Usnea barbata (L.) dry acetone extract (F-UBA) and to investigate their anticancer potential for possible use in oral cancer therapy. U. barbata dry acetone extract (UBA) was solubilized in ethanol: isopropanol mixture and loaded in a formulation containing hydroxypropyl methylcellulose (HPMC) K100 and polyethylene glycol 400 (PEG 400). The UBA influence on the F-UBA pharmaceutical characteristics was evidenced compared with the references, i.e., mucoadhesive oral films containing suitable excipients but no active ingredient loaded. Both films were subjected to a complex analysis using standard methods to evaluate their suitability for topical administration on the oral mucosa. Physico-chemical and structural characterization was achieved by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Pharmacotechnical evaluation  proved that F-UBAs are suitable for oral mucosal administration. The brine shrimp lethality (BSL) assay was the F-UBA cytotoxicity prescreen. Cellular oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBA in blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line were investigated through complex flow cytometry analyses. Moreover, F-UBA influence on both cell type division and proliferation was determined. Finally, using the resazurin-based 96-well plate microdilution method, the F-UBA antimicrobial potential was explored against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019. The results revealed that each UBA-loaded film contains 175 µg dry extract with a usnic acid (UA) content of 42.32 µg. F-UBAs are very thin (0.060 ± 0.002 mm), report a neutral pH (7.01 ± 0.01), a disintegration time of 146 ± 5.09 s, and an ex vivo mucoadhesion time of 85 ± 2.33 min, and they show a swelling ratio after 6 h of 211 ± 4.31%. They are suitable for topical administration on the oral mucosa. Like UA, they act on CLS-354 tumor cells, considerably increasing cellular oxidative stress, nuclear condensation, and autophagy and inducing cell cycle arrest in G0/G1. The F-UBAs inhibited the bacterial and fungal strains in a dose-dependent manner; they showed similar effects on both Candida sp. and higher inhibitory activity against P. aeruginosa than S. aureus. All these properties lead to considering the UBA-loaded mucoadhesive oral films suitable for potential application as a complementary therapy in OSCC.Oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high death rate and an inadequate response to conventional chemotherapeutic drugs. Medical research explores plant extracts’ properties to obtain potential nanomaterial-based anticancer drugs. The present study aims to formulate, develop, and characterize mucoadhesive oral films loaded with  Approximately 3–10% of all cancer mortality is known to be contributed by oral cancer , betel qConsequently, global medical research focuses on discovering and developing alternative therapies against cancer, investigating the properties of plant extracts to obtain potential nanomaterial-based anticancer drugs. According to Dehelean et al. , the antOcimum sanctum L., Curcuma longa L., Vaccinium corymbosum L., Vaccinium macrocarpon Aiton, Momordica charantia L., Azadirachta indica A. Juss, Senegalia Catechu (L.f.) P.J.H. Hurter & Mabb., Dracaena cinnabari Balf.f., Piper nigrum L., and Zingiber officinale Roscoe) and their active secondary metabolites . Their list could be completed with Melissa officinalis L.  mg/mL against S. aureus is similar to CTR of [0.755–0.023] mg/mL. On P. aeruginosa, F-UBA of 3.487 mg/mL acts similarly with CTR of 1.603 mg/mL; lower film concentrations of [1.749–0.055] mg/mL have similar effects with CTR of [1.511–0.023] mg/mL.The inhibitory activity of UBA-loaded mucoadhesive oral films is higher on Candida sp. [Data from dida sp.  in the eCandida sp. in a dose-dependent manner. After 24 h incubation with 3.497 mg/mL F-UBA, both Candida sp. strains are partially dead [Candida sp.  shows 282.78 µg/g of usnic acid from a total phenolic content of 862.843 µg/g. Using a complex UHPLC-ESI-OT-MS-MS analysis, Salgado et al. [U. barbata dry acetone extract was investigated first on A. salina larvae as cytotoxicity prescreen and then on the tongue squamous cell carcinoma (CAL-27 cell line) [U. barbata dry acetone extract displayed dose-dependent cytotoxicity; high doses promoted apoptosis and DNA damage in human blood cells by enhancing ROS levels [In the plant kingdom, lichens are promising sources of antimicrobial and anticancer drugs , and Usnmedicine . For almo et al.  identifio et al. ,77. The ll line) ,25,77. CS levels . Only WBS. aureus, S. pneumoniae, S. pyogenes, Enterococcus sp., other Gram-positive bacteria isolated from oral cavity and pharynx  and P. aeruginosa [C. albicans and C. parapsilosis [Moreover, we evaluated the UBA antimicrobial potential on bacterial and fungal strains responsible for opportunistic oral cavity infections in elderly and/or immunocompromised patients ,79,80: Sruginosa ,81,82,832 tensile strength and 31.85–54.64% elongation, reported by Kraisit et al. [All UBA pharmacological activities could have promising applications in oral medicine; therefore, it was incorporated into mucoadhesive oral films. Through physico-chemical and pharmacotechnical analyses, F-UBA properties were compared with reference ones to evaluate their suitability for application to the oral mucosa. A slight weight difference was observed due to the UBA load in the R formulation, and an appropriate uniformity of the films belonging to the same batch was registered, proving that a homogenous drug loading was achieved. The F-UBA thickness allows application on the oral mucosa, being easily acceptable by patients, and its values are included in the optimal range of 0.005–0.200 mm . This prt et al.  for theiThe efficiency of a plasticizer agent in mucoadhesive film formulations is primarily determined by the amount used and the polymer–plasticizer interaction. When it comes to an aqueous dispersion, the proportion and amount of partition are influenced by the plasticizer’s solubility in water and its affinity for the polymer. The moisture content affects the mucoadhesive film’s physical stability, preventing them from being brittle. The film has