The human retina, located in the innermost layer of the eye, plays a decisive role in visual perception. Dissecting the heterogeneity of retinal cells is essential for understanding the mechanism of visual development. Here, we performed single-cell RNA-seq to analyze 194,967 cells from the donors of infants and young children, resulting in 17 distinct clusters representing major cell types in the retina: rod photoreceptors (PRs), cone PRs, bipolar cells (BCs), horizontal cells (HCs), amacrine cells (ACs), retinal ganglion cells (RGCs), Müller glial cells (MGs), microglia, and astrocytes (ASTs). Through reclustering, we identified known subtypes of cone PRs as well as additional unreported subpopulations and corresponding markers in BCs. Additionally, we linked inherited retinal diseases (IRDs) to certain cell subtypes or subpopulations through enrichment analysis. We next constructed extensive intercellular communication networks and identified ligand-receptor interactions that play crucial roles in regulating neural cell development and immune homeostasis in the retina. Intriguingly, we found that the status and functions of PRs changed drastically between the young children and adult retina. Overall, our study offers the first retinal cell atlas in infants and young children dissecting the heterogeneity of the retina and identifying the key molecules in the developmental process, which provides an important resource that will pave the way for research on retinal development mechanisms and advancements in regenerative medicine concerning retinal biology. The retina, composed of a complicated array of cell subpopulations, is the system responsible for light perception and visual signal transmission. In general, several retinal cell types compose the layered structure of the retina . The outin vivo features of the development of the human NR and RPE and offering insightful clues for further functional studies  techniques has allowed us to study the retina in greater detail than before, revealing a tremendous number of subtypes within the traditionally defined classification. In human, 18 distinct cell clusters, including well-known retinal cells and novel rod cell subpopulations, have been identified from three post-mortem donors . Voigt e studies . Lu et a studies .In this research, we analyzed 194,967 single-cell transcriptomic profiles from the infant and young child in different developmental stages. Unsupervised clustering defined 17 clusters, all of which were assigned to corresponding major retinal cell types. Communication network analysis connected the receptor-ligand pair with certain biological processes in the retina. Subsequently, we identified known subtypes of cone PRs as well as previously unknown subpopulations and corresponding markers in BCs by performing reclustering. By hypergeometric enrichment analysis, we further associated inherited retinal diseases (IRDs) with certain cell subtypes or subpopulations. Next, the differences between the retinas of the young child and adult were investigated. Intriguingly, we found that the status and functions of PRs changed between the retinas of the young child and adult. Our study provides a valuable data resource for retinal development research and the advancement of retinal regenerative medicine.The collection of human retinas was approved by the Ethics Committee of the Eye and ENT Hospital of Fudan University (2019025) and in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for medical research involving human subjects. All protocols were performed according to the “Interim Measures for the Administration of Human Genetic Resources” administered by the Chinese Ministry of Health. Written informed consent was obtained from the minors’ next of kin for the publication of any potentially identifiable images or data included in this article.The retinal tissues came from a 10-month-old (TenM) and a 2-year-old (TwoY) postmortem human donors. The donors’ eyeballs were collected by Eye and ENT Hospital of Fudan University for corneal transplantation, and eyeball samples were acquired within 12 h after the death of the donors. The remaining eyeball tissues were used to extract retinal tissues. The retinal tissues were exposed by removing the iris, lens, and vitreous successively. Both whole retinas from each donor were carefully dissociated and placed in precooled HBSS  without a retinal pigment epithelium (RPE)/choroid layer. The whole retina from one of the eyeballs of the two-year-old donor was digested into a single-cell suspension in trypsin-EDTA solution  (containing 0.25% trypsin and 0.02% EDTA) at 37°C for 20 min, and the remaining retinal tissues were immediately frozen in liquid nitrogen. Then, the single-cell suspension was filtered through a 40 μm cell strainer  to eliminate any clumped cells. The freshly prepared cell suspension was immediately used for the construction of single-cell sequencing libraries.2, 0.1% NP40, and protease inhibitors) and then homogenized on ice in an RNase-free 2 ml glass Dounce homogenizer  15× with a loose pestle and 15× with a tight pestle. The homogenate was passed through a 40 µm filter to remove the block mass. The cell filtrate was subjected to density gradient centrifugation. Then, 400 µL of cell filtrate was mixed with 400 µL of 50% iodixanol solution  in 2 ml lo-Bind tubes . After 29% iodixanol solution and 35% iodixanol solution were carefully layered into the bottom, the tube was centrifuged at 3,000 × g at 4°C for 30 min.The freshly prepared cell suspensions were used to generate cDNA libraries with a Single Cell 3′ Library and Gel Bead Kit V2  according to the manufacturer’s instructions. Flash-frozen tissues were homogenized in 2 ml ice-cold lysis buffer  containing 0.04% BSA  and centrifuged at 500 × g for 5 min. The supernatant was discarded, and then 50 μL ice-cold 1 × PBS containing 0.04% BSA and 0.2 U/µl RNase Inhibitor  was combined with the cell pellet by gentle pipetting using a regular-bore pipette tip. The nuclear concentration was determined using a hemocytometer . Next, the Nuclei were loaded on a Chromium Single Cell Controller  to generate single-cell gel beads in emulsion (GEMs) by using a Single Cell 3′ Library and Gel Bead Kit V2. Captured cells released RNA and barcoded in individual GEMs. Libraries were generated from each donor sample according to the manufacturer’s instructions. Indexed libraries were converted with an MGIEasy Lib Trans Kit  and then sequenced on the MGISEQ 2000  platform with paired-end 26 bp+100 bp+8 bp (PE26 + 100+8).We first used Cell Ranger 3.0.2 (10×Genomics) to process raw sequencing data, and then Seurat  was appl1) Filtering Low-quality genes.Data quality control is carried out as follows:2) Filtering low quality cells.Genes that are expressed in insufficient cells contribute little to the analysis of cell types and population, resulting in low capture sensitivity, low expression quantity, and low reliability, so these genes should be filtered.a. High proportion of mitochondrial genes.If the proportion of mitochondrial genes is high and the cell types are not a particularly mitochondria-rich cell, it indicates those cells capture insufficient genes and should be removed. The reason for the high proportion of mitochondrial genes could be that the cell activity is weak and tends to apoptosis, or cell membrane is damaged, which leads to mRNA dissociation and less mRNA is captured.b. too low or too high total gene number and counts.During library preparation, RNA may be lost due to cell lysis or low efficiency of cDNA capture and amplification. Cells with a low gene number and counts are considered low quality cells and should be removed. Also, cells with too high gene number or counts may indicate doublet cells (capture two cells as one) and therefore should be filtered.Seurat is the current mainstream analysis tool for single-cell data. The heterogeneity of the retina was determined using the Seurat R package . We perfp value less than 0.01 and KEGG terms with a p value less than 0.05 were considered