Background: The combination of probiotics and traditional Chinese medicine (TCM) is a prospective therapy for ulcerative colitis (UC), and its efficacy and safety need to be urgently evaluated.Objective: This study aims to comprehensively assess the efficacy and safety of probiotics combined with TCM for the treatment of UC.Methods: The Pubmed, EMBASE, Cochrane library, China Academic Journals (CNKI), Wan-fang database, Chinese biomedical literature service system (CBM), and Chinese Science and Technology Journals (CQVIP) were searched. Subgroup analysis were designed in accordance with different control drugs, treatment courses, and types of probiotics. The Review Manager software (version 5.4.1) was utilized for statistical analysis.Results: 14 original studies containing 1,154 patients were analyzed and showed that probiotics with TCM was more effective than 5-aminosalicylic acid (5-ASA), probiotics or TCM used individually. Moreover, probiotics combined with TCM could inhibit the intestinal inflammation, reduce the recurrence rate and the incidence of adverse events. The subgroup analysis showed that a mixture of different probiotics was more effective than a single strain.Conclusion: It is suggested that probiotics combined with TCM could effectively control clinical symptoms, inhibit intestinal inflammatory response, and finally slow down the disease progress and reduce the disease recurrence with less adverse events. The mixture of different probiotics used in conjunction with individually tailored TCM is a potential clinical strategy for UC. Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) that caused by inappropriate immune responses to intestinal commensal microbes . It is cAkkermansia muciniphila, Roseburia intestinalis and Bifidobacterium infantis, can improve acute and chronic experimental colitis by regulating the microbiota and inhibiting the inflammatory response  statements checklist .Pubmed, EMBASE, Cochrane library, China Academic Journals (CNKI), Wan-fang database, Chinese biomedical literature service system (CBM), Chinese Science and Technology Journals (CQVIP), and other databases were systematically searched from the founding to November, 2021. No language constraints were considered for publications. The search keywords were following: “traditional Chinese medicine OR Chinese medicine OR Herbal medicine OR Herb” AND “Probiotic OR Probiotics” AND “Ulcerative colitis.”Studies were included if they met the PICOS criteria. Specifically, patients with a definite diagnosis of UC were included in the study and those with Crohn’s disease or unspecified IBD subtypes were excluded. The original literature was included only if probiotics combined with TCM were used a mean of intervention, and excluded if other therapeutic measures were added. To comprehensively assess whether the combined use of probiotics and TCM is effective and necessary, original studies were included, including 5-ASA or probiotics or TCM alone as control measures. The clinical effective rate is the primary outcome in this meta-analysis. The recurrence rate, inflammatory cytokines of proteins, including C-reaction protein (CRP), tumor necrosis factor-α (TNF-α), Interleukin (IL)-6, IL-8, IL-10 and adverse events are the secondary outcomes. Primary outcome must be reported in the original literature, while secondary outcomes are not mandatory. At last, only the randomized controlled trials (RCTs) were included for analysis.The included studies were reviewed independently by two authors (ZY and YS) to extract the following data: first author, publication year, number of patients, probiotic species, control drugs, course, outcomes, follow-up times and adverse events. Any differences were resolved through discussion between the two authors.The Cochrane risk of bias tool was performed to assess the risk of bias of the included studies . The risThe primary outcome was the clinical effective rate. In order to clarify the therapeutic potential of probiotics and TCM and provide theoretical support for their practical application, we set up subgroups analysis of clinical effective rate of different control drugs, treatment courses, and different types of probiotic species. The secondary outcomes were concluded recurrence rate, inflammatory cytokines  and adverse events. Similarly, the subgroup analysis of recurrence rate in different courses was set to analyze the effectiveness of long-term use of probiotics combined with TCM. In addition, subgroups analysis of adverse events was set up to further evaluate the safety of probiotics combined with TCM with the course of treatment and specific symptoms as classification criteria.2. When I2>50%, it is considered to have significant heterogeneity and a random-effects model is used to estimate the pooled effect. In contrast, a fixed-effects model was used. p < 0.05 was considered significant. The funnel plot was conducted to the evaluation of publication bias.The Review Manager software (version 5.4.1) was utilized for statistical analysis. The risk ratio (RR) and 95% confidence interval (CI) were used to analyze the dichotomous variables. The standardized mean difference (SMD) and a 95% CI were applied to assess the continuous variables. Heterogeneity was estimated by Cochran’s Q test and assessed using IAs shown in The general characteristics of 14 included studies were shown in As shown in p < 0.00001, I2 = 54%)  .p = 0.0002, I2 = 0%)    . AdditioI2 = 0%) . In the 2 = 85%) .p < 0.0001, I2 = 0%)  .p = 0.002, I2 = 0%)   . It is w2 = 90%) .Bacillus licheniformis) was used in three studies, but there was no difference in clinical effective rate between the probiotics combined with TCM group and control group  (Bacillus subtilis and Enterococcus Faecium)       . HoweverI2 = 0%) , species2 = 60%)  and prob2 = 57%)  used.p < 0.00001, I2 = 0%)  .p = 0.003, I2 = 0%)    and 1 yeI2 = 0%)  after thp < 0.00001, I2 = 88%)  .p < 0.00001, I2 = 97%)  .p < 0.00001, I2 = 92%)     and incr2 = 99%) . Neverth2 = 99%) .p = 0.0001, I2 = 32%)  .p = 0.0003, I2 = 33%)    but had I2 = 0%) .p = 0.002, I2 = 0%)   . However2 = 80%) .Publication bias was assessed by funnel plots for the included 15 studies which reported the primary outcome. The funnel plots were asymmetrical, suggesting a possible publication bias .in vitro does not directly colonize the host intestine as beneficial bacteria, but regulates the intestinal microbiota via multiple mechanisms to exert therapeutic effects on UC. Probiotics can adhere to epithelial cells during colonization and compete directly with pathogenic bacteria to inhibit their invasion and proliferation. In addition, probiotics can stimulate the production of nutrients needed by beneficial bacteria, such as short-chain fatty acids, thus helping to restore the homeostasis of commensal beneficial bacteria and whole microorganisms in the intestinal tract  in mice  in rats  signaling pathway and inhibiting the release of pro-inflammatory cytokines , has attracted much attention due to its potent anti-inflammatory effects. Curcumin can inhibit the activation of NF-κB signaling pathway and alleviate intestinal inflammation by inhibiting the levels of IL-1, IL-2 and TNF-α (Strobilanthes cusia (Nees) Kuntze (qīng dài), the dried rhizome of Scutellaria baicalensis Georgi (huáng qín), and the dried aboveground part of Andrographis paniculata (Burm.f.) Nees (chuān xīn lián), which are commonly used in the treatment of UC, have been demonstrated to have the effect on the inhibition of the NF-κB signaling pathway (Dà Huáng Mŭ Dān Tāng) can regulate the balance of Th17/Treg cells to improve UC. Another formula, Scutellaria Decoction (Huáng Qín Tāng) can restore the drift of Th1/Th17 cells and the proliferation of Treg cells to improve experimental colitis, which also indicate the advantages and potential of TCM in immune regulation , has therapeutic effect on experimental colitis by restoring intestinal microbial dysbiosis , Coptis Toxin-Resolving Decoction (Huáng Lián Jiĕ Dú Tāng), and Stomach-Calming Powder (Píng Wèi Săn), can inhibit experimental colitis by restoring microbial diversity, increasing the abundance of beneficial bacteria, and inhibiting the growth of pathogenic bacteria  and the dried aboveground part of Portulaca oleracea L. (mă chĭ xiàn). The extracts of the dried root of Codonopsis pilosula (Franch.) Nannf. (dăng shēn), the dried root and rhizome of steamed Panax ginseng C.A.Mey. (hóng shēn) and the dried ripe kernel of Coix lacryma-jobi L. (yì yĭ rén) can also increase the abundance of Lactobacillus and Bifidobacterium, while dăng shēn can also promote the abundance of another probiotic, A. muciniphila, further inhibit the growth of pathogenic bacteria and alleviate colonic inflammation in mice , can also regulate epithelial cell tight junction protein and promote the repair of epithelial barrier by increasing the abundance of A. muciniphila  had better protective effects on intestinal epithelial cells damaged by lipopolysaccharide . Some probiotic bacteria, such as Bifidobacterium and Bacillus, can convert protopanaxadiol ginsenosides Rg3 and Rg5 into ginsenosides Rh2 and Rh3, and promote the partial conversation of protopanaxadiol ginsenosides Rb1, Rb2, and Rc cleavage sugars into monoglycosylated ginsenoside compound K, which ultimately improves the anti-inflammatory and antitumor activities of ginseng , have been proved to have therapeutic effects on UC , the dried rhizome of Atractylodes macrocephala Koidz. (bái zhú), the dried rhizome of Coptis chinensis Franch. (huáng lián), the dried root of Astragalus mongholicus Bunge (huáng qí), the dried ripe seed of Dolichos lablab L. (bái biăn dòu), the dried root of Paeonia lactiflora Pall. (bái sháo), the dried sclerotium of Poria cocos (Schw.) Wolf (fú líng), the dried root of Pulsatilla chinensis (Bunge) Regel (bái tóu wēng), the dried root and rhizome of Salvia miltiorrhiza Bunge (dān shēn), the dried root of Codonopsis pilosula (Franch.) Nannf. (dăng shēn), the dried processed product of leaf or stem and leaf of Strobilanthes cusia (Nees) Kuntze (qīng dài) and the dried rhizome of Dioscorea oppositifolia L. (shān yào), with frequencies of 8, 6, 5, 5, 4, 4, 4, 3, 3, 3, 3, 3, respectively. We further divided these high-frequency medicinals into categories based on their effects. The dried root and rhizome of Glycyrrhiza glabra L. (gān căo), bái zhú, dăng shēn, huáng qí, the dried ripe seed of Dolichos lablab L. (bái biăn dòu), the dried sclerotium of Poria cocos (Schw.) Wolf (fú líng) and the dried rhizome of Dioscorea oppositifolia L. (shān yào) are all Qi-boosting and spleen-strengthening medicinals, which are suitable for UC patients with spleen deficiency and dampness accumulation.To determine the characteristics and patterns of specific herbal medicinals used in the included studies , the dried root of Pulsatilla chinensis (Bunge) Regel (bái tóu wēng), dān shēn, the dried root of Paeonia lactiflora Pall. (bái sháo) and qīng dài are suitable for UC patients with damp-heat syndrome in the large intestine, which have the effects of clearing heat, drying dampness, cooling blood, and detoxication. TCM is characterized by the treatment based on syndrome differentiation (The dried rhizome of ntiation . Accordintiation . Consequntiation .There are still several limits in our study. Firstly, the number of original studies included in this meta-analysis was limited, and some outcomes, such as CRP, IL-8, and IL-10, were not recorded in sufficient trials, potentially biasing the results of meta-analysis. Secondly, the lack of a substantial number of high-quality, multicenter standard RCTs in the included studies, as well as the lack of explanations of random assignment procedures and blinding in some studies, may be the risk factors affecting the quality of our meta-analysis. Moreover, despite using SMD to reduce the impact of diverse data measurement units and methods on the results, the results of the inflammatory cytokines in this meta-analysis still had a high level of heterogeneity. At last, due to a lack of necessary data, we could not comprehensively evaluate the long-term efficacy and safety of probiotics combined with TCM. It is hoped that more relevant studies will be conducted in the future to promote the rational use of probiotics combined with TCM in the treatment of UC.Probiotics combined with TCM can improve the clinical efficiency of UC patients, reduce the recurrence rate and adverse events, suggesting that the combination has the potential for the treatment of UC and has significant clinical promotion value. The interaction between probiotics and TCM improve their therapeutic effects on UC. The combination of different probiotic with TCM guided by syndrome differentiation is a valuable protocol to increase clinical efficacy. More high-quality RCTs are still needed to evaluate the long-term efficacy and safety of probiotics combined with TCM in the treatment of UC.


