Regulation of PKR-dependent RNA translation inhibition by TRIM21 upon virus infection or other stress.

The host always employs various ways to defend against viral infection and spread. However, viruses have evolved their own effective strategies, such as inhibition of RNA translation of the antiviral effectors, to destroy the host's defense barriers. Protein synthesis, commonly controlled by the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), is a basic cellular biological process among all species. In response to viral infection, in addition to inducing the transcription of antiviral cytokines by innate immunity, infected cells also inhibit the RNA translation of antiviral factors by activating the protein kinase R (PKR)-eIF2α signaling pathway. Regulation of innate immunity has been well studied; however, regulation of the PKR-eIF2α signaling pathway remains unclear. In this study, we found that the E3 ligase TRIM21 negatively regulates the PKR-eIF2α signaling pathway. Mechanistically, TRIM21 interacts with the PKR phosphatase PP1α and promotes K6-linked polyubiquitination of PP1α. Ubiquitinated PP1α augments its interaction with PKR, causing PKR dephosphorylation and subsequent translational inhibition release. Furthermore, TRIM21 can constitutively restrict viral infection by reversing PKR-dependent translational inhibition of various previously known and unknown antiviral factors. Our study highlights a previously undiscovered role of TRIM21 in regulating translation, which will provide new insights into the host antiviral response and novel targets for the treatment of translation-associated diseases in the clinic.

The RNA Surveillance Factor UPF1 Represses Myogenesis via Its E3 Ubiquitin Ligase Activity.

UPF1 is an RNA helicase that orchestrates nonsense-mediated decay and other RNA surveillance pathways. While UPF1 is best known for its basal cytoprotective role in degrading aberrant RNAs, UPF1 also degrades specific, normally occurring mRNAs to regulate diverse cellular processes. Here we describe a role for UPF1 in regulated protein decay, wherein UPF1 acts as an E3 ubiquitin ligase to repress human skeletal muscle differentiation. Suppressing UPF1 accelerates myogenesis, while ectopically increasing UPF1 levels slows myogenesis. UPF1 promotes the decay of MYOD protein, a transcription factor that is a master regulator of myogenesis, while leaving MYOD mRNA stability unaffected. UPF1 acts as an E3 ligase via its RING domain to promote MYOD protein ubiquitination and degradation. Our data characterize a regulatory role for UPF1 in myogenesis, and they demonstrate that UPF1 provides a mechanistic link between the RNA and protein decay machineries in human cells.

Multidimensional engineering of Saccharomyces cerevisiae for the efficient production of heme by exploring the cytotoxicity and tolerance of heme.

Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H2O2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD600), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22, REE1, PHO84, HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts.

A study on loading multiple epitopes with a single peptide.

Epitopes, the basic functional units of antigens, hold great significance in the field of immunology. However, the structure and composition of epitopes and their interactions with antibodies remain unclear, which limits in-depth studies on epitopes and the development of subunit vaccines. In a previous study on the localization of anti-influenza HA monoclonal antibodies (mAbs), three strains with different characteristics reacted with the same peptide. In this study, by conventional immunological assays, computer homology modeling, and molecular docking simulations, we found that (1) the peptide could bind to three strains of mAbs with different reaction characteristics utilizing different combinations of immunodominant groups. (2) By computer molecular docking and simulation methods, the immunodominant groups on the two peptides could be combined into a multi-epitope peptide bound to six strains of mAbs. We established a method for multi-epitope peptide recombination from these immunodominant groups. (3) The immune effect of the recombinant multi-epitope peptide was better than that of a single peptide. Our findings facilitate the understanding of the composition of antigen epitopes and provide a theoretical and experimental basis for developing polyvalent vaccines and understanding immune responses at the molecular level.

Cross-reaction mediated by distinct key amino acid combinations in the complementary-determining region (CDR) of a monoclonal antibody.