significantly enriched.The functional enrichment analysis included two parts: Gene Ontology (GO) terms and KEGG pathways. Lists of genes were analyzed using the clusterProfiler R package, and the Benjamini-Hochberg (BH) method was used to correct for multiple comparisons. GO terms with a p-value < 0.05. The parameter “--threshold” was set to 0.01, which indicates that Ligands/Receptors encoding genes were considered to be expressed by the cell type with higher than 1% cell expressing the gene in the corresponding cell types.The cellular communication network was constructed by utilizing CellPhoneDB database . The sigTo reduce noise, we averaged the expression of every 100 cells within the cluster and then calculated the pairwise Pearson correlation coefficients between pairs of dots based on their average expression profiles. Interdot relationships were shown if their Pearson correlation was greater than 0.8. Visualization of the correlation network was achieved using Cytoscape .Monocle is currently the mainstream tool for pseudotime analysis, which is to construct the change trajectory between cells by sorting the information of cell transcript abundance, so as to reshape the change process of cells over time. Monocle can be used for the simulation of cell differentiation lineages during development and cytopathic trajectories in disease progression. Also, the Monocle2 R package (version 2.10.1) was performed to construct single-cell pseudotime trajectories to identify developmental transitions . We usedp value less than 0.01 were considered to be significant.Monocle uses BEAM to calculate the changes in gene expression as cells pass from an early developmental stage through the branch and to identify the DEGs between different branches. We used the “BEAM” function with default parameter settings, and genes with a Homo sapiens TF list from AnimalTFDB 3.0  and then performed the hypergeometric test (“dhyper” function in R) using the risk genes and DEGs of each cluster. A value of 0.05 was used as a threshold to define significance.For disease enrichment analysis, we first retrieved retinal disease risk genes from the RetNet database  and dissociated them into single-cell or single-nucleus suspensions without surface marker preselection . For eacPDE6G ; these cone types are responsible for the perception of colors with different wavelengths  and M-cones  were identified  in goldfish  and performed a hypergeometric test to determine whether these disease-related genes were enriched among the DEGs of certain clusters, aiming to discover putative disease-associated cell types and thus provide new insights for retinal diseases. Most of the disease types we investigated in this study are caused by malfunctions of PRs. Retinitis pigmentosa (RP), whose symptoms generally include trouble seeing at night and decreased peripheral vision, is characterized by retinal cell degeneration, beginning with rods and then progressing to cones ] over 1.5  and adult retinal data from a previous study  and thenchildren , and thechildren . More imntiation . Meanwhinse gene . NR4A2,  neurons . Activatntiation . Moreoveh arrest .TMSB4X, JUNB, FOS, CRABP1, and MT2A) were significantly overexpressed in MALAT1+_1 or MALAT1+_2 subtypes (MALAT1 is a long-chain noncoding RNA that plays a role in retinal homeostasis and disease. Lukowski et al. have found that the differential expression of MALAT1 can be used to identify rod cells in different cellular states, namely, healthy or degenerating rod PRs. It has indicated that the rod subpopulations with low MALAT1 expression represent putative early degenerating rod PRs, and MALAT1 can be considered as a potential target to enhance the survival of rod PRs . In our subtypes . Volcanosubtypes . In addisubtypes . MALAT1+KCNIP4, RDH8, MYO9A, and TUBB4B were specifically expressed in the cone PRs of young children, while MRPL41, RABL3, and GNGT2 were highly expressed in adults (GNAT1, ROBO2, NRXN3, and DLG2 were highly expressed in young children, while CPLX3, SLC25A6, and PCP2 were differentially expressed in adults (Finally, cone PRs and BCs in adults and young children were further analyzed, revealing the specific expression of genes . For insn adults . In addin adults . For BCsn adults . As showThis complex intrinsic composition of retinal cells highlights the necessity of single-cell technologies for the detailed dissection of transcriptome signatures. Although several papers regarding fetal, postnatal and adult retinas have been published, no group has yet published an atlas of the retina from the infant and young child. The maturation and aging of the retina are of significant value in the study of its development and pathology. We believe that the data resources and insights obtained in our study could be a very useful addition to this field.Here, we report the first single-cell atlas of the human retina at different developmental stages. Our dataset consisted of 194,967 cells and nuclei, achieving an unprecedentedly large scale for human retina research. We also identified microglia, which are considered the resident immune cells in the human retina, and found that microglia showed putative interactions primarily with rod PRs, BCs, and MGs through ligand-receptor communication network analysis, implying that rod PRs, BCs, and MGs might play an important role in immune homeostasis.GRIK1 and GRM6. We further described the different properties of each subpopulation by assessing the expression pattern of function-related ion channels. The above results suggested that functional subtypes of BCs might be identified in early human retinas from snRNA-seq data, further experimental verification needs to be conducted.We next sought to identify subtypes and subpopulations of PRs and BCs through reclustering. Among cone PRs, we identified S-cones, M-cones, and L-cones. For BCs, reclustering resulted in 11 subpopulations; 5 of these subpopulations were classified as OFF BCs, while the 3 subpopulations were thought to be ON BCs according to the expression of The distinctions between the retinas of young children and adults were also illuminated in this study, with a focus on the differences in the function and gene expression of retinal cells at different stages of development and the determination of specific cell subtypes with characteristic genes. First, our analysis data revealed that the primary function of MGs in the retina of young children was sensory system development and visual system development, while those of adults were mainly involved in transmembrane transport and ion transport. Based on the differences in MALAT1 expression patterns, rod PRs were divided into three cell subtypes, namely, MALAT1+_1 and MALAT1+_2, with higher MALAT1 expression, and MALAT1-, with lower MALAT1 expression. The differences in gene expression and cellular function among these three subclusters were confirmed through system analysis. This enabled us to form a deeper understanding of the cell characteristics of these three subtypes. In general, the gene expression patterns of PRs and BCs in the retina of young children were significantly different from those in the adult retina. Our results revealed significant differences in cell state between the retinas of young children and adults.IRDs are a group of retinal disorders caused by inherited gene mutations and can result in vision loss or blindness . CurrentIn conclusion, we constructed the first transcriptome profiles of the human retina from infants and young children at single-cell resolution, which provided valuable clues regarding the postnatal development of the human retina. The subtypes of retinal cells with specifically expressed genes were also identified. In addition, we linked IRDs to certain cell subpopulations along with corresponding molecular markers. By evaluating the similarities and differences between the retinas of adults and young children, we reported differentially expressed genes and various patterns of functional enrichment among different stages of development in the human retina.


