In vitro biological evaluation and in silico insights into the antiviral activity of standardized olive leaves extract against SARS-CoV-2.

There is still a great global need for efficient treatments for the management of SARS-CoV-2 illness notwithstanding the availability and efficacy of COVID-19 vaccinations. Olive leaf is an herbal remedy with a potential antiviral activity that could improve the recovery of COVID-19 patients. In this work, the olive leaves major metabolites were screened in silico for their activity against SARS-CoV-2 by molecular docking on several viral targets such as methyl transferase, helicase, Plpro, Mpro, and RdRp. The results of in silico docking study showed that olive leaves phytoconstituents exhibited strong potential antiviral activity against SARS-CoV-2 selected targets. Verbacoside demonstrated a strong inhibition against methyl transferase, helicase, Plpro, Mpro, and RdRp (docking scores = -17.2, -20, -18.2, -19.8, and -21.7 kcal/mol.) respectively. Oleuropein inhibited 5rmm, Mpro, and RdRp (docking scores = -15, -16.6 and -18.6 kcal/mol., respectively) respectively. Apigenin-7-O-glucoside exhibited activity against methyl transferase and RdRp (docking score = -16.1 and -19.4 kcal/mol., respectively) while Luteolin-7-O-glucoside inhibited Plpro and RdRp (docking score = -15.2 and -20 kcal/mol., respectively). The in vitro antiviral assay was carried out on standardized olive leaf extract (SOLE) containing 20% oleuropein and IC50 was calculated. The results revealed that 20% SOLE demonstrated a moderate antiviral activity against SARS-CoV-2 with IC50 of 118.3 µg /mL. Accordingly, olive leaf could be a potential herbal therapy against SARS-CoV-2 but more in vivo and clinical investigations are recommended.

Native Mass Spectrometry Dissects the Structural Dynamics of an Allosteric Heterodimer of SARS-CoV-2 Nonstructural Proteins.

Structure-based drug design, which relies on precise understanding of the target protein and its interaction with the drug candidate, is dramatically expedited by advances in computational methods for candidate prediction. Yet, the accuracy needs to be improved with more structural data from high throughput experiments, which are challenging to generate, especially for dynamic and weak associations. Herein, we applied native mass spectrometry (native MS) to rapidly characterize ligand binding of an allosteric heterodimeric complex of SARS-CoV-2 nonstructural proteins (nsp) nsp10 and nsp16 (nsp10/16), a complex essential for virus survival in the host and thus a desirable drug target. Native MS showed that the dimer is in equilibrium with monomeric states in solution. Consistent with the literature, well characterized small cosubstrate, RNA substrate, and product bind with high specificity and affinity to the dimer but not the free monomers. Unsuccessfully designed ligands bind indiscriminately to all forms. Using neutral gas collision, the nsp16 monomer with bound cosubstrate can be released from the holo dimer complex, confirming the binding to nsp16 as revealed by the crystal structure. However, we observed an unusual migration of the endogenous zinc ions bound to nsp10 to nsp16 after collisional dissociation. The metal migration can be suppressed by using surface collision with reduced precursor charge states, which presumably resulted in minimal gas-phase structural rearrangement and highlighted the importance of complementary techniques. With minimal sample input (∼µg), native MS can rapidly detect ligand binding affinities and locations in dynamic multisubunit protein complexes, demonstrating the potential of an "all-in-one" native MS assay for rapid structural profiling of protein-to-AI-based compound systems to expedite drug discovery.

Desulfovibrio desulfuricans and its derived metabolites confer resistance to FOLFOX through METTL3.