In immunology, cross-reaction between antigens and antibodies are commonly observed. Prior research has shown that various monoclonal antibodies (mAbs) can recognize a broad spectrum of epitopes related to influenza viruses. However, existing theories on cross-reactions fall short in explaining the phenomena observed. This study explored the interaction characteristics of H1-74 mAb with three peptides: two natural peptides, LVLWGIHHP and LPFQNI, derived from the hemagglutinin (HA) antigen of the H1N1 influenza virus, and one synthetic peptide, WPFQNY. Our findings indicate that the complementarity-determining region (CDR) of H1-74 mAb comprised five antigen-binding sites, containing eight key amino acid residues from the light chain variable region and 16 from the heavy chain variable region. These critical residues formed distinct hydrophobic or hydrophilic clusters and functional groups within the binding sites, facilitating interaction with antigen epitopes through hydrogen bonding, salt bridge formation, and π-π stacking. The study revealed that the formation of the antibody molecule led to the creation of binding groups and small units in the CDR, allowing the antibody to attach to a variety of antigen epitopes through diverse combinations of these small units and functional groups. This unique ability of the antibody to bind with antigen epitopes provides a new molecular basis for explaining the phenomenon of antibody cross-reaction.

Focused ultrasound versus the loop electrosurgical excision procedure to treat women with cervical high-grade squamous intraepithelial lesions under 40: a retrospective study.

BACKGROUND: This study aimed to compare the efficacy of focused ultrasound (FUS) and the loop electrosurgical excision procedure (LEEP) for the treatment of cervical high-grade squamous intraepithelial lesions (HSILs) among women of reproductive age. METHODS: Case records of patients aged < 40 years who were treated for cervical HSILs using either FUS or LEEP from September 1, 2020 to May 31, 2022 were retrospectively reviewed. Patients were followed up for cure, recurrence, human papillomavirus (HPV) clearance, and complications within 1 year of treatment. Odds ratios and 95% confidence intervals were determined using univariate and multivariate logistic regression models to analyze the association between disease evidence or HPV clearance and treatment modalities or other covariates. RESULTS: Of the 1,054 women who underwent FUS or LEEP, 225 met our selection criteria. Among the selected women, 101 and 124 received FUS and LEEP, respectively. There was no significant difference between the FUS and LEEP groups in the cure rate during the 3-6 months of follow-up (89.11% vs. 94.35%, P = 0.085) and recurrence rate during the 6-12 months follow-up (2.22% vs. 1.71%, P = 0.790). Both groups exhibited enhanced cumulative HPV clearance rates; however, the rates were not significantly different between the FUS and LEEP groups (74.23% vs. 82.79%, P = 0.122 during the 3-6 months follow-up; 84.95% vs. 89.17%, P = 0.359 during the 6-12 months follow-up). Furthermore, the incidence of complications caused by the FUS and LEEP techniques was comparable (5.0% vs. 5.6%, P = 0.818). CONCLUSIONS: We found that FUS and LEEP have similar efficacy, safety, and reliability in treating women (aged < 40 years) with HSILs.

De novo biosynthesis of betulinic acid in engineered Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae, and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla-derived lupeol C-28 oxidase (BPLO) and Arabidopsis-derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae.

Pharmacologically significant constituents collectively responsible for anti-sepsis action of XueBiJing, a Chinese herb-based intravenous formulation.

Sepsis, a life-threatening health issue, lacks effective medicine targeting the septic response. In China, treatment combining the intravenous herbal medicine XueBiJing with conventional procedures reduces the 28-day mortality of critically ill patients by modulating septic response. In this study, we identified the combined active constituents that are responsible for the XueBiJing's anti-sepsis action. Sepsis was induced in rats by cecal ligation and puncture (CLP). The compounds were identified based on their systemic exposure levels and anti-sepsis activities in CLP rats that were given an intravenous bolus dose of XueBiJing. Furthermore, the identified compounds in combination were assessed, by comparing with XueBiJing, for levels of primary therapeutic outcome, pharmacokinetic equivalence, and pharmacokinetic compatibility. We showed that a total of 12 XueBiJing compounds, unchanged or metabolized, circulated with significant systemic exposure in CLP rats that received XueBiJing. Among these compounds, hydroxysafflor yellow A, paeoniflorin, oxypaeoniflorin, albiflorin, senkyunolide I, and tanshinol displayed significant anti-sepsis activities, which involved regulating immune responses, inhibiting excessive inflammation, modulating hemostasis, and improving organ function. A combination of the six compounds, with the same respective doses as in XueBiJing, displayed percentage survival and systemic exposure in CLP rats similar to those by XueBiJing. Both the combination and XueBiJing showed high degrees of pharmacokinetic compatibility regarding interactions among the six active compounds and influences of other circulating XueBiJing compounds. The identification of XueBiJing's pharmacologically significant constituents supports the medicine's anti-sepsis use and provides insights into a polypharmacology-based approach to develop medicines for effective sepsis management.