The accurate germline gene assignment and assessment of somatic hypermutation in antibodies induced by immunization or infection are important in immunological studies. Here, we illustrate issues specific to the construction of comprehensive immunoglobulin (IG) germline gene reference databases for outbred animal species using rhesus macaques, a frequently used non-human primate model, as a model test case. We demonstrate that the genotypic variation found in macaque germline inference studies is reflected in similar levels of gene diversity in genomic assemblies. We show that the high frequency of IG heavy chain V (IGHV) region structural and gene copy number variation between subjects means that individual animals lack genes that are present in other animals. Therefore, gene databases compiled from a single or too few animals will inevitably result in inaccurate gene assignment and erroneous SHM level assessment for those genes it lacks. We demonstrate this by assigning a test macaque IgG library to the KIMDB, a database compiled of germline IGHV sequences from 27 rhesus macaques, and, alternatively, to the IMGT rhesus macaque database, based on IGHV genes inferred primarily from the genomic sequence of the rheMac10 reference assembly, supplemented with 10 genes from the Mmul_051212 assembly. We found that the use of a gene-restricted database led to overestimations of SHM by up to 5% due to misassignments. The principles described in the current study provide a model for the creation of comprehensive immunoglobulin reference databases from outbred species to ensure accurate gene assignment, lineage tracing and SHM calculations. Macaca mulatta) being the most common model. Central for studies of B cell function is the isolation of antigen-specific monoclonal antibodies (mAbs), which facilitates detailed biochemical, structural, and functional analyses of the response   used genHere, we analyzed the KIMDB and IMGT rhesus macaque IGHV databases for overlapping allele and gene content to identify sequence gaps that may deleteriously affect germline gene assignment and SHM determination. We examined available genomic and inferred haplotype-based evidence to identify structural variation that may affect gene content between individual macaques. Finally, we examined how gene databases constructed using either the single genomic assembly model or the multiple animal model influenced the analyses of SHM using an IgG library from a test animal that had not been used to generate either of the two databases.Macaca mulatta (Rhesus monkey) were downloaded from NCBI with identifiers GCA_003339765.3 (rheMac10), GCA_008058575.1 (RheMacS), as well as GCA_005453305.1 and GCA_005453525.2 (ASM545330v1 and ASM545352v2) representing a single animal. Therefore, genes present on either of latter two assemblies are treated in this analysis as one genome, denoted Cirelli et al., without a version number. Only the chromosome 7 sequence was extracted as this contains the macaque IGH locus, which was used for exact searches of sequences in both orientations, except for Cirelli et al. , including IgM paired-end reads (ERR2856338) and IgG paired-end reads (ERR2856339) from animal D20 . Databasi et al. , for whiThe preprocessing and germline inference from the D20 IgM and IgG libraries were performed with IgDiscover (v0.12.4) using default settings, except for 3’ end barcode length set to 21 nucleotides (nts), species set to rhesus_monkey and number of iterations set to one to produce an individualized IGHV database from the IgM library. After obtaining the germline IGHV genotype, three IgDiscover assignment runs were performed on the D20 IgG test library, using either the rhesus macaque KIMDB, rhesus macaque IMGT or the D20 individualized genotype with the same settings as previously described. The filtered tab from these runs were used to compare mutation levels obtained when assigning to the different databases.Databases were compared in two ways. The first approach aimed to identify identical sequences in the two databases. This included sequences that occurred within the string of longer sequences, accounting for the identification of potential truncated matches. The results were illustrated in a Venn diagram using ggVenDiagram  (v1.1.4)The second approach aimed to compare gene content of the two databases and involved complete alignments. Here, we considered exact matching to be insufficient since different alleles of a given gene could be present in the two databases. For each IGHV sequence in KIMDB, we then identified the most similar IMGT sequence by computing all vs all pairwise alignments, picking the best scoring alignment and computing the number of differences between two aligned sequences. Since the comparison concerned sequences that are highly similar, we aligned their entire length using a global alignment method. All vs all alignments were computed using BioPython’s  (v1.76) To define the IGHV genotype and generate an individualized database for animal D20, an IgM library was generated as described , 26 and For the comparison of SHM levels, we used an IgG library from rhesus macaque D20, an animal that had not been used to generate the KIMDB database or the IMGT database. Three analyses were performed with the D20 IgG library. First, the library was assigned to the individualized D20 database to identify the appropriate germline assignment for each sequence read. For each assigned gene from D20 run, read identifiers were extracted and used to obtain SHM related values from the D20 IgG runs with KIMDB and IMGT as starting databases. All sequences assigned to a germline sequence in the initial individualized run were grouped into allele specific bins. The same allele specific binned sequences could then be traced in the subsequent KIMDB and IMGT assigned analyses, thereby enabling the SHM per gene to be determined based on the V_errors column of the filtered table output. Genes and their median V mutations, determined as number of differences reported by IgBLAST were compared to the closest IGHV allele in the database.Inferred haplotype analysis of the D20 IgM library was performed using the IgDiscover plotallele module as described  using IGRheMac10  and rheMStructural and copy number gene variation in the IGH locus has been demonstrated previously in both human and macaques , 17. ThiThe first step in antibody VDJ sequence analysis involves assigning sequences to a germline gene database to define gene and allele usage of each antibody. Here, we compared two immunoglobulin IGHV germline gene databases for rhesus macaque antibody analysis, IMGT  and KIMDThe two databases were first compared for exact allelic matches, accounting for the potential presence of sequences that were not full-length in one of the databases. As shown in the Venn diagram in We next grouped the alleles according to genes and compared the two databases for best matching gene content. The result for each KIMDB gene is shown as distance to the closest IMGT gene by comparing the best-matching alleles from each gene . These mWe also performed the analysis in a reciprocal manner, asking if there were any functional IMGT genes that could not be matched with genes in KIMDB. We identified 5 F-marked IMGT genes that that did not have a closely related sequence in KIMDB: IGHV1-69, IGHV3-12, IGHV3-35, IGHV3-108 and IGHV3S40 . Of the 91 alleles, 77 were identical to alleles already present in KIMDB and 14 alleles were novel  and identified 91 IGHV alleles, representing 66 IGHV genes. The full allelic content of D20 is shown in a phylogenetic tree . We then extracted all 14108 IgG sequences that were assigned to IGHV3-128 in KIMDB and compared their mutation level to the levels obtained when assigning the same sequences to KIMDB-3-128. The analysis illustrated clearly that the median number of mutations in the IgG sequences was higher (median of 30 instead of 22 mutations) if the database was missing this gene, since 99% of those reads instead get assigned to the next closest gene, IGHV3-88 , 14 (4.7% SHM) and 10 (3.3% SHM), 8 (2.7% SHM) nucleotides respectively when the IMGT database is used in contrast the individualized D20 database. The results demonstrate that the level of SHM of many genes expressed in D20 would be misinterpreted as having SHM levels overestimated by approximately 5% when an incomplete database is used  approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), Rh1-6 at the CEA, France and A01-05 in the Oregon National Primate Research Center (OHSU), USA. Ethical permits for the work in this study are as follows: Samples from AFL/KI and Primgen: Stockholms Norra Djurförsöksetiska nämnd: N275/14 - Animals: D7, D8, D11, D15, D16 and D19, Stockholms Norra Djurförsöksetiska nämnd: N193/16 - Animals: E01-E10; Samples from CEA: APAFIS#3132-2015121014521340v2 - Animals: Rh1-Rh6; Samples from OHSU, IACUC: TR01_IP00000028 - Animals: A01-A05.Conceptualization, MK, MC, and GKH. Methodology: MK, Analysis: MK, MC and GKH, Manuscript writing: MK, MC, and GKH. All authors contributed to the article and approved the submitted versionThis work was funded by a Distinguished Professor grant from the Swedish Research Council (2017-00968), grants from the National Institutes of Health (HIVRAD 1P01AI157299 and CHAVD UM1Al144462), and a European AIDS Vaccine Initiative (EAVI) 2020 (681137) grant.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