retained water due to the plasticizer hygroscopic attributes, and less than 5% moisture loss is required for better physical strength . As expeM. communis had a disintegration time from 3 h to over 24 h and a swelling ratio between 136–272%.The neutral pH confirms that mucoadhesive oral films should not irritate the oral mucosa . In addiThe presence of hydroxyl groups in the HPMC molecule ensures the matrix stability of the swollen hydrophilic polymer . Soon afAll pharmacotechnical parameters indicated no substantial differences between F-UBA and R, confirming that UBA-loaded mucoadhesive oral films are suitable for application to the oral mucosa. Numerous authors, knowing the bioactivities of plant extracts incorporated in various polymeric films, described only their physico-chemical and pharmacotechnical properties ,31,102.In our study, UBA-loaded mucoadhesive oral films have neutral pH, and fast disintegration permitted pharmacological potential evaluation through in vivo and in vitro studies.U. barbata dry acetone extract. Thus, the cytotoxicity prescreen was performed on A. salina larvae. Anticancer activity was assessed on a similar OSCC cell line . Blood cell cultures were used as normal cells because buccal mucosa has a substantial blood supply and is permeable for most blood cells [The F-UBA investigation was performed using similar studies evaluating od cells . The twood cells . Migratood cells . In addition, various substances go through the oral mucosa by simple diffusion, reaching the capillary area under normal conditions. The UBA-loaded mucoadhesive films are projected to remain on oral mucosa different lesions for 85 min. Therefore, it is essential to know their effects on blood cells because sensitization and bleeding are much more frequent in the buccal mucosa. The blood samples were collected from a single donor .The UBA has a considerable UA content, and F-UBA action is expected to be similar to that of the positive control (C2UA). Data analysis shows that F-UBA selectively affected tumor cells. Compared with C1DMSO negative control, it increased ROS levels, nuclear condensation, autophagy, and cell cycle arrest in G0/G1 and diminished DNA synthesis in the CLS-354  tumor cell line. Inducing high oxidative stress, F-UBA triggered the previously mentioned processes that lead to CLS-354 cancer cells’ death. The F-UBA exhibited a protective effect on normal blood cells, diminishing the apoptotic processes due to 1% DMSO: caspase 3/7 activation, cell cycle arrest in G0/G1, nuclear condensation, and autophagy.Usnea lichens, is soluble in acetone and is found in a significant amount in the U. barbata dry acetone extract [A. salina larvae, used as an animal model. After 24 h of exposure to F-UBA, the lipids accumulation in the digestive tube and their progressive penetration in neighboring tissues lead to massive cellular damage and brine shrimp larvae death after 48 h.Usnic acid, the main bioactive metabolite in the  extract . Assessi extract  reportedUsnea sp. extracts, the primary quantified metabolite is usnic acid; the others are not commonly determined individually and are quantified as total phenolic content. The dry acetone extract loaded in F-UBA contains total phenolic compounds of 862.84 mg PyE/g, from which 241.83 mg/g is usnic acid [Nevertheless, UA exhibits beneficial bioactivities , displaying a dual redox behavior  on the surface of their mucoadhesive oral patches. After incubation, the patches were examined by direct imprinting on a solid culture medium; the growth of bacteria and yeast strains was observed and expressed as the number of colony-forming units (CFU) on the surface of the imprint. Chiaoprakobkij et al. [S. aureus and E. coli, counting the living bacterial cells on the films loaded with Garcinia mangostana L. extract. Other authors also selected S. aureus and E. coli for antibacterial activity evaluation of their poly/plant extracts films [The antimicrobial activity of oral films containing plant extracts is evaluated through various methods. For example, Gajdziok et al.  applied j et al.  performets films . They mets films .In a great measure, these qualificatives are similar to the resazurin dye chart for a rapid evaluation of antibacterial  and antiS. aureus, P. aeruginosa, and Candida sp., which are responsible for opportunistic oral infectious diseases in immunocompromised patients [C. albicans, F-UBA can maintain a low risk because this fungal species is implied in OSCC generation, according to Prakash et al. [Directly applied to the oral malignant lesion, the UBA-loaded mucoadhesive oral films could exert a topical anticancer effect, reducing the progression of oral squamous cell carcinoma. This formulation could also help through other mechanisms . Becausepatients ,79. Thesh et al. . The thih et al. , could bh et al. ; usnic ah et al. . Dar et h et al.  considerUsnea lichens were consumed as tea or decoction for liver detoxification [None of the numerous analyzed mucoadhesive film compositions included lichen extracts, even though lichens are known for their antitumor, antibacterial, antifungal, antiviral, and enzyme inhibitory properties. Recognized in Traditional Chinese Medicine (TCM) as “Song Lo,” fication . The typfication  and assoU. barbata dry acetone extract were manufactured using HPMC K100 as a polymer matrix and PEG 400 as a plasticizer. Complex physico-chemical and pharmacotechnical analyses proved their suitability for oral administration.In this study, mucoadhesive oral films containing The pharmacological evaluation confirmed F-UBA in vitro anticancer activity on oral squamous cell carcinoma based on high oxidative stress induced in CLS-354 cells. The results also revealed F-UBA cytoprotective action on normal cells and the dose-dependent growth inhibition of bacterial and fungal pathogens involved in immunosuppressed patients’ oral infections.Therefore, the present study suggests that UBA-loaded mucoadhesive oral films could be a helpful phytotherapeutic formulation in the complementary treatment of oral squamous cell carcinoma. Further in vivo and clinical research could be following steps in the F-UBA analysis, aiming to confirm their medical benefits.


Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae and Pseudomonadaceae were the most abundant mosquito-associated bacterial families. Our data suggest that the mosquito gut microbial composition varies among host species. In addition, we found a correlation between the microbiome composition and the presence of microsporidians. The prediction of metagenome functional content from the 16S rRNA gene sequencing suggests that microsporidian infection is characterised by some bacterial species capable of specific metabolic functions, especially the biosynthesis of ansamycins and vancomycin antibiotics and the pentose phosphate pathway. Moreover, we detected a positive correlation between the presence of microsporidian DNA and bacteria belonging to Spiroplasmataceae and Leuconostocaceae, each represented by a single species, Spiroplasma sp. PL03 and Weissella cf. viridescens, respectively. Additionally, W. cf. viridescens was observed only in microsporidian-infected mosquitoes. More extensive research, including intensive and varied host sampling, as well as determination of metabolic activities based on quantitative methods, should be carried out to confirm our results.The animal gut microbiota consist of many different microorganisms, mainly bacteria, but archaea, fungi, protozoans, and viruses may also be present. This complex and dynamic community of microorganisms may change during parasitic infection. In the present study, we investigated the effect of the presence of microsporidians on the composition of the mosquito gut microbiota and linked some microbiome taxa and functionalities to infections caused by these parasites. We characterised bacterial communities of 188 mosquito females, of which 108 were positive for microsporidian DNA. To assess how bacterial communities change during microsporidian infection, microbiome structures were identified using 16S rRNA microbial profiling. In total, we identified 46 families and four higher taxa, of which The online version contains supplementary material available at 10.1007/s00248-021-01944-z. Microsporidians (Microsporidia) are among the most widespread, obligate intracellular parasites, causing mainly zoonotic or waterborne infections in humans, but able to infect almost all animal phyla . Among 1Plasmodium infectivity in mosquitoes [Wolbachia spp. [Aedes and Culex mosquitoes [Mosquitoes can act as vectors for many disease-causing viruses and parasites and even carry and transmit multiple pathogens in a single host, creating numerous opportunities for interactions among vertebrate hosts, vectors, and pathogenic organisms. These interactions can occur on multiple levels and may ultimately affect transmission patterns and disease pathogenesis. One example is the possible immunosuppressive effect of filarial nematodes on malaria leading to reduced squitoes . The lowsquitoes  or the bhia spp. . Moreovesquitoes .Plasmodium parasites [Vavraia culicis impairs the development of Plasmodium berghei by priming the immune melanisation response in the adult mosquito [Anopheles arabiensis with a vertically transmitted microsporidian species prevented P. falciparum transmission because it reduced the establishment of Plasmodium oocysts in the Anopheles midgut and impaired the colonisation of Anopheles salivary glands by Plasmodium sporozoites [Nosema ceranae is to some extent associated with changes in gut microbiome structure and its presence is strongly correlated with some gut microbiome members  [The effects of microsporidians on the development of disease-causing organisms in mosquitoes have been studied mainly for malaria parasites for developing novel strategies to control mosquito populations or their capability to transmit arasites –12. It hmosquito . Howevermosquito  and behamosquito  or flighmosquito . Interesmosquito , 17. A rrozoites . Moreovellifera) .Tenebrio molitor) in association with tapeworm (Hymenolepis diminuta) infection [It has been increasingly recognised that microorganisms may participate in host–parasite interactions . The gutnfection . Like thnfection , 24 or pnfection , 26. Thenfection , 28.Many studies have shown that sex, stage of development, host environment, diet type and pathogenic infection can influence the mosquito microbiota , 29–37. Serratia marcescens as a microbiome member may enhance dengue virus infection [P. berghei infection [Wolbachia spp. may repress [P. falciparum [Anopheles gambiae. Although mosquitoes are common hosts of microsporidia, there is a lack of data on their effects on the mosquito microbiota. Therefore, the present study aimed to detect changes in mosquito gut microbiota associated with microsporidian infection. As a model, we used mosquitoes collected from natural populations.Mosquito–microbiome interactions play an important role in mosquito biology, including the development of pathogens , 39. Symnfection  or inhibnfection . It was  repress  or inhiblciparum  in AnophAedes vexans (19), Coquillettidia richiardii (16), Ochlerotatus annulipes (63), O. cantans (77) and O. sticticus (13). Among them, 108/188 (58%) were positive for one or more microsporidian species  and V4R (ATGGACTACCAGGGTATCTAA) primers targeting the V4 region in prokaryotic 16S rRNA gene . PrimersDNA concentration and fragment length distribution of the library were established using the High Sensitivity D1000 Screen Tape assay on a 2200 Tape Station system . Clonal template amplifications were performed using the Ion Torrent One Touch System II and the Ion Torrent OT2 Kit (Life Technologies) according to the manufacturer’s instructions. Sequencing was carried out using the Ion 540 Kit-OT2 and Ion Torrent S5 system on the Ion 540 chip (Life Technologies) according to the manufacturer’s instructions.fastx_uniques and sortbysize algorithms [Raw sequence data were pre-filtered using the Ion Torrent Suite software version 5.10.1 (Life Technologies) to remove polyclonal and low-quality sequences. Further bioinformatic analyses were conducted using fastq data and custom workflow. Sequence reads shorter than 200 bp were removed from the dataset using Geneious R11.1.5 (Biomatters Ltd.). Leading and trailing low-quality bases were removed using Trimmomatic version 0.39 . FASTX-Tgorithms . Chimeragorithms .denoising steps [unbias algorithm. Phylogenetic affiliations were analysed by the usearch sintax algorithm using a confidence threshold of 0.8 [otutab_rare algorithm [Operational taxonomic unit (OTU) clustering at 97% similarity was done in USEARCH version 11.0.667 . Sequencng steps . The ZOTd of 0.8 –53. ZOTUd of 0.8 –56. ZOTUd of 0.8 . Then, tlgorithm  to compaThe functional potential of prokaryotic communities in all samples was predicted using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) version 2.4.1 software package . The ZOTWeissella identified by NGS, a near-complete Weissella 16S rRNA gene was amplified using genus-specific S-G-Wei-0121-a-S-20 (CGTGGGAAACCTACCTCTTA) and S-G-Wei-0823-a-A-18 (CCCTCAAACATCTAGCAC) primers [To confirm the taxonomic position of the  primers . PCR reaEscherichia coli exonuclease I and FastAP Alkaline Phosphatase  and sequenced using the BigDye v3.1 Kit and ABI Prism 3130XL Genetic Analyzer , following the manufacturer’s instructions. Sequence chromatograms were checked for accuracy in Geneious R11.1.5. Then, sequences were compared to GenBank using blastn [Samples containing visible bands were purified with g blastn  optimizeg blastn .Spiroplasma species isolated from mosquitoes, all 16S sequences assigned to the genus Spiroplasma published in GenBank (www.ncbi.nlm.nih.gov) were used. As close outgroups, the 16S sequences of Clostridium ramosum and five Mycoplasma spp. were used. The bacterial strains and GenBank accession numbers for DNA sequences used in the Spiroplasma spp. phylogenetic analysis are provided in Supplementary Table 1.To identify the Sequences were aligned using the L–INS–I algorithm in MAFFT version 7.388 , as implalpha_div algorithm [r was used, defined as r =|Z| / √N, where “Z” is the WMW test statistic and “N” is the number of observations. The relationship between the two variables was tested using the Spearman correlation coefficient .The Chao1 index and Shannon diversity in individual samples were calculated using the