Association between negative life events through mental health and non-suicidal self-injury with young adults: evidence for sex moderate correlation.

BACKGROUND: Non-suicidal self-injury (NSSI) has exhibited an increasing trend in recent years and is now globally recognized as a major public health problem among adolescents and young adults. Negative life events (NLEs) are positively associated with NSSI. We sought to explore (1) whether sex plays a role in the risk of NLEs leading to NSSI and (2) the role played by mental health (MH). METHODS: We adopted a multi-stage cluster sampling method to select college students across four grades from May to June 2022. Generalized linear models were used to evaluate the relationships between NLEs, sex, MH and NSSI, presented as incidence-rate ratios (RRs) with 95% confidence intervals (CIs). We examined the complex relationship between these variables using the PROCESS method for moderation analysis. RESULTS: Following the exclusion of data that did not meet the study requirements, data from 3,578 students (mean age: 20.53 [± 1.65] years) were included. Poisson regression results indicate that high-level NLEs (RR = 0.110, 95%CI: 0.047-0.173) are associated with increased NSSI. Furthermore, interaction effects were observed among sex, NLEs and NSSI. MH and sex moderated the relationship between NLEs and NSSI. CONCLUSION: Identifying risk factors for NSSI is also important when exploring the interaction between NLEs and MH given the potential for NSSI to significantly increase the risk of later psychopathological symptoms and substance abuse problems. In addition, the significance of sex differences in risk factors for NSSI should be determined. This study evaluated how the impact of NLEs on NSSI can be reduced among adolescents from multiple perspectives.

The increased longitudinal basal-to-apical strain ratio in the right ventricular free wall is associated with neonatal pulmonary hypertension.

It has been a challenging work to identify and assess neonatal pulmonary hypertension (PH). Right ventricular longitudinal strain (RVLS) is primarily used in evaluating right ventricular (RV) systolic function. This study aimed to investigate the association of the changes in segmental and global RVLS with neonatal PH, hoping to provide a new marker for indicating neonatal PH other than obtaining information on RV function. This was a cross-sectional study with 62 neonates, generally divided into PH and non-PH groups confirmed by echocardiography. For 30 infants later diagnosed with bronchopulmonary dysplasia (BPD), specific analysis was conducted by subdividing them into BPD with and without PH subgroups. Conventional echocardiography markers and the global and segmental RVLS were measured and compared. Their diagnostic performance in evaluating PH was analyzed. Regardless of grouping, the biventricular function of all infants was similar and in normal range. No significant difference was found in global strain parameters, either. In the case of PH, tricuspid regurgitant velocity (TRV), left ventricle systolic eccentricity index (LVsEI), and the basal-to-apical strain ratio (Ratio bas/api) of RV free wall (RVFW) were significantly higher (P < 0.001, P < 0.05, P < 0.05). By contrast, the magnitude of apical segmental strain reduced significantly (P < 0.05) and was significantly lower than that of basal segmental strain in BPD with PH subgroup (P = 0.024). The area under the curve values for Ratio bas/api was highest (0.846), followed by LVsEI (0.746) and apical segmental strain (0.272). CONCLUSION: As a relatively standardized parameter, Ratio bas/api of RVFW was significantly higher in the case of neonatal PH with normal cardiac function and could be regarded as a new indicator for PH. WHAT IS KNOWN: • It has been challenging work to diagnose neonatal pulmonary hypertension (PH), and conventional echocardiography has been widely applied, though it is not sufficient enough. • RV longitudinal strain (RVLS) is primarily used to assess RV systolic function, and its role in diagnosing PH was rarely considered. WHAT IS NEW: • The basal-to-apical strain ratio (Ratio bas/api) of RV free wall increased significantly in all infants with PH regardless of causes. • As a relatively standardized parameter, Ratio bas/api could be regarded as a new indicator for diagnosing PH, apart from conventional echocardiographic parameters.