BACKGROUND: Chemoresistance is a critical factor contributing to poor prognosis in clinical patients with cancer undergoing postoperative adjuvant chemotherapy. The role of gut microbiota in mediating resistance to tumour chemotherapy remains to be investigated. METHODS: Patients with CRC were categorised into clinical benefit responders (CBR) and no clinical benefit responders (NCB) based on chemotherapy efficacy. Differential bacterial analysis using 16S rRNA sequencing revealed Desulfovibrio as a distinct microbe between the two groups. Employing a syngeneic transplantation model, we assessed the effect of Desulfovibrio on chemotherapy by measuring tumour burden, weight, and Ki-67 expression. We further explored the mechanisms underlying the compromised chemotherapeutic efficacy of Desulfovibrio using metabolomics, western blotting, colony formation, and cell apoptosis assays. FINDINGS: In comparison, Desulfovibrio was more abundant in the NCB group. In vivo experiments revealed that Desulfovibrio colonisation in the gut weakened the efficacy of FOLFOX. Treatment with Desulfovibrio desulfuricans elevates serum S-adenosylmethionine (SAM) levels. Interestingly, SAM reduced the sensitivity of CRC cells to FOLFOX, thereby promoting the growth of CRC tumours. These experiments suggest that SAM promotes the growth and metastasis of CRC by driving the expression of methyltransferase-like 3 (METTL3). INTERPRETATION: A high abundance of Desulfovibrio in the intestines indicates poor therapeutic outcomes for postoperative neoadjuvant FOLFOX chemotherapy in CRC. Desulfovibrio drives the manifestation of METTL3 in CRC, promoting resistance to FOLFOX chemotherapy by increasing the concentration of SAM. FUNDING: This study is supported by Wuxi City Social Development Science and Technology Demonstration Project (N20201005).

Genome-wide identification, expression profiling, and protein interaction analysis of the CCoAOMT gene family in the tea plant (Camellia sinensis).

BACKGROUND: The caffeoyl-CoA-O methyltransferase (CCoAOMT) family plays a crucial role in the oxidative methylation of phenolic substances and is involved in various plant processes, including growth, development, and stress response. However, there is a limited understanding of the interactions among CCoAOMT protein members in tea plants. RESULTS: In this study, we identified 10 members of the CsCCoAOMT family in the genome of Camellia sinensis (cultivar 'HuangDan'), characterized by conserved gene structures and motifs. These CsCCoAOMT members were located on six different chromosomes (1, 2, 3, 4, 6, and 14). Based on phylogenetic analysis, CsCCoAOMT can be divided into two groups: I and II. Notably, the CsCCoAOMT members of group Ia are likely to be candidate genes involved in lignin biosynthesis. Moreover, through the yeast two-hybrid (Y2H) assay, we established protein interaction networks for the CsCCoAOMT family, revealing 9 pairs of members with interaction relationships. CONCLUSIONS: We identified the CCoAOMT gene family in Camellia sinensis and conducted a comprehensive analysis of their classifications, phylogenetic and synteny relationships, gene structures, protein interactions, tissue-specific expression patterns, and responses to various stresses. Our findings shed light on the evolution and composition of CsCCoAOMT. Notably, the observed interaction among CCoAOMT proteins suggests the potential formation of the O-methyltransferase (OMT) complex during the methylation modification process, expanding our understanding of the functional roles of this gene family in diverse biological processes.

Deciphering alternative splicing events and their therapeutic implications in colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors with complex molecular regulatory mechanisms. Alternative splicing (AS), a fundamental regulatory process of gene expression, plays an important role in the occurrence and development of CRC. This study analyzed AS Percent Spliced In (PSI) values from 49 pairs of CRC and normal samples in the TCGA SpliceSeq database. Using Lasso and SVM, AS features that can differentiate colorectal cancer from normal were screened. Univariate COX regression analysis identified prognosis-related AS events. A risk model was constructed and validated using machine learning, Kaplan-Meier analysis, and Decision Curve Analysis. The regulatory effect of protein arginine methyltransferase 5 (PRMT5) on poly(RC) binding protein 1 (PCBP1) was verified by immunoprecipitation experiments, and the effect of PCBP1 on the AS of Obscurin (OBSCN) was verified by PCR. Five AS events, including HNF4A.59461.AP and HNF4A.59462.AP, were identified, which can distinguish CRC from normal tissue. A machine learning model using 21 key AS events accurately predicted CRC prognosis. High-risk patients had significantly shorter survival times. PRMT5 was found to regulate PCBP1 function and then influence OBSCN AS, which may drive CRC progression. The study concluded that some AS events is significantly different in CRC and normal tissues, and some of these AS events are related to the prognosis of CRC. In addition, PRMT family-driven arginine modifications play an important role in CRC-specific AS events.

Beta-elemene: A phytochemical with promise as a drug candidate for tumor therapy and adjuvant tumor therapy.