lgorithm . Indexeslgorithm . Visualilgorithm . Statistlgorithm . CompariThe correlation between microbiota composition and mosquito species, in addition to the effects of microsporidian infection and their interaction with mosquito microbiome composition, was tested using the analysis of similarities (ANOSIM) with 999 permutations . VariatiWeissella and mosquito species were calculated based on the chi-square test. Any p-values of ≤ 0.05 were considered significant.Comparisons of the two groups and correlation analysis were tested considering mosquitoes belonging to different species using stratified permutation tests. Additionally, statistical significances among p > 0.848; Supplementary Fig. p > 0.758; Supplementary Fig. After quality-filtering the samples, 18,006,087 reads were yielded. The average read number per sample was 95,777 (SD = 9 343). The clustering of ZOTUs across samples produced 1437 unique ZOTUs. The median of ZOTUs was 96 and ranged between 84 and 112. There were no differences in species richness , Flavobacteriaceae  and Comamonadaceae . Diversity analysis of mosquito microbiota based on the cluster tree using UPGMA showed that the diversity of bacterial communities was determined by mosquito species rather than microsporidian infection  and mosquito species . By contrast, the results of PCoA for separate host species showed that the occurrence of microsporidians clearly differentiated the observed microbiome compositions , which did not group the bacterial communities according to microsporidian infection Fig. , the ANOons Fig. .Fig. 2GuAurantimonadaceae, Conexibacteraceae, Cytophagaceae, Dermabacteraceae and Hydrogenophilaceae displayed high values for the exclusive taxon component (A > 0.9). Additionally, the Actinomycetaceae, Halomonadaceae, Methylobacteriaceae, Moraxellaceae, Mycobacteriaceae and Sphingomonadaceae were noticed in all mosquitoes without microsporidians (B = 1). Mosquitoes infected with microsporidians were characterised by the exclusive occurrence of the Leuconostocaceae (A = 1). Moreover, the Acetobacteraceae, Bradyrhizobiaceae, Pseudomonadaceae and Rhizobiaceae were noticed in all infected mosquitoes (B = 1), while the Spiroplasmataceae was associated with the presence of microsporidians due to a high B-component value (B > 0.9).Indicator species analysis for infected and non-infected mosquitoes Table  showed tSpiroplasmataceae, only one ZOTU, Spiroplasma sp. PL03, was clustered. The sequence represented a Spiroplasma species that does not have a reference sequence published in databases. Phylogenetic analysis grouped this sequence with other endosymbiotic Spiroplasma spp.    Fig. . This reLeuconostocaceae was assigned to the genus Weissella, while the almost-complete 16S rRNA gene (GenBank acc. nos. MW892051–MW892067) allowed species identification using BLASTn search, revealing the highest similarity to Weissella viridescens (100% identities). This bacterium was detected only in microsporidian-positive mosquitoes , but a strong positive correlation  between microsporidian and W. cf. viridescens frequency was observed   between microsporidian-positive and non-infected mosquitoes. For both infected and non-infected mosquitoes specifically, carbohydrate metabolism, metabolism of terpenoids and polyketides, and amino acid metabolism were the most abundant pathways (> 10%) at level 2 , the biosynthesis of vancomycin group antibiotics (p < 1e−15) and the pentose phosphate pathway (p < 1e−15), when compared to the microbiome of non-infected mosquitoes  and Bacteroidetes and Firmicutes (each of about 20%) These results are broadly consistent with the findings of a large-scale study conducted in South Korea in which the gut bacteria of 305 insects, including mosquitoes, were examined . Similarcrobiota –80.Ae. aegypti, a high diet abundance was positively correlated with Enterobacteriaceae and Flavobacteriaceae and negatively with Sphingomonadaceae [An. gambiae: bacteria belonging to the SAR11 clade were abundant in sugar-fed mosquitoes, while an increased presence of Enterobacteriaceae, Yersiniaceae and Pseudomonadaceae was noticed in mosquitoes after blood feeding [The mosquito gut microbiome composition depends on factors that mainly affect the larval stages, such as environmental microbes, feeding behaviours, or biotic agents . Mosquitnadaceae . The cha feeding . HoweverAe. japonicus, Ae. triseriatus, Culex coronator, Cx. nigripalpus and Cx. restuans [Reports on whether the mosquito microbiome composition can be species-dependent are sparse. Our ANCOM and UPGMA analyses suggest that adults in natural populations host microbial communities that vary among mosquito species. Similar observations have been provided for other Culicidae species, such as restuans , 87. It restuans ); howeveO. cantans, for which we observed a partial overlap between the microbial compositions of infected and microsporidian-free individuals. Further studies, including quantitative analyses, should be performed to determine whether this result is associated with the infecting microsporidian species or the level of infestation.Although PCoA considering all mosquito microbiota as one pooled sample suggested that microsporidians do not affect the gut bacterial compositions, the analyses carried out for each species separately grouped mosquito microbiota according to the infection. An exception was Alphaproteobacteria decreased in Ap. mellifera honey bees infected with N. ceranae and that this effect was stronger when the infected bees were chemically exposed to insecticides or fungicides; additionally, the Gammaproteobacteria abundance increased in the Nosema-infected bees, but significantly only when hosts were co-exposed to pesticides [Alphaproteobacteria in microsporidian-infected Co. richiardii, O. cantans and O. sticticus. In addition, we found an increased contribution of Gammaproteobacteria in infected O. annulipes and O. sticticus.Our data show that infection by microsporidians is associated with a change in the gut microbial composition of the host. To our knowledge, no prior studies have examined the impact of microsporidian infections on the mosquito microbiota, while reports about correlations between microsporidians and microbiota of other invertebrate hosts are sparse and mainly concern honeybees. For instance, it has been noted that the abundance of sticides . Our resW. cf. viridescens was unequivocally correlated with infection. The genus Weissella belongs to lactic acid bacteria [Weissella spp. are also found as components of insect gut microbiota, for example, of Hymenoptera and Orthoptera [Weissella spp. can survive in an acidic environment [Our results suggest that microsporidian infections are correlated with specific gut microbiome members. The occurrence of bacteria –96. Althbacteria –96, Weisthoptera , 97. Morthoptera . Our resironment , 94–96.Spiroplasma sp. PL03 and W. cf. viridescens in microsporidia-positive mosquitoes suggests associations between these bacteria and parasitic infection. Spiroplasma is broadly distributed among invertebrate hosts, such as insects and spiders [Spiroplasma spp. in insects can confer resistance to a range of parasites, including fungi, nematodes and parasitoids [Spiroplasma species isolated, to date, from mosquitoes are not virulent to mosquitoes  , 110. ThSpiroplasma sp. PL03 and W. cf. viridescens, two bacterial species found in this study, represent bacteria whose participation in the mosquito gut is highly dependent on microsporidian infection. Our results imply that the gut microbiome may also affect pathogens vectored by mosquitoes. For example, the previously observed resistance of microsporidian-infected mosquitoes to Plasmodium transmission [In this paper, we suggest that microsporidian infection shapes the microbial community structure and microbial activity in infected mosquitoes, especially the biosynthesis of antibiotics and the pentose phosphate pathway. This result confirms previous findings that parasites modulate the gut microbiome in insects and that this is an important agent during modulated host- and parasite-associated microbiome interactions. Endosymbiotic smission  might beSupplementary file1 (PDF 837 KB)Below is the link to the electronic supplementary material.


