras had a similar pH range and optimum pH as wild TrLipE but a narrower temperature range of 40-80°C, and TrL17 and the other chimeras showed lower optimum temperatures of 70°C and 60°C, respectively. In addition, the half-lives of the chimeras were lower than those of TrLipE under optimum temperature conditions. Molecular dynamics simulations indicated that chimeras had high RMSD, RMSF, and B-factor values. When p-nitrophenol esters with different chains were used as substrates, compared with TrLipE, most of the chimeras had a low
and high
value. The chimeras TrL2, TrL3, TrL17, and TrL18 could specifically catalyze the substrate 4-nitrophenyl benzoate, with TrL17 showing the highest
/
value of 363.88 ± 15.83 L⋅min
⋅mmol
. Mutants were then designed by investigating the binding free energies of TrL17 and 4-nitrophenyl benzoate. The results indicated that single, double, and triple substitution variants (M89W and I206N; E33W/I206M and M89W/I206M; and M89W/I206M/L21I and M89W/I206N/L21I, respectively) presented approximately 2- to 3-fold faster catalysis of 4-nitrophenyl benzoate than the wild TrL17. Our observations will facilitate the development of the properties and industrial applications of TrLipE.
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