In this study, the Fenton oxidized lignin was prepared to investigate the effect of Fenton oxidation modification on the activity of lignin immobilized β-glucosidase (β-GL). The results demonstrated that Fenton oxidation could significantly improve the activity and stability of immobilized β-GL. This is because the Fenton oxidation increased the electrostatic, hydrogen bonding, and hydrophobic forces between lignin and β-GL, resulting in increased lignin adsorption onto β-GL. The Fenton oxidation also changed the chemical structure of lignin, altering the lignin-β-GL binding site and reducing the negative effect of lignin on the β-GL catalytic domain. This research will improve understanding of the effect of Fenton lignin oxidation on immobilized β-GL activity and expand the use of lignin in enzyme immobilization.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Candidatus Liberibacter spp., the bacteria associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. HLB management has heavily relied on insecticide applications to control the ACP, although there are efforts towards more sustainable alternatives. In previous work, our group assessed the potential bioactivity of different strains of Bacillus thuringiensis (Eubacteriales: Bacillaceae) (Bt) containing cry/cyt genes as feasible tools to control ACP nymphs. Here, we report an attempt to use the cry11A gene from Bt to produce transgenic sweet orange plants using two promoters. For the genetic transformation, 'Hamlin' and 'Valencia' sweet orange seedlings were used as sources of explants. Transgenic plants were detected by polymerase chain reaction (PCR) with specific primers, and the transgene copy number was confirmed by Southern blot analyses. Transcript expression levels were determined by qPCR. Mortality assays of D. citri nymphs were carried out in a greenhouse, and the effect of the events tested ranged from 22% to 43% at the end of the five-day exposure period. To our knowledge, this is the first manuscript reporting the production of citrus plants expressing the Bt cry11A gene for the management of D. citri nymphs.

Centella asiatica (L.) Urban is an herbaceous perennial plant of the Apiaceae family that has diverse medicinal uses. Its active components are saponin, phenolics, and polyacetylenes. Plant cell cultures have been exploited for the efficient production of metabolites with pharmacological activity. In this work, we prepared adventitious root cultures of C. asiatica and analyzed their content and biological activity. Adventitious root extracts were found to increase glucose uptake by differentiated L6 skeletal muscle cells and to be more efficient than the extract of whole plants. Chromatographic fractionation of the extracts from adventitious roots of C. asiatica led to the isolation of two known polyacetylenes, araliadiol (1) and 8-acetoxy-1,9-pentadecadiene-4,6-diyn-3-ol (2), in addition to a new polyacetylene, which we have named centellidiol (3). All the three polyacetylenes stimulated glucose uptake in a dose-dependent manner. The methanol extract of adventitious roots contained 0.53% and 0.82% of compounds 1 and 2, respectively, which are values that were 15 and 21 times higher that are found in mother plants. We therefore suggest that the high content of these polyacetylenes contributes to the high efficacy of C. asiatica adventitious root cultures. Overall, adventitious root cultures of C. asiatica can be part of a secure supply of effective ingredients including polyacetylenes.

Oplopanax elatus is a valuable medicinal plant, but its plant resource is lacking. Adventitious root (AR) culture of O. elatus is an effective way for the production of plant materials. Salicylic acid (SA) exerts enhancement effect on metabolite synthesis in some plant cell/organ culture systems. To clarify the elicitation effect of SA on fed-batch cultured O. elatus ARs, this study investigated the effects of SA concentration, and elicitation time and duration. Results showed that flavonoid and phenolic contents, and antioxidant enzyme activity obviously increased when the fed-batch cultured ARs were treated with 100 μM SA for 4 days starting on day 35. Under this elicitation condition, total flavonoid and phenolic contents reached 387 rutin mg/g DW and 128 gallic acid mg/g DW, respectively, which were significantly (p < 0.05) higher than those in the SA-untreated control. In addition, DPPH scavenging and ABTS+ scavenging rates, and Fe2+ chelating rate also greatly increased after SA treatment, and their EC50 values were 0.0117, 0.61, and 3.34 mg/L, respectively, indicating the high antioxidant activity. The findings of the present study revealed that SA could be used as an elicitor to improve the flavonoid and phenolic production in fed-batch O. elatus AR culture.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an "enhanced specificity" SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.

Protein bound uremic toxins (PBUTs) are small substances binding to larger proteins, mostly human serum albumin (HSA), and are challenging to remove by hemodialysis (HD). Among different classes of PBUTs, p-cresyl sulfate (PCS) is the most widely used marker molecule and major toxin, as 95 % is bound to HSA. PCS has a pro-inflammatory effect and increases both the uremia symptom score and multiple pathophysiological activities. High-flux HD to clear PCS leads to serious loss of HSA, which results in a high mortality rate. The goal of the present study is to investigate the efficacy of PCS detoxification in serum of HD patients using a biocompatible laccase enzyme from Trametes versicolor. Molecular docking was used to gain an in-depth understanding of the interactions between PCS and the laccase to identify the functional group(s) responsible for ligand-protein receptor interactions. UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS) were used to assess the detoxification of PCS. GC-MS was used to identify the detoxification byproducts and their toxicity was assessed using docking commutations. In situ synchrotron radiation micro-computed tomography (SR-µCT) imaging available at the Canadian Light Source (CLS) was conducted to assess HSA binding with PCS before and after detoxification with laccase and undertake the corresponding quantitative analysis. GC-MS analyses confirmed the detoxification of PCS with laccase at a concentration of 500 mg/L. The potential pathway of PCS detoxification in the presence of the laccase was identified. Increasing laccase concentration led to the formation of m-cresol, as indicated by the corresponding absorption in the UV-Vis spectra and a sharp peak on the GC-MS spectra. Our analysis provides insight into the general features of PCS binding on Sudlow site II, as well as insights into PCS detoxification product interactions. The average affinity energy for detoxification products was lower than that of PCS. Even though some byproducts showed potential toxicity, the level was lower than for PCS based on toxicity indexes (e.g., LD50/LC50, carcinogenicity, neurotoxicity, mutagenicity). In addition, these small compounds can also be more easily removed by HD compared to PCS. SR-µCT quantitative analysis showed adhesion of the HSA to a significant reduced extent in the presence of the laccase enzyme in bottom sections of the polyarylethersulfone (PAES) clinical HD membrane tested. Overall, this study opens new frontiers for PCS detoxification.