Forthis evaluation, 2-mm-thick sections of bovine brains were prepared andplastinated with domestic polyester. Slices were compared before impregnationand after curing using standardized photographs taken after dehydration andafter curing. Plastination followed the standard protocol: fixation,dehydration, forced impregnation, and curing. Fifteen brain slices wereplastinated with each polyester . There was no significantdifference in the percent shrinkage between groups after plastination of P18 andP40, but the curing time of Cristalan© polymer was too short forimpregnation. Therefore, no initiator was used for C polymers impregnation.Thus, domestic polyester P18 was a viable option for the process.Plastination is a technique used to preserve biological tissues while retainingmost of their original appearance. In the technique, developed by Dr. Gunthervon Hagens in 1977, specimens were impregnated with a polymer, such as silicone,epoxy, or polyester. Considered the most suitable material for brainplastination, polyester has a wide application in teaching and research comparedwith imaging techniques. The materials for plastination are usually importedfrom Germany and more expensive than domestic products. If domestic polymerswere to enter the market it would favor the expansion of plastination in Brazil.Hence, this study evaluated the feasibility of using domestic polyesters toreplace the usual Biodur Using biological materials for teaching, research, and university extension requiresstabilization to preserve tissue structure and avoid natural decomposition. Overtime, several substances have been discovered and developed for tissue fixation andconservation, such as heavy salts, tannins, glycerin, alcohols, phenols, andaldehydes ,2.Discovered in 1867 by German chemist August Wilhelm von Hofmann, formaldehyde became the fixation and preservation icon for biological tissues andanatomical specimens and is commonly used in morphology laboratories due to its lowcost, rapid tissue penetration, and long preservation capacity . It is hIn the search for a substitute for formaldehyde conservation, a new technique(plastination) emerged in the late 1970s. Plastinated specimens are odorless,moisture-free, durable, non-toxic, maintenance-free ,5, and pPlastination is a process in which body fluids and fat are replaced by apolymerizable resin. According to von Hagens et al. , plastinWorldwide, polyester plastination mainly uses the P40 resin from the German companyBiodur ,10, as i® P40 polyester in slice plastination.Hence, this study evaluated the feasibility of using Brazilian polyesters to replacethe Biodur®and Arazyn 1.0 #08 (P18) from Redelease®. The viscosity range (<900mPa.s) was selected according to studies on the relationship between polymerviscosity and tissue shrinkage were selected for testing: 3 Cristalans C1, C2, and C3) from Novapol, and C3 Unlike P40 resin, which is polymerized by ultraviolet light, the domestic polymerscan be cured by adding a chemical initiator. Manufacturers suggest using 1% (v/v) ofthe initiator for a 30-min complete curing time. The resin and initiator start achemical reaction, and the liquid resin begins to increase in viscosity entering agel phase within 20-25 min, which is too viscous for routine impregnation. Becausethis time is too short for a satisfactory impregnation ,11, it wvs time curve)  initiator and allowed to cure to confirmpolymerization time.Polymerization begins as soon as the initiator is mixed with the resin, with agradual and slow increase in viscosity until the moment preceding the gel state inthe final period of curing, when viscosity increases at exponential rates(inflection point of the viscosity e curve) . To estaResults were recorded and examined to determine which concentration of initiator andpolyester remained fluid for >2 days but <3 days (enough time for impregnationand polymerization). Results indicated that only P18 with ≤0.125% of initiator hadthe potential to allow complete impregnation. The short curing time of all the “Cresin mixes”, even with small amounts of initiator, were not acceptable forimpregnation.Five bovine heads, donated by the Mafrical meat-packaging facility, located inCariacica, Espírito Santo, Brazil were used for this study. The research wasapproved by the Animal Ethics Committee of the Federal University of Espírito Santo(CEUA-UFES), under No. 31/2019. Immediately after receiving the heads, they wereopened with the aid of a circular saw and the brains were carefully removed. Thespecimens were washed under running water to remove blood and clots, fixed by weeklyimmersion in formalin baths with increasing concentrations of 2, 5, 7, and 10%, andrefrigerated (5-7°C). Brains were stored in 10% formalin for at least 5 months toensure complete fixation .®  conventional deli meat slicer, set at 2-mm cutting thickness. Finally, they were protected/containedwith custom-made wire mesh  around the perimeter andeach group of 5 was secured with string, forming a “sandwich”. Dehydration of thesections (tied groups of 5) was performed with four consecutive cold (-25°C) weeklyacetone baths of 95, 95, 100, and 100%, inside a freezer. The grids containing thespecimens were positioned vertically during dehydration to prevent the slices fromweighing each other down and to facilitate the escape of acetone bubbles  ratio of acetone tobiological material . DehydraAfter dehydration, the sections were randomly distributed into five experimentalgroups defined by the polyester manufacturer: Redelease (P18 polyester), Biodur (P40polyester), and Novapol  . This stEach group of 5 dehydrated slices was removed from the acetone and submerged into oneof the five polyester containers in the vacuum chamber. For the P18 polyester,0.125% (w/w) Butanox initiator was added with a micropipette as part of theimpregnation mixture. For resins C1, C2, and C3 and for P40 polyester, no initiatorwas added . The res3/h, USA) wasturned on for five minutes before starting impregnation, so that it reached workingtemperature , creating a “glass × silicone cord × glass” sandwich held together bymetal clips. One side was left open until the chamber was filled with the specimenand resin mixture. Each assembled flat chamber was filled with an impregnated sliceand the corresponding impregnation resin and initiator .The P18The vertical positioning of the flat chamber allowed air bubbles in the fillingmixture to rise to the surface and then be removed using a small syringe with ahypodermic needle. After this procedure, the chamber was closed with the siliconegasket and metal clips.The assembled flat chambers containing P40 were cured under UV light, whereasspecimens impregnated with domestic polyesters were cured without UV light. After 24h of initial curing, the latter were placed in a 40°C oven for 48 h to acceleratefull cure. Since both the photochemical and chemical curing processes are highlyexothermic, fans were used for temperature control .After curing, the flat chamber was dismantled, and the finished specimen was removed.This was done by removing the metal clips and using the tip of a scalpel blade toaid in detachment at the junction of the polyester plate and the glass .The suitability of the domestic polyester for plastination of nervous tissue and thequality of the final specimen were verified. The parameters for verifying resinsuitability/compatibility included: resin-mix viscosity as a function ofimpregnation time, behavior of the resin-mix in relation to the forced impregnationsteps, curing and disassembly of the flat chambers, transparency of the curedpolymers, and specimen shrinkage. Shrinkage was assessed by measuring and recordingthe percentage shrinkage of brain sections. To evaluate the final specimen, thefinal stiffness of the cured polyester slices , differentiation ofwhite and gray matter, and visual transparency were qualitatively compared with P40and recorded.Shrinkage is best calculated by volume measurement , which iTo standardize analysis, all sections were identified and photographed immediatelyafter dehydration and after curing. A scale tray was used to hold the camera at apredetermined angle and focal length.The total surface area of the top of the section was measured using the free softwareImageJ (USA), which calculates the area from the number of pixels in the photograph,using a specific scale as a parameter.t-test for independent samples. A P<0.05 significance level wasused in all tests. Calculations and statistical analysis were performed usingIBM® SPSS® version 26.0 (USA).In the homoscedasticity analysis of all data sets, statistical assumptions wereevaluated by the Levene's test; the Kolmogorov-Smirnov test was used to evaluate thenormality of all scalar variables to determine the subsequent statistical tests. AWilcoxon test was performed to indicate possible differences in section shrinkagewithin the same group, considering the area before impregnation and after curing.Comparisons between two different groups were performed by the2) was calculated using Microsoft Excel 2019 .For initiator concentration as a function of curing time test, the coefficient ofdetermination  is extracted at this pressure. Styrene is known todamage vacuum pumps over time . HoweverCuring time required for each domestic polyester with Butanox initiator at differentconcentrations was observed and recorded . Only thSlice impregnation should be completed within 48 h, so that the impregnation mixturemust be in a liquid state for casting. Of the domestic polyesters tested, only theP18 polyester sample with 0.125% (m/m) initiator met these requirements: liquid atend of impregnation and curing after 48 h. Therefore, this initiator concentrationwas chosen for the impregnation mixture for the P18 polymer. Furthermore, noincrease in viscosity was noticed in the impregnation mixture after 10 h ofimpregnation. This occurred because the polymer only enters a gel state (suddenincrease in viscosity) closer to the complete curing time.The Novapol resin mixture samples cured within minutes to a few hours; from anextrapolation calculation, the amount of initiator needed to keep the polymer fluidfor an optimum period of time (long enough to thoroughly impregnate the tissue) wastoo small even for a volumetric pipette, making it unfeasible to measure for mixinginto the polymer.Given these results, the Novapol samples failed to achieve satisfactory results underthe circumstances evaluated in this assay. Thus, it was unfeasible to add theinitiator in the impregnation step, as was done with P18, restricting its use to thecuring stage. Therefore, no initiator was used in the Novapol (C's) impregnationbatches.After curing, the flat chambers were dismantled to obtain the finished sections.Sections impregnated with Novapol resins  were not satisfactory.When disassembling the flat chambers, the resin adhered to the glass plates atvarious points, preventing slice removal and occasionally leading to breakage of thefinal specimen and the chamber glass . The poiMechanical and optical properties of the final specimens  seemed to be as transparent and stiff as the reference polymer(P40). The visual differentiation between the white and gray matter was alsoexcellent. When touching the sections, no wet or sticky areas were noted, whichindicated a complete cure in both P40 and P18 specimens. This suggested that thelatter may be a substitute for P40.Tissue shrinkage is an important factor in plastination studies. Regardless of thepolymer used , the process generates some shrinkage,which is a slight drawback of the technique. Considerable shrinkage can distort theinitial shape of the specimen, which is essential for morphometric measurements andimaging comparison .Among the several factors affecting shrinkage, von Hagens  and Brow2) of eachsection was measured after dehydration (immediately before impregnation) and aftercuring. 2)  .Size differed between the impregnated sections depending on the position of thebrain: some were more rostral (anterior) and some more caudal (posterior). However,this difference did not influence the shrinkage calculation, since the statisticalanalyzes did not show a significant difference between the initial volumes of thegroups  because sections were randomly distributed intogroups.The area of the sections immediately before impregnation (after dehydration) andafter curing were statistically different for both groups analyzed (P=0.001), whichshowed a shrinkage during plastination , as expet=1.229, df=28, P=0.229) (When comparing the reference polyester (P40) to the alternative (P18) under thetesting conditions, the PSR showed no significant difference(P=0.229) , which cDespite the great disparity between viscosities of the polyesters used in thisresearch , the tisDe-bureaucratizing and cost reduction of the technique by using domestically marketedpolymers would help disseminate the technique in Brazil, providing numerousadvantages to teaching, research, and university outreach. As discussed,plastination eliminates the need to maintain specimens in toxic preservativesolutions. Moreover, only plastination allows research on 2-3 mm sections for a longperiod of time, as it produces sturdy specimens that can be manipulated forobservation from all angles. Other preservation methods produce extremely fragileand brittle specimens. In neuroanatomy, for example, brain structures themselves areextremely brittle, overly complex, and closely spaced, leading to the need for thinspecimens for demonstration so that information is not lost . PlastinOf the domestic polyesters examined, only P18 resin showed no significantdifference compared to the reference resin (P40) regarding tissue shrinkage.Visually, color and appearance of the final specimens impregnated with P18 weresimilar to those plastinated with P40 .P18 resin plastination resulted in high quality material, allowing visualizationand practical handling. P18 was chosen because of its “plastinic” properties:colorless, rigidity, durability, easy handling, and lower cost compared to thereference polyester. Thus, P18 polyester proved to be a viable alternative toP40, with excellent visual results.Good reproducibility as well as the good preservation of the slices and theirdurability are the main qualities for their use in museums, in the teaching ofhuman anatomy, and for comparison with imaging techniques. Compared with otherartificial anatomical models, plastinates stand out for their accuraterepresentation of anatomical structures.