The ribosome has long been thought to be a homogeneous cellular machine that constitutively and globally synthesises proteins from mRNA. However, recent studies have revealed that ribosomes are highly heterogeneous, dynamic macromolecular complexes with specialised roles in translational regulation in many organisms across the kingdoms. In this review, we summarise the current understanding of ribosome heterogeneity and the specialised functions of heterogeneous ribosomes. We also discuss specialised translation systems that utilise orthogonal ribosomes. Saccharomyces cerevisiae, ribosomes are composed of an SSU (40S) with 33 RPs and 18S rRNA and an LSU (60S) with 46 RPs and 3 rRNAs  that together form an 80S ribosome [Escherichia coli, 70S ribosomes consist of two subunits, which are designated as the 30S subunit (SSU), comprising 16S rRNA and 21 RPs, and the 50S subunit (LSU), comprising 23S rRNA, 5S rRNA, and 33 RPs [Ribosomes translate genetic information from mRNAs to proteins. They are fundamental multi-subunit macromolecules comprising ribosomal RNA (rRNA) and ribosomal proteins (RPs). Since the discovery of ribosomes in the mid-1950s, researchers have attempted to elucidate the structure and function of this large ribonucleoprotein complex. Ribosomes consist of two asymmetric subunits, a small subunit (SSU) and a large subunit (LSU); however, the internal composition of these subunits and their macromolecular size vary among organisms. For example, in the yeast ribosome . In Eschd 33 RPs . Both rRd 33 RPs . Furtherd 33 RPs ,14. Seved 33 RPs , and difd 33 RPs . Thus, aE. coli after infection [Since the discovery of electron microscopy in 1955, ribosome composition has been thought to vary based on the observation of differences in the size and shape of ribosomes ,21. Consnfection .Plasmodium berghei was shown to differentially express two divergent rRNA operons (rrn) over different phases of its life cycle [Arabidopsis thaliana [et al. also proposed the concept of the ‘ribosome code’ for functionally distinct forms of ribosomes in yeast [In the next two decades, researchers generally assumed that all ribosomes are the same in the cell; thus, the ribosomes were assumed to be passive translation machines. However, the concept of specialised ribosomes became prominent again in the 1980s, because studies indicated the differential expression of individual ribosome components in multiple, diverse invertebrate model organisms. For example, the malaria-causing parasite fe cycle ,24. In athaliana ,26. In 2thaliana . This mothaliana . Komili in yeast . Based oin yeast ,14,29–32The rRNA operon is generally found in multiple copies, ranging from 1 to 15 copies in bacteria, 1–4 copies in archaea, and thousands in eukarya . PhylogeP. berghei [Streptomyces coelicolor, a model actinomycetes strain, differentially expresses heterogeneous rRNAs during various stages of morphological development [Haloarcula marismortui contains three 16S rRNA operons: rrnA, rrnB, and rrnC. The sequence of rrnC is virtually identical to that of rrnA, whereas rrnB shows a high divergence in nucleotide sequences [H. marismortui rRNA operon can contribute rapid growth over a wide range of temperatures, because of their optimal functional structures at various temperatures [The first case of rRNA heterogeneity was reported in the malaria parasite  berghei ,24  with similar but not identical sequences [E. coli rRNA operons, rrnH is highly expressed in response to nutrient limitation-induced stress [rrnH operon affect the expression of functionally coherent gene sets, including the master regulators of the general stress response, RpoS, and stress-related protein factors, RelA and RelE [Vibrio vulnificus CMCP6 [V. vulnificus CMCP6, ribosomes bearing rRNAs encoded by the rrnI operon (I-ribosomes) are responsible for preferential translation of specific mRNAs [V. vulnificus cells adapt to environmental changes, such as temperature shifts and variations in nutrient availability. In this case, I-ribosome-mediated preferential mRNA selection appears to use a non-canonical mRNA selection mechanism, not Shine–Dalgarno (SD)-anti-SD (ASD) interactions [In equences ,53. Theyequences ,54. Specus CMCP6 . Among tic mRNAs . This prractions .The widespread, persistent existence of divergent rRNA genes throughout evolution suggests an adaptive advantage. Further studies are required to dissect the cellular functions of ribosomes containing species-specific genome-encoded divergent rRNAs in other organisms and their evolutionary relationships.E. coli. The stress-induced free toxin MazF, a sequence-specific endoribonuclease, cleaves target mRNAs specifically at certain single-stranded ACA-sites, leading to the rapid arrest of cell growth [Δ43 ribosomes [Δ43 ribosomes remain controversial [E. coli MazF, a Mycobacterium tuberculosis orthologue of MazF (MazF-mt6) can cleave 23S rRNA in the evolutionarily conserved helix/loop 70, resulting in production of modified 50S subunit that is defective in ribosomal subunit association [Specialised ribosomes bearing heterogeneous rRNA generated post-transcriptionally by ribonucleases have been reported in l growth  Fig. 1)E. coli. ibosomes . These moversial ,60. Unliociation . These fE. coli endoribonuclease, RNase G, also participates in the generation of heterogeneous 16S rRNAs in E. coli cells when exposed to aminoglycoside antibiotics, such as kanamycin and neomycin [E. coli cells showed the accumulation of 16S rRNA precursors containing 3–8 extra nucleotides at the 5’ terminus due to incomplete processing by RNase G. A subpopulation of heterogeneous ribosomes bearing incompletely processed 16S rRNA has low affinity for aminoglycoside antibiotics and, consequently, renders E. coli cells resistant to these antibiotics [Another neomycin ,63. Analibiotics .rrn operon disruption (referred to as ‘unlinked rrn operons’) is widespread in prokaryotes [Helicobacter pylori, independent maturation of the 16S and 23S-5S precursors that were separately transcribed from the unlinked rrn operon was observed [rrn operons are symbiotic bacteria, indicating specific roles of rRNA species transcribed from them in symbiosis [Notably, karyotes . In Heliobserved ; which pymbiosis .These studies suggest that subpopulations of ribosomes bearing heterogeneous rRNAs generated by differential processing have important biological roles in response to various stress conditions.E. coli rRNAs [In all organisms, rRNAs undergo extensive post-transcriptional modifications that facilitate the diversity of their composition and ribosome activity . To dateli rRNAs . Among tli rRNAs . These mli rRNAs ,71.E. coli under oxidative stress [In many cases, the precise roles of rRNA modifications are largely unknown. However, many of these modifications are conserved and clustered in functionally important regions of rRNAs, such as the peptidyl transferase centre (PTC) or ribosomal subunit interface ,73. Thuse stress . In yease stress . Additioe stress .Uneven rRNA modifications, such as partial and altered modification