EUD modeling are consistent across tumour sizes, configurations, and treatment depths. These results suggest that the developed reference tables can be used as a practical clinical resource for clinical decision-making on GRID therapy and to facilitate heterogeneity dose estimates in clinical patients when this commercially available GRID device is used.Dose prescription for the inhomogeneous dosing in spatially fractionated radiation therapy (SFRT) is challenging, and further hampered by the inability of several planning systems to incorporate complex SFRT dose patterns. We developed dosing reference tables for an inventory of tumour scenarios and tested their accuracy with water phantom measurements of GRID therapy, delivered by a standard commercial GRID collimator. We find that dose heterogeneity parameters and EUD between different tumour sizes at the same depth were within 5% of the prescription dose. PVDR from profile measurements at the tumour center differed from D10/D90, but D10/D90 variations for the same tumour depths were within 11%. Three approximation equations were developed for calculating EUDs of different prescription doses for three radiosensitivity levels for 3-cm deep tumours. Dosimetric parameters were consistent and predictable across tumour sizes and depths. Our study results support the use of the developed tables as a reference tool for GRID therapy.Computations of heterogeneity dose parameters in GRID therapy remain challenging in many treatment planning systems (TPS). To address this difficulty, we developed reference dose tables for a standard GRID collimator and validate their accuracy. The .decimal Inc. GRID collimator was implemented within the Eclipse TPS. The accuracy of the dose calculation was confirmed in the commissioning process. Representative sets of simulated ellipsoidal tumours ranging from 6–20 cm in diameter at a 3-cm depth; 16-cm ellipsoidal tumours at 3, 6, and 10 cm in depth were studied. All were treated with 6MV photons to a 20 Gy prescription dose at the tumour center. From these, the GRID therapy dosimetric parameters (previously recommended by the Radiosurgery Society white paper) were derived. Differences in D5 through D95 and  Spatially fractionated radiation therapy (SFRT) is a specially designed radiation therapy modality characterized by the delivery of a high and intentionally heterogeneous dose using megavoltage x-ray or proton beams for the management of patients with bulky tumours. Photon beam GRID therapy commonly employs commercially available GRID block collimators or multi-leaf collimator (MLC) modulation to generate the heterogeneous dose pattern by converting an open photon treatment field into multiple small beamlets, which are collimated into a well-defined pattern.An ample body of literature has shown drastic and often rapid tumour responses to SFRT, with unexpectedly low toxicity rates in the treatment of bulky, recurrent, or therapy-refractory tumours ,3,4,5,6.Most of the clinical SFRT experience is derived from GRID block collimator  techniques. While a complete understanding of the radiobiological underpinnings of the observed SFRT responses is still an area of continued investigation, both shallow and deep-seated bulky tumours have consistently shown high symptomatic response rates to collimator-based GRID therapy ,14,15,16GRID therapy is generally delivered with commercially available GRID block collimators. While SFRT techniques other than GRID, such as Lattice (a 3D form of GRID therapy), have been developed, due to the fixed geometric properties, dosimetric consistency, and convenience of straightforward delivery, GRID collimator-based SFRT treatment continues to be widely practiced and prevalent in most treatment centers per an RSS GRID/Lattice, Microbeam, and FLASH Working Groups survey. With these properties, GRID collimators are an attractive platform for both high-volume practices and for those lacking access to highly advanced technologies, as well as an entry technology for institutions that are starting new SFRT programs.However, for clinical physicists and radiation oncologists, the profound departure of SFRT from the well-established dosimetric and planning principles poses new and unfamiliar challenges. In view of the inherent complexity associated with the heterogeneous dose patterns in SFRT and the potential difficulty in adapting current treatment planning systems (TPS) to GRID dosimetry, there is a need to further facilitate dosimetric computations and treatment planning while remaining clinically effort- and time-efficient, particularly for entry-level centers that seek to establish a GRID program.Dosimetric reference tables have been widely employed in radiation oncology for treatment planning, for example, the prostate seed implant nomograms and eye-plaque seed activity look-up tables. We have adapted this overall concept to the use of GRID collimator-based SFRT. Collimator-based GRID therapy lends itself very well to the concept of reference tables because commercially available GRID collimators are generalized devices with standard physical and geometric parameters. We therefore hypothesize that reference tables of dosimetric parameters generated from an array of representative treatment delivery scenarios can provide a robust and validated representation of the complex GRID dosimetry and may thereby be useful as a tool for practical clinical application in GRID therapy.The purpose of this study was to develop and validate practically applicable GRID dosimetry reference tables for use by physicists, dosimetrists, and radiation oncologists for formulating GRID therapy treatment plans. Specifically, we studied the consistency of the developed reference tables across different tumour geometry, depth, and field size characteristics. The accuracy of a TPS for calculating GRID therapy dose distributions was verified by experimental testing first. The dose–volume histograms (DVH) of sample tumours were used to derive SFRT heterogeneity and modeling metrics; subsequently, the reference tables were generated.PVDR), the doses covering 95%, 90%, …, 10%, and 5% of tumour volume  and equivalent uniform dose (EUD).The reference tables were developed and tested based on a commercially available GRID collimator. The Eclipse planning system  was used. Tables were derived for the array of tumour depths, sizes, and geometries from the established planning system Eclipse, and subsequently calculated for their peak/valley dose ratio    a, and thFL, USA) b. BecausTM GRID collimator is an 18 kg brass collimator. The thickness of the brass block is 7.5 cm. The diameter of the holes is 0.80 cm on the upstream side and 1.00 cm on the downstream side, and the center-to-center separation of the holes on the block is 1.40 cm on the lower surface. With this design, approximately 50% of the tissue in the collimated areas is irradiated by the collimated primary beam; the remainder is shielded by the brass block. All holes in the GRID are divergent. Dosimetric characteristics of the .decimal Inc.TM are shown in The .decimal Inc.The specifications of the .decimal Inc. collimator differ from that of the original GRID collimator by Radiation Products Design and consist of a 7.5 cm thick Cerrobend block with a hexagonal pattern a,b of ciTM  water scanning system was used to perform in-water scans. The profiles were taken using a PTW  microDiamond Type 60019 detector having a sensitive volume of 0.004 mm3. Water tank measurements indicate that, although the percent depth dose (PDD) curve has noticeably changed compared to the open field, the depth of dose maximum (dmax) remains the same for this hexagonal pattern GRID design. Thus, when we generate plans and perform quality assurance (QA) measurements, we can still use the dmax depth measured from the open field  can be defined as follows:From the scanned dose profiles at different depths, the peak/valley  dose ratio , when the depth was changed from 1 cm to 10 cm. For the Radiation Products DesignsTM Cerrobend GRID collimator using a 6 MV beam, it was reported that when depth was increased from 1.5 cm to 10 cm, the PVDR decreased from 7.4 to 5.0 (or VPDR increased from 0.14 to 0.20) [The to 0.20) .(a).GRID therapy planning without a TPSMU) needed for delivering the prescribed peak dose  are calculated based on the output factor of the central hole, usually near or passing through the beam’s central axis. The approach is the same for multileaf collimator formed GRID fields, in which an experimental measurement is involved to determine the output factor for delivering the prescribed peak dose at the tumour center depth.To develop and test the reference tables, we simulated a scenario where the GRID collimator could not be implemented within the TPS for dose calculation. In this case the monitor units (max (OUTmaxd) will vary with the field size it needs to be measured. If the dose pD is prescribed at the depth of dmax, the MU is calculated according to the following:In a patient treatment, the jaws and/or multileaf collimator (MLC) can be used to reduce the field size to create a conformal field adapted to the tumour size. Because the output factor of the GRID field central hole at dd) other than dmax. In addition, if two opposed GRID fields are used [d. If the output factor of the GRID field at the depth d is dOUT, then,However, the depth of the tumour center may be variable, and the treating physician may prescribe the dose to a depth .It should be noted that because of the dosimetric characteristics of megavoltage X-ray beams, if the dose prescription depth is beyond d(b).GRID therapy planning using a TPSTo develop the reference tables, we generated plans for different tumour sizes, shapes  and depths in a flat phantom. The Eclipse system is one of the few TPS that can perform dose computations for commercial GRID collimators. In order to do this, a DICOM file containing the specific geometric features of the GRID collimator, generated by the vendor, had been installed earlier