Aspergillus niger, Aspergillus glaucus, Penicillium in the Aspergillaceae family, and Talaromyces and Rasamsonia emersonii in Trichocomaceae were the key microorganisms involved in the formation of the characteristic qualities of Qingzhuan tea.In order to analyze the changes in the microbial community structure during the pile fermentation of Qingzhuan tea and their correlation with the formation of quality compounds in Qingzhuan tea, this study carried out metagenomic and metabolomic analyses of tea samples during the fermentation process of Qingzhuan tea. The changes in the expression and abundance of microorganisms during the pile fermentation were investigated through metagenomic assays. During the processing of Qingzhuan tea, there is a transition from a bacterial dominated ecosystem to an ecosystem enriched with fungi. The correlation analyses of metagenomics and metabolomics showed that amino acids and polyphenol metabolites with relatively simple structures exhibited a significant negative correlation with target microorganisms, while the structurally complicated B-ring dihydroxy puerin, B-ring trihydroxy galloyl puerlin, and other compounds showed a significant positive correlation with target microorganisms.  Aspergillus, Cyberlindnera, Rasamsonia, Bacillus, Debaryomyces, Aspergillus_fumigatus, and Bacillus_subtilis participate in the formation of the main quality components of dark tea [Pile fermentation is a key process in the formation of the characteristic quality of dark tea. This process is characterized by the growth and succession of microbial communities ,2,3. Durdark tea ,6,7. Durdark tea ,6,8,9. Tdark tea . Many phdark tea ,11. Theadark tea .Aspergillus, Penicillium, Debaryomyces, Bacillus, Lactococcus, and Enterobacter, and others [Aspergillus has been identified as the dominant microorganism in the early stage of pile fermentation of Pu’er tea and it is mainly involved in multiple biological processes such as the transformation from tea polyphenols to theabrownin and the degradation of caffeine [Aspergillus genus, such as cellulases, hemicellulases, proteases, and α-amylases, could catalyze the major metabolites in Pu’er tea during the fermentation process [Aspergillus is considered to be the most prevalent genus during the microbial fermentation of Pu’er tea, while Aspergillus niger has been often documented as the most dominant species [Pseudomonas, Bacillus, and Brevibacterium, and a variety of thermophilic bacterial species have been isolated during microbial culture [Firmicutes and Proteobacteria are the dominant bacterial phyla. Ascomycota, Trichomycetes, and Aspergillus are the dominant fungi, and fungi have been reported to make greater contributions to tea polyphenol metabolism compound conversion and tea quality formation [Aspergillus has been regarded as the predominant genus in the pile fermentation of Qingzhuan tea, followed by the Penicillium, Debaryomyces, Thermomyces, Rasamsonia, and Byssochlamys genera [The main microbial genera in the pile fermentation of dark tea include d others ,14. Aspecaffeine ,16. More process . The fun species ,18. Comp culture . Fungal ormation ,6,20,21.ormation ,23. In rs genera ,25. Howes genera ,27,28. Xs genera . Xiao etIn this study, the metagenomic method was used to analyze the dynamic changes in the microbial community structure during the pile fermentation stage of Qingzhuan tea and metabolomics was employed to investigate the dynamic changes in the quality compounds of Qingzhuan tea. This study aimed to explore the correlation between the dominant strains and key quality compounds during the pile fermentation process and to reveal the effect of microorganisms on the transformation and formation of key metabolites. Our findings will provide new perspectives for analyzing the quality formation mechanism of Qingzhuan tea.The experimental samples used in this study were obtained from the Zhaoliqiao Tea Factory in Chibi city, Hubei province, China. Sampling was started at the beginning of the pile fermentation of raw tea, and sampling was conducted every other day 10 times during entire pile fermentation. The samples from the first sampling were named QZT1, and the samples obtained from the 1st to the 10th were successively named QZT1~QZT10. The fermentation piles were turned three times. QZT1~QZT4 were the samples obtained from the first pile turning, QZT5~QZT7 from the second pile turning, and QZT8~QZT10 from the third pile turning. Each sample was randomly selected from 10 points in different layers of the large fermentation pile. About 1 kg of the sample was mixed, sealed in a sterile sampling bag, and quickly frozen on dry ice for metagenomic and metabolomic sequencing analyses. The LC-MC analysis and metagenomic analysis were conducted with three biological replicates.The 60 g tea samples were added into 800 mL sterile water and stirred thoroughly to obtain a mixture solution. After filtering with sterile gauze, the filtrate was centrifuged at 1200 rpm for 10 min at 4 °C. Afterwards, the supernatant was collected and centrifuged at 10,000 rpm for 10 min, and then the precipitate was collected and centrifuged twice at 120,000 rpm for 10 min each time, followed by DNA extraction.2O to dissolve the DNA sample. (7) An amount of 1 μL RNase A was added to digest RNA. The sample solution stood at 37 °C for 15 min and was stored at −20 °C.DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method with the following steps. (1) The 1000 μL CTAB lysate was sucked into a 2.0 mL EP tube. An amount of 20 μL of lysozyme was added and an appropriate amount of tea sample was added into the lysate. The samples were subjected to a water bath at 65 °C, inversion mixed evenly, and lysed thoroughly. (2) After centrifugation, 950 μL supernatant was supplemented with an equal volume of the mixture of phenol (pH = 8.0), chloroform, and isoamyl alcohol (at the ratio of 25:24:1), inversion mixed, and centrifuged at 12,000 rpm for 10 min. (3) The supernatant was collected, supplemented with an equal volume of chloroform: isoamyl alcohol (24:1), inversion mixed, and centrifuged at 12,000 rpm for 10 min. (4) The supernatant was sucked into a 1.5 mL centrifuge tube, supplemented with 3/4 (supernatant) volume of isopropanol, shaken up and down, and precipitated at −20 °C for 5 min. (5) After centrifugation at 12,000 rpm for 10 min, the supernatant was removed and the precipitate was washed twice with 1 mL of 75% ethanol, and the remaining small amount of liquid was collected and centrifuged again, and then sucked out with a pipette. (6) The obtained precipitate was blow-dried on an ultra-clean bench and supplemented with 50 μL ddHAn amount of 1 μg of genomic DNA was used to construct a metagenomic library. Specifically, the library was constructed using the NEBNext Ultra DNA Library Prep Kit for Illumina, and library construction steps included sample fragmentation, end repair and A-tailing addition, adapter connection, and library PCR amplification. Subsequently, PCR reaction product was purified with 0.6 times Agencourt AMPure XP (nucleic acid purification magnetic beads) and the concentration of the library was detected by Qubit2.0. Gene sequencing was conducted using Illumina PE150 from Nuohezhiyuan Company .https://www.github.com/cjfield/readfq, accessed on 4 July 2022) was used to preprocess the raw data obtained from the Illumina HiSeq sequencing platform to obtain clean data for subsequent analysis. SOAPdenovo V. 2.04 software  was used to analysis the genome assembly based on the obtained clean data.The Readfq  was the platform used to predict the open reading frame (ORF) of the scaffolds of the samples and information with a length < 100 nt was removed from the prediction results. CD-HIT software  was used to remove redundancy from ORF prediction results so as to obtain a non-redundant initial gene catalogue. Then, Bowtie 2 software (Bowtie 2.2.4) was used to align the clean data of the samples with their scaffolds. Based on the abundance information in the gene catalog, core–pan gene analysis, correlation analysis between samples, and gene number Venn diagram analysis were performed.MetaGeneMark  was used to align the unigenes to the functional databases including the KEGG database , eggNOG , and CAZy 24 151 . The best blast hit result was selected from the alignment results of each sequence for subsequent analysis. Based on the abundance table at each taxonomic level, annotated gene number was counted; relative abundance overview was displayed; abundance cluster heatmap was plotted; and metabolic pathway comparative analysis, PCA , NMDS  dimensionality reduction analysis, and LEfSe  analysis of functional differences between groups (with LDA score set as 4) were performed.DIAMOND software  were put into the vial. The samples from each group were subjected to LC-Orbitrap-MS, and 10 μL samples from each group were mixed to establish a quality control sample (QC) for instrument stability monitoring and data deviation correction.The liquid chromatography analysis conditions were as follows: Hypersil GOLD C18  column was adopted; the column temperature was 40 °C; the sample plate temperature was 4 °C; the mobile phase A was water +0.1% formic acid; the mobile phase B was acetonitrile +0.1% formic acid; the flow rate was 0.3 mL/min; and the injection volume was 2 μL. The gradient elution conditions are shown in m/z; the resolution was 60,000, the number of data-dependent scanning triggers was 10, and the fragmentation voltage was 20 V ± 50%.Mass spectrometry analysis conditions were as follows: Q-Extractive Focus high-resolution combined mass spectrometer was adopted and the mass spectrometer was equipped with a heatable electrospray ion source (HESI-II). The full-scan mode settings were as follows: For data collection in positive ion mode, the sheath gas flow rate was 45 arb; the auxiliary gas flow rate was 15 arb; the exhaust gas flow rate was 1 arb; the capillary temperature was 350 °C; the capillary voltage was 3.8 kv; and the RF (radio-frequency) energy level of the lens was 60%. For Controid data acquisition mode, the mass range of the acquisition was 50~1000 The chemical component data of each sample were collected using Xcalibur 2.1.x software, and the raw data in .RAW format were inputted into Compound Discoverer 2.0 software for peak identification and peak integration. Subsequently, retention time correction, peak alignment, background subtraction, and devolution were performed to obtain two-dimensional data matrix with the peak area, retention time, mass/charge ratio, and sample name of each group.metlin.scripps.edu, accessed on 13 September 2022), HMDB , and KEGG . According to the obtained secondary mass spectrum information of possible compounds and retention time, the secondary spectra of the corresponding compounds were searched in Metlin , Mzcloud , and Massban . According