patterns, further imply the existence of additional roles under certain physiological conditions ,83–85. AE. coli and eukaryotic ribosomes, respectively [Dictyostelium discoideum, significant changes in RP composition were observed at various stages of development. Twelve distinct RPs appear at specific stages of cell differentiation (between vegetative amoebae and spores). Among them, two RPs were specific to ribosomes of vegetative amoebae, and three were specific to spores, suggesting that RP paralogs may contribute to the different regulation of protein synthesis during growth and development [S. cerevisiae, 59 of the 78 RPs retain two genomic copies [ASH1 mRNA in yeast, Komili et al. showed that duplicated RPs have distinct functional roles in translational regulation, including ribosomal assembly and paralog-specific aberrant localisation [Ribosome heterogeneity may also originate from the incorporation of different core RP paralogs . RPs areectively ,87,88. Relopment . In S. cc copies . Notablyc copies . In a stlisation . In addilisation .E. coli, two ribosomal core proteins, bL31 and bL36, have two paralogs: bL31A and bL31B, and bL36A and bL36B, respectively [E. coli [E. coli at lower temperatures and maintained the translation reading frame more efficiently than ribosomes with bL31B in vivo [Approximately 46.5% of bacterial genomes have paralogous genes for one to three RPs . Some RPectively . A study[E. coli . In the  in vivo . In addi in vivo .Drosophila melanogaster, some RP paralogs show differential expression in the adult male germline [Tissue-specific expression of paralogs of core RP has also been observed at the transcript and protein levels . For exagermline . eL22 isgermline . uL16L agermline . Similargermline .Further studies are required to identify the functional redundancy or specificity of core RP paralogs and how they act jointly to create a specialised ribosome.S. cerevisiae and mouse embryonic stem cells (mESCs), suggesting that stoichiometry among core RPs depends on both growth conditions and the number of ribosomes bound per mRNA [Another possibility for creating heterogeneous ribosomes is to alter the relative abundance of core RPs . Ribosomper mRNA . These fFurthermore, ribosomes lacking specific RPs are present in cells. For instance, using the selected-reaction monitoring (SRM)-based MS method, the absolute abundance of RPs in translating ribosomes and profiled transcripts enriched or depleted from subsets of ribosomes were measured in mouse ESCs . In thisThese findings indicate that alterations in the RP composition can result in ribosome heterogeneity, contributing to translational control.E. coli, comparative proteomic analysis has revealed that six RPs were methylated , three proteins were acetylated , and protein uS12 was methylthiolated [E. coli [E. coli varies with cell growth and nutrient deprivation, increasing the stability of the ribosomal stalk complex under stress [A. thaliana, 23 of the 80 cytosolic RP families contain residue-specific covalent modifications that represent potential differential modification sites [A. thaliana showed an increase in the day/night phosphorylation ratio of RPs, suggesting that differential phosphorylation of RPs may contribute to the modulation of diurnal protein synthesis in plants [Heterogeneity in ribosomes due to the PTM of RPs, including phosphorylation, ubiquitination, methylation, acetylation, and SUMOylation, has been postulated to generate ‘specialised ribosomes’. In many organisms, most RPs are post-translationally modified. In hiolated ,105. Sev[E. coli ,107. Notr stress . In yeasr stress . For exar stress . In A. ton sites . The extn plants . PatternS. cerevisiae [In addition to the composition of rRNAs and core RPs, the heterogeneity of ribosomes can result from other protein factors associated with the ribosome . Proteomrevisiae . Cells wS. cerevisiae [RACK1, the receptor for activated protein C kinase 1, is a highly conserved scaffold protein that can interact with several signalling molecules, either directly or as part of a larger complex . Crrevisiae . Furtherrevisiae ,119. Therevisiae . In addirevisiae . These sFragile X mental retardation protein (FMRP) is a well-known ribosome-associated factor. FMRP is a broadly expressed RNA-binding protein that associates with mRNAs and other proteins to form large ribonucleoprotein complexes (mRNPs). These complexes have been proposed to participate in the transport, localisation, and translation of target mRNAs . FMRP inD. melanogaster, the apoptotic regulator, Reaper, inhibits translation initiation by directly and specifically binding to the 40S ribosomal subunit. Specifically, the direct binding and regulation of ribosome function by Reaper allows the selective translation of mRNAs initiating at alternative start codons or from certain internal ribosome entry site elements [In elements . In humaelements . These fE. coli [In the process of bacterial translation initiation, base pairing between an SD sequence in the 5’ untranslated region of mRNA and a complementary ASD sequence at the 3’ end of 16S rRNA plays a crucial role in mRNA selection by ribosomes  Fig. 2)Fig. 2). E. coli . FurtherE. coli  /orthogonal-mRNA (O-mRNA) pairs were developed by gene duplication, followed by a new positive and negative selection method [in vivo efficiency of unnatural amino acid incorporation, Wang et al. evolved an orthogonal ribosome (ribo-X) that enhanced tRNACUA-dependent decoding of amber codons placed in orthogonal mRNA in E. coli [CUA pairs to substantially increase the efficiency of site-specific unnatural amino acid incorporation in E. coli. This study enabled the synthesis of proteins that incorporate unnatural amino acids at specific sites in vivo. Chubiz and Rao developed a computational model of orthogonal ribosomes by enumerating all possible SD/ASD pairs and then selecting those that minimally interfered with the translation of native mRNA without toxic effects on the cell [For selectively and preferentially producing recombinant proteins of interest in n method ,138. Thin method . To incr E. coli   ribosomes, whose subunits are covalently linked through an optimised RNA staple, were discovered to have a new intrinsic polymerisation function ,148 Fig. CarlsonE. coli ribosomes, revealing structural details and providing insights into their composition [Over the past few decades, evidence of gene expression regulation by specialised translation machinery has accumulated. In this review, we focused on heterogeneity in ribosome composition and its functional roles, as well as engineered orthogonal specialised ribosome systems. Although the biological roles of most naturally occurring specialised ribosomes are largely unknown, technological advances in high-throughput analyses and genetic engineering methods combined with bioinformatic analyses would improve the accurate assessment of ribosome heterogeneity and its physiological role. For example, high-resolution native MS analysis provided sufficient resolution to identify differences