BACKGROUND: ß-Elemene (IUPAC name: (1 S,2 S,4 R)-1-ethenyl-1-methyl-2,4-bis(prop-1-en-2-yl) cyclohexane), is a natural compound found in turmeric root. Studies have demonstrated its diverse biological functions, including its anti-tumor properties, which have been extensively investigated. However, these have not yet been reviewed. The aim of this review was to provide a comprehensive summary of ß-elemene research, with respect to disease treatment. METHODS: ß-Elemene-related articles were found in PubMed, ScienceDirect, and Google Scholar databases to systematically summarize its structure, pharmacokinetics, metabolism, and pharmacological activity. We also searched the Traditional Chinese Medicine System Pharmacology database for therapeutic targets of ß-elemene. We further combined these targets with the relevant literature for KEGG and GO analyses. RESULTS: Studies on the molecular mechanisms underlying ß-elemene activity indicate that it regulates multiple pathways, including STAT3, MAPKs, Cyclin-dependent kinase 1/cyclin B, Notch, PI3K/AKT, reactive oxygen species, METTL3, PTEN, p53, FAK, MMP, TGF-ß/Smad signaling. Through these molecular pathways, ß-elemene has been implicated in tumor cell proliferation, apoptosis, migration, and invasion and improving the immune microenvironment. Additionally, ß-elemene increases chemotherapeutic drug sensitivity and reverses resistance by inhibiting DNA damage repair and regulating pathways including CTR1, pak1, ERK1/2, ABC transporter protein, Prx-1 and ERCC-1. Nonetheless, owing to its lipophilicity and low bioavailability, additional structural modifications could improve the efficacy of this drug. CONCLUSION: ß-Elemene exhibits low toxicity with good safety, inhibiting various tumor types via diverse mechanisms in vivo and in vitro. When combined with chemotherapeutic drugs, it enhances efficacy, reduces toxicity, and improves tumor killing. Thus, ß-elemene has vast potential for research and development.

Suxiao Jiuxin Pill alleviates myocardial ischemia/reperfusion-induced autophagy via miR-193a-3p/ALKBH5 pathway.

BACKGROUND: Myocardial ischemia/reperfusion injury (MIRI) poses a formidable challenge to cardiac reperfusion therapy due to the absence of effective clinical interventions. Methylation of N6-methyladenosine (m6A), which is the most common post-transcriptional modifications occurring within mammalian mRNA, is believed to be involved in MIRI by modulating autophagy. MicroRNAs (miRNAs) play a crucial role in regulating gene expression at the post-transcriptional level and have been implicated in the regulation of m6A methylation. Suxiao Jiuxin Pill (SJP) is extensively used in China for the clinical treatment of angina pectoris and confers benefits to patients with acute coronary syndrome who have received percutaneous coronary intervention. However, the precise mechanisms underlying SJP intervention in MIRI remain unclear. PURPOSE: This study aimed to demonstrate, both in vivo and in vitro, that SJP could alleviate autophagy in MIRI by regulating miR-193a-3p to target and upregulate the demethylase ALKBH5. METHODS: An in vitro hypoxia/reoxygenation model was established using H9c2 cells, while an in vivo MIRI model was established using Wistar rats. A lentivirus harboring the precursor sequence of miR-193a-3p was employed for its overexpression. Adeno-associated viruses were used to silence both miR-193a-3p and ALKBH5 expressions. Cardiac function, infarct size, and tissue structure in rats were assessed using echocardiography, triphenyl tetrazolium chloride (TTC) staining, and HE staining, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was employed to detect the levels of apoptosis in rat cardiac tissue. m6A methylation levels were assessed using colorimetry. GFP-RFP-LC3B was used to monitor autophagic flux and transmission electron microscopy was used to evaluate the development of autophagosomes. Western Blot and qRT-PCR were respectively employed to assess the levels of autophagy-related proteins and miR-193a-3p. RESULTS: SJP alleviated autophagy, preserved cardiac function, and minimized myocardial damage in the hearts of MIRI rats. SJP attenuated autophagy in H/R H9C2 cells. Elevated levels of miR-193a-3p were observed in the cardiac tissues of MIRI rats and H/R H9C2 cells, whereas SJP downregulated miR-193a-3p levels in these models. ALKBH5, a target gene of miR-193, is negatively regulated by miR-193a-3p. Upon overexpression of miR-193a-3p or silencing of ALKBH5, m6A methylation decreased, and the autophagy-attenuating effects of SJP and its components, senkyunolide A and l-borneol, were lost in H/R H9C2 cells, whereas in MIRI rats, these effects were not abolished but merely weakened. Further investigation indicated that the METTL3 inhibitor STM2475, combined with the silencing of miR-193a-3p, similarly attenuated autophagy in the hearts of MIRI rats. This suggests that a reduction in m6A methylation is involved in autophagy alleviation. CONCLUSION: We demonstrated that SJP mitigates autophagy in MIRI by downregulating miR-193a-3p, enhancing ALKBH5 expression, and reducing m6A methylation, a mechanism potentially attributed to its constituents, senkyunolide A and l-borneol.