While the currently-known cellular functions of these genes are diverse, we find that they share similar expression patterns along the neural tube, pharyngeal arches, and later craniofacial structures. As these genes show co-expression in the pharyngeal arches where NCCs reside, we sought to elucidate the effect of individual gene depletion on craniofacial development and NCC migration. We find that reduction of several 16p12.1 genes significantly disrupts craniofacial and cartilage formation, pharyngeal arch migration, as well as NCC specification and motility. Thus, we have determined that some of these genes play an essential role during vertebrate craniofacial patterning by regulating specific processes during NCC development, which may be an underlying mechanism contributing to the craniofacial defects associated with the 16p12.1 deletion.Copy number variants (CNVs) associated with neurodevelopmental disorders are characterized by extensive phenotypic heterogeneity. In particular, one CNV was identified in a subset of children clinically diagnosed with intellectual disabilities (ID) that results in a hemizygous deletion of multiple genes at chromosome 16p12.1. In addition to ID, individuals with this deletion display a variety of symptoms including microcephaly, seizures, cardiac defects, and growth retardation. Moreover, patients also manifest severe craniofacial abnormalities, such as micrognathia, cartilage malformation of the ears and nose, and facial asymmetries; however, the function of the genes within the 16p12.1 region have not been studied in the context of vertebrate craniofacial development. The craniofacial tissues affected in patients with this deletion all derive from the same embryonic precursor, the cranial neural crest, leading to the hypothesis that one or more of the 16p12.1 genes may be involved in regulating neural crest cell (NCC)-related processes. To examine this, we characterized the developmental role of the 16p12.1-affected gene orthologs,  Embryonic development requires the proper function of thousands of genes in order for cells to proliferate and divide, differentiate, migrate long distances to their final destination, and communicate with one another appropriately. Disruption of protein function due to mutations of genes that are required for these processes during embryogenesis can lead to severe developmental defects and neurodevelopmental disorders such as intellectual disabilities (ID) or Autism spectrum disorder (ASD) . GeneticCraniofacial defects are one of the most prevalent congenital defects that can severely affect quality of life . As cranPOLR3E, MOSMO, UQCRC2, and CDR2  RNA polymerase III subunit E (POLR3E) is important for catalyzing the transcription of small RNAs, though the exact function of POLR3E in relation to RNA polymerase III activity is unknown (MOSMO) is a negative regulator of Sonic Hedgehog (Shh) signaling by participating in the degradation of the Frizzled Class receptor, Smoothened  is a component of the mitochondrial respiratory chain complex and is essential for the production of ATP (CDR2) is an onconeural protein that is known to be ectopically expressed in breast or ovarian tumors, resulting in the generation of autoantibodies that leads to cerebellar degeneration , suggesting that they may influence NCC development and migration. Knockdown (KD) strategies were then utilized to assess the contribution of each 16p12.1-affected gene to facial and cartilage development. We previously showed that depletion of polr3e, mosmo, and uqcrc2 led to smaller facial features  using standard methods , polr3e , uqcrc2 , cdr2 , or standard control MO (5-cctcttacctcagttacaatttata-3); purchased from Gene Tools . In knockdown (KD) experiments, all MOs were injected at either the 2-cell or 4-cell stage with embryos receiving injections 2 or 4 times total. Mosmo and control MOs were injected at 12ng/embryo for 50% KD and 20ng/embryo for 80% KD; polr3e and control MOs were injected at 5ng/embryo for 30% KD, 10ng/embryo for 50% KD and 20ng/embryo for 80% KD; uqcrc2 and control MOs were injected at 35ng/embryo for 50% KD and 50ng/embryo for 80% KD; cdr2 and control MOs were injected at 10 ng/embryo for 50% KD. Splice site MOs were previously validated through a Reverse transcriptase polymerase Chain Reaction (RT-PCR), as described (Morpholinos (MOs) were targeted to early splice sites of escribed   and gateway-cloned into pCSF107mT-GATEWAY-3′GFP destination vectors. Constructs used were mosmo-gfp, polr3e-gfp, uqcrc2-gfp, cdr2-gfp, and gfp in pCS2+. In rescue experiments, MOs were injected with mRNA  in the same injection solution.Rescues were performed with exogenous mRNAs co-injected with their corresponding MO strategies  Pizzo e. Xenopusin situ hybridization, performed as previously described  formamide for 20–45 min before prehybridization. During hybridization, probe concentration was 0.5ug/mL.Embryos were fixed overnight at 4°C in a solution of 4% paraformaldehyde in PBS, gradually dehydrated in ascending concentration of methanol in PBS, and stored in methanol at−20°C for a minimum of 2 hours, before escribed . After bXenopus sox9 hybridization probe was a kind gift from Dr. Dominique Alfandari , and the Xenopus twist hybridization probe was a kind gift from Dr. Richard Harland and Dr. Helen Willsey . The templates for making antisense probes for polr3e, mosmo, uqcrc2, and cdr2 were PCR amplified from the reverse transcribed cDNA library, using the following primer sets: polr3e forward, 5′—GGATAGTCGCTCAGAACACG—3′, polr3e reverse, 5′—GGGTCAGCTTTGTCTGGATC—3′, mosmo forward, 5′—TCTGGATGTTTGTTTCTGGCTGC—3′, mosmo reverse, 5′—GGGTAATTTGTAGGGTTGGCCTC—3′, uqcrc2 forward, 5′—TCCTCTCTAGGAGGCTTTACTCTG—3′, uqcrc2 reverse, 5′—GGGAGCCAATTTCACCAATCAG—3′, cdr2 forward,The cdr2 reverse, 5′—GCGCAGATCATACAGCTCCTTC—3′. The antisense digoxigenin-labeled hybridization probes were transcribed in vitro using the T7 MAXIscript kit. Embryos were imaged using a Zeiss AxioCam MRc attached to a Zeiss SteREO Discovery. V8 light microscope. Images were processed in ImageJ.5′—GACAGCAACGTGGAGGAGTTC—3′, and Xenopus embryos were anesthetized with benzocaine and fixed in 4% paraformaldehyde in PBS overnight. Alcian blue staining of embryos was performed based on the Harland Lab protocol. Before ethanol dehydration, embryos were bleached under a fluorescent light in 1.8x saline-sodium citrate, 1.5 H2O2, and 5% (vol/vol) formamide for 30 min. Embryos were imaged in PBS, using a Zeiss AxioCam MRc attached to a Zeiss SteREO Discovery V8 light microscope. Images were processed in ImageJ. Analysis of cartilage structures was performed in ImageJ utilizing the polygon, area, and line functions. Measurements included the average ceratohyal area (outlined cartilage in t-test using Graphpad (Prism).At stage 42, X. laevis embryos were unilaterally injected two times with either control MO or 16p12.1 gene-specific MO and a gfp mRNA construct. The other blastomere was left uninjected. Embryos were raised in 0.1X MMR through neurulation, then sorted based on left/right fluorescence. For NCC specification experiments, embryos were fixed at stage 16, and for pharyngeal arch (PA) visualization, embryos were fixed between stage 25–30. Whole-mount in situ hybridization was then performed according to the previously described procedure. Analysis of NCC specification markers from in situ experiments was performed on dorsal view images in ImageJ by measuring the total area of expression using the polygon tool. Analysis of PAs from in situ experiments was performed on lateral view images in ImageJ. Measurements were taken to acquire: 1) arch area, the area of individual PA determined using the polygon tool, 2) arch length, the length of the distance between the top and bottom of each PA, and 3) arch migration, the ventral most part of the PA to the neural tube. All measurements were quantified by taking the ratio between the injected side versus the uninjected side for each sample, respectively. Statistical significance was determined using a student’s unpaired t-test in Graphpad (Prism).Half KDs were performed at the two-cell stage. 2CO3, 4.25 mM K Gluc, 2 mM MgSO4, 1 mM CaCl2, 17.5 mM Bicine, with 50 ug/mL Gentamycin Sulfate, pH 8.3), before being stripped of vitelline membranes and imbedded in clay with the anterior dorsal regions exposed. Skin was removed above the NCC using an eyelash knife, and NCCs were excised. Explants were rinsed, and plated on fibronectin-coated coverslips in imaging chambers filled with fresh DFA. Tissues were allowed to adhere for forty-five minutes before being moved to the microscope for time-lapse imaging of NCC motility. Microscopy was performed on a Zeiss Axio Observer inverted motorized microscope with a Zeiss 20x N-Achroplan 0.45 NA Phase-contrast lens, using a Zeiss AxioCam camera controlled with Zen software. Images were collected using large tiled acquisitions to capture the entire migratory field. Eight to ten explants, from both control and experimental conditions, were imaged at a six-minute interval, for 6 hours. Data was imported to ImageJ, background subtracted, and cropped to a uniform field size. Migration tracks of individual cells were collected manually using the Manual Tracking plug-in. Mean speed rates were imported to Graphpad (Prism), and compared between conditions using student’s unpaired t-tests. Three independent experiments were performed for each condition.Embryos at stage 17 were placed in modified DFA solution , Alexa Fluor568 phalloidin , and Hoechst 33342 solution . Microscopy was performed on a Zeiss AiryScan inverted motorized microscope with a Zeiss 20X lens, using a Zeiss AxioCam camera controlled with Zen software. Images were acquired using large tiled acquisitions to capture the entire migratory field. Images of five to seven explants, from both control and experimental conditions were imported to ImageJ, and the total number of PH3-labeled positive cells versus the total number of cells were quantified using an automated particle counter after thresholding each image. Cell counts were imported to Graphpad (Prism), and compared between conditions using student’s unpaired t-test. Three independent experiments were performed for each condition.For NCC proliferation, NCC tissue explants were allowed to adhere and migrate on fibronectin-coated coverslips for 4 hours before being fixed in 4% paraformaldehyde in PBS. Explants were permeabilized in 0.1% Triton-X 100 in PBS, blocked with a solution containing 2% bovine serum albumin, 0.1% Triton-X 100 in PBS, and incubated in Phospho-Histone H3 (Ser10) , goat anti-rabbit Alexa FluorOne of the more prominent symptoms in patients with the 16p12.1 deletion are craniofacial dysmorphisms with varying severity . Childrepolr3e, mosmo, uqcrc2, and cdr2, across multiple stages of development in Xenopus laevis embryos. To examine this, we performed whole-mount in situ hybridization with DIG-labeled antisense RNA probes against these four genes , we observed overt defects on cartilage morphology compared to controls , leading to our hypothesis that these genes may impact processes important for the embryonic progenitors of these tissues.Reduced dosage of controls , includicontrols . Surprisial size , it