in the copy number of uL7 and uL12 RPs on intact position . Shi et position . High-thposition . Single-position .Although the functional consequences of differences in ribosome composition remain challenging, reports based on these advanced technologies provide compelling evidence in support of the specialised function of ribosomes in various organisms. For example, studies on genome-encoded divergent rRNAs in bacteria have demonstrated that ribosomes bearing these divergent rRNAs modulate the expression of genes that contribute directly to bacterial stress adaptation, highlighting a novel function of specialised ribosomes ,56. Furtin vivo to produce recombinant proteins and explore the poorly understood functions of the ribosome. These studies have developed methods for parallel genetic circuits [Significant progress has been made in the research on engineered specialised ribosomes through functionally orthogonal ribosomes and mRNA circuits  and the circuits , expandecircuits ,142, andcircuits . Despitecircuits . Second,circuits . For exacircuits . Extensi


Pedicularis cephalantha  and P. nigra , endemic to southwestern China. Both plastomes have typical quadripartite structures with one large-single copy region, one small-single copy region, and two inverted repeat regions. Both plastome sequences contained 37 tRNA genes and eight rRNA genes, but they differed in the numbers of protein-coding genes: P. cephalantha had 76 functional genes and 12 pseudogenes while P. nigra had 74 functional genes and 13 pseudogenes. Phylogenetic analysis shows that P. cephalantha and P. nigra are closely related, then sister to P. oederi in the family Orobanchaceae.We report the complete chloroplast genome (plastome) sequences of  Pedicularis L. (Orobanchaceae) is the largest genus of hemiparasitic plants  Vaniot ex Bonati 1921. We then reconstructed a plastome phylogeny within the genus by combining available plastome data of Pedicularis species in GenBank to determine the phylogenetic positions of both species. Voucher specimens of P. cephalantha  and P. nigra  were collected from the Yulong Mountain, Lijiang, Yunnan, China  and from Gaolushan, Yuxi, Yunnan, China , respectively. Voucher specimens of the two species were deposited at the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences .kers Ree . HoweverP. cephalantha and 9,481,360 for P. nigra) were obtained. These raw data were used to assemble the complete plastome sequences de novo using the GetOrganelle toolkit  and P. nigra (NCBI accession: OL544940) showed typical quadripartite structures with one large-single copy region, and one small-single copy region separated by two inverted repeat regions. Plastome sizes of P. cephalantha and P. nigra were 147,087 bp  and 14,726 bp , respectively. The total gene contents were 133 in P. cephalantha and 132 in P. nigra. Both plastomes had 37 tRNA genes and eight rRNA genes, but the numbers of protein-coding genes differed: P. cephalantha had 76 functional genes and 12 pseudogenes while P. nigra had 74 functional genes and 13 pseudogenes.Total genomic DNA was extracted from leaves of both species dried with silica gel using a modified CTAB method  Kanitz (Orobanchaceae) was selected as the outgroup. A maximum-likelihood tree was reconstructed using raxmlGUI (Edler et al. P. cephalantha and P. nigra formed a clade with 100% bootstrap values (P. oederi Vahl in the Clade 7. The complete chloroplast genomes and phylogenetic results in this study provide new insights into the phylogenetic backbone and evolutionary biology of Pedicularis.To clarify the phylogenetic positions of these two species, we downloaded an additional 29 published plastomes of 22 p values , represep values . Our new


Melilotus officinalis (L.) Pall. in skeletal muscles is still unknown. In this study, we explored the possible regulatory targets of M. (L.) Pall. that affects the repair patterns in chronic muscle injury. We analyzed the potential target genes and chemical composition of M. (L.) Pall. and constructed a “drug-component-disease target genes” network analysis. Five active ingredients and 87 corresponding targets were obtained. Muscle-tendon junction (MTJ) cells were used to perform receptor-ligand marker analysis using the CellphoneDB algorithm. Targets of M. (L.) Pall. were screened further for the cellular ligand-receptor protein action on MTJs. Enrichment analysis suggests that those protein-associated ligand receptors may be associated with a range of intercellular signaling pathways. Molecular docking validation was then performed. Five proteins  may be regulated by the active ingredient luteolin and scoparone. Finally, molecular dynamics simulations revealed that luteolin can stably target binding to MMP2. M. (L.) Pall. influences skeletal muscle repair patterns by affecting the fibroblast interactions in the muscle-tendon junctions through the active ingredients luteolin and scoparone.Information regarding the function of  Melilotus officinalis (L.) Pall. (M. (L.) Pall.) is a traditional Chinese medicine that is widely distributed and has broad prospects for development and utilization [lization . M. (L.)lization –3. In anlization . M. (L.)Skeletal muscle injuries are one of the most common sports injuries, accounting for approximately 40% of sports-related injuries in older people . MusclesLiu et al. isolated 29 compounds from M. (L.) Pall. alcoholic extracts. They have good antioxidant activity and play an important role in anti-inflammatory and antioxidant functions . Recent In this study, network pharmacology and molecular docking approaches have been used to predict the possible regulatory targets of M. (L.) Pall. in muscle repair patterns, to reveal the potential molecular mechanisms of this compound in regulating muscle repair patterns and provide new ideas for the treatment of skeletal muscle injury.http://www.uniprot.org/).The active ingredients of M. (L.) Pall. were obtained from previous research results . The actA “drug-component-target” network structure was constructed with the active ingredient and corresponding target genes of M. (L.) Pall. using the Cytoscape (version 3.7.2) software . Each noP < 0.05, key intercellular interactions were identified. The results were visualized using the dot_plot function in the CellphoneDB and the R software.Seurat results from our previous study of single-cell data analysis were used to perform ligand-receptor maker analysis . Muscle-R language and VennDiagram packages were used to obtain the M. (L.) Pall. targets on the MTJ cells . The actTo perform molecular dynamics simulations (MD), the force field parameters of luteolin were generated in this study using ACPYPE Server, an online tool , 27, 28.homo sapiens, and molecular function (MF), biological process (BP), and cellular component (CC) were used for GO enrichment analysis as previous researches [P < 0.05 to search for the major functional and in vivo pathways significantly enriched by the active ingredient targets. Bar graphs of the pathways in the GO and KEGG pathway enrichment analysis