Applications of magnetic solid-phase extraction in the sample preparation of natural product analysis (2020-2023).

Sample preparation, including extraction, separation, and purification, is a vital process for natural product analysis. As an attractive sample pretreatment method, magnetic solid-phase extraction (MSPE) has gained plenty of attention, mainly due to its simpler operation, less consumption of organic solvents, and shorter processing time than traditional SPE. This updated review is devoted to summarizing the applications of MSPE based on different magnetic nanomaterials in the analysis of various natural products in complex matrixes, such as biological samples, plants, and Chinese herbal preparations in the past four years (2020-2023). The preparation and fabrication of different materials are briefly introduced. Furthermore, the extraction mechanism and interaction forces between adsorbent and analytes are elaborated, and the advantages and disadvantages of different adsorbents coupled with various analytical methods for MSPE of different natural products are summarized. Moreover, the future trends and opportunities for MSPE in the natural product analysis are discussed. It is expected that this work can provide updated information for future research on the applications of MSPE in such fields.

Implementation of social needs screening for minoritized patients newly diagnosed with breast cancer: a mixed methods evaluation in a pragmatic patient navigation trial.

BACKGROUND: Social needs inhibit receipt of timely medical care. Social needs screening is a vital part of comprehensive cancer care, and patient navigators are well-positioned to screen for and address social needs. This mixed methods project describes social needs screening implementation in a prospective pragmatic patient navigation intervention trial for minoritized women newly diagnosed with breast cancer. METHODS: Translating Research Into Practice (TRIP) was conducted at five cancer care sites in Boston, MA from 2018 to 2022. The patient navigation intervention protocol included completion of a social needs screening survey covering 9 domains (e.g., food, transportation) within 90 days of intake. We estimated the proportion of patients who received a social needs screening within 90 days of navigation intake. A multivariable log binomial regression model estimated the adjusted rate ratios (aRR) and 95% confidence intervals (CI) of patient socio-demographic characteristics and screening delivery. Key informant interviews with navigators (n = 8) and patients (n = 21) assessed screening acceptability and factors that facilitate and impede implementation. Using a convergent, parallel mixed methods approach, findings from each data source were integrated to interpret study results. RESULTS: Patients' (n = 588) mean age was 59 (SD = 13); 45% were non-Hispanic Black and 27% were Hispanic. Sixty-nine percent of patients in the navigators' caseloads received social needs screening. Patients of non-Hispanic Black race/ethnicity (aRR = 1.25; 95% CI = 1.06-1.48) and those with Medicare insurance (aRR = 1.13; 95% CI = 1.04-1.23) were more likely to be screened. Screening was universally acceptable to navigators and generally acceptable to patients. Systems-based supports for improving implementation were identified. CONCLUSIONS: Social needs screening was acceptable, yet with modest implementation. Continued systems-based efforts to integrate social needs screening in medical care are needed.

CircDDX21 alleviates trophoblast dysfunction and Treg differentiation in recurrent spontaneous abortion via miR-520a-5p/ FOXP3/PD-L1 axis.

BACKGROUND: Recurrent spontaneous abortion (RSA) is a common complication during pregnancy, which is a burden to patients both physically and mentally. Circular RNAs (circRNAs) play important roles in RSA. However, the roles of circDDX21 in RSA development remain unknown. METHODS: Decidual samples were harvested from healthy pregnant women and RSA patients. In HTR-8/SVneo and Bewo trophoblast cells, proliferation and migration were analyzed by cell counting kit-8 (CCK-8)/5-ethynyl-2'-deoxyuridine (EdU) staining and transwell/wound healing assays, respectively. CD4+ T cells from peripheral blood mononuclear cells of patients were incubated with trophoblast-conditioned medium. Regulatory T cells (Treg) proliferation was detected by carboxyfluorescein succinimidyl ester (CFSE) assay. Treg proportion, Treg/T helper 17 cells (Th17) ratio, and cytokines were measured using flow cytometry. The association among genes was validated using dual-luciferase assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP). RESULTS: CircDDX21 and Forkhead box P3 (FOXP3) decreased, while miR-520a-5p increased in the decidual tissues of RSA patients. CircDDX21 overexpression promoted trophoblast proliferation and migration, and facilitated CD4+ T cell differentiation into Treg. CircDDX21 targeted miR-520a-5p to elevate FOXP3. MiR-520a-5p overexpression reversed the promoted trophoblast cell function of circDDX21 overexpression in HTR-8/SVneo cells. FOXP3 overexpression reversed the repressed trophoblast cell function elicited by miR-520a-5p overexpression in HTR-8/SVneo cells. FOXP3 promoted Treg differentiation by transcriptionally upregulating programmed cell death ligand 1 (PD-L1). CONCLUSION: CircDDX21 ameliorated trophoblast dysfunction and Treg differentiation in RSA via miR-520a-5p/FOXP3/PD-L1 axis.