into our TPS per the vendor’s specific instructions.2 field size. After the GRID dose calculation accuracy is confirmed, the GRID collimator can be used in the TPS for creating patient treatment plans. Similar to intensity modulated radiation therapy (IMRT), a patient-specific treatment plan QA can be performed to verify the delivered dose.Following installation of the GRID collimator in the TPS, its calculation accuracy was verified. For this verification, water tank scans of the percentage depth dose curves and dose profiles were performed at different depths for various field settings and compared with the TPS-generated counterparts. A calibrated ion-chamber suitable for small field dosimetry or film was used to verify the absolute dose delivered via a GRID field, and a ≤3% difference, which is generally considered acceptable for radiation therapy plan point dose measurement, was set as a tolerance limit. The radiochromic film was calibrated and its accuracy was verified prior to use using a previously published protocol . The sof(c).Tumour size and geometry selection for generating reference tablesTo determine the dosimetric impact of tumour sizes and depths, we chose ellipsoidal tumours ranging from 6. to 20 cm in diameter located either at 3 cm, 6 cm or 10 cm depth . These g(d).EUD) calculationEquivalent uniform dose  model, we calculated the average surviving fraction, and then derived the corresponding t volume . The MLQt volume ,23,24. Aequation . Becauseef below  to obtaiThe MLQ equation is as follows:iSF is the survival fraction at the dose iD.α and β are radiosensitivity parameters of the cell, λ is the repair rate , assumed to be T = 5 min. The SF calculation is not very sensitive to T.1/2= 1 h ; δ = 0.1−1, and β = 0.0502 Gy−2; for semisensitive cancer cells, α = 0.289 Gy−1, and β = 0.0289 Gy−2; for radioresistant cancer cells, α = 0.149 Gy−1, and β = 0.0149 Gy−2, respectively. Similar assumptions can be made for normal tissue using α/β = 3 Gy. All radio-response parameters of the MLQ model are summarized in For cancer cells, we used a consensus value of α/β = 10 Gy. By assuming cancer cells as radiosensitive (SF(2Gy) = 0.3), semisensitive (SF(2Gy) = 0.5) and radioresistant (SF(2Gy) = 0.7), the individual α and β values of these three types of cancer cells can be derived from the LQ model. Therefore, for radiosensitive cancer cells, α = 0.502 GyThe average survival fraction if is the fraction of target volume receiving dose iD. The average survival fraction was then utilized to solve the MLQ Equation (6) to determine the equivalent uniform dose (EUD) by solving the following equation for EUD:pD, we can calculate a list of corresponding EUDs. For a tumour located at 3 cm depth and treated with GRID therapy with different prescription doses, a 2nd order polynomial approximation equation is given in the following Equation (7):a0, a1 and a2 are the fitting coefficients.For different prescription doses TM GRID collimator and generated plans for different tumour sizes, shapes , and depths in a flat phantom. The reference tables and dose–volume histogram (DVH) curves were derived based on recommendations of the “RSS GRID, Lattice, Microbeam and FLASH working group white paper” [We carried out a series of studies using a .decimal Inc.e paper” .The PDDs calculated using the TPS were compared to those measured with the water tank and were found to be in excellent agreement. This is illustrated in Comparisons of TPS and measured data were assessed with respect to variations in tumour size and depth. PVDRs. PVDR remains constant across varying tumour sizes, and decreases with increasing depth. Due to internal scattering, which will increase the dose in the shielded valley dose zones with increasing depth, PVDR decreased from 6.3 at 3 cm depth to 4.5 at 8 cm depth. Because a larger tumour tends to have a smaller ratio of D10/D90, mainly due to significant increase in D90 (the dose covering 90% of target volume), in the case of a 20 cm tumour centered at 8 cm depth, D10/D90 is found to be close to the PVDR (4.33 vs. 4.50).The DVH-derived dose tables were evaluated with respect to D10/D90, an important metric in SFRT. The respective peak-valley features calculated from the dose profiles and the D10/D90 and D5/D95 ratios are presented in EUDs in GRID therapy, the EUDs were calculated for different tumour scenarios. EUDs calculated using different radiosensitivities. The EUD of the radioresistant cancer cells was 30–40% higher than that of radiosensitive cancer cells for all tumour geometry scenarios.In view of the importance of reporting EUDs calculated for the three types of normal tissue cells. Radioresistant normal tissue showed a greater EUD than radiosensitive normal tissue.EUDs of cancer cells at different prescription doses using a 16-cm tumour located at 3 cm depth as an example. EUDs of radiosensitive, semisensitive, and radioresistant cancer cells, respectively.We calculated the EUDs for different tumour sizes and averaging them at the same prescription dose, the approximate equations to estimate EUDs for different radio-sensitivities  at different prescription doses pD were best fitted by three 2nd order polynomial functions as follows:When calculating Spatially fractionated radiation therapy is increasingly used in clinical practice. However, the clinical application of SFRT parameters that are profoundly different from familiar dosing concepts, and the variable capabilities of treatment planning systems to manage SFRT computations, present major challenges for clinical practitioners, particularly those who seek to newly establish an SFRT practice. Our results present proof of principle—based on our demonstration example of GRID collimator-based SFRT—that standardized reference tables to guide SFRT prescriptions can be developed, are robust, and thus may provide a practically applicable tool to assist GRID prescription and estimation of SFRT parameters.The GRID collimator is an ideal scenario for the development and testing of these reference tables because it is a standard device that generates consistent dose heterogeneity properties. This also allows several dosimetric parameters to be directly derived from the proposed reference tables, as shown in our results.Validation testing of our TPS-calculated dose profiles by water tank measurements showed a high accuracy with differences between TPS-calculated and measured GRID field doses within 3% at depths beyond the buildup region. Larger discrepancies were noted within the buildup region, as it is typically not accurately modeled by the TPS. Because the accuracy of small-field dose calculation is a longstanding concern , this ovAs we hypothesized, the commercially available GRID collimator showed consistent dose metrics for the range of studied treatment parameters. Regardless of tumour size, when located at the investigated 3 cm depth the standard deviations for the coverage doses of D95, D90, D80, … D20, D10, and D5 were less than 0.5 Gy (2.5% of prescription dose). Our results support that the proposed reference table data  can be dPVDR. PVDR is an important dosimetric parameter recommended in GRID therapy documentation as a measure of dose heterogeneity based on recent guidelines [PVDR adds significant complexity to clinical practice. The peak/valley ratio varies inversely with depth, and it has been unclear at which particular depth to report the PVDR when characterising GRID therapy.Our reference tables also provide a good estimate of the idelines ,29. The idelines ,31. In 3PVDR. We believe a peak-valley dose metric determined from the 3D target volume’s DVH ratio of D10/D90 better reflects the dose heterogeneity of GRID therapy across the tumour volume than PVDR derived from the dose profile at a single depth. We provided D10/D90, along with D5/D95, and profile-based PVDR in a reference table , the D10/D90 and the peak/valley dose ratio from a dose profile at the tumour center are very similar, indicating that for large tumours the D10/D90 can represent the peak/valley ratio at the center.Our results support this view. The EUD, a concept that is highly relevant but particularly challenging for clinical physicists and clinicians. Our EUD results calculated for cancer cells of the following three different radiosensitivities are interesting: GRID therapy delivers different EUDs to different cells showing different radiosensitivities; we found that the EUD of radioresistant cancer cells was greater than the EUD of radiosensitive cancer cells  parameters. It may further assist in the implementation of GRID therapy programs in institutions that newly implement this technique. Our tables may also serve to provide a reexamination of previously published GRID therapy clinical data, in which only tumour size, depth, and prescription dose information were reported. Such capability is of great importance for correlating clinical outcomes from previously treated patient cohorts with respect to specific dosimetric parameters that are postulated to relate to SFRT response . This isTM GRID collimator and a 6 MV beam. The reference tables  who face the common situation of lacking TPS availability for GRID planning. Observed variations in dosimetric parameters from different tumour sizes and depths are consistent and predictable. The resulting reference tables, graphs, and approximation equations developed in this study may serve as a guide for clinical decision-making on treatment selection, facilitate reporting requirements for GRID collimator-based SFRT in bulky tumours, and assist in the dose analysis of previously published GRID therapy clinical outcome data, which commonly lacks TPS-generated analysis of the heterogeneous tumour dose.