to the fragmentation energy, the corresponding fragment ions were aligned and the candidate compounds were further listed. Based on the retrieval information from TMDB (pcsb.ahau.edu.cn:8080/TCDB/f) database and the related literature, the candidate compounds were screened and characterized.By using the OPLS-DA  model, the key difference compounds of samples in different processing procedures were analyzed. After an accurate mass/charge ratio list (primary mass spectrometry information) was obtained from the raw data matrix, the relevant secondary mass spectrometry information was obtained using LC-Orbitrap-MS. According to the primary mass spectrum information of differential compounds, the possible compounds were retrieved and listed by searching online databases Metlin  and orthogonal partial least squares–discriminant analysis (OPLS-DA).Samples with three replicates from the pile fermentation process were used as the experimental materials. In total, 570,421.25 Mbp of raw data were obtained; after removing reads with low-quality bases , those with a lower proportion of N bases (default was set to 10 bp), and those that exceeded the threshold (default was set to 15 bp), 567,343.62 Mbp of clean data were obtained from sequencing by Illumina HiSeq. After assembly, a total of 6,682,219,438 bp of scaffolds were obtained. After gene prediction by MetaGeneMark Linux 64 software, a total of 6,353,703 ORFs were obtained. After redundancy removal, a total of 740,943 ORFs were obtained with a total length of 324.59 Mbp, of which the number of complete genes was 368,400, accounting for 49.72%.Based on the abundance table of genes in each sample, the gene number information of each sample was obtained. Different numbers of samples were randomly selected to obtain the number of genes in the combinations of different numbers of samples, based on which the dilution curves of the core and pan genes were constructed and plotted . The cloBased on the relative abundance of microorganisms, principal component analysis (PCA) and Bray–Curtis distance cluster analysis of the samples during pile fermentation were performed. The results showed that, during the Qingzhuan tea pile fermentation process, the samples clustered into three groups: QZT1, QZT2, QZT3, and QZT4 in stage 1 belonged to group 1; QZT5, QZT6, and QZT7 in stage 2 to group 2; and QZT8, QZT9, and QZT10 in stage 3 to group 3. These clustering results were basically consistent with the three pile turning time points during pile fermentation. Stage 2 and stage 3 were clearly separated from stage 1, and stage 2 tended to move to stage 3. The fungal community was scattered in the early stage of pile fermentation and the microbial community of QZT6 in stage 2 exhibited the greatest changes, and then the microbial community in stage 2 tended to be consistent with that in stage 3 of pile fermentation .In order to better demonstrate the differences in the microbial community structure during the Qingzhuan tea pile fermentation process, the relative abundances of fungi and bacteria in the macrogene data were analyzed. As shown in Lichtheimia and Helicocarpus , the abundance of Aspergillus was significantly increased, while the abundance of Pichia remained relatively stable.Subsequently, this study analyzed the changes in the relative abundance of microorganisms at kingdom levels. The results showed that, in the early stage of Qingzhuan tea pile fermentation (stage 1), the dominant microorganisms were cocarpus B. With t−5). A total of 13,759 (1.86%) ORFs were aligned to the CAZy database, 264,663 (35.72%) ORFs to the KEGG database, and 268,864 (36.29%) ORFs to the eggNOG database. Based on the resistance gene database (CARD), the non-redundant gene set was annotated  and 516 genes were aligned to the CARD database , Lactobacillaceae contributed the most to stage 2 (middle stage), and Aspergillaceae and Trichocomaceae contributed the most to stage 3 (late stage).In this study, linear discriminant analysis (LDA) in LEfSe was used to investigate the samples in the early (stage 1), middle (stage 2), and late (stage 3) three stages so as to identify differential microorganisms, and an LDA value > 4 and ectively B,C. The Proteobacteria phyla and two Actinobacteria phyla, except one fungus . In Proteobacteria microorganisms, there were two α-Alphaproteobacteria class, Sphingomonadales order, Sphingomonadaceae family, Sphingomonas genus, and one γ-Gammaproteobacteria class and Xanthomonadales order. The two Actinomycetes phyla belonged to the Actinobacteria class.As shown in Firmicutes phyla and 7 Proteobacteria phyla as well as bacterial communities at various levels of order, family, and genera contained therein: Firmicutes and its Lactobacillus genus (5) and Weissella genus (1), Proteobacteria and its Enterobacteriaceae family (4) and Acetobacter genera (3). The 4 differential fungal communities significantly enriched in stage 2 included Mucoromycota phylum (1), Ascomycota phyla (3), and fungal communities at various levels of the order, family, and genera contained therein: Ascomycota phylum and its Peltigerales order (2) and Pichia genus (1).Twenty differential microorganisms significantly enriched in the middle stage of pile fermentation (stage 2) included 14 bacteria, 4 fungi, and 2 viruses. The 14 differential bacteria significantly enriched in stage 2 were composed of 6 Actinobacteria bacterial classes and 68 Ascomycota fungal phyla as well as the communities at various levels of order, family, and genus contained therein. Differential microorganisms in bacteria included Saccharopolyspora genera (8), Actinopolysporaceae families (4), and Streptomycetales orders (2). Except for 2 Agaricomycetes classes from the Basidiomycota phylum, the other 68 significantly enriched differential fungal microorganisms were all from the Ascomycota phylum, including Eurotiomycetes classes (28), Lecanoromycetes classes (10), Sordariomycetes classes (9), Dothideomycetes classes (8), Leotiomycetes classes (7), and Pezizomycetes classes (4). The Eurotiomycetes class with the most differential microorganisms included 9 Onygenales orders and 19 Eurotiales orders. The Aspergillaceae family (8 species), Trichocomaceae family (5 species), and Elaphomycetaceae family (3 species) were the main microorganisms in the Eurotiales order. The species Aspergillus niger, Aspergillus_wentii, Aspergillus_turcosus, Aspergillus_clavatus, Penicillium_subrubescens, and Penicilliopsis_zonata in the Aspergillaceae family, and S-Rasamsonia_emersonii, Talaromyces_marneffei, and Talaromyces_verruculosus in the Trichocomaceae family were found.The differential microorganisms significantly enriched in stage 3 (the late stage of pile fermentation) included 15 p < 0.05 as screening thresholds. Most of the screened compounds were enriched in amino acid and polyphenol metabolic pathways. With the pile fermentation progression, the contents of polyphenolic metabolites were decreased to varying degrees mainly including amino acids such as theanine , chlorogenic acid , GCG , puerin of the two types of B-ring dihydroxyl , galloyl puerin B-ring trihydroxyl , and others. However, the contents of tannin  and amino acid analogs  in the polyphenol metabolic pathway were increased to varying degrees with the progression of pile fermentation.Further, we also conducted metabolomic analysis on the same batch of Qingzhuan tea processing samples  and scre2 = 0.52, p < 0.05) revealed that there was a significant correlation between microorganisms and the metabolic profile of Qingzhuan tea. Correlation analysis was conducted between 53 metabolites with VIP > 1.5 identified by Qingzhuan tea metabolomics method and 106 microorganisms screened by metagenomic LEfSe analysis (Sphingomonadaceae family and two strains of Glomeromycetes classes) exhibited the pattern opposite to that between VIP compounds and the other 88 target microorganisms, which might be closely related to their different growth characteristics and nutritional requirements. Subsequent analysis found that, during the process of Qingzhuan tea pile fermentation, the relative expression patterns of 23 metabolites (11 annotated) were the same as those of most screened microorganisms, while the relative expression patterns of 28 metabolites were opposite to those of most screened microorganisms.Proctor’s analysis examined the correlation between the microorganism and metabolite profiles of Qingzhuan tea. As shown in analysis . The resThe metabolites with the same expression pattern as the screened microorganisms were mainly from polyphenol metabolism-related pathways (8/11), chlorogenic acid , GCG , phenolic acids , phenolic amides , two kinds of B-ring dihydroxy pu-er , B-ring tri-hydroxyl galloyl pu-er , and other compounds. Three other amide metabolites  and the target microorganism exhibited the same relative expression pattern. The metabolites whose expression patterns were opposite to those of screened microorganisms mainly involved four amide compounds composed of , , fatty amides , and cyclic peptides ; four amino acids including theanine , glutamine , amino acid analogs , and ; two polyphenol metabolites including tannins  and phenolic acid , and two disaccharide metabolites,  and .Sphingomonas genus, which mainly showed a significant positive correlation with amino acids  and B-ring dihydroxy puertin  metabolism compounds, and the Aspergillaceae family and Trichocomaceae family contributed greatly to stage 3. Among the Aspergillaceae family, Aspergillus_fumigatus was only significantly correlated to compound , while Aspergillus_tubingensis and Aspergillus_niger were significantly correlated with . Aspergillaceae family members such as Paecilomyces_Penicilliopsis, Aspergillus_wentii, and Aspergillus_clavatus, and the Trichocomaceae family showed correlations with almost all metabolites. Among them, GCG showed a significant positive correlation with the Ajellomycetaceae family of the Eurotiales order, Aspergillus_wentii of the Aspergillaceae order, and Verticillium_dahliae of the Sordariomycetes class. However, purine and catechin showed a positive correlation with most of the target microorganisms. The correlation pattern between microorganisms and GCG or B-ring trihydroxy galloyl puerlin  was completely opposite to that between microorganisms and all identified amino acids. These completely opposite correlation patterns might regulate the carbon/nitrogen balance in tea leaves to some extent during the pile fermentation process of Qingzhuan tea. Amino acids and flavonoids represent nitrogen-containing and carbon-rich metabolites, respectively, and thus their metabolisms are regulated by the carbon/nitrogen balance. Glu and ethylamine are the precursors synthesized by theanine, while phenylalanine is the starting unit of flavonoid metabolism. All the carbon skeletons used for synthesizing Glu, ethylamine, and Phe