Co-augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.

Two new anti-inflammatory trachylobane diterpenoids from Euphorbia atoto.

Two new rare trachylobane euphoratones A-B (1-2), together with five known diterpenoids (compounds 3-7), were isolated from the aerial parts of Euphorbia atoto. Their structures were unambiguously elucidated through HRESIMS, 1D and 2D NMR spectral analysis. Compounds 1, 3, 4 and 7 showed weak anti-inflammatory activities (IC50 77.49 ± 6.34, 41.61 ± 14.49, 16.00 ± 1.71 and 33.41 ± 4.52 µM, respectively), compared to the positive control quercetin (IC50 15.23 ± 0.65 µM).

Influence of Bi3+ Doping on Electrochemical Properties of Ti/Sb-SnO2/PbO2 Electrode for Zinc Electrowinning.

Bi3+ doped Ti/Sb-SnO2/PbO2 electrode materials were fabricated by electrodeposition to improve their electrochemical performance in zinc electrowinning. The surface morphology, chemical composition, and hydrophilicity of the as-prepared electrodes were characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and contact angle. An electrochemical measurement and an accelerated lifetime experiment were also conducted to investigate the electrocatalytic performance and stability of the electrodes. The results show that the Bi3+ modification electrode has an important effect on the coating morphology, the crystal structure, the surface hydrophilicity, the electrocatalytic activity, and the stability. The electrode prepared from the solution containing 2 mmol·L-1 Bi(NO3)3 (marked as the Ti/Sb-SnO2/2Bi-PbO2 electrode) exhibits the best hydrophilicity performance (θ = 21.6°) and the longest service life (1196 h). During the electrochemical characterization analysis, the Ti/Sb-SnO2/2Bi-PbO2 electrode showed the highest oxygen evolution activity, which can be attributed to it having the highest electroactive surface (qT* = 21.20 C·cm-2) and the best charge-transfer efficiency. The DFT calculation demonstrated that the doping of Bi3+ leads to a decrease in the OER reaction barrier and an increase in the DOS of the electrode, which further enhances the catalytic activity and the conductivity of the electrode. Moreover, the simulated zinc electrowinning experiment demonstrated that the Ti/Sb-SnO2/2Bi-PbO2 electrode consumes less energy than other electrodes. Therefore, it is expected that the Bi3+ modified electrode will become a very promising electrode material for zinc electrowinning in the future.

Applications of Nanozymes in Chiral-Molecule Recognition through Electrochemical and Ultraviolet-Visible Analysis.

Chiral molecules have similar physicochemical properties, which are different in terms of physiological activities and toxicities, rendering their differentiation and recognition highly significant. Nanozymes, which are nanomaterials with inherent enzyme-like activities, have garnered significant interest owing to their high cost-effectiveness, enhanced stability, and straightforward synthesis. However, constructing nanozymes with high activity and enantioselectivity remains a significant challenge. This review briefly introduces the synthesis methods of chiral nanozymes and systematically summarizes the latest research progress in enantioselective recognition of chiral molecules based on electrochemical methods and ultraviolet-visible absorption spectroscopy. Moreover, the challenges and development trends in developing enantioselective nanozymes are discussed. It is expected that this review will provide new ideas for the design of multifunctional chiral nanozymes and broaden the application field of nanozymes.

Detection of Arsenic(V) by Fluorescence Sensing Based on Chlorin e6-Copper Ion.