did ial size , nor wasial size . Howevertwist expression patterns between wild-type or KD sides, and we tracked the progress of migratory NCCs  area, length of each individual pharyngeal arch , we hypothesized that depletion of one or more of these genes may disrupt NCC migration and motility. To test this, we utilized single-blastomere injection strategies to generate left-right chimeric embryos, allowing for a side-by-side comparison of ory NCCs . Followiin vitro migration assays were performed, as previously described  antibody as a marker for cell proliferation , NCC motility (polr3e and uqcrc2), and NCC specification (polr3e and mosmo), revealing new basic roles for these genes during embryonic development.To functionally explore the basis of the craniofacial and cartilage defects associated with the 16p12.1 deletion, we analyzed craniofacial phenotypes and cellular mechanisms underlying decreased dosage of four genes affected within this region. Our results show that three genes impacted by this CNV can contribute to normal craniofacial morphogenesis in s laevis , in a mopolr3e and mosmo knockdowns severely impacted nearly all examined aspects of craniofacial morphogenesis and NCC development across early developmental stages, uqcrc2 and cdr2 knockdowns produced very minimal or no phenotypes in these areas.As the 16p12.1 deletion is multigenic in nature, we sought to determine each gene’s contribution to a craniofacial phenotype. Although we have narrowed our studies to focus on how each individual 16p12.1-affected gene contributes to facial patterning, our findings align with the idea that the presentation of symptoms associated with the deletion is a cumulative product of the impacted region. While polr3e, mosmo, and uqcrc2 in both craniofacial and cartilage development. Individual depletion of these genes narrowed facial width and area, and decreased eye size in a way that appears analogous to the smaller head and smaller eye size phenotypes observed in children with the 16p12.1 deletion , or it could also be that, within the 16p12.1 region, polr3e and mosmo are more sensitive to fluctuations of dosage levels than uqcrc2 and cdr2. We also note that there were no other obvious abnormalities in the embryos beyond the craniofacial and brain phenotypes that we report. However, we have not specifically tested other tissue and organ systems such as cardiac or GI, and some children with 16p12.1 deletion do also have cardiac and GI symptoms. Thus, it would be worth examining these systems in the future.While we show that several 16p12.1-affected genes are important for regulating NCC development and subsequent formation of their tissue derivatives, it remains unclear as to why any of these genes may be exceptionally critical in these tissues. Given that they are all generally expressed in the pharyngeal arches and NCC regions, it might be somewhat surprising that only two were associated with major effects. This could be due to compensation from other closely related genes for polr3e significantly affected NCC specification, suggesting that it may be important for regulating transcription of genes necessary in maintaining the identity and subsequent differentiation of NCCs. Studies suggest that loss-of-function of other subunits of RNA polymerase III, POLR1C and POLR1D, are also associated with craniofacial disorders, and specifically with cartilage hypoplasia and cranioskeletal anomalies, due to deficient ribosome biogenesis, increases in cellular death, and deficiencies in NCC migration . Given that POLR3E has been shown to interact with POLR1C and POLR1D, it is plausible that it may function in a similar capacity.One question is how our findings fit within the known roles of these genes, and how they may mechanistically influence NCC-related processes. POLR3E is a subunit of RNA polymerase III, important for regulating the transcription of small RNAs, such as 5S rRNA and tRNAs . In our mosmo can directly impact NCC specification, highlighting a new cell biological role for this gene.Recent work in cell culture suggests that MOSMO acts as a negative regulator of Sonic hedgehog signaling (Shh) by degrading the Frizzled class receptor, Smoothened , and ouruqcrc2 produced both craniofacial and cartilage phenotypes, potentially due to decreased NCC motility. This could be especially damaging in the context of multipotent NCCs, as metabolism is increasingly demonstrated to perform a commanding role in determination of cell fate and subsequent motility (UQCRC2 is a component of the mitochondrial respiratory chain complex III that is required for its assembly and is important for normal mitochondrial activity to produce ATP . Reducedmotility .Cdr2 mRNA can be found in almost all cell types, however, its protein expression is limited to Purkinje neurons, some brainstem areas, and spermatogonia (Xenopus did not produce significant craniofacial or cartilage defects, nor affect NCC developmental processes. It is also possible that other closely related genes, such as cdr1, cdr3, or cdr2l, can functionally compensate for the lack of cdr2 levels.CDR2 is an oncogenic protein that is strongly expressed in the cerebellum . CDR2 isatogonia . This lapolr3e and mosmo showed an enhancement of the brain development phenotype, thus highlighting that the combined impact of loss of function of these genes could have an important effect (Altogether, it is clear that our current knowledge of how these genes ultimately contribute to embryonic craniofacial and cartilage morphogenesis is lacking, and further basic cell biological examination of 16p12.1-affected gene function within a developmental context is necessary for a better mechanistic understanding of the 16p12.1 deletion etiology. Finally, it will also be essential to explore how these genes ultimately synergistically or epistatically regulate the pathology associated with the 16p12.1 deletion. To this aim, our model provides a unique advantage by being a moderate throughput, titratable, and inexpensive system to combinatorially deplete numerous genes simultaneously. For example, our previous work using combinatorial depletion of t effect . Togethe


Saturniidae is one of the most emblematic families of moths, comprising nearly 3000 species distributed globally. In this study, DNA barcode analysis and comparative morphology were combined to describe three new species within the genus Automeris, which is the most diverse genus in the family. Automerisllaneros Decaëns, Rougerie & Bonilla, sp. nov., Automerismineros Decaëns, Rougerie & Bonilla, sp. nov., and Automerisbelemensis Decaëns, Rougerie & Bénéluz, sp. nov. are described from the Colombian Orinoco watershed, the Colombian Eastern Cordillera, and the area of endemism of Belém in the Brazilian Amazonia, respectively. They all belong to the Automerisbilinea  species subgroup, which comprises a number of species that are sometimes difficult to distinguish from each other using morphology alone. Here, the description of these three new species is based on significant differences from their closest relatives, either in terms of wing patterns, genitalia, DNA barcodes or a combination of these features.The  Saturniidae family, popularised as the wild silkmoths, is one of the most emblematic families of moths, because of the giant size, colourful patterns, or tailed hindwings of some of its species. In the latest published checklist  to very large ones approaching a wingspan of 20 cm ; wing patterns can be cryptic, mimicking leaves , can harbour large eyespots , or can be aposematic in colour ; wings can be rounded  or elongated , or some taxa have spectacular tailed hindwings .The hecklist , as manyHemileucinae, the genus Automeris comprises species whose size ranges from small to very large; their main feature is the presence of a large eyespot on the dorsal surface of the hindwings. It is the most diverse genus within the family. In his monograph of the subfamily Hemileucinae, Automerisbilinea , which was defined by Automerisillustris .Within subfamily MV) bulb powered by a small portable generator. A white sheet of 2 m height × 3 m width was used as a reflector. Collecting took place throughout each entire night, i.e., from 18:30 h to 06:30 h, in order to increase the probability of detecting species with different flight behaviours ; in December 2002 in the Boyacá department, Colombia, in an area of humid Andean forest (1500 m in elevation) with moderate level of forest fragmentation (G. Lecourt and DB leg.); and from April to July 2008 in the state of Pará, Brazil, in an area of Amazonian forest with moderate to high levels of forest fragmentation (TD leg.). Moths were attracted by a Mercury Vapour ; 16 ♂♂ and 1 ♀ of Automeriscinctistriga ; 5 ♂ of Automerisfieldi Lemaire, 1969; 5 ♂♂ of Automerisgodartii ; 3 ♂♂ of Automerislemensis Lemaire, 1972; 28 ♂♂ and 1 ♀ of Automerismidea .Morphological features of the prepared specimens were compared with those of the species represented in Automerisllaneros sp. nov., five specimens of Automerismineros sp. nov. and 17 specimens of Automerisbelemensis sp. nov., and we also included sequences of closely related species obtained from the Barcode of Life Data Systems . DNA was extracted using a routine silica-based 96-well extraction automation protocol  within dataset DS-AUTONSP (https://doi.org/10.5883/DS-AUTONSP). An unrooted neighbour joining (NJ) tree was computed on BOLD V4 using p-distances and the BOLD aligner option to compare the sequences obtained from the specimens of the three new species and those of closely related taxa. BOLD was used to calculate uncorrected p-distances between newly described species and their closest relatives. We also used the barcode identification numbers (BINs), i.e., clusters of barcode sequences automatically generated in BOLD which have a high concordance with species, as an additional source of information for species discrimination ;CDB collection of Diego Bonilla ;CFB collection of Frédéric Bénéluz ;CTD collection of Thibaud Decaëns ;IAvH Instituto de Investigación de Recursos Biológicos Alexander von Humboldt ;MNHNMuséum national d’Histoire naturelle of Paris (France);MPEGMuseu Paraense Emilio Goeldi .Taxon classificationAnimaliaLepidopteraSaturniidaeDecaëns, Rougerie & Bonillasp. nov.E69CFE8B-3FA2-5408-83CC-7352B315C882http://zoobank.org/CE1EAA15-8197-4B98-A2F4-062C40A83153Holotype. Colombia • ♂ . Colombia • 15 ♂♂ and 5 ♀♀, all same data as holotype; all specimens collected at MV light except one pair ab ovo, reared on Pyracantharegersiana in Rouen (France) by TD, and 3 ♂♂ and 2 ♀♀ ab ovo, reared on Quercus sp. in Bogotá (Colombia) by L.D. Ramirez and DB. Deposited as follow: 2 ♂♂ and 2 ♀  in IAvH, 5 ♂♂ in MNHN , 6 ♂♂ and 1 ♀ in CTD, 1 ♂ in CFB, 4 ♂♂ and 2 ♀♀ in CCGM, 1 ♂ and 1 ♀ in CDB.Automerismineros sp. nov. is similar to the reddish forms of A.midea, a species with a large and essentially Amazonian distribution . The nearest neighbour is A.belizonensis (1.8% minimum p-distance), from which it can be distinguished by the more rounded shape of the forewings and the more vivid coloration of the hindwings.ion Fig. . Howeverdea Fig. . The apeWingspan: 77–84 mm. Head: dark brown, labial palpi and antennae orange brown. Thorax: dorsally