Directed Biosynthesis of Mitragynine Stereoisomers.

Mitragyna speciosa ("kratom") is used as a natural remedy for pain and management of opioid dependence. The pharmacological properties of kratom have been linked to a complex mixture of monoterpene indole alkaloids, most notably mitragynine. Here, we report the central biosynthetic steps responsible for the scaffold formation of mitragynine and related corynanthe-type alkaloids. We illuminate the mechanistic basis by which the key stereogenic center of this scaffold is formed. These discoveries were leveraged for the enzymatic production of mitragynine, the C-20 epimer speciogynine, and fluorinated analogues.

Phylogeny-Aware Chemoinformatic Analysis of Chemical Diversity in Lamiaceae Enables Iridoid Pathway Assembly and Discovery of Aucubin Synthase.

Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus, and the outgroup Paulownia tomentosa, further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi-iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery.

Reactivity of an Unusual Amidase May Explain Colibactin's DNA Cross-Linking Activity.

Certain commensal and pathogenic bacteria produce colibactin, a small-molecule genotoxin that causes interstrand cross-links in host cell DNA. Although colibactin alkylates DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and ß-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line and incorporating two electrophilic cyclopropane warheads into the final natural product scaffold. This proposed transformation was supported by the detection of a colibactin-derived cross-linked DNA adduct. Overall, this work provides a biosynthetic explanation for colibactin's DNA cross-linking activity and paves the way for further study of its chemical structure and biological roles.

Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring.

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.

HDAC3 controls gap 2/mitosis progression in adult neural stem/progenitor cells by regulating CDK1 levels.

The maintenance of the resident adult neural stem/progenitor cell (NSPC) pool depends on the precise balance of proliferation, differentiation, and maintenance of the undifferentiated state. Identifying the mechanisms that regulate this balance in adult hippocampal NSPCs can provide insight into basic stem cell self-renewal principles important for tissue homeostasis and preventing tumor formation. Pharmacological inhibition of histone deacetylases (HDACs), a class of histone-modifying enzymes, have promising effects in cancer cells, yet the specific roles of individual HDACs in stem cell proliferation is unclear. Here using conditional KO (cKO) mice and in vitro cell culture, we show that histone deacetylase 3 (HDAC3) is required for the proliferation of adult NSPCs. Detailed cell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defect in cell cycle progression through the gap 2/mitosis (G2/M) but not the S phase. Moreover, HDAC3 controls G2/M phase progression mainly through posttranslational stabilization of the G2/M cyclin-dependent kinase 1 (CDK1). These results demonstrate that HDAC3 plays a critical role in NSPC proliferation and suggest that strategies aimed at pharmacological modulation of HDAC3 may be beneficial for tissue regeneration and controlling tumor cell growth.

Inducible knockout of Mef2a, -c, and -d from nestin-expressing stem/progenitor cells and their progeny unexpectedly uncouples neurogenesis and dendritogenesis in vivo.

Myocyte enhancer factor (Mef)-2 transcription factors are implicated in activity-dependent neuronal processes during development, but the role of MEF2 in neural stem/progenitor cells (NSPCs) in the adult brain is unknown. We used a transgenic mouse in which Mef2a, -c, and -d were inducibly deleted in adult nestin-expressing NSPCs and their progeny. Recombined cells in the hippocampal granule cell layer were visualized and quantified by yellow fluorescent protein (YFP) expression. In control mice, postmitotic neurons expressed Mef2a, -c, and -d, whereas type 1 stem cells and proliferating progenitors did not. Based on this expression, we hypothesized that Mef2a, -c, and -d deletion in adult nestin-expressing NSPCs and their progeny would result in fewer mature neurons. Control mice revealed an increase in YFP(+) neurons and dendrite formation over time. Contrary to our hypothesis, inducible Mef2 KO mice also displayed an increase in YFP(+) neurons over time-but with significantly stunted dendrites-suggesting an uncoupling of neuron survival and dendritogenesis. We also found non-cell-autonomous effects after Mef2a, -c, and -d deletion. These in vivo findings indicate a surprising functional role for Mef2a, -c, and -d in cell- and non-cell-autonomous control of adult hippocampal neurogenesis that is distinct from its role during development.

AI model to detect contact relationship between maxillary sinus and posterior teeth.