are derived from the intermediates  produced from glycolysis and the tricarboxylic acid (TCA) cycle. The shared carbon backbones may be a key factor regulating the compound flow between the amino acid pathway and the flavonoid pathway [The metagenomic analysis showed that the microorganism with the largest contribution to stage 1 was the  pathway ,31.Actinomycetes and molds, and yeast was the least [Mucor racemosa from the pile fermentation process of Qingzhuan tea and simulated fermentation, and found that this strain was an important strain involved in pile fermentation process of Qingzhuan tea. The evolution of the microbial community structure may be due to changes in metabolite contents during the pile fermentation [During the fermentation process of Qingzhuan tea, the microbial community plays an important role in the quality formation and material transformation process. Previous studies have shown that the enzymatic reaction during the pile fermentation process is mainly responsible for the reduction in chemical components ,28. Aminhe least . Wang etentation .Aspergillus niger, Aspergillum gloucus, and Penicillium were identified as the dominant strains in the fermentation process of Pu’er tea [Talaromyces (three species) and Rasamsonia (two species) of the Trichocomaceae family were also identified as differential microorganisms in this study, and they were detected in Liubao tea and Puer tea [Talaromyces is a thermotolerant genus that mostly grows and reproduces in high-temperature, high-humidity, aerobic organic matter clumps and it can secrete high temperature-resistant hydrolytic enzymes. Rasamsonia is a new genus including thermotolerant and thermophilic Talaromyces and Geosmithia species and it has a strong activity of decomposing cellulose at about 50 °C, and the rate of degrading cellulose of thermophilic Sporotrichum is five times that of the normal temperature strain Trichoderma [Our results showed that fungi were more important than bacteria in the pile fermentation of dark tea, which was similar to the previous report on Fuzhuan tea ,21. Aspeu’er tea ,19, Fuzhu’er tea , and Qinu’er tea . TalaromPuer tea ,35, but choderma ,35.Aspergillus can secrete α-amylase, glucoamylase, cellulase, pectinase, and other proteases, and it plays an important role in decomposing proteins and lipids, and producing the unique flavor of tea [Aspergillus niger, Aspergillum gloucus, Penicillium of the Aspergillaceae family, and Talaromyces and Rasamsonia of the Trichocomaceae family were identified as the dominant strains in the process of pile fermentation of Qingzhuan tea, and they were important participants in the formation of the sensory qualities of Qingzhuan tea such as the unique flavor and taste. However, a series of scientific issues such as how these dominant microorganisms, the Aspergillus family and the Trichocomaceae family, interact with the tea components and how they participate in the formation of quality compounds need to be further explored.Molds are the most common microorganisms found in the fermentation process of Pu-er tea. As the dominant strain, r of tea ,19. In tTrichocomaceae, Elaphomycetaceae, Aspergillaceae in the Eurotiomycetes family and Eurotiales order were the main contributors. Puerin extracts from Pu-er tea have been confirmed to significantly reduce the levels of blood sugar, total cholesterol, and triglycerides in plasma, and their hypoglycemic effect might be derived from their inhibition of α-glycosidase activity [One previous study has shown that, under the action of fungal fermentation, Puerins I~VIII formed from the condensation transformation of catechin and theanine were detected in Pu’er tea and Fuzhuan tea, and their (-)-epicatechin or (-)-epigallocatechin parts were replaced by N-ethylpyrrolidone . Two pueactivity . There aAspergillaceae, Trichocomaceae, and Elaphomycetaceae. The Aspergillaceae family included Aspergillus niger, Aspergillum gloucus, Aspergillus wentii, Aspergillus turcosus, Aspergillus clavatus, and Penicillium. Amino acids and polyphenol metabolites with relatively simple structures showed a significant negative correlation with target microorganisms, while the complexes with complicated structures such as catechin–amino acid and catechin–purine showed a significant positive correlation with target microorganisms. Aspergillus niger, Aspergillums gloucus, and Penicillium in the Aspergillaceae family, and Talaromyces and Rasamsonia emersonii in the Trichocomaceae family were the key microorganisms for the characteristic quality formation of Qingzhuan tea.In the early stage (stage 1) and the middle stage (stage 2) of Qingzhuan tea pile fermentation, the dominant microorganisms were bacteria. In the late stage (stage 3) of pile fermentation, the dominant fungi were mainly


DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-β, are potential biomarkers for dystrophic muscle characterization.Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the mdx and wild-type mice. Metabolite changes were analyzed using 1-H Nuclear Magnetic Resonance, while TNF-α and TGF-β were assessed using Western blotting to examine inflammation, and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups.To investigate disease progression and aging, we utilized young (1-month old) and old (21–25 months old) mdx animals of the same age. The metabolites alanine, methionine, 3-methylhistidine were higher, and taurine and glycerol were lower in young animals in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine and the proteins TNF-α and TGF-β had no difference in the analysis between groups (p > 0.05).The histological analysis of the tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild type or mdx animals is protected from severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-β do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.Surprisingly, histological and protein analysis reveals that the tongue of young and old  DMD gene 6].[6].5][6].[6].5]. Arou. Aroumdxh on DMD . The mdxd fibers .mdx mice show changes in metabolites related to the progression of muscle degeneration and aging  14] [14]mdx  [14]mdx mdx mice, the tongue muscles (TON) show an unusual behavior as the disease progresses compared to other muscles. In 3-month mdx mice, inflammatory cells were hardly found in the TON, unlike other masticatory muscles. In addition, the expression of collagen did not change in the TON of these mdx, while it was three times higher in masseter muscle (MAS), when compared to the control . In. In16].  control . These r control  describemdx’s diaphragm and quadriceps muscles, compared to the wild-type 32].[32].mdx .[32].mdxnd soleus is also mdx mice, compared to wild type, has already been seen in other muscles, indicating the possibility of a deficient taurine synthesis by the mdx muscles 27] [27] 18][ [27] 18]mdx mice 44]. In . In mdx  muscles  studied mdx diaphragm muscles, in comparison with the control and over age, with 1, 4 and 9 months. The intrinsic muscles of the larynx (ILM) were identified as muscles protected from dystrophinopathy in the mdx mice, as they did not show signs of muscle damage, degeneration or regeneration, during the course of the disease [mdx animals did not differ from the wild type animals, in young and old. Messina et al. (2011) [TNF-α is a pro-inflammatory protein produced by activated macrophages, mast cells, endothelial cells and some other cell types. It stimulates the expression of adhesion molecules in endothelial cells, increasing leukocyte recruitment and adhesion. TNF-α is secreted at the site of inflammation and can enter the bloodstream . The TNF disease . The con disease . These r. (2011)  analyzedmdx mice. Glycine is a non-essential amino acid involved in the production of DNA, phospholipids and collagen, in addition to being involved in the release of energy [mdx mice and dystrophin/utrophin double knockout mice can attenuate the progression of dystrophic pathology, as well as improve the effectiveness of prednisolone, the current gold standard treatment for DMD. Anderson and Skrabek [mdx mice treated with deflazacort, from a metabolomic perspective. They showed that the concentration of glycine in the mdx heart decreased with disease progression, but with a 2-week high-dose treatment with deflazacort, glycine levels increased above the levels of the mdx placebo group. Glycine supplementation has already been shown to protect against loss of myotubes in nutrient restricted/growth factor restriction models with C2C12 muscle cells in vitro. This in vitro protection has been shown to be dependent on mammalian target of rapamycin complex 1 (mTORC1) signaling [mdx muscle, close to the 20th day degeneration peak, may be related to the high levels of glycine presented in our study.The metabolites glycine and glutamic acid were different only in dystrophic animals, being more concentrated in young f energy . D.J. Haf energy  demonstr Skrabek  studied ignaling  and alsoignaling . Thus, tmdx mice, such as improvement in dystrophinopathy with decreased necrosis [mdx were equivalent to those of control animals of the same age, indicating protection against myonecrosis on the tongue.Glutamic acid, also known as glutamate (the anion), is a non-essential amino acid, one of the 20 proteinogenic amino acids and the most abundant rapid excitatory neurotransmitter in the nervous system . Glutathnecrosis . In a menecrosis . Lafertenecrosis  observednecrosis  and its These results corroborate the protection observed in histology and the use of these as muscle biomarkers of degeneration. Other studies on affected muscles can elucidate their specificities as biomarkers for the progression of DMD, without the interference of aging.mdx animals remains protected from the intense and progressive myonecrosis described in other muscles, as evidenced by histological analysis. This protection was verified by the biomarker proteins for inflammation and fibrosis, and by the concentrations of the metabolites 3-methylhistidine, acetic acid, glutamic acid, alanine, creatine, phosphocreatine, glycerol, glycine, isoleucine, methionine, succinic acid and taurine already used for dystrophic muscle differentiation and in this work presented similar results between wild type and mdx.The tongue of young (1 month) and old (21 to 25 months) Among the possible biomarkers analyzed, the metabolites alanine, methionine, 3-methylhistidine, taurine and glycerol may be efficient for specific assessments, but care should be taken when using them to monitor the progression of the disease, since they change throughout life.Muscle degeneration biomarkers, which in other studies proved to be efficient  did not show any difference with aging and in the spared muscles. Other studies on affected muscles can elucidate their specificities as biomarkers for the progression of DMD, without the interference of aging.