dark brown with red orange piliform scales on the ventral side; legs dark brown. Abdomen: dark orange brown with dark brown piliform scales on the dorsal side; eighth abdominal segment lacking any remarkable sclerotised structure. Forewings: length 40–42 mm, slightly elongated, rounded apex, straight outer margin; dorsal ground colour orange brown, suffused with yellow scales in the ante- and postmedian areas and, to a lesser extent, by pink scales in the median area; antemedial line faint, almost indistinct, only visible as yellow scales bordering its distal edge; postmedial line barely preapical (1–3 mm), slightly convex from apex to vein CuA2, then bent toward the anal margin, yellow in colour, lined distally by a line of black scales; discocellular mark rectangular, darker than the surrounding wing surface, with a dark brown spot in its centre, and three to four small spots of the same colour at its corners. Ventral side with a large dull orange area extending on the main basomedian area, with veins marked in distinctive orange scales; apical area dark brown, extending along outer margin and toward tornus. Postmedial line well marked, dark reddish brown; marginal band diffuse, suffused with yellow scales and disappearing toward apex. Discocellular mark large and black, with a white discal spot in its centre and surrounded by a thin diffuse ring of yellow scales. Hindwings: basomedian area vivid orange-red with a 7–12 mm × 6–8 mm eyespot in its centre formed by, from its centre: a small well marked white pupil surrounded by a dark brown iris, a first large black periocellar ring, a second large yellow ring of the same width, and finally a barely visible line of black scales enclosing the eyespot. Postmedial line black and lunular, distally bordered by a thin line of yellow scales, and proximally by another thin line of yellow and black scales; postmedian area formed by a thin vivid orange-red band and bordered by a large orange-brown marginal band covered with yellow scales. Ventral side light brown, suffused with yellow scales, particularly on the marginal band; venation distally marked with orange scales. Postmedial line dark brown, becoming faint toward the anal margin; discal cell with a small white spot.♂ Fig. . WingspaWingspan ♀ . Ventral side light orange brown, the venation marked by orange scales, marginal band suffused with orange scales; postmedial line dark brown; large black discocellular mark, with a large white discal spot in its centre. Hindwings: Basomedian area brownish red; eyespot with the same structure as in the male, slightly duller in colour; black and lunular postmedial line, bordered by a line of yellow scales on both edges; postmedian area brownish red; marginal band covered with yellow scales. Ventral side light brown, suffused with yellow to pink scales; venation distally marked by orange scales; discal point small and white.n ♀ Fig. : 94 mm. Genitalia ♂  in France (rearing #17 by TD) and on Quercus sp. (Fagaceae) in Colombia (rearing by L. D. Ramirez and DB). Native foodplants remain unknown. Rearing was successful in plastic boxes, feeding larvae with fresh branches changed every 2–4 days. Larvae completed six instars within two months on P.regersiana and pupated in a brown cocoon.Eggs were obtained from a wild collected female. Larvae hatched 22 days after and readily fed on Eggs are white with a black micropyle, laterally flattened, 2 mm diameter × 0.8 mm height, laid in dense cluster of several dozens. First larval instar: head black. Body 4 mm upon hatching, 6 mm maximal length; pale yellow with black scoli and spines. Second instar: Head black. Body 7–8 mm maximal length; brownish yellow dorsally, dull yellow ventrally; scoli and spines dark brown. Third instar: Head black. Body: 14 mm maximal length; brownish yellow dorsally, green yellow ventrally; scoli and spines dark brown. Fourth instar: Head black. Body 19–20 mm maximal length; dark brown dorsally with fine light green stripes, light green ventrally; scoli and spines dark brown to black. Fifth instar: Head green. Body: 35–40 mm maximal length; light green colour with pink dorsal ornamentation, a lateral ivory strip ventrally and dorsally bordered with a thin black line; scoli and spines light green. Sixth instar: Same colour and ornamentation as previous instar; 35–40 mm maximal length. Pupa and cocoon: Last instar larvae spin a thin and supple cocoon of beige silk. Pupa 24–37 mm long, dark brown. Reared adults emerged from the cocoon early in the morning one to two months after pupation.Automerismineros sp. nov. is known form the type locality only, in the Oriental Cordillera of Colombia near Muzo .• ♂ Fig. ; Pará stParatypes (16 ♂♂). Brazil • 13 ♂♂, same data as holotype with different sampling locations in the same area: 4.7990°S, 49.3630°W; 4.8110°S, 49.3670°W; 4.8050°S, 49.3690°W; 4.8040°S, 49.3230°W. Brazil • 1 ♂; Pará state, Pacajá; June 2008; 3.7060°S, 51.0390°W; at MV light; TD leg. Brazil • 2 ♂♂; Maranhão state, Reserva Biologica do Gurupi; 18 Apr. 2010; 4.0014°S, 46.8372°W; at MV light; TD leg. Deposited as follow: 3 ♂♂ in MPEG , 4 ♂♂ in the MNHN , 3 ♂♂ in CFB , 2 ♂♂ in CCGM , 4 ♂♂ in CTD .Automerisbelemensis sp. nov. is closely related to A.cinctistriga and A.godartii from which it is difficult to separate based on wing patterns, particularly if we consider the phenotypic variability that characterises these species  with A.lemensis as the nearest neighbour with 2% minimum uncorrected p-distance  than in A.cinctistriga (4–7 mm). The DNA barcodes of A.llaneros sp. nov. are assigned to a different BIN than those of A.cinctistriga (see discussion), and the two species are very clearly separated in the DNA barcode tree, bringing additional support to their treatment as two distinct species.A.llaneros sp. nov. near A.belizonensis, A.mineros sp. nov., and A.fieldi on the NJ tree , A.belizonensis, and A.fieldi ; postmedial line black, lunular, bordered proximally and distally by thin lines of yellow scales; postmedian area dull orange; marginal band large and grey. Ventral side uniformly orange brown, with a weak oblique postmedial line interrupted before it reaches the costal margin, a vestigial premarginal line forming darker U-shaped marks between veins, and a small white discal point.♂ Fig. . WingspaFemale unknown.Genitalia ♂ . This led us to further explore the question, first by a comparison of male genitalia, used conventionally but whose discriminating characters between closely related species in this group sometimes remains uncertain or even equivocal , perfectly fitting the distribution of A.cinctistriga as described in the literature (BIN (BOLD:ABZ3239) was represented by the two Colombian specimens used herein as type material for the description of A.llaneros sp. nov. This position is also consistent with wing ornamentation, since the lectotype represented by A.cinctistriga.In the case of ree Fig. , making  species . In the terature , 2002. TAutomeris does not represent a surprising finding. The cryptic diversity of Automeris has already been highlighted by recent taxonomic studies, in which traditionally recognised species with large geographical distributions have been divided into several new species based primarily on differences in DNA barcodes (Saturniidae, which nevertheless is among the best studied within the “Heterocera”.Overall, the discovery of new species, including some cryptic ones, in a highly diverse genus such as barcodes . This unThe fact that species traditionally recognised as having a wide distribution prove to be in fact complexes of cryptic species with more restricted distributions also raises new and interesting questions concerning the specificity of the different biogeographical areas which constitute the neotropics. The three regions from which the species described in our study originate are a perfect example. The saturniid fauna is considered to be made up of a mixture of endemics and widely distributed species, with variable proportions depending on the regions. For instance, the Eastern Cordillera of Colombia is considered to be a hotspot of diversity, harbouring a significant proportion of endemics , whereas


In this study, we have therefore evaluated the influence of these factors on the hydroxycinnamic acid and luteolin derivative levels in three categories of artichoke: Seed-propagated open-pollinated cultivars; vegetatively propagated cultivars; and seed-propagated hybrids. The hydroxycinnamic acids and luteolin derivatives were quantified by RP-HPLC-DAD. The average flower head weight was the lowest in tertiary heads and GA3-treated artichokes, followed by secondary and main heads. Moreover, the hydroxycinnamic acid and luteolin derivatives levels were significantly higher in tertiary heads than in secondary or main heads. In addition, the GA3 treatment significantly reduced the hydroxycinnamic acid content and, in contrast, improved luteolin derivatives levels. These effects depended on the flower head order and cultivar. Knowledge of the effects of flower head order and GA3 treatment is therefore key in order to achieve the greatest health-benefits from artichoke consumption.Flower head orders and the use of GA Asteraceae family. Artichoke is a rich source of antioxidant compounds and contains high amounts of total phenolic compounds [Globe artichoke is a perennial plant belonging to the ompounds , mainly ompounds ,3. Althoompounds . The exiompounds . Polypheompounds . Differeompounds ,8.The production of globe artichokes is largely based on vegetatively propagated clones of basal and lateral offshoots, as is the case in the ‘Blanca de Tudela’ cultivar. The most widely cultivated artichoke cultivar in Spain, ‘Blanca de Tudela’ has two production periods due to its reflowering ability: The first one coincides with the autumn-winter season (early production), and the second one, with the spring season (late production). ‘Blanca de Tudela’ has been characterised as an early cultivar. However, vegetatively propagated genotypes have several disadvantages, including susceptibility to pathogen transmission, physiologically heterogeneous plant material, a low rate of multiplication and limited flexibility in the transplant schedule, high planting costs and a high percentage of planting failures . Differe3), which stimulates both cell elongation and division in addition to the development of lateral heads. The exogenous supply of GA3 is used to accelerate and synchronize flowering, promote early harvest, and enhance yield [3 treatment is influenced by genotype, the sowing/planting date, concentrations added, and the frequency and number of applications [3 application was critical for decreasing time to flowering and altering the cynarin and chlorogenic acid levels in globe artichoke.In the same vein, the use of different chemical regulators has also been studied for controlling growth and earliness. The most widely used compound is gibberellic acid . Therefore, the main aim of this research has been to study the influence of flower head order and GA3 treatment on the individual phenolic content of globe artichoke cultivars obtained by different propagation methods in order to fill this knowledge gap.In our previous work , we stud2 for each cultivar