Forage palm is extremely suitable as animal fodder due to its hightolerance to the climatic rigors of the semiarid region and its abilityto withstand the harsh physical–chemical limitations of poor soils. Thus,in this study, the effects of the partial replacement  of a molasses- or oat-based commercialconcentrate with forage palm bran (FPB)on the acceptability, apparent digestibility and glycemic response of horsesat maintenance were evaluated. The ratio of concentrate to roughage(Tifton 85 hay) was  Forage palm use asfeed for maintaining various domestic species , including horses , during prolonged droughts is becoming increasingly more common.In its natural form, this roughage is an excellent source of water for animals; however, in branform, it may be considered as an energy concentrate due to the lowconcentration of its cell-wall constituents . The mostwidespread forage palm species is the small sweet palm  Salm-Dyck) has an average of 11.9 % dry matter (DM) as well as (based on DM) 4.4 % crudeprotein (CP), 27.3 % neutral detergent fiber (NDF), 17.3 % aciddetergent fiber (ADF) and 1.8 % ether extract (EE) . Palm bran also has a low starch concentration and a high content of soluble carbohydrate; pectin; vitamins A, B, E and C; andminerals such as calcium, magnesium and potassium .Recent studies have highlighted that the These characteristics indicate the great potential of using palm bran in thediet of horses that, as functional cecum herbivores, are able to efficientlyutilize its carbohydrates through cecal microbial fermentation . In addition, horses are susceptible to several metabolicdisorders caused by excess starch in their diet; therefore, the use of feedscontaining low starch becomes essential for maintaining the health of thedigestive tract . Excess starch also promotes elevatedpostprandial glycemic responses, which can cause diseases such as insulinsensitivity . Another advantage of the inclusion ofpalm bran in the diet is its lower cost compared with corn bran .There are few studies reporting the use of palm bran  in horse diets, especially with respect to its acceptability anddigestibility. Horses are highly selective animals: they select foodsbased on factors such as visual characteristics, odor, flavor, availability, texture andvariety . Thus, when evaluating the use of analternative feed, variables such as intake, digestibility and carbohydratemetabolism should be optimized, as they impact performance directly. The following two hypotheses were made in this study: (1) the inclusion of palm bran in the concentrate ofequine diets does not decrease intake, and (2) thedigestibility coefficient and the postprandial glycemic index are similarbetween diets containing different levels of palm bran inclusion and controldiets . Thus, the objective this pilot study was toevaluate the effects of the partial replacement of a commercial concentrate withforage palm bran on acceptability, apparent digestibility and glycemicresponse in maintenance equine diets.2All experimental procedures were approved by the Ethics Committee inResearch with Production Animals of the Federal University of Sergipeaccording to protocol no. 05/15.2.1Nopalea cochenillifera (L.) Salm-Dyck) forage palm cultivar was used. The palmwas obtained from the Embrapa Coastal Tablelands experimental station, located in FreiPaulo, Sergipe, Brazil  and a commercial pelleted concentrate, in thequantities recommended for adult horses at maintenance (NutriEquiPMSE®15.6), were offered daily, and water andmineral mix were offered ad libitum  .The experimental diets were formulated to meet the nutritional requirementsof adult horses at maintenance according to the NRC (2007) recommendations.Free access to Tifton 85 hay (Treatment 1 (control) – no FPB;Treatment 2 – inclusion of 5 % (50 g/kg) FPB as a substitute for commercialconcentrate;Treatment 3 – inclusion of 10 % (100 g/kg) FPB as a substitute for commercialconcentrate;Treatment 4 – inclusion of 15 % (150 g/kg) FPB as a substitute for commercialconcentrate.For the preference test and digestibility test, four treatments wereapplied:2.3Cynodon dactylon (L.) Pers.).Animals scored from 4 (moderately thin) to 6 (moderately conditioned)according to a body condition scale (BCS) developed by Henneke et al. (1983), which ranges from 1 (extremely thin) to 9 (extremely obese). Mareswere allocated to treatments in a completely randomized pattern.For the intake preference test, 10 barren, healthy, 10 The preference test was divided into two steps: in Step I, the commercialconcentrate was offered in pelleted form; in Step II the concentrate wasoffered as bran after processing in a 3 mm sieve mill. The four treatmentswere the same in both steps. The step interstitial period was 30 d.The diets were provided in a single concrete trough that was 3 m in length. Thetreatments  were arranged so as to be equidistant from eachother along the trough and in a random manner, minimizing the possibility ofan animal choosing any specific region .The preference test was performed according to the methodology proposed byRivera et al. (2019). For both stages, the experimental period lasted 6 d: 3 d for adaptation and 3 d for observations. Allanimals were evaluated on each of the 3 observation days, resulting in30 observations for each stage. The instantaneous focal methodology was used, and each animal was observed for 15 min. Theorder of the animals' choice of treatments was recorded – the treatment that was effectively ingested, not justsmelled by the animal, was considered to be the one that waschosen or preferred.The animals were removed from the paddock and taken into the individualobservation stalls. Before the test, all mares received 300 g of pelleted(Step I) or bran (Step II) commercial feed without palm forage in order toreduce anxiety . There was then a 10 min intervalbetween the end of this meal and the start of the preference test.During the preference test, 300 g of feed from each treatment was provided.Each experimental diet was randomly placed in the trough (so as to be equidistant fromthe other treatments), and the treatments were offered simultaneously to the animals, resulting in a total feed mass of 1.2 kg.At the end of each observation, in Step I, orts were weighed in order todetermine the intake ratio (IR). The IR percentage was obtained by the ratiobetween the amount actually consumed in a diet in relation to the totalamount of feed offered, considering the four treatments ; this was calculated as follows:2.4For the digestibility test, four healthy 13 Animals were kept in individual stalls  was stored and frozen at The apparent digestibility coefficient (DCApparent digestible energy (DE2.5® Works) was used the determine the grossenergy (GE), with benzoic acid as the standard calibrator. The analyses ofneutral detergent fiber (NDF) and acid detergent fiber (ADF) were performedas described by Van Soest et al. (1991).Nutrient dry matter , mineral matter, ether extract , crude protein , mineralmatter (MM) and ether extract (EE) were determined using the methodologiesdescribed in AOAC (2012). An adiabatic calorimeter  were determined according toHoffman (2001) (Table 1).The digestible energy values were estimated according to the formulasdescribed in the NRC (2007) for concentrate and roughage:2.6Blood glucose samples were taken during the digestibility test, on the lastday of the total feces collection. The collection periods were 30 minbefore and 30, 60, 90, 120, 180 and 240 min after the experimental feedswere given, according to the methodology described by Rodiek and Stull(2007).After local antisepsis, blood samples were obtained through venipuncture ofthe jugular using disposable needles, in 2 mL vacuum collection tubescontaining anticoagulant ethylenediaminetetraacetic acid (EDTA)–sodium fluoride, forplasma glucose assessment; 4 mL of blood was collected and centrifuged, and 2 mLof plasma was stored in microtubes and frozen at For the glycemic test, mean