Objectives: To establish a novel deep learning networks (MSF-MPTnet) based on panoramic radiographs (PRs) for automatic assessment of relationship between maxillary sinus floor (MSF) and maxillary posterior teeth (MPT), and to compare accuracy of MSF-MPTnet, dentists and radiologists identifying contact relationship. Study design: A total of 1035 PRs and 1035 Cone-beam computed tomographys （CBCT）images were collected from January 2018 to April 2022. The relationships were classified into class I and II by CBCT. Class I represents non-contact group, and class II represents contact group. 350 PRs were randomly selected as test dataset and accuracy of MSF-MPTnet, dentists, and radiologists was compared. Results: The intraclass correlation coefficient of dentists was 0.460-0.690 and it was 0.453-0.664 for radiologists. Sensitivity and accuracy of MSF-MPTnet were 0.682-0.852and 0.890-0.951, indicating that the output performance of MSF-MPTnet was reliable. Accuracy of maxillary premolars and molars were 79.7%-90.3 %, 76.2%-89.2 % and 72.9%-88.3 % in MSF-MPTnet model, dentists and radiologists. Accuracy of class I relationship in the MSF-MPTnet model (67.7%-94.6 %) was higher than that of dentists (56.5%-84.6 %) in maxillary first premolars and right second premolar, and accuracy of class I relationship in the MSF-MPTnet model is also higher than radiologists (40.0%-78.1 %) in all teeth positions (p < 0.05). Conclusions: MSF-MPTnet model could increase detecting accuracy of the relationship between MSF and MPT, minimize pseudo contact relationship and reduce frequency of CBCT use.

Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule.

Adult neurogenesis occurs throughout life in the mammalian hippocampus and is essential for memory and mood control. There is significant interest in identifying ways to promote neurogenesis and ensure maintenance of these hippocampal functions. Previous work with a synthetic small molecule, isoxazole 9 (Isx-9), highlighted its neuronal-differentiating properties in vitro. However, the ability of Isx-9 to drive neurogenesis in vivo or improve hippocampal function was unknown. Here we show that Isx-9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult-generated dentate gyrus neurons. Isx-9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx-9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization. Molecular exploration of Isx-9-induced regulation of neurogenesis (via FACS and microarray of SGZ stem and progenitor cells) suggested the involvement of the myocyte-enhancer family of proteins (Mef2). Indeed, transgenic-mediated inducible knockout of all brain-enriched Mef2 isoforms (Mef2a/c/d) specifically from neural stem cells and their progeny confirmed Mef2's requirement for Isx-9-induced increase in hippocampal neurogenesis. Thus, Isx-9 enhances hippocampal neurogenesis and memory in vivo, and its effects are reliant on Mef2, revealing a novel cell-intrinsic molecular pathway regulating adult neurogenesis.

Glycosylation enhances peptide hydrophobic collapse by impairing solvation.

Post-translational N-glycosylation of proteins is ubiquitous in eukaryotic cells, and has been shown to influence the thermodynamics of protein collapse and folding. However, the mechanism for this influence is not well understood. All-atom molecular dynamics simulations are carried out to study the collapse of a peptide linked to a single N-glycan. The glycan is shown to perturb the local water hydrogen-bonding network, rendering it less able to solvate the peptide and thus enhancing the hydrophobic contribution to the free energy of collapse. The enhancement of the hydrophobic collapse compensates for the weakened entropic coiling due to the bulky glycan chain and leads to a stronger burial of hydrophobic surface, presumably enhancing folding. This conclusion is reinforced by comparison with coarse-grained simulations, which contain no explicit solvent and correspondingly exhibit no significant thermodynamic changes on glycosylation.