was used, and nine artichoke plants were distributed at 1.67 × 0.8 m . The experimental design was carried out in randomised block trials with two replicates for each cultivar and treatment. An experimental plot of 12 m × 0.8 m . The see × 0.8 m : Two vegw/v. The doses applied were variable depending on the artichoke cultivar. ‘Lorca’ and ‘Olympus F1’ were treated with 30 ppm GA3 (1.875 cc L−1), and ‘Calicó’, ‘Shymphony F1’, ‘Madrigal F1’, and ‘Sambo F1’ were treated with 60 ppm (3.75 cc L−1). Gibberellic acid was applied every 15 days, when the plant had produced 7–8 true leaves, and a total of three treatments were applied to the plants during the crop cycle . No GA3 treatment was applied to the ‘Blanca de Tudela’ cultivar during the cycle due to its high degree of earliness. For the hormonal treatment, a commercial product was used in soluble concentrate form with a gibberellic acid concentration of 1.6% 3 ranged from 14 November 2018 to 9 May 2019, with a total of 24 harvest dates along the entire crop cycle. For control or untreated artichokes, the harvest dates ranged from 4 December 2018 to 9 May 2019 with 22 harvest dates in total. Artichokes were harvested every 7–10 days following commercial export criteria, when flower heads reached their average commercial size. Next, the artichokes were classified according to the flower head order: Main, secondary, and tertiary heads. After harvest, the artichokes were transported to the laboratory and analysed on the same day. Ten artichokes per head order and cultivar were analysed individually.Crop management was performed according to the standard commercial practices used by growers in southeastern Spain. The harvest dates for artichokes treated with GAArtichokes of each cultivar, flower head order, and treatment were harvested and weighed with 15–20 cm of the floral stem using a Radwag WLC 2/A2 Precision Balance  with accuracy to two decimal places. The average flower head weight was expressed as the mean ± SE in grams (g).® (IKA TP 18) and then centrifuged at 10,000× g for 15 min at 4 °C. The homogenate was then filtered through 0.45 µm CHROMAFIL® filters , according to the method described by Martínez-Esplá et al. [For the phenolic extraction, 5 g of fresh receptacle was ground with 15 mL of methanol 80% containing 2 mM NaF for 2 min in an Ultra-Turraxá et al. , for use−1. The gradient started with 1% solvent to reach 15% B at 15 min, 30% at 30 min, 40% at 40 min, and 95% at 45 min, which was maintained to 50 min and then returned to 1% up until 60 min. Chromatograms were recorded at 320 and 360 nm for hydroxycinnamic acids and luteolins, respectively. Hydroxycinnamic acids were identified by their mass, spectra, and retention time, as can be seen in O-caffeoylquinic acid calibration curve. Luteolin derivatives were identified using the available literature [O-rutinoside, as it was reported previously [The quantification of phenolic compounds was carried out using RP-HPLC-DAD. Phenolic extracts (20 µL) were analysed using a 1200 Infinity series HPLC coupled with diode array detection . The chromatographic separation was performed using a Luna C18 column . The mobile phase was 1% formic acid in water (solvent A) and in acetonitrile (solvent B) with a flow rate of 1 mL minterature ,27 and ieviously ,27. An eO-caffeoylquinic acid ; 5-O-caffeoylquinic acid or chlorogenic acid ; 1,3-di-O-caffeoylquinic acid or cynarin ; 3,4-di-O-caffeoylquinic acid ; 3,5-di-O-caffeoylquinic acid ; and 4,5-di-O-caffeoylquinic acid . As for luteolin derivatives, the following three were identified: Luteolin 7-O-glucuronide 3-O-glucoside ; luteolin 7-O-glucuronide ; and luteolin 7-O-glucoside . The results for the major hydroxycinnamic acids and luteolins were expressed as g·kg−1, and the minor hydroxycinnamic acids were expressed as mg·kg−1 of fresh weight (FW). The total phenolic concentration was calculated as the sum of individual phenolics and was expressed as g·kg−1. Data are the mean ± SE.The following hydroxycinnamic acids were identified: 3-p < 0.05) among artichoke head orders for each cultivar and treatment. For each cultivar and flower head order, significant differences at p < 0.05 between the treatments were analysed using Student’s t-test. All of the analyses were performed using the SPSS software package, version 22 .Data were subjected to the analysis of variance (ANOVA). The sources of variation detected were the artichoke head order and treatment. Mean comparisons were carried out using a multiple range test (Tukey’s HSD test) to find significant differences , but smaller ones can often be found weighing 150–200 g) . Heads a0–200 g .3. As far as we know, this is the first time that the influence of flower head orders on average weight has been reported in artichoke. In all of these cultivars studied, the main head orders showed a significantly higher average weight than the secondary and tertiary heads, and the tertiary heads were significantly smaller than the secondary ones . In the 3) greatly affects the vernalisation of artichokes when applied to plants with 10 or more true leaves [3 influences the leaves initiation and stem elongation, as well as the development of lateral heads [3 treatment negatively affected the average weight of ‘Calicó’, ‘Sambo’, ‘Madrigal’, ‘Symphony’, and ‘Olympus’ cultivars. Specifically, this treatment significantly reduced the average weight of the tertiary heads in the vegetatively propagated cultivars studied and the main, secondary or tertiary heads in the seed-propagated hybrids, depending on the cultivar. The GA3 treatment significantly decreased the average weight of tertiary heads in most of the seed-propagated hybrids studied, with the exception of the ‘Symphony’ cultivar. Although applying GA3 to artichokes primarily affects flower initiation and early harvest, it can have a negative impact on head size, weight, and total yield [3 has a great influence on the average weight of some flower head orders, but this influence directly depends on the cultivar , ‘Madrigal’ and ‘Symphony’ (with treatment), and ‘Calicó’ (with and without treatment) A,B. Specultivars A. On the3-treated ‘Calicó’ and ‘Sambo’ artichokes and untreated ‘Tupac’ artichokes. ‘Tudela’, GA3-treated ‘Tupac’, and untreated ‘Olympus’ showed significant differences between heads, and the 3-CQA content increased from the main to the tertiary heads. In the ‘Tupac’ and ‘Symphony’ cultivars, the GA3 treatment mainly increased the 3-CQA content in the three flower head orders. The amounts of the following minor hydroxycinnamic acids were also determined in the flower head orders: 3-CQA, 1,3-diCQA, and 4,5-diCQA. Data are shown in 3. The 4,5-diCQA content was significantly higher in tertiary heads than in secondary heads, however, and the secondary heads showed more 4,5-diCQA than the main heads in the GA3-treated ‘Olympus’ artichokes and in the untreated ‘Tupac’ and ‘Tudela’ artichokes. The GA3 treatment significantly reduced the 4,5-diCQA levels in the three flower head orders of the ‘Tupac’, ‘Calicó’, and Madrigal’ cultivars.On the other hand, 1,3-diCQA is derived from 3,5-diCQA by an unknown enzyme. Most of the cultivars studied, ‘Lorca’, ‘Tupac’, ‘Sambo’, ‘Symphony’, ‘Madrigal’, and ‘Olympus’, showed significant differences in the 1,3-diCQA content between the three flower head orders. In these cultivars, the 1,3-diCQA content was significantly higher in untreated artichokes and in the tertiary heads than in the main and secondary heads. The 4,5-diCQA is derived from the same biosynthesis pathway as 1,3-diCQA from the biosynthesis of 3,5-diCQA mediated by the CQT enzyme (hydroxycinnamoyl-CoA quinate cinnamoyl transferase). No significant differences were observed in the 4,5-diCQA content among flower head orders of the ‘Calicó’, ‘Sambo’, and ‘Symphony’ cultivars treated with GAArtichoke is highly abundant in polyphenols, especially hydroxycinnamic acid derivatives, which represent a significant fraction of the whole secondary metabolites. In agreement with several studies performed on artichoke crops ,35,36,37This variability in caffeoylquinic acid derivative levels in artichoke heads has also been reported in relation to different harvest times represented by the different classes of heads  in the ‘Violetto di Provenza’ cultivar . In this3 decreased the levels of all hydroxycinnamic acids compared to the levels found in untreated artichokes, with the exception of two minor phenolic compounds—3-CQA and 1,3-diCQA—whose levels slightly increased in the treated head orders , and ‘Tupac’ and ‘Olympus’ showing the lowest levels (≈2.50–6.50 g·kg−1 FW). Our results concerning the total phenolic content of the ‘Symphony’ cultivar agree with the results of Turkiewicz et al. [−1 DW). In the present work, however, ‘Sambo’ artichoke showed higher total phenolic levels than those reported by Turkiewicz et al. [The total phenolic content, calculated as the sum of individual phenolic compounds, varied among genotypes, with ‘Tudela’, ‘Sambo’, ‘Symphony’, and ‘Madrigal’ displaying the highest levels  [As seen in the present study and observed previously ,8,43, th(FuFoSe) . Indeed, phenolic compounds are reported to have beneficial effects in the treatment of hepatobiliary diseases, hyperlipidaemia, dropsy, rheumatism, and cholesterol metabolism . These b3-treated plants [3 treatments decreased the phenolic content in the edible portion of the ‘Imperial Star’ heads. Our results highlight the fact that the functional quality of artichokes is strongly influenced by genotype, with high variability in individual phenolic content influenced by two main factors: Flower head orders and gibberellic acid treatment.Similar results were obtained in a previous study in which the edible receptacles of flower heads produced by control artichoke plants had a higher phenolic content than those of the GAd plants . These aOur results report, for the first time, that the levels of individual phenolic compounds  in seed-propagated open-pollinated, vegetatively propagated, and seed-propagated hybrid globe artichoke cultivars are highly influenced by both the flower head order and gibberellic acid treatment. Firstly, tertiary head orders showed the highest individual phenolic content, followed by secondary and main heads, and this effect was cultivar-dependent. Secondly, the gibberellic acid treatment affected the content of these bioactive compounds in different ways. Artichokes treated with gibberellic acid showed lower hydroxycinnamic acid concentrations than the untreated artichokes, but greater luteolin derivative content in those gibberellic acid-treated artichokes levels. The influence of gibberellic acid depended on the flower head order and artichoke cultivar. In conclusion, the results of this study are an attempt to fully characterise the individual phenolic influence in certain globe artichoke cultivars. Our aim is to contribute knowledge to the study of the functional aspects of this important traditional plant food and to gain a better understanding of their potential health-promoting properties. Moreover, this study could be of use for improving the use of gibberellic acid treatments in artichoke crops so that the inflorescences have greater beneficial effects for human health.