METTL3-dependent N6-methyladenosine modification is involved in berberine-mediated neuroprotection in ischemic stroke by enhancing the stability of NEAT1 in astrocytes.

Ischemic stroke (IS) is one of the principal causes of disability and death worldwide. Berberine (BBR), derived from the traditional Chinese herbal medicine Huang Lian, has been reported to inhibit the progression of stroke, but the specific mechanism whereby BBR modulates the progression of ischemic stroke remains unclear. N6-methyladenosine (m6A) modification is the most typical epigenetic modification of mRNA post-transcriptional modifications, among which METTL3 is the most common methylation transferase. During the study, the middle cerebral artery occlusion/reperfusion (MCAO/R) was established in mice, and the mice primary astrocytes and neurons induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was simulated in vitro. Level of LncNEAT1, miR-377-3p was detected via RT-qPCR. The levels of Nampt and METTL3 were measured by Western blot. CCK8 and LDH assay was performed to detect cell viability. Here, we found that berberine alleviates MCAO/R-induced ischemic injury and up-regulates the expression of Nampt in astrocytes, miR-377-3p inhibits the expression of Nampt in astrocytes after OGD/R, thus promoting neuronal injury. NEAT1 binds to miR-377-3p in OGD/R astrocytes and plays a neuronal protective role as a ceRNA. METTL3 can enhance NEAT1 stability in OGD/R astrocytes by modulating m6A modification of NEAT1. Taken together, our results demonstrate that berberine exerts neuroprotective effects via the m6A methyltransferase METTL3, which regulates the NEAT1/miR-377-3p/Nampt axis in mouse astrocytes to ameliorate cerebral ischemia/reperfusion (I/R) injury.

Comparative Analysis of Two NNMT Bisubstrate Inhibitors through Chemoproteomic Studies: Uncovering the Role of Unconventional SAM Analogue Moiety for Improved Selectivity.

Unconventional S-adenosyl-L-methionine (SAM) mimics with enhanced hydrophobicity are an adaptable building block to develop cell-potent inhibitors for SAM-dependent methyltransferases as targeted therapeutics. We recently discovered cell-potent bisubstrate inhibitors for nicotinamide N-methyltransferase (NNMT) by using an unconventional SAM mimic. To delve into the selectivity implications of the unconventional SAM mimic, we employed a chemoproteomic approach to assess two potent NNMT inhibitors LL320 (Ki, app = 6.8 nM) and II399 (containing an unconventional SAM mimic, Ki, app = 5.9 nM) within endogenous proteomes. Our work began with the rational design and synthesis of immobilized probes 1 and 2, utilizing LL320 and II399 as parent compounds. Systematic analysis of protein networks associated with these probes revealed a comprehensive landscape. Notably, NNMT emerged as the top-ranking hit, substantiating the high selectivity of both inhibitors. Meanwhile, we identified additional interacting proteins for LL320 (38) and II399 (17), showcasing the intricate selectivity profiles associated with these compounds. Subsequent experiments confirmed LL320's interactions with RNMT, DPH5, and SAHH, while II399 exhibited interactions with SHMT2 and MEPCE. Importantly, incorporating the unconventional SAM mimic in II399 led to improved selectivity compared to LL320. Our findings underscore the importance of selectivity profiling and validate the utilization of the unconventional SAM mimic as a viable strategy to create highly selective and cell-permeable inhibitors for SAM-dependent methyltransferases.