To assess the prevalence of psychiatric disorders in patients with type 1 diabetes mellitus (T1D) and to compare patients with and without psychiatric disorder.We made a cross-sectional study including patients with T1D assisted in the outpatient clinics of the Brazilian National Health System. To assess depression and anxiety, we used the PHQ-9 questionnaire and the DSM-5th edition criteria, respectively. B-PAID evaluated the level of emotional distress associated with diabetes; EAT-26, eating disorders; SCI-R, adherence to the proposed clinical treatment.We analyzed 166 patients aged 33 (22-45.2) years, 53.6% female. The prevalence of depression and anxiety was 20.5% and 40.4%, respectively. HbA1c was worse in the depressed , in the anxious ones  and in the patients with high levels of B-PAID . There was no difference in the prevalence of complications related to diabetes.The prevalence of psychiatric disorders and emotional distress related to diabetes was high in our population of T1D patients, and depression and high levels of B-PAID were associated with the worse glycemic control. Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease that causes absolute deficiency in insulin production. People with the disease need daily insulin doses and glycemic self-monitoring several times a day, in addition to healthy living habits, with a routine of diet and physical activity. This demand for continuous self-care is especially difficult in childhood and adolescence. The transition to adulthood, in addition to impacting psychological maturity and increasing independence, commonly leads to worsening of the metabolic control of diabetes .According to WHO data, depressive disorder affects about 4.4% of the world population, while the estimated prevalence of anxiety is 3.6% ,8, has aThe aim of this study was to evaluate the prevalence of psychiatric disorders in patients with T1D and its relationship with adherence to treatment, emotional distress related to diabetes control, metabolic rate and increased risk of related complications in the short and long term in a cohort of patients in southern Brazil.Cross-sectional study carried out in specialized care clinics at two university centers in the cities of Pelotas and Porto Alegre, southern Brazil – Federal University of Pelotas and Federal University of Rio Grande do Sul. Patients with T1D who had performed more than 2 consultations registered in medical records, the last service being performed in the previous 12 months. The recruitment of patients took place from March 2016 to October 2019.Patients under 10 years of age, those with other types of diabetes, those who had no registered consultation in the last 12 months or who did not agree to participate in the study were excluded.The diagnosis of T1D was made by a specialist doctor using clinical and laboratory criteria, such as a history of diabetic ketoacidosis, exclusive treatment with insulin, presence of positive glutamic acid anti-decarboxylase antibody or C peptide below the reference value.Hospital de Clínicas de Porto Alegre . CAAE number 91767018.9.1001.5327.The research project was submitted to and approved by the ethics committees of the Medical School of the Federal University of Pelotas  and the All participants signed an informed consent form.The variables under study were collected by previously trained interviewers, and data collection took place in three phases: 1. application of a face-to-face questionnaire to obtain socio demographic data, 2. three self-administered questionnaires previously standardized and translated into Portuguese –17 addreThe clinical information obtained from the medical record was the presence of macrovascular disease , systemic arterial hypertension, dyslipidemia, kidney disease, diabetic neuropathy and retinopathy and hypoglycemia.The laboratory tests evaluated in medical records within the last 12 months, were: glycated hemoglobin, the most recent , urine sample albuminuria (immunoturbidimetry), serum creatinine (colorimetric method), thyroid function (immunoassay of microparticles by chemiluminescence), antibodies to celiac disease (chemiluminescence method), anti-glutamic acid decarboxylase antibody  and C peptide (microparticle chemiluminescent immunoassay).Those ones with a medical record of the event were considered with macrovascular disease. Patients admitted as hypertensive and dyslipidemic were using antihypertensive and hypolipidemic medication, respectively. Participants who had albuminuria in isolated urine sample > 30 mg/g creatinine and/or estimated glomerular filtration (eGFR) < 60 mL/min/1.73 m²  in two different times were considered renal patients ,19. The Participants were asked about their previous diagnosis of psychiatric disorder indicating which disease(s) they had: depression, anxiety, panic disorder, bipolar disorder, “I don't know” – when they didn't know their psychiatric diagnosis – and “other”, when the pathology was not included in the presented relation. For objective evaluation, the Brazilian Problem Areas In Diabetes Scale (B-PAID) questionnaires, related to the level of emotional distress associated with the routine of living with diabetes, with values ≥ 40 indicating high level of emotional distress, considered “Severe B-PAID” . Eating For statistical analysis and graphic representation, we used the program Statistical Package for the Social Sciences  for Windows (version 22). Dichotomous variables were described as number and percentage, and quantitative variables were defined as mean, standard deviation, median and interquartile range according to the Gaussian distribution. Comparisons were made using t test for variables with normal distribution, and Mann-Whitney test for non-Gaussian distribution for continuous variables, and chi-square and Fisher's exact for qualitative variables. Logistic and linear regression models were used to assess possible effects of other additional risk factors. The level of significance adopted was 5%.The sample calculation was based on a Brazilian study with T1D patients, whose prevalence of depression was 13.6% with a sample size of 181 patients being defined, assuming 5% as the maximum difference between the prevalence of real depression and estimated, with a 95% confidence level.Altogether, 191 patients met the inclusion criteria, with 25 being excluded for not completing the clinical interview. Among the participants, 120 came from the Porto Alegre outpatient clinic and 46 from Pelotas. Regarding the patients excluded from the study (n = 25), they were younger with a median of 21 years (15.0-27.5), had shorter disease duration and a higher level of HbA1c 9.4% [7.5%-12.1%], (79 mmol/mol [58-109]), than the patients included in the study. The other variables were not different. (Supplement).The analysis of the 166 patients included showed that 89 53.6%) were women, with a median of 14 years of disease duration, HbA1c median of 8.5% (7.8%-9.4%), (69 mmol/mol [62-79]), and monthly family income of 2 , with 67 (40.4%) patients having positive screening for anxiety and 34 (20.5%) for depressive disorder, exclusively . Of the A comparative analysis was performed between the groups of patients with and without depression assessed by PHQ-9, which are described in The comparison between groups of patients with and without anxiety , on the As for the eating disorder, 79 (47.6%) patients had positive EAT screening. The group of patients with positive screening had an older age, were more frequently female, had a higher rate of clinical comorbidities and a higher level of suffering from B-PAID .The B-PAID instrument diagnosed 79 (47.6%) patients with high emotional load, with glycemic control being worse in this group. The group with the greatest emotional distress had a higher percentage of women and more patients with positive screening for eating disorder. The individuals most adherent to the treatment were in the group with the lowest emotional distress and those who practiced regular physical activity had significantly lower levels of emotional distress . B-PAID Treatment adherents totaled 91 (54.8%) using the SCI-R questionnaire. The group with adhesion showed better levels of glycated hemoglobin , (68 mmol/mol [58-76] vs. 73 mmol/mol [64-81]), more frequent physical activity , more self-blood glucose monitoring  and lower frequency of psychiatric medication use  in relation to patients poorly adherent. Smoking was more frequent in T1D patients with low adherence to treatment when compared with adherents  (Supplement).Our study describes a cohort of T1D patients in two tertiary care centers for patients with diabetes in southern Brazil. Our findings show that depressed patients with high suffering related to diabetes had worse glycemic control, although we did not observe an association between psychiatric disorder and the presence of chronic complications of T1D.In the present study, depressed patients are also more prone to severe hypoglycemia and a greater need for hospitalization due to the disease. Depressed patients were more prone to eating disorders. They also had less adherence to the proposed treatment and high levels of emotional distress related to T1D (B-PAID). In addition, they had a significant suicidal behavior or ideation, mainly in women.Anxiety was related to sedentary behavior and low adherence to daily glycemic self-monitoring. It was more frequent in females. Those with anxiety disorder had statistically higher emotional distress related to diabetes (B-PAID), which perhaps explains the 72.2% of anxious patients with some thought of ideation and in some cases even attempted suicide. In addition, anxiety was strongly related to low adherence to diabetes treatment, although not significant, which can probably be related to our sample size.Regarding to prevalence, we observed 20.5% and 40.4% of depressed and anxious patients, respectively. While Maia and cols. reported only 13.6% of depressed T1D patients, using the Hospital Anxiety and Depression Scale (HADS) tool for diagnosis  and dataWe found almost 55% of the screened sample with a positive result for some psychiatric disorder, of which 68% had already been evaluated by psychologists/psychiatrists at some point in their lives and only 27% were using pharmacological treatment. It is known that underdiagnosed and, consequently, undertreated patients worsen the prognosis of diabetes-related outcomes . In thisMore adherent patients had better glycemic control, while high levels of diabetes-related suffering were associated with an increased risk of depression and anxiety. In anxious patients, the prevalence of physical inactivity was higher than in controls. In the context of evaluating psychiatric comorbidities, sedentary patients with high levels of emotional distress seem to be more likely to develop such pathologies, even before they express symptoms of depression or anxiety. Therefore, the active search for such a condition should be encouraged and even should be part of the screening for chronic complications of diabetes.According to the ADA, physical exercise is recommended for every T1D patient  and shouThe demands for self-care of T1D increase the risk of developing psychiatric disorders –6. A freRegarding the limitations, we carried out a cross-sectional study, collecting some clinical data in medical records. Due to the design, we are limited in the definition of some vascular complications of diabetes, such as macrovascular disease and other comorbidities . The diagnostics made by simple record in the medical record. This may certainly have underestimated the actual number of patients with vascular complications and the potential risk of inadequate data filling. However, since both clinics are university centers specialized in diabetes, the medical record information is reliable. Furthermore, we do not estimate the relevance of neuropathic pain in the psychological health of the participants. When using self-administered questionnaires that assess diabetes-related stress, we assumed the risk of misdiagnosing patients as depressed, which may have increased the frequency of depression, according to Roy and Lloyd . In addiThe strengths of our study were the multicenter design, a broad evaluation of psychiatric disorders, the use of well-validated questionnaires and the finding of a huge number of patients with T1D have underdiagnosed and undertreated psychiatric disorders, which can impair glycemic control. Moreover, we found a high prevalence of eating disorders, psychiatric disorders and emotional distress related to type 1 diabetes. In particular, depression and elevated B-PAID were associated with worse glycemic control. Sedentary behavior and emotional distress or illness-related stress were associated with the presence of these pathologies. Therefore, medical care for psychiatric disorders should be encouraged by current guidelines and screening for these morbidities should be performed actively and early.


