A scaffold protein manages the biosynthesis of steroidal defense metabolites in plants.

Solanaceae plants produce two major classes of valuable sterol derived natural products-steroidal glycoalkaloids and steroidal saponins-from a common cholesterol precursor. Attempts to heterologously produce these molecules have consistently failed, although the genes responsible for each biosynthetic step have been identified. Here we identify a cellulose synthase like protein, an unexpected biosynthetic component that interacts with the early pathway enzymes, enabling steroidal scaffolds production in plants. Moreover, knockout of this gene in black nightshade, Solanum nigrum resulted in plants lacking both steroidal alkaloids and saponins. Unexpectedly, these knockout plants also revealed that steroidal saponins deter serious agricultural insect pests. This discovery provides the missing link to engineer these high value steroidal molecules, and also pinpoints the ecological role for the steroidal saponins.

Genome-based discovery of pachysiphine synthases in Tabernaemontana elegans.

Plant-specialized metabolism represents an inexhaustible source of active molecules, some of which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range of valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case for the pachysiphine derivatives which show interesting antitumor and anti-Alzheimer activities but accumulate at very low levels in several Tabernaemontana species. Unfortunately, genome data in Tabernaemontanaceae are lacking and knowledge on the biogenesis of pachysiphine-related MIAs in planta remains scarce, limiting the prospects for the biotechnological supply of many pachysiphine-derived biopharmaceuticals. Here, we report a raw version of the toad tree (Tabernaemontana elegans) genome sequence. These new genomic resources led to the identification and characterization of a couple of genes encoding cytochrome P450 with pachysiphine synthase activity. Our phylogenomic and docking analyses highlight the different evolutionary processes that have been recruited to epoxidize the pachysiphine precursor tabersonine at a specific position and in a dedicated orientation, thus enriching our understanding of the diversification and speciation of the MIA metabolism in plants. These gene discoveries also allowed us to engineer the synthesis of MIAs in yeast through the combinatorial association of metabolic enzymes resulting in the tailor-made synthesis of non-natural MIAs. Overall, this work represents a step forward for the future supply of pachysiphine-derived drugs by microbial cell factories.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

Oncological outcomes of tumor ablation compared to surgical resection in early-stage hepatocellular carcinomas: a systematic review with meta-analysis.

BACKGROUND: The optimal choice of treatment for early-stage hepatocellular carcinomas (HCC) remains controversial, with recent conflicted guidelines. This systematic review evaluated whether ablation is oncologically non-inferior to surgical resection. METHODS: We performed a systematic search of the EMBASE, MEDLINE, CENTRAL and Web of Science databases to identify randomized controlled trials comparing tumor ablation and surgical resection for early-stage HCCs. A non-inferiority margin of 5% (RR 0.93) for overall survival (OS) was considered, following a consensus of clinical experts. RESULTS: We identified 5829 citations from which 11 trials (n = 1736) were included. The non-inferiority of tumor ablation was not observed for OS (RR 0.92; 95%CI 0.85-1.00,I2 = 33%). Recurrence-free survival was reduced with ablation (RR 0.80; 95%CI 0.69-0.93,I2 = 49%). There was no difference in terms of extra-hepatic recurrence and minor complications. Tumor ablation was associated with decreased overall morbidity (RR 0.43; 95%CI 0.30-0.62,I2 = 31%) and major complications (RR 0.22; 95%CI 0.07-0.71,I2 = 66%). Intra-hepatic recurrence was higher with ablation (RR 1.28; 95%CI 1.10-1.48,I2 = 12%). Certainty of evidence was low to moderate. CONCLUSION: We did not observe the oncological non-inferiority of tumor ablation when compared to surgical resection. Nevertheless, most analyses were of low quality of evidence, including the overall survival. We cannot exclude that the true effect of tumor ablation is different than the currently observed one.

Incorporation of nitrogen in antinutritional Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids.

Geographic Variation in First Lower Extremity Amputations Related to Diabetes and/or Peripheral Arterial Disease.