Efficacy of Yisui granule on myelodysplastic syndromes in SKM-1 mouse xenograft model through suppressing Wnt/ß-catenin signaling pathway.

OBJECTIVE: To unmask the underlying mechanisms of Yisui granule (, YSG) for the treatment of Myelodysplastic syndromes (MDS). METHODS: Our study used an SKM-1 mouse xenograft model of MDS to explore the anti-tumor potential of YSG and its safety, assess its effect on overall survival (OS), and evaluate whether its mechanism is associated with the demethylation of the secreted frizzled related protein 5 (sFRP5) gene and suppressing Wnt/ß-catenin pathway. Bisulfite amplicon sequencing was applied to detect the level of methylation of the sFRP5 gene; western blotting, immunofluorescence staining, and real-time Polymerase Chain Reaction were performed to detect DNA methyltransferase 1 (DNMT1), sFRP5, and other Wnt/ß-catenin pathway-related mRNA and protein expression. RESULTS: The results showed that high-dosage YSG exerted an anti-tumor effect similar to that of decitabine, improved OS, and reduced long-term adverse effects in the long term. Mechanically, YSG reduced the expression of DNMT1 methyltransferase, decreased the methylation, and increased the expression of the Wnt/ß-catenin pathway antagonist-sFRP5. Furthermore, components of the Wnt/ß-catenin pathway, including Wnt3a, ß-catenin, c-Myc, and cyclinD1, were down-regulated in response to YSG, suggesting that YSG could treat MDS by demethylating the sFRP5 gene and suppressing the Wnt/ß-catenin pathway. CONCLUSIONS: Our findings demonstrated that YSG could be used alone or in combination with decitabine to improve outcomes in the MDS animal model, providing an alternative solution for treating MDS.

Genome-wide characterization of COMT family and regulatory role of CsCOMT19 in melatonin synthesis in Camellia sinensis.

BACKGROUND: Caffeic acid O-methyltransferase (COMT) is a key enzyme that regulates melatonin synthesis and is involved in regulating the growth, development, and response to abiotic stress in plants. Tea plant is a popular beverage consumed worldwide, has been used for centuries for its medicinal properties, including its ability to reduce inflammation, improve digestion, and boost immune function. By analyzing genetic variation within the COMT family, while helping tea plants resist adversity, it is also possible to gain a deeper understanding of how different tea varieties produce and metabolize catechins, then be used to develop new tea cultivars with desired flavor profiles and health benefits. RESULTS: In this study, a total of 25 CsCOMT genes were identified based on the high-quality tea (Camellia sinensis) plant genome database. Phylogenetic tree analysis of CsCOMTs with COMTs from other species showed that COMTs divided into four subfamilies (Class I, II, III, IV), and CsCOMTs was distributed in Class I, Class II, Class III. CsCOMTs not only undergoes large-scale gene recombination in pairs internally in tea plant, but also shares 2 and 7 collinear genes with Arabidopsis thaliana and poplar (Populus trichocarpa), respectively. The promoter region of CsCOMTs was found to be rich in cis-acting elements associated with plant growth and stress response. By analyzing the previously transcriptome data, it was found that some members of CsCOMT family exhibited significant tissue-specific expression and differential expression under different stress treatments. Subsequently, we selected six CsCOMTs to further validated their expression levels in different tissues organ using qRT-PCR. In addition, we silenced the CsCOMT19 through virus-induced gene silencing (VIGS) method and found that CsCOMT19 positively regulates the synthesis of melatonin in tea plant. CONCLUSION: These results will contribute to the understanding the functions of CsCOMT gene family and provide valuable information for further research on the role of CsCOMT genes in regulating tea plant growth, development, and response to abiotic stress.

A chromosome-scale genome of Peucedanum praeruptorum provide insights into Apioideae evolution and medicinal ingredient biosynthesis.