plasma glucose concentrations, peak responsesand the time to reach the glucose peaks were calculated. Mean glucoseconcentrations were analyzed using the area under the curve (AUC),considering the baseline and postprandial moments (0 to 240 min)according to Borghi et al. (2017).2.7All data were analyzed using the Sisvar system . Intakepreference tests were evaluated using the Friedman test, with data from thetable of Newell and Macfarlane . Intake ratio (IR)data were subjected to an analysis of variance and regression analysis. Forintake, apparent digestibility and glycemic responses, the fixed effects oftreatments , period and random animalwithin the Latin square and residual were considered. The first value foreach variable observed within the experimental period was used as a covariate.Variance and regression analyses used the 5 % level of significance, andmeans were compared using a Tukey test.33.1In Step I (pelleted concentrate), the preference test indicated that thediets with low FPB inclusion (0 % and 5 %) were preferred  was associated with alower preference for the 10 % . Blood glucose remained high until 180 min afterfeeding, when the peak plasma glucose concentration occurred, converging tobaseline values at 240 min in all treatments. The lowest value of theglucose peak was observed 180 min after feeding in the treatmentcontaining 7.42 % FPB inclusion .This preference for diets containing less forage palm was only found in StepI, when the concentrate was offered in pelleted form. In Step II, when theconcentrate was offered in bran form, no difference in preferencewas observed.Some studies show that horses have developed a feed intake strategy,selecting their diet based on visual characteristics, odor, flavor, texture,availability and variety, always giving preference to what is familiar.It is possible that the horses, who were used to ingesting the commercial pelleted feed,reacted negatively to the addition of the palm bran because it modifiedthe structure of the pellet, decreasing its firmness over time. It is alsopossible that the high pectin content in palm bran may havealtered the texture of the commercial diet feed due to its highagglutination capacity , negatively influencingthe preference for diets containing higher levels of FPB.The overall mean DM intake was 1.8 % of the body weight, which is withinthe NRC (2007) recommendations of 1.5 %–3.0 % for the maintenance of adulthorses.According to Frape (2008), the main factors that regulate DM intake inhorses are the capacity of the different segments of the gastrointestinaltract, the rate of digestion passage, the concentration of nutrients in thediet, and, especially, the fulfillment of energy and protein requirements. Itis noteworthy that the overall DE and CP intakes met the NRC (2007)requirements, suggesting that this may have been a factor responsible forkeeping the DM intake as expected (Table 3).The higher intake of MM followed the increase in the level of inclusion ofFPB, a characteristic related to the high mineral content of this feed(25.40 %) compared with the concentrate (8.03 %). The inverse was observedfor the EE intake, which decreased as FPB inclusion increased, due to thelow concentration of this nutrient in the bran (0.98 %) compared with thatof the concentrate (6.07 %).As apparent digestibility coefficients were similar among treatments, itis possible to infer that the inclusion of FPB provides an excellent source of nutrients in the diet. This is possibly due to the chemical composition of FPB,which has high concentrations of NDF (51.69 %) and hemicellulose(27.28 %). These nutrients are converted to volatile fatty acids duringcecal microbial fermentation providing energy that is rapidly available to the animal .Therefore, even with a reduction in the amount of energy in the diet, as thelevel of inclusion of FPB increased, there was no decrease in the apparentdigestibility coefficients, probably due to the large presence offermentable carbohydrates in the FPB which compensated for its low density.Digestibility coefficients lower than those observed were found byCasalecchi et al. (2012) when evaluating a concentrate of extruded corn foradult mares: 44.7 % for NDFDCBaseline glucose concentrations were consistent with the physiologicalconcentrations reported in the literature for fasting horses, between 80 and100 mg/dL, which reached 150 mg/dL at 3 h following the consumption of a starchy diet. However, peak glucose concentrations did not reachthis value. The peak glucose value, considering treatments containing FPB,was 122.7 mg/dL.When evaluating diets composed of grass hay and concentrate containingdifferent starch sources  in the equine diet,Gobesso et al. (2009) found peak glucose concentrations of 161.63 mg/dL forcorn and 123.23 mg/dL for oats 90 min after ingestion of the experimentaldiets, which are values higher than those observed in the present study (Table 6).The occurrence of a relatively low value at the time of peak plasma glucoseconcentration may indicate that the absorption of this nutrient occurreduniformly over time, which is beneficial to the animal in view of its highpredisposition to become insulin resistant when fed high starch diets.The area under the curve (AUC) indicated that the inclusion of FPB did notincrease blood glucose concentration over time. The AUC assists in thequantification of the glycemic response to a certain feedstuff with a knowncarbohydrate concentration: the sooner the glucose is removed from thebloodstream, the lower the AUC will be .Postprandial glycemic responses are regulated according to several factors,such as chyme viscosity, passage rate prolongation, delayed The current results demonstrate the potential of FPB as a low-glycemic-indexfeed for horses, which are usually susceptible to numerous metabolicdisorders, such as insulin resistance, obesity, colic and laminitis,resulting from the excessive supply of hydrolyzable carbohydrates in theirdiet . The use of feeds with a low glycemic index is astrategy to reduce the occurrence of these serious nutritional disorders.5Nopalea cochenillifera (L.) Salm-Dyck), which could replace up to 15 % of commercial concentrates for horseswithout reducing their feed intake or decreasing the digestibility of dietarynutrients, keeping the postprandial glycemic response low. Thus, palm branproved to be an alternative feedstuff of great potential for equinemaintenance diets.The horses had a positive acceptance to forage palm bran (


Lactobacillus helveticus was found occurring as the dominant species in NWS cultures and Levilactobacillus brevis as codominant in the cheese factories where the temperature of NWS production was mainly lower than 40°C, suggesting that the variability in the parameters of the NWS culture preparation could differently modulate the bacterial species in NWS cultures. Using turbidity test approach on 303 bacterial isolates from the NWS cultures, 120 distinct phages were identified. L. helveticus phage contamination of NWS cultures was revealed in most of the analyzed samples, but despite the great recovery of bacteriophage contamination cases, the microbial quality of NWS cultures was high. Our results support the presence of natural bacteriophage resistance mechanisms in L. helveticus. The use of NWS cultures probably creates an ideal environment for the proliferation of different L. helveticus strains balanced with their phages without a clear dominance. It is evident, from this study, that the presence of a high biodiversity of NWS bacterial strains is relevant to avoid phages dominance in NWS cultures and consequently to keep a good acidification ability.This study focused on the microbial and bacteriophages identification and characterization in cheese-production facilities that use natural whey starter (NWS) cultures for Trentingrana production. Bacterial and phage screening was carried out on cooked not acidified whey and NWS samples isolated from six dairy factories, for 