BACKGROUND: To assess trends of first cases of lower extremity amputation (LEA) related to diabetes and/or peripheral arterial disease (PAD), according to areas of residency and neighbourhood material and social deprivation quintiles, in the province of Quebec, Canada. METHODS: Using the Quebec Integrated Chronic Disease Surveillance System, we calculated crude and age-standardized annual incidence rates of first LEA (total, minor, and major) among adults 40 years of age and older with diabetes and/or PAD in fiscal years 2006 and 2019. Area of residency was compiled in 3 categories: (1) Montreal and other census metropolitan areas; (2) midsize agglomerations (10,000-100,000 inhabitants); and (3) small towns and rural areas (< 10,000 inhabitants). We also stratified according to neighbourhood material and social deprivation quintiles. One-year and 5-year all-cause mortality after first LEA were compared according to area of residency. RESULTS: Among the 10,275 individuals who had a first LEA, age-standardized LEA rates remained stable between 2006 and 2019, whereas major LEA rates declined in all geographical areas and minor LEA rates increased (31.6%) in small towns and rural areas. In 2019, age-standardized LEA rates were higher in midsize agglomerations and small towns and rural areas compared with census metropolitan areas. Age-standardized LEA rates in 2019 were higher among the most deprived quintile compared with the most privileged quintile for material and social deprivation. No difference was observed in mortality after first LEA according to area of residency. CONCLUSIONS: There are health disparities in the burden of diabetes and PAD related to first LEA in the province of Quebec. To improve preventive care and reduce the burden of LEA, targeted actions should be taken among the most deprived groups and rural settings.

Streamlined screening platforms lead to the discovery of pachysiphine synthase from Tabernanthe iboga.

Plant-specialized metabolism is largely driven by the oxidative tailoring of key chemical scaffolds catalyzed by cytochrome P450 (CYP450s) enzymes. Monoterpene indole alkaloids (MIAs) tabersonine and pseudo-tabersonine, found in the medicinal plant Tabernanthe iboga (commonly known as iboga), are tailored with oxidations, and the enzymes involved remain unknown. Here, we developed a streamlined screening strategy to test the activity of T. iboga CYP450s in Nicotiana benthamiana. Using multigene constructs encoding the biosynthesis of tabersonine and pseudo-tabersonine scaffolds, we aimed to uncover the CYP450s responsible for oxidative transformations in these scaffolds. Our approach identified two T. iboga cytochrome P450 enzymes: pachysiphine synthase (PS) and 16-hydroxy-tabersonine synthase (T16H). These enzymes catalyze an epoxidation and site-specific hydroxylation of tabersonine to produce pachysiphine and 16-OH-tabersonine, respectively. This work provides new insights into the biosynthetic pathways of MIAs and underscores the utility of N. benthamiana and Catharanthus roseus as platforms for the functional characterization of plant enzymes.

Quantitative Single-Cell Mass Spectrometry Provides a Highly Resolved Analysis of Natural Product Biosynthesis Partitioning in Plants.

Plants produce an extraordinary array of natural products (specialized metabolites). Notably, these structurally complex molecules are not evenly distributed throughout plant tissues but are instead synthesized and stored in specific cell types. Elucidating both the biosynthesis and function of natural products would be greatly facilitated by tracking the location of these metabolites at the cell-level resolution. However, detection, identification, and quantification of metabolites in single cells, particularly from plants, have remained challenging. Here, we show that we can definitively identify and quantify the concentrations of 16 molecules from four classes of natural products in individual cells of leaf, root, and petal of the medicinal plant Catharanthus roseus using a plate-based single-cell mass spectrometry method. We show that identical natural products show substantially different patterns of cell-type localization in different tissues. Moreover, we show that natural products are often found in a wide range of concentrations across a population of cells, with some natural products at concentrations of over 100 mM per cell. This single-cell mass spectrometry method provides a highly resolved picture of plant natural product biosynthesis partitioning at a cell-specific resolution.

Roles of three cytochrome P450 monooxygenases in triterpene biosynthesis and their potential impact on growth and development.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that 3 cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive 3-step oxidation reaction at the C28 position of ß-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of ß-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6ß position of ß-amyrin/oleanolic acid. The genes encoding these 3 CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps.