In type 2 diabetes, hyperuricemia is associated with cardiovascular disease (CVD) and the metabolic syndrome (MetS), but associations in type 1 diabetes (T1D) have not been well-defined. This study examined the relationships between serum urate (SU) concentrations, clinical and biochemical factors, and subsequent cardiovascular events in a well-characterized cohort of adults with T1D. In 973 participants with T1D in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study (DCCT/EDIC), associations were defined between SU, measured once in blood collected 1997–2000, and (a) concurrent MetS and (b) incident ‘any CVD’ and major adverse cardiovascular events (MACE) through 2013. SU was higher in men than women , and was associated with MetS features in both . During follow-up, 110 participants (11%) experienced “any CVD”, and 53 (5%) a MACE. Analyzed by quartiles, SU was not associated with subsequent CVD or MACE. In women, SU as a continuous variable was associated with MACE  even after adjustment for age and HbA1c . Predominantly normal range serum urate concentrations in T1D were higher in men than women and were associated with features of the MetS. In some analyses of women only, SU was associated with subsequent MACE. Routine measurement of SU to assess cardiovascular risk in T1D is not merited.Trial registration clinicaltrials.gov NCT00360815 and NCT00360893. The MetS can also occur in type 1 diabetes (T1D) and is associated with increased risk for micro- and macrovascular complications4. Regardless of cause, hyperuricemia has been implicated in gout, cardiovascular disease (CVD), hypertension, and renal injury6. SU concentrations are higher in men than women, yet for reasons not fully elucidated, associations with macrovascular disease are stronger in women7. The sex-specific associations between SU and subsequent vascular events in T1D remain poorly defined. To address this deficit, we utilized the remarkable long-term data collected by the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study.Elevated serum concentrations of urate (SU) may be caused by inherited disorders of metabolism and/or acquired conditions, such as the metabolic syndrome (MetS), renal impairment, obesity, and type 2 diabetes (T2D)11, then related SU to ‘any CVD’ event and ‘major adverse cardiovascular events’ (MACE) occurring over a median of 14 years of follow-up. Due to known sex differences in SU concentrations and associations with CVD7, and of sex differences in risk factors such as CRP, insulin sensitivity and lipoproteins14, sex-specific analyses were performed.For SU determination, we used samples from DCCT/EDIC participants who had no clinically evident macrovascular disease at “baseline”, defined for this purpose as 1997–2000. We determined sex-specific associations of SU with concurrent clinical status including features of the MetS16. Briefly, the DCCT aimed to determine if intensive diabetes management could prevent or delay the onset and/or progression of microvascular complications in T1D. Between 1983 and 1989, 1441 participants with T1D aged 13–39 years were randomized to conventional (n = 730) or intensive (n = 711) diabetes therapy16. In parallel, two cohorts were defined: a primary prevention cohort (n = 726) with 1–5 years duration of T1D, no retinopathy and urinary albumin excretion rate (AER) < 40 mg/24 h; and a secondary intervention cohort (n = 715) with 1–15 years duration of T1D, mild-to-moderate non-proliferative retinopathy and urinary AER < 200 mg/24 h. Baseline exclusion criteria included hypertension (≥ 140/90 mmHg or medication use), hyperlipidemia (fasting serum cholesterol level ≥ 3 SD above age- and sex-specific means or medication use), other CVD, and other significant medical conditions. In 1993, after an average of 6.5 years follow-up, the DCCT was terminated early because of clear-cut benefit of intensive treatment on the primary end-point, retinopathy, and similar benefits on nephropathy and neuropathy16. In 1994, the majority (96%) of the surviving cohort entered the observational follow-up EDIC study15, which continues today with 94% of survivors continuing participation. During DCCT, the mean HbA1c in the intensive vs. conventional treatment arms were 7.2% and 9.1%, respectively, but throughout EDIC the two treatment arms have maintained similar HbA1c of 8.0%15.The study met the Declaration of Helsinki guidelines and was approved by the MUSC Institutional Review Board and all participating DCCT/EDIC centers. Each participant was over 18 years of age and provided written informed consent. The design, methods, and outcomes of the DCCT/EDIC study have been detailed previouslyIn 1996, a collaboration between the DCCT/EDIC and the Medical University of South Carolina (MUSC) was established to identify markers and mechanisms for vascular complications of diabetes, and 25 of 28 DCCT/EDIC clinical centers participated. Among 987 participants, 973 (98.6%)  were free of CVD at the time of blood collection for the present study (1997–2000). These subjects represented 71% of the 1375 EDIC participants and were representative of the entire DCCT/EDIC cohort..17. For the present study, SU was measured on a single occasion in fasting serum samples collected 1997–2000.During annual EDIC assessments, height, weight, blood pressure, and pulse rate were recorded, and venous blood was collected and sent to the DCCT/EDIC Central Biochemistry Laboratory  for measurement of HbA1c by high performance liquid chromatography. Fasting lipids  and AER were measured in alternate years. Fasting blood was also drawn for the MUSC study and serum aliquots were prepared and shipped overnight on ice to MUSC, then stored (− 80 °C) until thawed for SU and other research measures. LDL-cholesterol was calculated using the Friedewald equation. Kidney disease was defined as microalbuminuria (i.e. AER ≥ 30 mg/24 on ≥ 2 consecutive visits) during DCCT/EDIC. Estimated glomerular filtration rates (eGFR) were calculated from serum creatinine measured annually during EDIC. A measure of insulin sensitivity, the estimated glucose disposal rate  was calculated using an equation developed by Williams et alHyperlipidemia was defined as LDL-C ≥ 130 mg/dl or use of lipid-lowering medications. Hypertension was defined as one or more of: systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, documented hypertension, or use of anti-hypertensive medications, including angiotensin converting enzyme (ACE) inhibitors or angiotensin II receptor blocker (ARB) drugs for any reason. Metabolic syndrome (MetS) was defined as having two or more of the following during EDIC: (1) systolic BP ≥ 130 mmHg, diastolic ≥ 85 mm Hg, or use of anti-hypertensive medications for any reason; (2) waist circumference ≥ 88 cm (women) or ≥ 102 cm (men); (3) HDL-cholesterol < 50 mg/dl (women) or < 40 mg/dl (men); and (4) fasting triglycerides ≥ 150 mg/dl.18.CVD events were identified through 2013 and were adjudicated by a committee masked to DCCT treatment assignment and HbA1c. “Any CVD” was defined as any of the following: non-fatal myocardial infarction (MI); stroke; death judged to be secondary to CVD; subclinical (“silent”) MI detected on an annual electrocardiogram; angina confirmed by ischemic changes with exercise tolerance testing or clinically significant obstruction on coronary angiography; congestive heart failure ; angioplasty and/or coronary artery bypass. MACE was defined as any of the following: CVD death, non-fatal MI, or stroke19. The normal reference range of SU is 1.5–6.0 mg/dl in women and 2.5–7.0 mg/dl in men. Elevated concentrations are defined as being above these upper limits7, or alternately, a combined level of > 6.8 mg/dl2. In the present work, sex-specific SU concentrations are presented as quartiles and as continuous variables.Serum urate was measured on first-thaw sera by an enzymatic/spectrophotometric assay detecting hydrogen peroxide using an auto-analyzer at the MUSC Central Laboratory12.C-Reactive Protein (CRP) was determined in sera by high-sensitivity nephelometry  as previously described, with the lower limit of detection being 0.2 mg/l, and intra- and inter-assay CVs < 2% and < 11%, respectivelyAnalyses were performed in SAS 9.4 . Participant characteristics at the time of sample acquisition (1997–2000) were reported by sex-specific SU quartiles. Comparisons between quartiles were made using the Kruskal–Wallis test for continuous variables and chi-square test for categorical variables. Cox proportional hazards regression models were used to evaluate the relationship between SU, as a fixed covariate, and subsequent “any CVD” and MACE  and 2.0% of men (> 7.0 mg/dl)7.As shown in Fig. Demographic, clinical, and biochemical characteristics for men and women according to serum urate quartiles are shown in Table The prevalence of MetS at the time of SU sampling did not differ significantly between prior DCCT randomization groups  but was higher in the secondary intervention than the primary prevention group . SU did not differ according to these prior DCCT categories. As shown in Table CRP levels were higher in women than men . In addition to the association between SU quartiles and CRP in women, mean (SD) SU also correlated with concurrent CRP in women , but not in men . The statistically significant SU-CRP correlation in women did not persist after adjustment for age, BMI and HbA1c (p = 