LncRNA HAND2-AS1 inhibits proliferation and promotes apoptosis of non-small cell lung cancer cells by inactivating PI3K/Akt pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is correlated with high incidence and mortality rate. Functionality of lncRNA HAND2-AS1 is only reported in endometrioid endometrial carcinoma and osteosarcoma. In our study, the role of HAND2-AS1 in NSCLC was investigated. METHODS: We first detected the expression of HAND2-AS1 in lung tissues and serum of both NSCLC patients and healthy controls by qRT-PCR. Correlation between HAND2-AS1 expression level and clinical data of NSCLC patients was analyzed by Chi-square test. NSCLC cells, and cell proliferation, cell apoptosis and expression of PI3K/Akt pathway-related proteins were detected by CCK-8 assay, cell apoptosis assay and Western blot, respectively. RESULTS: HAND2-AS1 expression was significantly down-regulated in NSCLC. HAND2-AS1 and tumor size of NSCLC patients were closely associated. Serum HAND2-AS1 can be used to effectively distinguish osteosarcoma patients from healthy controls, and it can also be used to predict prognosis of osteosarcoma patients. HAND2-AS1 overexpression inhibited osteosarcoma cell proliferation, promoted cell apoptosis, and down-regulated phosphorylation of PI3K/Akt pathway-related proteins. PI3K/Akt pathway inhibitor showed no significant effects on HAND2-AS1 expression, but reduced its effects on cell proliferation and apoptosis. CONCLUSION: We conclude that HAND2-AS1 may suppress the proliferation of NSCLC cells by targeting PI3K/Akt pathway.

The human gut bacterial genotoxin colibactin alkylates DNA.

Certain Escherichia coli strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin's chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing E. coli Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing E. coli and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis.

Is New Zealand water fluoridation justified?

Public health programmes extend beyond the clinical context and focus on measures that affect the lives of large subgroups or the population as a whole. An example of this is community water fluoridation (CWF), the altering of fluoride levels in the water supply with the aim of preventing the initiation and slowing the progression of dental caries lesions for the benefit of entire populations. Despite the unfeasibility of randomised controlled trials of CWF, a large volume of evidence is available on the topic. However, CWF remains a polarising and keenly contested issue. CWF is also an intervention where it is difficult to provide everyone affected with a choice. The Nuffield Council on Bioethics is an independent body that examines and reports on ethical questions, and they have provided a useful ethical framework for considering CWF via the 'stewardship' model. This commentary aims to discuss each of the public health aims and how they can be applied and weighed to reach a justified position about CWF.

Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum.

TroR is a putative metal-dependent regulatory protein that has been linked to the virulence of the human pathogen Treponema pallidum. It shares high homology with the well-known iron-dependent regulatory protein DtxR from Corynebacterium diphtheriae, as well as the manganese-dependent MntR from Bacillus subtilis. However, it has been uncertain whether manganese or zinc is the natural cofactor of TroR to date. Herein, we established an efficient method named "double-fusion tagging" to obtain soluble TroR for the first time. A series of studies, including ICP, CD, fluorescence, ITC, and electrophoresis mobility shift assay (EMSA), were performed to resolve the discrepancies in its metal-binding specificity. In addition, bioinformatic analysis as well as mutation studies were carried out to find the genetic relationships of TroR with its homology proteins. In conclusion, our findings indicate that TroR is a manganese-dependent rather than a zinc-dependent regulatory protein.

[Overexpression of eIF4E gene in acute myeloid leukemia and its relation with disease progression].

The aim of this study was to detect the expression level of eIF4E gene in patients with non-treated, remission and non-remission/relapse acute myeloid leukemia (AML), and other non-malignant haematologic diseases so as to analyze and reveal the relationship of eIF4E gene expression with AML progression. SYBR Green I RT-PCR was used to assay the expression level of eIF4E mRNA extracted from bone marrow mononuclear cells in 30 patients with AML (6 in M2, 5 in M3, 8 in M4, 10 in M5, 1 in M6) and 20 patients with non-malignant hematologic diseases. The ß2-microglubin(ß2M) was used as internal reference and the formula 2(-ΔCt)×100% was applied to calculate the expression level of eIF4E gene. The results showed that the eIF4E expression level (7.098 ± 5.544)% in patients with non-treated and non-remitted/relapsed AML was significantly higher than that in patients with remission (0.964 ± 0.312)% (P < 0.01) and non-malignant hematologic diseases (0.248 ± 0.163)% (P < 0.01). There was no difference between latter two group patients, even though the expression level of eIF4E gene in patients with M4 and M5 was higher. As compared with non-malignant hematologic diseases, the expression level of eIF4E gene of patients with remission patients showed no significant difference. It is concluded that the over-expression of eIF4E gene has been found in patients with AML, and its level obviously decreases along with remission of disease, thus the eIF4E gene may be a surveillance parameter for disease progression.