The high toxicity of arsenic (As) can cause irreversible harm to the environment and human health. In this study, the chlorin e6 (Ce6), which emits fluorescence in the infrared region, was introduced as the luminescence center, and the addition of copper ion (Cu2+) and As(V) provoked a regular change in fluorescence at 652 nm, whereas that of As(III) was 665 nm, which was used to optionally detect Cu2+, arsenic (As(III), and As(V)). The limit of detection (LOD) values were 0.212 µM, 0.089 ppm, and 1.375 ppb for Cu2+, As(III), and As(V), respectively. The developed method can be used to determine Cu2+ and arsenic in water and soil with good sensitivity and selectivity. The 1:1 stoichiometry of Ce6 with Cu2+ was obtained from the Job plot that was developed from UV-visible spectra. The binding constants for Cu2+ and As(V) were established to be 1.248 × 105 M-1 and 2.35 × 1012 M-2, respectively, using B-H (Benesi-Hildebrand) plots. Fluorescence lifetimes, B-H plots, FT-IR, and 1H-NMR were used to postulate the mechanism of Cu2+ fluorescence quenching and As(V) fluorescence restoration and the interactions of the two ions with the Ce6 molecule.

[Gene cloning and functional analysis of an iridoid oxidase gene in Rehmannia glutinosa].

Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, which has activities of heat-clearing,blood-cooling, Yin-nourishing, and body fluid-promoting. Iridoid glycosides are the main bioactive in R. glutinosa. Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides. In this study, an iridoid oxidase gene Rg IO was screened based on the transcriptome data, followed by bioinformatics analysis, expression characteristic detection, and subcellular localization analysis. The results show that the coding region of Rg IO is 1 536 bp, with 511 amino acids encoded, and the molecular weight is about 58 258. 01. The protein sequence of Rg IO contains the conserved domains and motifs of cytochrome P450 oxidases. Rg IO has the highest sequence identities with its ortholog proteins in Striga asiatica, Striga hermonthica, and Centranthera grandiflora and has good sequence identities(77. 28%) with Catharanthus roseus Cr IO. Rg IO shows specific expression in the leaf of R. glutinosa. In response to MeJA induction, the expression of MeJA in leaves and roots after treatment increases by 3. 15 and 1. 3 times at 3 h and 6 h,respectively. The result of subcellular localization shows that Rg IO is distributed in the endoplasmic reticulum. Agrobacterium-mediated transient expression of Rg IO gene in leaves of R. glutinosa makes the content of catalpol increase by 0. 82 times compared with the transient expression of the empty vector. This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R. glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.

Comparative genomics of the medicinal plants Lonicera macranthoides and L. japonica provides insight into genus genome evolution and hederagenin-based saponin biosynthesis.

Lonicera macranthoides (LM) and L. japonica (LJ) are medicinal plants widely used in treating viral diseases, such as COVID-19. Although the two species are morphologically similar, their secondary metabolite profiles are significantly different. Here, metabolomics analysis showed that LM contained ~86.01 mg/g hederagenin-based saponins, 2000-fold higher than LJ. To gain molecular insights into its secondary metabolite production, a chromosome-level genome of LM was constructed, comprising 9 pseudo-chromosomes with 40 097 protein-encoding genes. Genome evolution analysis showed that LM and LJ were diverged 1.30-2.27 million years ago (MYA). The two plant species experienced a common whole-genome duplication event that occurred ∼53.9-55.2 MYA before speciation. Genes involved in hederagenin-based saponin biosynthesis were arranged in clusters on the chromosomes of LM and they were more highly expressed in LM than in LJ. Among them, oleanolic acid synthase (OAS) and UDP-glycosyltransferase 73 (UGT73) families were much more highly expressed in LM than in LJ. Specifically, LmOAS1 was identified to effectively catalyse the C-28 oxidation of ß-Amyrin to form oleanolic acid, the precursor of hederagenin-based saponin. LmUGT73P1 was identified to catalyse cauloside A to produce α-hederin. We further identified the key amino acid residues of LmOAS1 and LmUGT73P1 for their enzymatic activities. Additionally, comparing with collinear genes in LJ, LmOAS1 and LmUGT73P1 had an interesting phenomenon of 'neighbourhood replication' in LM genome. Collectively, the genomic resource and candidate genes reported here set the foundation to fully reveal the genome evolution of the Lonicera genus and hederagenin-based saponin biosynthetic pathway.