were plotted using the clusterProfiler toolkit in R and ggplot [The study species was searches –33. The d ggplot , 34.P < 0.05. Spearman's method was used for correlation analysis, and the results of the analysis were presented as chord plots using the circlize package of the R software (version 0.4.12) [Statistical analyses were performed using the R software (version 3.6.3), and differences were significant at  0.4.12) .Five of the six active ingredients had 87 corresponding targets (after excluding duplicates). The “herbal-active-component-disease target gene” regulatory network was constructed . The redHelicobacter pylori infection, rheumatoid arthritis, endocrine resistance; ErbB, MAPK, PI3K−Akt, Rap1, HIF−1, relaxin, estrogen, GnRH, and AGE−RAGE signaling pathways in diabetic complications; and so on). This suggests that M. (L.) Pall. may target some intercellular signaling pathways, such as neuropeptide-related, tumor-related, and stress direction-related pathways  . The int Figures . These g Figures .Pharmacological database analysis suggests that scoparone, the active ingredient of M. (L.) Pall. targets CCL2 and luteolin targets VEGFA, MMP2, MET, and EGFR . CCL2, VIn the above molecular docking analysis, MMP2 and luteolin had the minimum binding free energy. The free energy of luteolin target binding energy for MMP2 is -10.8 kJ/mol. To further assess the binding efficacy of MMP2 and luteolin, MD simulations were performed . Due to To the best of our knowledge, this study is the first to assess the impact of M. (L.) Pall. on the musculotendinous junction. According to the TCMSP database, M. (L.) Pall. has five active ingredients (OB ≥ 30% and DL ≥ 0.18) potentially acting on 87 targets. Therefore, the “herb-active ingredient-disease target gene” network was constructed. We found that M. (L.) Pall. influences the interaction of fibroblasts in muscle-tendon junctions and affects muscle repair patterns through the modulation of five ligand receptor-related proteins  using the active ingredients luteolin and scoparone. And MD simulations revealed that luteolin can stably target binding to MMP2.Scoparone is a naturally occurring coumarin found in green plants. It is purified from a lipid-lowering herb that reduces the proliferation of human peripheral blood mononuclear cells, scavenges reactive oxygen species, inhibits tyrosine kinase, and enhances the production of prostaglandins . Recent Recent studies have found that luteolin protects skeletal muscles from attrition caused by inflammation and downregulates the expression of proteins associated with muscle catabolism . LuteoliLuteolin was found to target MMP2 in MD simulations. Elevated levels of MMP2 expression are associated with insulin resistance due to extracellular matrix (ECM) remodelling in skeletal muscle . This suThis study provides new data supporting the treatment of sports injuries using M. (L.) Pall. and provides a theoretical basis for clinical application. All the core components of M. (L.) Pall. were screened and docked successfully with their key targets. The core components of M. (L.) Pall. have good binding activities to their key targets, suggesting that they can effectively treat skeletal muscles. However, more Chinese medicine databases need to be used, and target prediction databases need to be improved. This study did not directly examine the chronic muscle injury dataset, but rather by examining the MTJ fibroblastic dataset, which may be biased. Furthermore, more in vivo and ex vivo clinical studies are also needed to validate the mechanisms of action M. (L.) Pall. in the treatment of chronic skeletal muscle injury.In summary, this study demonstrates that M. (L.) Pall. can skeletal muscle injury by acting on CCL2, VEGFA, MMP2, MET, and EGFR, through luteolin and scoparone. They are the key active ingredients of M. (L.) Pall. and affect intercellular signaling, such as neuropeptide-related, tumor-related, and stress-related pathways. M. (L.) Pall. may influence the interaction of fibroblasts in muscle tendon junctions to affect muscle repair patterns. Molecular docking analysis validated some of the network pharmacology results and confirmed the multicomponent, multitarget, and multipathway characteristics of M. (L.) Pall. in the treatment of skeletal muscle injury.


Importantly, a larger degree of internal circadian misalignment was robustly associated with more depression-like  and anxiety-like  mood levels during simulated night work. These findings offer a proof-of-concept demonstration of an evidence-based meal timing intervention that may prevent mood vulnerability in shift work settings. Future studies are required to establish if changes in meal timing can prevent mood vulnerability in night workers.Shift workers have a 25 to 40% higher risk of depression and anxiety partly due to a misalignment between the central circadian clock and daily environmental/behavioral cycles that may negatively affect mood and emotional well-being. Hence, evidence-based circadian interventions are required to prevent mood vulnerability in shift work settings. We used a stringently controlled 14-d circadian paradigm to assess mood vulnerability during simulated night work with either daytime and nighttime or daytime-only eating as compared with simulated day work (baseline). Simulated night work with daytime and nighttime eating increased depression-like mood levels by 26.2% ( SI Appendix).Shift workers have a 25 to 40% higher risk of depression and anxiety, with mental health disorders contributing to an estimated global cost of ∼US$1 trillion/y in lost workforce productivity alone –5. The ipFDR = 0.001; effect-size r = 0.78). In the DNMC group, simulated night work increased depression-like mood levels by 26.2% relative to each participant’s baseline  (P = not significant [n.s.]) . In the DNMC group, simulated night work increased anxiety-like mood levels by 16.1% relative to each participant’s baseline  (P = n.s.)   C. Conver [n.s.]) D. Simila= 0.005) E. Conver = n.s.) F. Importctively) G and H.except for the timing of meals. The relevance of diet on sleep, circadian rhythms and mental health is receiving growing awareness with the emergence of a new field, nutritional psychiatry .We found evidence that meal timing had moderate to large effects on depression-like and anxiety-like mood levels during simulated night work, and that such effects were associated with the degree of internal circadian misalignment. These effects were unlikely due to differences in study design, as the laboratory protocol of both groups was identical , ychiatry , 10. A rychiatry . Importaychiatry . In contychiatry , 13. Whiychiatry . Howeverychiatry . TherefoHyperglycemia is a risk factor for depression . SupportSI Appendix.The protocol was approved by the Partners HealthCare’s institutional review board and performed in accordance with the principles of the Declaration of Helsinki, and participants provided written informed consent. Detailed descriptions, protocol approval, and data availability are in Supplementary File