Peucedanum praeruptorum Dunn, a traditional Chinese medicine rich in coumarin, belongs to the Apiaceae family. A high-quality assembled genome of P. praeruptorum is lacking, which has posed obstacles to functional identification and molecular evolution studies of genes associated with coumarin production. Here, a chromosome-scale reference genome of P. praeruptorum, an important medicinal and aromatic plant, was first sequenced and assembled using Oxford Nanopore Technologies and Hi-C sequencing. The final assembled genome size was 1.83 Gb, with a contig N50 of 11.12 Mb. The entire BUSCO evaluation and second-generation read comparability rates were 96.0 % and 99.31 %, respectively. Furthermore, 99.91 % of the genome was anchored to 11 pseudochromosomes. The comparative genomic study revealed the presence of 18,593 orthogroups, which included 476 species-specific orthogroups and 1211 expanded gene families. Two whole-genome duplication (WGD) events and one whole-genome triplication (WGT) event occurred in P. praeruptorum. In addition to the γ-WGT shared by core eudicots or most eudicots, the first WGD was shared by Apiales, while the most recent WGD was unique to Apiaceae. Our study demonstrated that WGD events that occurred in Apioideae highlighted the important role of tandem duplication in the biosynthesis of coumarins and terpenes in P. praeruptorum. Additionally, the expansion of the cytochrome P450 monooxygenase, O-methyltransferase, ATP-binding cassette (ABC) transporter, and terpene synthase families may be associated with the abundance of coumarins and terpenoids. Moreover, we identified >170 UDP-glucosyltransferase members that may be involved in the glycosylation post-modification of coumarins. Significant gene expansion was observed in the ABCG, ABCB, and ABCC subgroups of the ABC transporter family, potentially facilitating the transmembrane transport of coumarins after bolting. The P. praeruptorum genome provides valuable insights into the machinery of coumarin biosynthesis and enhances our understanding of Apiaceae evolution.

Differential molecular mechanisms of substrate recognition by selenium methyltransferases, INMT and TPMT, in selenium detoxification and excretion.

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase, revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of dimethylselenide to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe.

GLI1 facilitates collagen-induced arthritis in mice by collaborative regulation of DNA methyltransferases.

Rheumatoid arthritis (RA) is characterized by joint synovitis and bone destruction, the etiology of which remains to be explored. Many types of cells are involved in the progression of RA joint inflammation, among which the overactivation of M1 macrophages and osteoclasts has been thought to be an essential cause of joint inflammation and bone destruction. Glioma-associated oncogene homolog 1 (GLI1) has been revealed to be closely linked to bone metabolism. In this study, GLI1 expression in the synovial tissue of RA patients was positively correlated with RA-related scores and was highly expressed in collagen-induced arthritis (CIA) mouse articular macrophage-like cells. The decreased expression and inhibition of nuclear transfer of GLI1 downregulated macrophage M1 polarization and osteoclast activation, the effect of which was achieved by modulation of DNA methyltransferases (DNMTs) via transcriptional regulation and protein interactions. By pharmacological inhibition of GLI1, the proportion of proinflammatory macrophages and the number of osteoclasts were significantly reduced, and the joint inflammatory response and bone destruction in CIA mice were alleviated. This study clarified the mechanism of GLI1 in macrophage phenotypic changes and activation of osteoclasts, suggesting potential applications of GLI1 inhibitors in the clinical treatment of RA.

Microbial production of the plant flavanone hesperetin from caffeic acid.

OBJECTIVE: Hesperetin is an important O-methylated flavonoid produced by citrus fruits and of potential pharmaceutical relevance. The microbial biosynthesis of hesperetin could be a viable alternative to plant extraction, as plant extracts often yield complex mixtures of different flavonoids making it challenging to isolate pure compounds. In this study, hesperetin was produced from caffeic acid in the microbial host Escherichia coli. We combined a previously optimised pathway for the biosynthesis of the intermediate flavanone eriodictyol with a combinatorial library of plasmids expressing three candidate flavonoid O-methyltransferases. Moreover, we endeavoured to improve the position specificity of CCoAOMT7, a flavonoid O-methyltransferase from Arabidopsis thaliana that has been demonstrated to O-methylate eriodictyol in both the para- and meta-position, thus leading to a mixture of hesperetin and homoeriodictyol. RESULTS: The best performing flavonoid O-methyltransferase in our screen was found to be CCoAOMT7, which could produce up to 14.6 mg/L hesperetin and 3.8 mg/L homoeriodictyol from 3 mM caffeic acid in E. coli 5-alpha. Using a platform for enzyme engineering that scans the mutational space of selected key positions, predicting their structures using homology modelling and inferring their potential catalytic improvement using docking simulations, we were able to identify a CCoAOMT7 mutant with a two-fold higher position specificity for hesperetin. The mutant's catalytic activity, however, was considerably diminished. Our findings suggest that hesperetin can be created from central carbon metabolism in E. coli following the introduction of a caffeic acid biosynthesis pathway.