Reduction hits the sweet spot: A revised biosynthesis of dolichol.

Dolichol is a lipid that is involved in protein glycosylation, a process that is essential for all eukaryotic life. In this issue of Cell, Wilson and coworkers1 report how a rare human genetic disorder led to the discovery of dolichol biosynthesis.

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo . Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statement: When cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo . Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

Steroidal scaffold decorations in Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato, and eggplant. Although it has been known that SGAs play important roles in defense in plants and "anti-nutritional" effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral, and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum) are reported to have potent anti-tumor, anti-proliferative, and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine, are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome-guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana, and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the starting cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine, and malonyl-solamargine SGA products. We further identified six enzymes from cultivated eggplant that catalyze the production of α-solasonine, α-solamargine, and malonyl-solamargine SGAs from solasodine aglycone via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high-value, steroidal bioactive molecules in heterologous hosts using synthetic biology.

Neuropsychology intervention for managing invisible symptoms of MS (NIMIS-MS) group: A pilot effectiveness and acceptability study.

BACKGROUND: People with MS (pwMS) commonly experience a range of hidden symptoms, including cognitive impairment, anxiety and depression, fatigue, pain, and sensory difficulties. These "invisible" symptoms can significantly impact wellbeing, relationships, employment and life goals. We developed a novel bespoke online group neuropsychological intervention combining psychoeducation and cognitive rehabilitation with an Acceptance and Commitment Therapy (ACT)-informed approach for pwMS in an acute tertiary hospital. This 'Neuropsychological Intervention for Managing Invisible Symptoms' in MS (NIMIS-MS) consisted of 6 sessions, each with a psychoeducation and ACT component. The content included psychoeducation around managing cognitive difficulties, fatigue, pain, sleep and other unpleasant sensations in MS with the general approach of understanding, monitoring, and recognising patterns and potential triggers. Specific cognitive rehabilitation and fatigue management strategies were introduced. The ACT-informed component focussed on three core ACT areas of the 'Triflex' of psychological flexibility (Harris, 2019): Being Present, Opening Up, and Doing What Matters. METHODS: 118 pwMS attended the NIMIS-MS group intervention which was delivered 14 times in six-week blocks over an 18-month period. To evaluate the effectiveness and acceptability, participants completed measures of depression and anxiety (HADS), functional impairment (WSAS), Values- Progress (VQ) and Values- Obstruction (VQ), and Acceptance of MS (MSAS) pre and post NIMIs-MS group intervention. Qualitative feedback was obtained during focus groups after the final session and via online feedback questionnaires RESULTS: Pre-post analysis showed that symptoms of depression and anxiety were significantly lower and acceptance of MS was significantly higher following completion of the NIMIS-MS group. Qualitative feedback showed that participants reported that they felt more equipped to manage the "invisible" symptoms of MS following completion of the group, and benefited from using ACT-based strategies and techniques. Participants highly valued the peer support that evolved during the NIMIS-MS groups. The online format was considered more accessible than in-person groups, due to less concerns of travel time, cost, fatigue, and comfort and infection. CONCLUSION: Evaluation suggests that our novel NIMIS-MS groups is an acceptable, beneficial and feasible approach for providing neuropsychological interventions to individuals with MS.

Legal Needs Arising in Mental Health Settings and Staff Capability and Support to Respond.