0.0939).20, respective serum urate concentrations  in women were: 3.4 ± 1.0 (n = 357), 3.6 ± 1.0 (n = 58), and 3.5 ± 1.0 (n = 18) mg/dl, p = 0.1325; and in men: 4.4 ± 0.9 (n = 509), 5.1 ± 1.3 (n = 26), and 5.0 ± 1.8 (n = 5), p = 0.0186. Thus, in men only, CRP-defined CVD risk category was associated with significantly higher SU.Using the same CRP-defined CVD risk categories for both sexes, There were 110 participants (11%) who experienced “any CVD” event, including 61 men (11%) and 49 women (11%). In the combined cohort (both sexes), there were no significant associations between baseline serum urate as a continuous variable and subsequent “any CVD” events  or MACE . This unstratified model assumes that the background cardiovascular risk is the same for the men and women. A sex-stratified model, allowing for the possibility that background risk differs, yielded similar results .2 at the time of SU measurement did not materially alter these findings.Sex-specific associations between baseline SU and subsequent CVD during EDIC are presented in Table There were no significant associations between SU quartiles and subsequent “any CVD” events or MACE for either sex Fig. . As onlyIn this study of middle-aged and older adults with T1D from the DCCT/EDIC cohort, we demonstrated generally normal serum urate concentrations, with higher values in men than women, as expected. SU concentrations were associated with features of the MetS, especially in women. In a longitudinal analysis through 2013, ‘baseline’ SU concentrations (as a continuous variable) were associated with subsequent MACE in women only, no association was found with “any CVD” in either sex. Results do not support the routine measurement of urate in T1D for CVD risk assessment.21. Relationships of urate with ACE/ARB medication may be explained by use of these agents in people with increased albuminuria and/or with recognized CVD risk, and by the association of renal dysfunction with elevated urate concentrations2 We also noted higher SU concentrations in those with than without MetS, and in both sexes, SU concentrations increased with the number of features of MetS present. Higher SU concentrations were associated with concurrent renal dysfunction, reflected by both lower eGFR and sustained increases in AER  were associated with coronary heart disease in women only, and this persisted after adjustment for hypertension and nephropathy23. In the other, the Steno Diabetes Center study (n = 676), higher SU concentrations were associated with CVD on univariate analysis, but the association was not independent of traditional CVD risk factors24.In T1D, links between the MetS and increased risk of renal/cardiovascular disease, and between renal dysfunction and cardiovascular disease or death are well recognizedWe noted sex differences in the relationship of SU to subsequent MACE (associated in women only), but not to “any CVD” (no association in either sex). The definition of “any CVD” encompasses a wider range of clinical scenarios than MACE, including revascularization procedures Table , and may27, even though SU levels are usually lower in women.Quartiles of SU were not significantly associated with subsequent “any CVD” or MACE in either sex. A more rigorous stratified analysis of SU in both sexes, which did not assume equal vascular event risk in men and women, demonstrated borderline statistical significance. In concert with these findings, other studies in the general population have noted sex disparities, with hyperuricemia predicting risk for CVD and mortality risk more effectively in women than men29. Also, they do not present sex-specific analyses but instead include sex as a variable in a cohort-wide analysis, if at all. In the Steno Diabetes Center study (n = 670) with median follow-up 5.2 and 6.2 years for cardiovascular events and mortality, respectively, higher baseline urate concentrations were associated with significantly higher risk of cardiovascular events, mortality and renal decline, even after adjustment for multiple risk factors28. Unlike the Steno Study, we reported results for each sex separately. However, using a more rigorous sex-stratified analysis, which did not assume similar event risk for men and women, and after adjustment for multiple covariates, our results did not reach statistical significance.Other T1D studies, from the Steno Diabetes Centre and the Preventing Early Renal Loss in Diabetes (PERL) trial that related serum urate to future hard clinical events are smaller, and/or have fewer events and/or shorter follow-up time than our DCCT-EDIC study29.The PERL trial (n = 530) used an entirely different approach, testing the effect of reducing SU concentrations with allopurinol. After ~ 3 years follow-up, there was no effect on the rate of decline in GFR (primary end-point) or the incidence of CVD events (secondary end-point)30. In another CACTI cohort analysis, baseline SU predicted CAC progression even after adjusting for many traditional risk factors, and addition of baseline SU concentrations to the model improved the related C-statistic and non-event reclassification index31. Sex-specific analyses were not presented31. Our study, which is not concordant, provides sex-specific analyses, uses ‘hard’ cardiovascular events rather than surrogate end-points, and has longer follow-up.With regard to associations of SU with surrogate measures of macrovascular disease in T1D, the Coronary Artery Calcification in Type 1 Diabetes (CACTI) Study found that SU predicted CAC progression independent of conventional CVD risk factors (including MetS) in those without renal diseaseOur study’s strengths include its focus on T1D, similar numbers of men and women, and especially its use of the well-characterized DCCT/EDIC cohort with long follow-up and hard clinical end-points. Limitations include the low numbers of subjects with elevated serum urate concentrations, low macrovascular event rates, large number of potential covariates, use of a single SU measure, lack of a non-diabetic comparator group, lack of detailed documentation of exposure to drugs that alter SU levels , and lack of genetic data. SU was measured several years after the randomization phase (DCCT) was completed; however, our analyses are adjusted for mean updated HbA1c minimizing the risk of bias in this regard. More data will be available in future as vascular events accrue in the DCCT/EDIC.In the well-characterized DCCT/EDIC cohort, we found generally normal serum urate concentrations that were higher in men than women. SU was strongly associated with features of the MetS and CRP in both sexes. Over a median of 14-years follow-up, baseline SU was associated with MACE in women only, but this association did not persist in a full multivariate analysis. Our study does not support the routine measurement of urate in early-middle aged people with T1D as a means to assess cardiovascular risk. Further studies in the DCCT/EDIC cohort will evaluate longer periods of follow-up as age advances and cardiovascular events accrue.


Of the 65 789 individuals who received care in Ahwaz, Iran, 59 085 were excluded, and of the 22 134 individuals who received care in Shiraz, Iran, 19 568 were excluded. This article has been corrected.1In the Original Investigation titled “COVID-19 Infection Risk Among Women in Iran Exposed vs Unexposed to Children Who Received Attenuated Poliovirus Used in Oral Polio Vaccine,”


For mid movies (1970-1999), Bombay/Mumbai should read 68, not 73; Delhi should read 45, not 46; Pune should read 8, not 9; Bangalore/Bengaluru should read 7, not 8; Nagpur should read 6, not 7; Kolkata/Calcutta (18), Simla/Shimla (12), Madras/Chennai (10) should be included; and Agra, Daman, and Rampur should be removed. For new movies (2000-2020), Bombay/Mumbai should read 83, not 91; Delhi should read 52, not 63; Bangalore/Bengaluru should read 9, not 11; Pune should read 8, not 10; Lucknow should read 7, not 8; Hyderabad should read 6, not 9; Kolkata/Calcutta (9) and Madras/ Chennai (6) should be included; and Agra and Chandigarh should be removed. The title of Table 9 has also been updated to include the full year  rather than a shortened form (1950-69).In Figure 11C, “No representation” should read “Least/No representation,” and “States never mentioned in 70 years” should read “Least/No State Mentions in the last 70 years”. Additionally, the legend (C) should now read “States with least or no representation (less than 0.2% movies in the entire corpus) in our corpus in the last 70 years” rather than “States never mentioned in our corpus in the last 70 years.”In the text, the sentence “As of 2021, India has 29 states and seven union territories” was incorrect and should read “As of 2021, India has 28 states and eight union territories.” Also, regarding under-representation in northeastern states, we inadvertently included Manipur and Tripura in the sentence “In fact, there have been zero mentions of the states of Manipur, Arunachal Pradesh, Meghalaya, Tripura, and Mizoram in over 700 movies across 70 years.” It should instead read “In fact, there have been zero mentions of the states of Arunachal Pradesh, Meghalaya, and Mizoram in over 700 movies across 70 years.”These errors have now been corrected online, the overall broad conclusions of the paper remain unaffected by the updated results, and the authors sincerely apologize for these errors.