Therefore, a systematic review and meta-analysis were conducted to comprehensively identify potential correlations.Ambient air pollution has been identified as a primary risk factor for mental disorders. In recent years, the relationship between exposure to ambient nitrogen dioxide (NOI2 over 50%).A literature search in 3 international databases was conducted before December 31, 2022. Relative risk (RR) and corresponding 95% confidence intervals (CI) were calculated to evaluate the strength of the associations. Summary effect sizes were calculated using a random-effects model due to the expected heterogeneity . However, the heterogeneity was high for the summary estimates, reducing the credibility of the evidence. In 2-pollutant models, results for NO2 increased by 0.3%, 0.2% and 2.3%, respectively, after adjusting for PM2.5, PM10 and SO2.A total of ten eligible studies were included in the meta-analysis, including 1,412,860 participants. The pooled analysis found that an increased risk of HAs for schizophrenia was associated with exposure to each increase of 10 μg/m2 exposure significantly increases the risk of hospital admission for schizophrenia. Future studies are required to clarify the potential biological mechanism between schizophrenia and NO2 exposure to provide a more definitive result.This study provides evidence that NO It affects more than 24 million people worldwide and is among the top 15 leading causes of disability in the world.,3 Empirical evidence shows that people with schizophrenia have impaired daily living skills and a higher risk of premature death than the general population. In addition to its impact on individuals, schizophrenia imposes a significant burden on society and the economy. People with schizophrenia, or those at risk of developing it, are more likely to engage in aggressive behavior towards others, potentially leading to violent crimes. Moreover, schizophrenia is one of the most socially and economically costly mental disorders. A comprehensive review of 56 studies indicated that the economic burden of schizophrenia ranged from 0.02% to 1.65% of GDP in different countries. Unfortunately, the prevalence of schizophrenia is on the rise in many countries, such as China and Korea.,8 Many studies suggest that the primary contributors to schizophrenia include genetic, socioeconomic, environmental, and behavioral risk factors.,9 The relationship between air pollution and schizophrenia has recently been highlighted.Schizophrenia is identified as a severe mental disorder characterized by abnormalities in thought and cognition. Exposure to particulate (PM2.5 and PM10) and gaseous pollutants  may trigger systemic and neuroinflammatory responses, possibly leading to psychopathology through altered neurotransmission and neurodegeneration.–13 Therefore, it has been plausibly hypothesized that air pollution exposure may contribute to schizophrenia pathogenesis. Nitrogen dioxide (NO2) is a gaseous air pollutant produced by fossil fuel combustion in transportation. In studies that determine the association between gaseous pollutants and schizophrenia, NO2 has received relatively high attention from researchers, but the results remain controversial. Some studies reported a positive correlation between them,,14 and others found no association.,16 We, therefore, performed a systematic review and meta-analysis of original research articles to further clarify the potential associations between NO2 exposure and schizophrenia.Air pollution represents the most significant environmental health risk globally. According to the World Health Organization, over 90% of the world's population resides in areas where air pollution levels exceed the recommended limits.2 on schizophrenia can be classified into short-term and long-term effects, where the latter relates to cumulative exposure while the former pertains to an individual acute response. This study examines the association between short-term NO2 exposure and schizophrenia, with daily NO2 concentrations serving as the independent variable, and the risk of hospital admission for schizophrenia as the dependent variable. Furthermore, the results from the 2-pollutant models reported in the included studies were analyzed separately to control for the potential impact of other pollutants  on the findings.The effects of NOThe systematic review and meta-analysis have been registered and published in PROSPERO, with the assigned registration number CRD42022378942.In light of the fact that all analyses were conducted on previously published studies, and no patient consent or ethical approval was required.2” OR air pollutants) AND (“schizophrenia” OR “schizophrenics”) AND . The reference lists of eligible studies and other related reviews were also screened to identify additional studies.This study was performed according to the preferred reporting items for systematic review and meta-analysis (PRISMA) statement (see Guidelines Checklist). The literature search of the PubMed, Embase and Web of Science electronic databases was independently conducted by 2 authors to identify any eligible epidemiological studies published up to December 31, 2022. A search strategy was detailed as following terms: (“nitrogen dioxide” OR “NO2 (<30 days); (C) the study provided quantitative effect estimates with 95% CIs (or standard errors) by comparing humans exposed to lower air pollution levels with humans exposed to greater levels; and (O) schizophrenia confirmed by clinical assessment of the International Classification of Diseases (ICD). Studies that did not report the effect of short-term NO2 exposure on the risk of hospital admission for schizophrenia were excluded. Reviews, non-English studies, and animal experiments were also excluded.Based on the predesigned PECO framework, the following eligibility criteria were selected in this study: (P) the study was performed among human participants; (E) the study focused on short-term exposure to NOAfter deduplication, 2 coauthors screened titles and abstracts for eligibility and retrieved the full texts of eligible studies for further assessment. Any disagreements in the process were resolved through group discussions until consensus was reached. The detailed process of eligible study selection is shown in Figure Two coauthors independently extracted the following elements from eligible studies. They entered the data into a predesign Excel spreadsheet, including first-author name, publication year, country of origin, study design, study period, outcome definition, study participants, exposure assessment, adjustment variables, and main results. Any discrepancy was resolved by discussion with a third coauthor. The establishment of this quality assessment scale is based on previous successful experience, in particular the selection of several items from the New Castle Ottawa and Cochrane Risk of Bias Tools. This scale evaluates 3 items: validation of schizophrenia , the quality of NO2 concentration measurements , the quality of the adjustment for confounders . A study with a score of 0 was categorized as “low quality,” and as “high quality” if it achieved a score of 5 (the highest score). All remaining study scores are classified as “medium quality.”The study design of eligible studies included in this analysis included both time-series and case-crossover studies. To the best of our knowledge, there are currently no valid scales to assess the quality of these 2 types of studies. The scale suggested by Mustafic was used to assess their quality. Additionally, the extraction effect estimates per unit increase in NO2 exposure to a 10 μg/m3 increment were standardized prior to formal analysis. When studies reported NO2 concentration in ppb, the data were converted using 1 ppb NO2 = 46/22.4 μg/m3. Afterwards, RR for each study was standardized according to the following formula.To ensure comparability of outcomes across different studies, it was necessary to transform various types of effect sizes into relative risks (RR). As not all studies reported RR values, we converted the outcome of change rate of morbidity by dividing it by 100 and adding 1, thus obtaining the RR value. For regression coefficients, exponential transformation was used to derive the RR.In short-term exposure studies, schizophrenia admission risks with different lag patterns  may be reported. To facilitate pooling across studies, lags were selected according to the following criteria: if only 1 lag estimate was provided, the estimate was selected; or if multiple lags were provided, in order of preference, the lag that researchers focused on or preferentially stated was chosen, the lag that was statistically significant, or the lag with the largest effect estimates.I2 statistics. A P value of Cochran Q <0.1 was considered statistically significant for heterogeneity. The I2 statistic provides quantitative indicators of heterogeneity and inconsistency between studies. According to the experience of previous studies, I2 is equal to 0% to 25%, 25% to 50%, 50% to 75%, and 75% to 100% representing no heterogeneity, mild heterogeneity, severe heterogeneity, and extreme heterogeneity, respectively. Due to the considerable heterogeneity of this study, a random-effects model was chosen to pool effect sizes. A subgroup analysis was then conducted to explain the source of heterogeneity. Publication bias was visually assessed using a funnel plot for asymmetry and Egger test (P < .1 indicated the existence of publication bias). Additionally, the trim-and-fill method was used to test for contributions of publication bias to the overall effect. The meta-analyses were performed using STATA version 13.0 .Standardized RR values and 95% confidence intervals for each eligible study were selected for meta-analysis. Heterogeneity between studies was assessed using Cochran Q and The search strategy identified 517 potentially relevant studies in 3 electronic databases. The full texts of 34 relevant articles were retrieved for further selection after removing duplicates, animal experiments, and reviews. Finally, eleven eligible studies were identified after excluding studies with non-short-term effects and did not report relevant outcomes , 2 in the USA, and 2 in South Korea. Most of these studies were performed in a time-series design (n = 9), and the remaining 2 used a case-crossover design, while the sample size of these studies ranged from 3218 to 649,052. All studies used data from national or regional ambient monitoring sites/centers for exposure measures. Three studies used chemiluminescence methods to measure NO2 concentration. Ten studies reported single-pollutant analysis (NO2) results, and 7 studies reported results from 2-pollutant models (NO2 plus other pollutants). For the diagnosis of schizophrenia, 7 and 3 studies used the ICD-10 and ICD-9 codes, respectively.Table 2 exposure with hospital admissions for schizophrenia. Under the random-effects model, NO2 exposure is significantly associated with an increased hospital admission risk for schizophrenia . In the subgroup analyses , PM10  and SO2 .Figure http://links.lww.com/MD/J691 respectively.All eligible studies were assessed for methodological quality. Six studies were of high quality, and 5 were of intermediate quality due to unadjusted holidays, influenza epidemics, etc. The quality assessment results and details are shown in Table P = .006 <0.1). The