Introduction: Legal issues are known to affect and be affected by mental health. But to what extent do legal issues surface in mental health settings and what do staff feel they need to support clients experiencing these issues? These questions were explored by a national mental health service interested in the potential for health justice partnership with local community based legal services. Methods: A survey of 999 frontline staff of a national mental health organisation. 146 staff (15%) responded from 70 service sites across Australia, including peer support workers (47%), support workers (20%), team leaders (17%) and clinicians (15%). Results: Staff identified a wide range of legal issues experienced by their clients (commonly referred to by staff as consumers), most commonly credit, debt and social security issues, housing, family law and family violence. Two-thirds (67%) of respondents indicated that they spent around 50% or more of their time 'responding to these types of issues'. Respondents indicated that they need more support to address legal issues facing their clients, particularly more knowledge of other services, connections with professionals in other organisations and connections with community. They also felt they could benefit from additional processes, tools, and resources, and time to manage their case load. Originality: While there is an emerging field of research exploring the legal capability of citizens, this study explores what mental health service staff feel they need to support consumers experiencing legal issues that can interact with mental health.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae, one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae, we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non-Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

Sex-Specific Cardiometabolic Determinants of Postoperative Atrial Fibrillation After Cardiac Surgery.

BACKGROUND: Cardiometabolic diseases increase the risk of postoperative atrial fibrillation (POAF), a complication leading to higher long-term risk of major cardiovascular events (MACE). It remains unknown whether the effect of these risk factors differs according to sex. We sought to evaluate the sex-specific predictors of POAF after coronary artery bypass grafting (CABG). METHODS: In a prospective registry of patients undergoing isolated CABG, we compared predictors of POAF between sexes with logistic regression models. Because of high prevalence of abdominal obesity in women, > 80% having a waist circumference (WC) ≥ 88 cm, median WC values were used to define abdominal obesity (men ≥ 102 cm, women ≥ 100 cm). RESULTS: This analysis included 6177 individuals (17% women). Mean age was 65.6 ± 8.9 years. POAF occurred in 32% of men and 28% of women (P < 0.05). Compared with men, women with POAF had similar WC; higher prevalence of hypertension and diabetes; lower high-density lipoprotein (HDL)-cholesterol; and higher glucose, triglyceride, low- density lipoprotein (LDL)-cholesterol, and C-reactive protein levels (all P < 0.05). After adjustment, age and abdominal obesity were associated with POAF in both sexes (P < 0.05). The interaction of WC with sex suggested a worse impact of WC on POAF risk among women (adjusted odds ratio [OR], 1.97; 95% confidence interval [CI], 1.48-2.62 vs in men 1.33; 95% CI, 1.17-1.50; P for interaction = 0.01). CONCLUSIONS: Abdominal obesity is a major predictor of POAF in both sexes, with higher risk in women. These results emphasize the need for enhanced strategies to manage abdominal obesity and its cardiometabolic consequences in the general population and the potential to develop sex-specific preventive interventions to reduce risk of POAF.

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Social determinants of long-term reported changes in physical activity and healthy eating during the COVID-19 pandemic in Canada: multiple cross-sectional surveys analysis from the iCARE study.

The long-term consequences of COVID-19 on healthy behaviours (physical activity practice and healthy eating) among Canadians remain largely unexplored. The objectives were (i) to describe the proportion of Canadians who reported a change in healthy behaviours, 9 and 20 months since the beginning of COVID-19; and (ii) to identify the social determinants associated with healthy behaviour changes. Using two representative Canadian surveys from the International COVID-19 Awareness and Responses Evaluation study (January 2021, n = 3000; November 2021, n = 3002), reported changes in healthy behaviours were assessed as follows: "In general, how have the following behaviours changed since the start of COVID-19?": (1) Increase; (2) No change; and (3) Decrease. The association between individual determinants and changes in healthy behaviours was analyzed using weighted univariate polytomous logistic regression models. In January 2021, 41% and 22% of respondents reported a decline in physical activity and healthy eating, respectively, while in November 2021, 34% and 20% of respondents reported a decline in physical activity and healthy eating, respectively. The main determinants associated with changes in healthy behaviours were younger age (18-25 years), area of residency, student status, changes in bodyweight, financial concerns/insecurity, anxiety/depression, and ethnicity. Changes in healthy behaviours were also associated with household composition, presence of chronic diseases, and occupation. In sum, this study depicted long-term changes in healthy behaviours during COVID-19, with differential changes according to social determinants of health. This study highlighted the presence of health inequalities in Canada during COVID-19 and supports the implementation of personalized programs in prevention of healthy behaviour degradation.