Genetic and pharmacological inhibition of METTL3 alleviates renal fibrosis by reducing EVL m6A modification through an IGF2BP2-dependent mechanism.

BACKGROUND: N6 -methyladenosine (m6A) is of great importance in renal physiology and disease progression, but its function and mechanism in renal fibrosis remain to be comprehensively and extensively explored. Hence, this study will explore the function and potential mechanism of critical regulator-mediated m6A modification during renal fibrosis and thereby explore promising anti-renal fibrosis agents. METHODS: Renal tissues from humans and mice as well as HK-2 cells were used as research subjects. The profiles of m6A modification and regulators in renal fibrosis were analysed at the protein and RNA levels using Western blotting, quantitative real-time polymerase chain reaction and other methods. Methylation RNA immunoprecipitation sequencing and RNA sequencing coupled with methyltransferase-like 3 (METTL3) conditional knockout were used to explore the function of METTL3 and potential targets. Gene silencing and overexpression combined with RNA immunoprecipitation were performed to investigate the underlying mechanism by which METTL3 regulates the Ena/VASP-like (EVL) m6A modification that promotes renal fibrosis. Molecular docking and virtual screening with in vitro and in vivo experiments were applied to screen promising traditional Chinese medicine (TCM) monomers and explore their mechanism of regulating the METTL3/EVL m6A axis and anti-renal fibrosis. RESULTS: METTL3 and m6A modifications were hyperactivated in both the tubular region of fibrotic kidneys and HK-2 cells. Upregulated METTL3 enhanced the m6A modification of EVL mRNA to improve its stability and expression in an insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2)-dependent manner. Highly expressed EVL binding to Smad7 abrogated the Smad7-induced suppression of transforming growth factor-ß (TGF-ß1)/Smad3 signal transduction, which conversely facilitated renal fibrosis progression. Molecular docking and virtual screening based on the structure of METTL3 identified a TCM monomer named isoforsythiaside, which inhibited METTL3 activity together with the METTL3/EVL m6A axis to exert anti-renal fibrosis effects. CONCLUSIONS: Collectively, the overactivated METTL3/EVL m6A axis is a potential target for renal fibrosis therapy, and the pharmacological inhibition of METTL3 activity by isoforsythiaside suggests that it is a promising anti-renal fibrosis agent.

Targeted drug delivery systems for elemene in cancer therapy: The story thus far.

Elemene (ELE) is a group of broad-spectrum anticancer active ingredients with low toxicity extracted from traditional Chinese medicines (TCMs), such as Curcumae Rhizoma and Curcuma Radix, which can exert antitumour activities by regulating various signal pathways and targets. However, the strong hydrophobicity, short half-life, low bioavailability and weak in vivo targeting ability of ELE restrict its use. Targeted drug delivery systems based on nanomaterials are among the most viable methods to overcome these shortcomings. In this review, we first summarize recent studies on the clinical uses of ELE as an adjunct antitumour drug. ELE-based combination strategies have great promise for enhancing efficacy, reducing adverse reactions, and improving patients' quality of life and immune function. Second, we summarize recent studies on the antitumour mechanisms of ELE and ELE-based combination strategies. The potential mechanisms include inducing pyroptosis and ferroptosis, promoting senescence, regulating METTL3-mediated m6A modification, suppressing the Warburg effect, and inducing apoptosis and cell cycle arrest. Most importantly, we comprehensively summarize studies on the combination of targeted drug delivery systems with ELE, including passively and actively targeted drug delivery systems, stimuli-responsive drug delivery systems, and codelivery systems for ELE combined with other therapies, which have great promise in improving drug bioavailability, increasing drug targeting ability, controlling drug release, enhancing drug efficacy, reducing drug adverse effects and reversing MDR. Our summary will provide a reference for the combination of TCMs such as ELE with advanced targeted drug delivery systems in the future.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants. In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch fermentation. ONE-SENTENCE SUMMARY: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).