Consistent with prior reports, our respondents were less physically active  and more sedentary (+11%) during the pandemic as compared to 6-months before. The pandemic also increased psychological stress (+22%) and brought on moderate symptoms of anxiety and depression. Respondents’ whose mental health deteriorated the most were also the ones who were least active . The majority of respondents were unmotivated to exercise because they were too anxious , lacked social support (+6%), or had limited access to equipment (+23%) or space (+41%). The respondents who were able to stay active reported feeling less motivated by physical health outcomes such as weight loss (-7%) or strength (-14%) and instead more motivated by mental health outcomes such as anxiety relief (+14%). Coupled with previous work demonstrating a direct relationship between mental health and physical activity, these results highlight the potential protective effect of physical activity on mental health and point to the need for psychological support to overcome perceived barriers so that people can continue to be physically active during stressful times like the pandemic.The COVID-19 pandemic has impacted the mental health, physical activity, and sedentary behavior of people worldwide. According to the Health Belief Model (HBM), health-related behavior is determined by perceived barriers and motivators. Using an online survey with 1669 respondents, we sought to understand why and how physical activity and sedentary behavior has changed by querying about perceived barriers and motivators to physical activity that changed because of the pandemic, and how those changes impacted mental health. The following results were statistically significant at  During the initial phase of the COVID-19 pandemic, governing bodies worldwide took decisive action to protect their citizens against the novel coronavirus by enforcing public lockdown and closing all non-essential services –8. A majmoderate-intensity aerobic exercise three times weekly may be equally effective at reducing psychological distress and decreasing symptoms of depression and anxiety without any of the drug-related side effects such as nausea, fatigue, or loss of appetite [One of the most effective interventions to prevent stress-induced mental illness is physical activity . People appetite , 16.However, maintaining a regular exercise program is difficult at the best of times and the conditions surrounding the COVID-19 pandemic may be making it even more difficult. The World Health Organization (WHO) recommends adults participate in 150 min/week of moderate-intensity aerobic physical activity  and 2 or more days per week of muscle-strengthening activities . GloballWhy has the COVID-19 pandemic exaggerated physical inactivity and sedentary behaviour? According to the Health Belief Model health-related behavior, and specifically PA, is related to perceived barriers and motivators . TypicalThe purpose of this study, therefore, was to determine what it is about the pandemic that is making people less active. Additionally, we aimed to unveil the most pertinent motivators reported by those continuing to meet daily PA targets. More specifically, we were interested in shifts in people’s perceived barriers and motivators to being physically active during the pandemic. Using an online survey, the present study gathered information from 1669 respondents pertaining to their physical activity, sedentary behavior and mental health before and during the initial lockdown of COVID-19. Results from this survey not only describe changes in barriers and motivators to PA, but provide concrete factors practitioners can explore with clients in an effort to promote PA behavior during a global pandemic.The ethics application and supporting documents for the study were reviewed and cleared by the McMaster University Research Ethics Board (MREB #3808) to ensure compliance with the Tri-Council Policy Statement and the McMaster Policies and Guidelines for Research Involving Human Participants. Consent was obtained via an online survey preamble. To achieve a small margin of error based on a population size of 37 million, we recruited a total of 1669 respondents over a two-month data collection period , for a 2% margin of error with 95% confidence intervals. The survey was open to all respondents at least 18 years of age, fluent in English, and able to complete the online survey. Respondents were recruited through the personal social media accounts of the research team and through local news sources (news articles by media at McMaster University and Hamilton Spectator). Respondents were also recruited via a link provided at the end of an op-ed piece published in The Conversation Canada, a national independent news source from the academic and research community.The survey consisted of 30 questions and used a mix of multiple-choice, single choice, and short answer questions to query respondents about their demographic information, and their current and past (prior to the pandemic) physical activity behaviour (minutes/week). Additionally, respondents were asked about their current and past mental health status . All questions pertaining to physical activity and mental health were designed using validated rating scales. Respondents were included in a draw for 20 cash prizes of $100 CAD as remuneration for their participation in the form of an emailed prepaid voucher.The Physical Activity and Sedentary Behavior Questionnaire (PASB-Q)  was adapRespondents were asked to report current and prior  barriers preventing them from being physically active using a multiple-choice list  and motivators encouraging them to be physically active  . The barAnxiety was measured using an adapted  version of the Generalized Anxiety Disorder 7-item Scale (GAD-7) . RespondDepression was measured using an adapted version of the Patient Health Questionnaire (PHQ-9) ; all butQuestion 3 from the Perceived Stress Scale (PSS)  was usedTo capture an overall change in mental health since the onset of COVID-19, respondents were asked to rate their overall mental health since COVID in relation to how it was in the six-months prior to COVID with the options of choosing “Much better”, “Better”, “No change”, “Worse”, or “Much worse”.® statistics software platform (Version 26) was used to carry out all analyses. Descriptive statistics  were computed to describe demographic characteristics, mental health, and physical activity levels. Normality was assessed using Shapiro-Wilkes tests and through visual inspection of histograms. For all analyses, significance was considered at p < 0.05, and nonparametric tests were chosen wherever data did not meet the assumption of normality.The IBM SPSSFor correlational analysis, all respondents who left 100% of the survey questions blank were removed (N = 166). Physical activity and mental health data were then screened for missingness which ranged from 8.2–11.8% and 10.2–17.3% respectively. Missing cells were subsequently imputed using expectation-maximization  for all Survey respondents were primarily female between 18–29 years of age, living in Canada and well-educated . Most reH = 2.85, p = < 0.01), 46–65  = 4.15, p = < 0.01), and 65+  = 7.85, p = < 0.01). The same pattern was seen in depression, wherein those aged 18–29 experienced higher depression than those 30–45  = 5.13, p = < 0.01), 46–65  = 5.92, p = < 0.01), and 65+  = 7.61, p = < 0.01). With respect to income, respondents making “less than enough” had significantly higher levels of anxiety and depression than those making “just enough”  = 6.93, p < 0.01; H = 9.19, p < 0.01) and “more than enough”  = 8.64, p < 0.01; H = 10.96, p < 0.01). As well, respondents making “just enough” had significantly higher anxiety and depression than those making “more than enough”  = -3.71 p < 0.01; H = -4.40, p < 0.01).Average anxiety  and depression  scores reflect moderate symptoms for both anxiety and depression. Tables Z = -17.00, p < 0.01) (p < 0.01) now felt stressed “often”  or “very often”  (p = 0.13) feeling stressed or feeling stressed “fairly often” (p = 0.28).To assess changes in self-perceived psychological stress, a Wilcoxon Signed Ranks Test was performed on ratings before and during the initial stages of the COVID-19 pandemic and McNemar’s tests were used to assess changes in frequencies. There was a significant increase in stress levels during the pandemic ( < 0.01) . Since t < 0.01) . The panp < 0.05), their strength-based activity decreased by 32 minutes , and their sedentary times increased by 33 minutes  , “very active” , or “moderately active”  before the pandemic, now identify as being “completely sedentary” . There was no change in the frequency of respondents who self-identified as “elite athletes” (p = 0.12)  . Tables p < 0.01) or “just enough”  = -2.96, p < 0.01) income to meet their needs had significantly lower levels of MVPA during COVID-19 than those making “more than enough”. Although this trend was seen overall, the effect was largest for the 18–29 age group. A similar trend was observed in sedentary behaviour wherein those who made “less than enough” experienced greater increases in daily sedentary time compared to those who made “more than enough”  = 95.14, p < 0.01), and those who made “just enough” also had elevated sedentary time compared to those who made “more than enough  = 56.61, p < 0.05). Similarly, those aged 18–29 reported the greatest sedentary time during the COVID-19 pandemic compared to any other age group.To identify respondents at higher risk for decreased physical activity during the initial stage of the COVID-19 pandemic, Kruskal-Wallis tests were conducted on between-group differences in physical activity change by income and age. Respondents who made “less than enough”  = -3.60, p < 0.01; H = 6.23, p < 0.01).When examining the change in total physical activity level split by the change in mental health status, respondents whose mental health got “worse” or “much worse” had greater reductions in physical activity since COVID-19 than those who experienced “no change” or got “better” or “much better” (r(1544) = -0.12, p < 0.01; r(1544) = -0.21, p < 0.01). This was not only reflected in their activity levels during the pandemic  = -0.18, p < 0.01; r(1540) = -0.31, p < 0.01; ST: r(1542) = -0.14, p < 0.01; r(1542) = -0.22, p < 0.01) but also before  = -0.11, p < 0.01; r(1537) = -0.06, p < 0.01). Similar patterns in anxiety and depression were observed for those who experienced greater increases in sedentary behaviour, such that greater changes in sedentary time were associated with greater anxiety and depression during the pandemic (r(1420) = 0.07, p < 0.01; r(1420) = 0.12, p < 0.01).Spearman’s rank-order correlations were conducted assessing relationships between physical activity  with mental health status Tables  and 3. Op < 0.01), ‘strength building’ , ‘enjoyment’ , ‘appearance goals’ , ‘social engagement’ , ‘sports training’ , and ‘healthcare provider recommended’ . In contrast, respondents reported being more motivated to be physically active for ‘stress reduction’ , ‘anxiety-relief’ , ‘improve sleep’ , and ‘no motivators’  (p > 0.05).To determine whether there were significant changes in the motivators and barriers to engage in physical activity due to COVID-19, McNemar’s tests were conducted. With respect to changes in motivators, respondents reported being less motivated to be physically active for ‘weight loss’  . Viewingp < 0.01), ‘no barriers’ , ‘lack of confidence’ , ‘recent injury’  and ‘insufficient finances’ . In contrast, respondents reported increases in ‘lack of motivation’ , ‘no facility access’ , ‘no equipment’ , ‘increased anxiety’ , and ‘lack of support’ . Barriers to engage in physical activity including ‘no access to childcare’, ‘lack of enjoyment’ and ‘fear of injury’ did not change because of the pandemic  = 29.97, p < 0.01, R2 = 0.02). Respondents who reported greater symptoms of anxiety were more likely to endorse ‘stress relief’ as a motivator  = 26.05, p < 0.01, R2 = 0.02) and ‘anxiety’ as a barrier  = 7.16, p < 0.01, R2 = 0.01) to engage in physical activity during the pandemic.As an exploratory analysis, we conducted a series of linear regressions to determine whether self-reported levels of anxiety and depression predicted self-perceived barriers and motivators to exercise, and they did. Unsurprisingly, respondents who reported greater depressive symptoms were more likely to endorse ‘lack of self-motivation’ as a barrier to engaging in physical activity during the pandemic  adopting a mode of physical activity that supports mental health, and 2) providing support to help minimize perceived psychological barriers to exercise . For exaNot surprisingly, government-mandated closure to gyms and other recreational training facilities made it more difficult for people to be physically active. This was realized as a lack of necessary space and equipment during the pandemic reported as major barriers to being physically active. The pandemic forced a shift in doing everything at home but not everyone’s home is large enough or well-equipped to support their physical activity needs. Indeed, income level was predictive of activity level during the pandemic. People who reported “just enough” or “less than enough” income experienced greater decreases in physical activity and worsening mental health, especially younger adults aged 18 to 29 years old. Interestingly, these findings do not mirror the common trend that physical activity level declines with age , althougOn top of being less active, our respondents reported spending significantly more time seated. The pandemic increased sedentary time by 10% or approximately 30 minutes per day. Although this may not seem like a lot, increasing sedentary time by just one hour has been associated with a 12% greater risk of mortality over a 6-year period . But sedDespite the valuable insights provided by this study; it is not without limitations. Our sample consisted mainly of young (18–29), highly educated (Bachelor’s degree or higher), female-identifying Canadian inhabitants which may limit the generalizability of the results. We recognize that this bias may have been partially attributed to one of our modes of recruitment being through an academic news source. On average, our respondents were meeting the physical activity recommendations , which iIn conclusion, our findings highlight the importance of physical activity in mental health while also capturing the most prevalent perceived barriers and motivators to exercise during a global pandemic. These findings have the potential to inform health and fitness practitioners as they navigate their practice during a pandemic. During stressful times, like the COVID-19 pandemic, people are especially motivated to be physically active for their mental health but may be too anxious or depressed to partake. Our results point to the need for additional psychological supports to help people maintain their physical activity levels during stressful times in order to minimize the psychological burden of the pandemic and prevent the development of a mental health crisis.S1 Appendix(DOCX)Click here for additional data file.