trim-and-fill analysis identified 4 missing studies for the analysis, and the adjusted estimate was not substantially changed .Funnel plots and Egger test were applied to assess potential publication bias Fig. . There i2 exposure and the risk of hospital admission for schizophrenia. Each 10 μg/m3 increase in NO2 concentrations is significantly associated with a 2.9% increased risk of hospital admission for schizophrenia. However, the results must be cautiously interpreted due to the high heterogeneity among the studies. By subgroup and trim-and-fill analyses, this correlation is unaffected by publication bias and heterogeneity. In a sense, this study provides synthesized evidence for previously inconsistent conclusions.In the present systematic review and meta-analysis, ten studies from different countries were investigated to examine the correlation between short-term NO2, providing essential evidence for the effects of NO2 exposure on mental disorders and the effect of environmental factors on schizophrenia. The results of the 2-pollutant model also confirm that the association was robust to co-pollutants  adjustment. Similarly, several previous meta-analyses found short-term NO2 exposure to be an independent risk factor for depression among air pollutants.,29 However, another previous meta-analysis reported that NO2 exposure was a protective factor for anxiety disorder. The results of these studies reveal that the effects of NO2 exposure on various mental disorders may differ. There was empirical evidence of the long-term effects of NO2 exposure on individuals developing schizophrenia. Two national cohort studies have demonstrated that higher concentrations of residential NO2 exposure during childhood were associated with an increased risk of later developing schizophrenia.,31 The underlying pathophysiological mechanisms linking environmental pollutant exposure to schizophrenia remain unclear. However, Alan S. Brown et al have proposed the gene-environment interplay hypothesis to elucidate this association. This hypothesis builds upon the premise that genetic factors are the primary cause of schizophrenia and suggests that environmental factors increase an individual risk of developing schizophrenia through intermediate pathways such as oxidative stress, apoptosis, inflammation, and the HPA axis. Potentially, 3 pathways can be considered, gene-environment interactions, genes as antecedents of environmental exposure, and epigenetic effects.This study is the first comprehensive analysis to identify the association between schizophrenia and short-term exposure to NO,34 Secondly, Tongling economy is comprised primarily of copper mining and planting, which means many outdoor employees are exposed to air pollution for extended times, thereby increasing the risk of hospital admissions for schizophrenia. Thirdly, exposure level estimation errors vary due to differences in the density of ambient monitoring stations across studies , which may be an essential source of heterogeneity.High heterogeneity is found among the ten studies included in this meta-analysis. After subgroup analysis, heterogeneity was primarily from the 6 studies within China. Studies in different countries also contributed a lower proportion of heterogeneity. Such heterogeneity is considered from the following perspective. The 6 studies from China (including 9 study cities), reports from Shenzhen (RR = 1.08), Hong Kong (RR = 1.08), and Tongling (RR = 1.12) had higher effect sizes than other cities. Firstly, socioeconomic factors between the different cities may contribute to this difference. In China, Shenzhen and Hong Kong are the main cities experiencing environmental stresses, including higher competitive pressures, more immigration, lower social cohesion and social support, and higher housing values, which have all been associated with developing schizophrenia. compared to 65% to 8.3% in China,37) and severe air pollution due to rapid industrialization. Secondly, although the methodological quality assessment scale employed in this study has been previously used in systematic reviews, its validity has not been scientifically established. Thirdly, publication bias may have resulted in an overestimation of the true effect size, as studies reporting non-significant effects may have remained unpublished. However, the trim-and-fill method did not reverse the initial outcomes. Fourthly, exposure assessment in all included studies was based on data from stationary ambient monitoring stations, which may have led to exposure misclassifications and information bias. Finally, differences in the intensity of air pollution exposure among individuals from different socioeconomic backgrounds within the same region may exist. Most of the included studies did not adjust for these factors, which may have influenced the study results.Several limitations need to be acknowledged in this study. Firstly, data were obtained from only 3 countries, with the majority of studies (7/11) being conducted in China, which may restrict the generalizability of the findings. This limitation may be attributed to local contextual factors and publication bias, where studies with negative results are less likely to be prioritized for publication. It is noteworthy that China has been reported to have a higher prevalence of schizophrenia (2.64% globally,39 Additionally, public health campaigns can raise awareness about the adverse effects of air pollution on mental health, encouraging individuals to take measures to reduce their exposure to pollutants. In terms of healthcare policy, our findings underscore the importance of early detection and treatment of schizophrenia. Policy-makers should prioritize the development of accessible and effective mental health services, including screening programs, early intervention programs, and evidence-based treatment options.This finding has significant policy implications for public health, as it highlights the need for preventive measures and interventions to reduce the incidence of this debilitating mental illness. Policy-makers should consider implementing targeted interventions aimed at reducing air pollution levels in areas with high population density or high levels of industrial activity. These interventions may include stricter regulations on industrial emissions, as well as the promotion of clean technologies and renewable energy sources, such as carbon neutrality policy.2 and HAs for schizophrenia. However, given the limitations identified in the study, further research is necessary, particularly from countries with high levels of ambient air pollution, such as Africa and India, to clarify the plausibility of this association. Moreover, it is recommended that future studies adjust for potential confounding factors, such as holidays, meteorological factors, and flu epidemics, to provide insight that could benefit relevant public and health policies.In summary, this systematic review and meta-analysis suggests an association between short-term exposure to NOConceptualization: Jiating Xu, Zhihua Zhang.Data curation: Zhiyong Lan, Penghao Xu.Formal analysis: Jiating Xu, Zhiyong Lan, Penghao Xu.Methodology: Zhiyong Lan, Penghao Xu, Zhihua Zhang.Visualization: Zhiyong Lan.Writing – original draft: Jiating Xu.Writing – review & editing: Penghao Xu, Zhihua Zhang.


Drosophila, the PcG/TrxG group proteins are recruited to the DNA regulatory sequences termed the Polycomb response elements (PREs). The PREs are composed of the binding sites for different DNA-binding proteins, the so-called PcG recruiters. Currently, the role of the PcG recruiters in the targeting of the PcG proteins to PREs is well documented. However, there are examples where the PcG recruiters are also implicated in the active transcription and in the TrxG function. In addition, there is increasing evidence that the genome-wide PcG recruiters interact with the chromatin outside of the PREs and overlap with the proteins of differing regulatory classes. Recent studies of the interactomes of the PcG recruiters significantly expanded our understanding that they have numerous interactors besides the PcG proteins and that their functions extend beyond the regulation of the PRE repressive activity. Here, we summarize current data about the functions of the PcG recruiters.The establishment and stable inheritance of individual patterns of gene expression in different cell types are required for the development of multicellular organisms. The important epigenetic regulators are the Polycomb group (PcG) and Trithorax group (TrxG) proteins, which control the silenced and active states of genes, respectively. In  During development of the multicellular organisms, individual patterns of gene expression are established in each cell type and stably transmitted through many consecutive cell divisions . The preDrosophila through studies on the Hox genes that specify the correct segmentation pattern of the body [The antagonism between the PcG and TrxG proteins was first demonstrated in the body ,5,6,7. Mthe body ,5,6,7. Tthe body ,11,12,13Drosophila, the PcG/TrxG group proteins are recruited to the specialized DNA regulatory sequences—the Polycomb response elements (PREs) [In s (PREs) ,14,15,16s (PREs) . In addis (PREs) , indicatA number of the PRE DNA-binding proteins, named PcG recruiters, which are implicated in the PRE repressive activity, have been identified ,15,16. HIn this review, we will summarize the known data on the participation of the PcG recruiters in the PRE repressive functions, as well as outside of the H3K27me3 domains, and will focus on the partners of the PRE DNA-binding proteins implicated in distinct regulatory activities.The majority of the known PcG proteins are organized into multisubunit complexes which are targeted to the chromatin and which mediate the repression of transcription ,26. The The PRC2 contains the Enhancer of zeste (E(z)), Suppressor of zeste 12 (Su(z)12), Extra sex combs (Esc), and the Chromatin assembly factor 1 (Caf1) subunits ,28. The ph-p and ph-d, giving rise to almost identical proteins. The Psc protein can be replaced in the complex by its homolog, the Su(z)2 protein [The Polycomb repressive complex 1 (PRC1) contains Polycomb (Pc), Polyhomeotic (Ph), Sex combs extra , and Posterior sex combs (Psc) subunits ,32,33. T protein . The cor protein ,37,38; t protein ,36,37,38 protein ,40.In addition to the core subunits, a number of minor proteins could be co-purified with the PRC1 and PRC2. One such PcG protein that co-purifies with both the PRC1 and PRC2 is the Sex comb on midleg (Scm) ,33,41. SThe TrxG proteins include subunits of different complexes involved in the activation of transcription . In partDrosophila has two complexes that belong to the SWI/SNF remodeler family—PBAP and BAP. The catalytic subunit of both complexes is the Brahma (Brm) ATPase [) ATPase ,48. Usin) ATPase . In addi) ATPase ,48.The Trithorax (Trx) and Trithorax-related (Trr) proteins are histone methyltransferases that catalyze the H3K4me1/2 modification characteristic for the active chromatin regions ,51,52 annejire gene, is an acetyltransferase that performs histone modifications at a subset of histone positions, including the H3K27ac modification, a hallmark of the active chromatin [The CBP protein, encoded by the hromatin . Histonehromatin .Drosophila, Trx, Trr, and CBP, as well as the H3K4me1/2 and H3K27ac modifications, are co-localized on the active enhancers and at the regions of PcG protein recruitment outside of the H3K27me3 domains [In  domains ,51,52,54PREs can be located at different distances from the target genes, both in the immediate vicinity of the transcription start site and at a distance of thousands of base pairs. Structurally, they are DNA fragments of several hundred base pairs in length. Like other regulatory elements, they are DNA regions with a reduced density of nucleosomes and are hypersensitive to DNase I. PREs contain binding sites for the PcG recruiters. The sites for the PcG recruiters in each particular PRE can be present in different combinations, in different numbers and positions relative to each other.The currently characterized PcG recruiters are Pleiohomeotic (Pho) ,57 and iSix of the eleven enumerated proteins, Pho, Phol, Combgap, Spps, Crol, and GAF, belong to the class of the C2H2-type Zinc finger (C2H2-type ZnFs) proteins . Pho andEach of the other five PcG recruiters has DNA-binding domains of distinct types . Psq conZeste contains the Myb/SANT-like DNA-binding domain that binds YGAGYG ,79,80.Fab7PRE in transgenic constructs [Dsp1 contains two High mobility group (HMG) motifs. Initially, Dsp1 was reported to bind to the GAAAA sequence in the nstructs . Howevernstructs . It has nstructs ,82,83, snstructs ,20, indiGrainy head (Grh) contains the Grh/CP2 DNA-binding domain that was first shown to bind to YNAACYGGTYYTGCGG in vitro ; a similpho mutants die at the pharate adult stage and have sex combs on the second and third legs, demonstrating a classic homeotic PcG phenotype [pho gene lead to a derepression of the Hox genes [pho mutations affect the ability of the transgenic PRE to repress reporter genes and so do mutations of its binding sites in the PRE [phol mutation does not have homeotic phenotype, but it enhances the pho mutation [bxdPRE is sensitive to the Pho knockdown by RNAi in Drosophila cells where Phol is absent, the double pho/phol mutations are required for the loss of Pc binding at the bxdPRE at the larval stage where both proteins are expressed [The henotype ,88. Accoox genes ,58,89. p the PRE ,15,16. H the PRE ,90 and, xpressed . Accordidsp1 mutants have a mixed PcG/TrxG phenotype, indicating that Dsp1 is most likely involved in both repression and activation of transcription [PcG core genes or pho, leading to more severe homeotic phenotypes. For example, the Trl [Psq [Adf1 [PcG mutants, and the grh [PcG and pho mutants. The cg (encoding Combgap) [Spps [pho mutants but enhance lethality.The cription . Mutatio the Trl ,64,70, PTrl [Psq , and Adfsq [Adf1  mutation the grh  mutationCombgap)  and Sppsp) [Spps  mutationcrol knockout and mutation of the Crol binding site decrease the silencing caused by transgenic evePRE and affect recruitment of Ph and Combgap [Trl mutations decrease the silencing caused by the Fab7PRE [Fab7PRE was shown to be sensitive to dsp1 mutation on larval polytene chromosomes, and the Dsp1 G(A)n site affected recruitment of PRC1 (Ph) and PRC2 (E(z)) [Combgap, Spps, Crol, GAF, Psq, Zeste, Dsp1, and Grh were confirmed to be important for the functioning of transgenic PREs either by genetic tests and/or by mutations of their binding sites ( and revi Combgap . Trl mut Fab7PRE . Dsp1 bi2 (E(z)) .Sfmbt gene lead to misexpression of the Hox genes [In immunoprecipitation experiments, Pho was shown to interact with the Scm-related gene containing four malignant brain tumor (MBT) domains protein (Sfmbt)  Figure . Structuox genes . Since box genes ,96,97 anox genes  (PRC1) aox genes  (PRC2). ox genes ,99.In addition to Pho, the Phol protein also interacts with Sfmbt. However, it was shown that Pho and Phol interact with Sfmbt in a mutually exclusive manner . A quantFor Spps, contacts with the PRC1 and PRC2 core subunits have not been described, but it is able to directly interact with the Scm protein .Of the other PcG recruiters, Combgap , Crol 6, GAF 9696, Psq , Zeste , Grh 9797, and Adsp1 mutants have a mixed PcG/TrxG phenotype [Ubx loss-of-function phenotype. Enhancement of the Ubx phenotype is also observed in genetic experiments with the Trl gene. In addition, hypomorphic Trl alleles have loss-of-function transformations in segment A6 [Fab7PRE fragment [bxdPRE element [Fab7PRE, Zeste sites were required for recruitment of the SWI/SNF TrxG Brm protein to the transgene [evePRE [Several observations indicate that Dsp1, GAF, Zeste, and Pho/Phol PcG recruiters have TrxG-related activities. As mentioned above, henotype  and, in gment A6 . Zeste wfragment  and play element . In the ransgene . In addi [evePRE .A number of PcG recruiters were shown to interact with the TrxG SWI/SNF ATP-chromatin remodelers. GAF was shown to co-purify both with BAP and PBAP ,109. PhoIn addition, PcG recruiters were shown to co-purify with several other TrxG proteins ,111. In The functional and physical connections of the PcG recruiters with the TrxG proteins indicate their importance for the PRE activating state and, potentially, a more global role in the activation of transcription. Accordingly, current data suggest that the binding of PcG core proteins, as well as PcG recruiters, is not restricted to the H3K27me3 domains ,23,24,61Drosophila S2 cells, approximately 80% of Combgap, Psq, and Zeste overlap with Ph. In the case of Adf1, the overlap with Ph is about 50% [If we evaluate the level of overlap of the PcG recruiters with the PcG proteins, then about 80% of the Pho, Combgap , and Crobout 50%  , then the intersection for Pho , CombgapAccordingly, over 80% of peaks for PRC1  and PRC2 (E(z)) fall outside of the H3K27me3 domains as well  Figure C. For PcUbx gene [Going back in history, GAF ,113,114,Ubx gene ,118,119.Ubx gene ,85,120. Ubx gene . Grh [85Ubx gene ,123 wereUbx gene .For several PcG recruiters, the estimated overlap was quantified. GAF binds to 20% of active promoters  and bindIn agreement with the functional overlap of the PcG recruiters with enhancers and promoters, the interactomes of the PcG recruiters contain many proteins implicated in the activities of these regulatory elements.The Pol II together with the general transcription factors , TFIIE, TFIIF, and TFIIH) are recruited to the promoters and they are assembled into the preinitiation complex (PIC). A successful formation of the PIC leads to a rapid initiation of transcription. Pol II pauses after it produces an RNA of 20–60 nucleotides in length. Current data suggest that the pausing of Pol II at the early elongation is an obligate part of the transcription cycle; a step that is universally encountered by Pol II for the majority of even highly expressed genes. The promoter pausing is primarily mediated by an action of the NELF and DSIF complexes that stabilize Pol II at the pause region ,125. In Of the PcG recruiters, GAF is well known to be implicated in pausing for a large subset of genes, most of which are developmentally regulated ,130,131.CG4562 and Pp2B-14D, which are actively transcribed, as well as to the Poxm gene, which is under the H3K27me3 repression [In a pull-down assay, Grh interacted with Taf2 and Taf6 TFIID subunits  Figure . In IP/Lpression .Pho can directly interact with the Spt5 subunit of DSIF ,136 Fig and showspt5 and nelf-A mutants enhance the PcG phenotype observed in pho mutant flies, suggesting an involvement of pausing factors in the PcG silencing [It was shown that, except for promoters, NELF ,137 and ilencing . Thus, bNot surprisingly, PRC1 is connected to promoter functioning as well. It was demonstrated that depletion of the PRC1 subunits by RNAi alters both phosphorylation of RNAP II and recruitment of the Spt5 subunit of the DSIF complex to the active genes  and decrMediator is a large co-activator complex consisting of about 30 subunits, named MED1–MED31, Cdk8, and cyclin C . MediatoAdf1 stands apart from other PcG recruiters in the way that it highly efficiently co-purifies with the Mediator complex . MoreoveThe architectural  proteins interact with the regulatory elements—boundaries that can limit enhancer–promoter interactions and block the spread of the heterochromatin ,145. TheOf the PcG recruiters, GAF was experimentally confirmed to be required for the boundary activity of particular insulators ,144. GAFIt was shown that 22–54% of the Combgap, Zeste, Psq, and Adf1 genome peaks overlap with the architectural protein CP190 in S2 cells . In regaWhile there is a huge overlap of Combgap, Zeste, Psq, and Adf1 with the boundary proteins genome-wide, it should be noted that CP190 frequently overlaps with promoters ,155, andThe study of the interactomes of the PcG recruiters revealed that they have many contacts with each other. The strongest of the known contacts in the co-IP experiments are observed between Crol and Combgap/Pho ; betweenDrosophila S2 cells, 88% of Psq peaks overlap with Combgap, while the majority of Zeste peaks overlapped with Combgap (90%) and Psq (88%) [In accordance with the interactions between the PcG recruiters, for some of them, Pho, Combgap, Spps, Crol, Psq, Zeste, and Adf1, a significant genome-wide overlap was reported ,20,59,61sq (88%) . The ovesq (88%) ,20,59 ansq (88%) .In addition, it should be noted that the extent of overlap can be different within and outside of the H3K27me3 domains. For Spps–Pho, it was reported to be lower outside of the H3K27me3 domains—about 90% and 70% of Spps peaks overlap with Pho, inside and outside of the H3K27me3 domains, respectively . AnotherSeveral observations support a co-operative activity of the PcG recruiters. It was shown that Pho protein binding to a chromatinized PRE in vitro requires the assistance of GAF or Zeste proteins . In anotIn addition to the co-operation, the activities of the PcG recruiters were proposed to be redundant and partially compensate for each other ,20.In connection with the potential network that can be formed by multiple interactions between the PRE DNA-binding proteins, their functional co-operation, and redundancy, we have proposed a model according to which the combination of the PRE DNA-binding factors at the PRE forms a “platform” that provides for an efficient recruitment of the core PcG/TrxG complexes . We propThe current data suggest that the PcG recruiters, as well as PcG core proteins, bind to regions inside and outside of the H3K27me3 domains and participate in both the repression and activation of genes. Although much progress has been made toward an understanding of the role of the PcG recruiters in assembly of the PcG protein complexes genome-wide, many questions remain to be elucidated.Multiple interactions between the PcG recruiters were identified and they show an extensive overlap with each other and with the PRC1 genome-wide. However, the currently described interactomes of the PcG recruiters, GAF, Combgap, Zeste, Psq, and Adf1, differ substantially from each other. This might suggest that each protein can attract a unique set of transcription factors to the DNA, and the presence of a binding site for a particular DNA-binding protein could subtly modulate the resulting activity of the DNA regulatory element. This can probably apply to both PREs and the other types of regulatory elements. The characterization of the interactomes of other PcG recruiters may provide a better understanding of the role of each of them in the genome-wide transcriptional regulation.