4 consecutive days in four different months. By means of a combined approach, using plate counts, bacterial isolation, and metataxonomic analysis  Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus and L. delbrueckii ssp. lactis  may represent a dealing factor able to negatively affect fitness and performance of dairy starter cultures . Phages L. helveticus and L. delbrueckii  characterized the dynamic of bacterial and phage community of the NWS collected to increase the existing knowledge and (ii) investigated if and how the cheese factory technology of NWS production affects the bacterial and phage community in the NWS cultures. The final goal is to maintain the high quality of Trentingrana production, avoiding economic loss associated with failure in milk fermentation and low cheese yield.cNAW and NWS culture samples used in Trentingrana manufacturing were collected for four consecutive days in four different months  from six dairy factories (A–F) located in the province of Trento  and operating into the Trentingrana Protected Designation of Origin cheese area of production .The Trentingrana production had stopped at cheese factory C for July and August months, so NWS and cNAW samples were collected in September only for cheese factory C.NWS titratable acidity was measured and expressed as Soxhlet–Henkel degrees (°SH); the pH was determined using a pH electrode . Samples were shipped to the laboratory under liquid nitrogen and stored at −80°C before the analysis.The temperature of the room and of the NWS tank has been determined using Testo 175 T2 . Temperature was recorded each minute for the overnight fermentation of the NWS.All the samples were diluted in sterile peptone water and plated onto whey agar medium (WAM) according to 2O2 drops on the colonies.Eight colonies grown onto WAM plates were randomly isolated from NWS samples (plates with a number of colonies in the range of 10–300). Each isolate was purified by subsequent culturing in whey broth (the broth version of the WAM used for plate counting). Pure cultures were stored at −80°C in glycerol (40% vol/vol) stocks. Cell morphology was determined by microscopic observation, Gram characterization was performed applying the KOH method , and catTM MicroPrep  following the manufacturer’s instructions.All bacterial isolates were grown 48 h in the whey broth culture at 45°C before DNA extraction. The bacterial DNA was isolated using Quick-gDNARandomly amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) was carried out in a total volume of 25 μL using primer M13 . ClusterTM kit  and sequenced in an ABI PRISM 3100 sequencer , using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems). The obtained sequences were compared using the BLAST algorithm available on the National Center for Biotechnology Information . All amplifications were run in a T100TM ThermalCycler .One isolate representative of each biotype was genotypically identified by 16S rRNA gene analysis. A fragment of the 16S rRNA gene was amplified using the primers 27F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and 1495R (5′-CTACGGCTACCTTGTTACGA-3′), designed by g for 15 min at 4°C. The genomic DNA was extracted from the pellet using the Power FoodTM Microbial DNA Isolation Kit  according to the manufacturer’s instructions. All DNA samples were purified by PowerClean DNA Clean-up Kit (Qiagen). The DNA quality and concentrations were determined by NanoDropTM 8000 Microvolume UV-Vis spectrophotometer (ThermoFisher Scientific).Illumina analysis was performed on 24 cNAW and 24 NWS samples (one cNAW and NWS sample for each month and cheese factory). Three milliliters of sample was centrifuged at 3,200 × Escherichia coli positions 341 to 805) was amplified. Unique barcodes were attached before the forward primers to facilitate the pooling and subsequent differentiation of samples. To prevent preferential sequencing of the smaller amplicons, the amplicons were cleaned using the Agencourt AMPure kit  according to the manufacturer’s instructions; subsequently, DNA concentrations of the amplicons were determined using the Quant-iT PicoGreen dsDNA kit  following the manufacturer’s instructions. In order to ensure the absence of primer dimers and to assay the purity, the generated amplicon libraries quality was evaluated by a Bioanalyzer 2100  using the High Sensitivity DNA Kit (Agilent). Following the quantitation, cleaned amplicons were mixed and combined in equimolar ratios.Amplicon library preparation, quality and quantification of pooled libraries, and pair-end sequencing using the Illumina MiSeq system  were carried out at the Sequencing Platform in Fondazione Edmund Mach . Briefly, for each sample, a 464-nucleotide sequence of the V3–V4 region , of the 1 and imported into Quantitative Insights Into Microbial Ecology . Sequences were quality filtered, trimmed, denoised, and merged using DADA2  database, which had been previously trimmed to the V4 region of 16S rDNA, bound by the 341F/805R primer pair, was applied to paired-end sequence reads to generate taxonomy tables. The data generated by MiSeq Illumina sequencing were deposited in the NCBI Sequence Read Archive and are available under Ac. Number PRJNA6951353 from sample SAMN17602423 to SAMN17602468.Raw paired-end FASTQ files were demultiplexed using idempng DADA2 . Chimeriogeny K. . α- and  emperor . Bacteri3 was included in tubes of 40-mL cNAW sample, acidified with 0.5 mL of sodium maleate buffer , and then inoculated with 1 mL of NWS from the same sampling day and dairy. After anaerobic incubation at 45°C for 72 h, the tubes were centrifuged at 4,000 revolutions/min for 15 min at 4°C. Supernatants obtained from the enrichment step were filtered with 45-μm membrane filter pore size  and checked for the presence of phages by means of turbidity test.All the 72 samples of NWS cultures were screened for the presence of bacteriophages. The related cNAW samples were used to isolate the virulent phages, which used the NWS isolated bacteria as host. Phage enrichment was performed as previously reported by 2) were added with 5% of the previously filtered supernatants and inoculated with 2% mL of host culture in the logarithmic growth phase. The tubes were incubated at 45°C and observed each 6 h until cell lysis occurred. The lysis was detected by the higher clearness of the broth, compared to controls (tubes without addition of the supernatants), which were more turbid due to bacteria growth. After 24 h, where no lysis was detected, the bacterial cultures were used for 2% inoculation of new tubes of WAM-Ca broth. After four rounds of inoculations without lysis, the bacterial isolate was considered as not suitable for the phage isolation. Where lysis was observed, the tubes were then filtered through 0.45-μm membrane filter pore size (Merck–Millipore), set to pH 7, and stored at 4°C until use, for no more than 7 days. Phage stocks were stored at −80°C with 40% vol/vol of glycerol.Tubes of WAM-Ca broth . The mixture was poured on the surface of WAM-Ca plates and then inoculated with 10 μL of phage dilutions in seven plate sectors. After anaerobic incubation at 45°C, clear plaques were checked. The phage titer was calculated using the formula titer (pfu/mL) = plaque number × dilution factor × 100.Phage titer was expressed as the number of plaque-forming units per mL of sample (pfu/mL). The suspension of the phage was diluted sevenfold by using phosphate-buffered saline added with CaCl+, 1 μg/mL); the second aliquot was not added with MytC+ and used as control. After 24 h of incubation at room temperature, in the dark, induced phages were detected as follows: 100 μL of each cell suspension was used to inoculate 7 mL of soft WAM  plated on a layer of 1.8% (wt/vol) agar and incubated for 24 h at the optimal conditions for each strain. The growth reduction onto MytC+ plates, after comparison with control plates, indicated the presence of phage.Each isolated bacterial biotype, characterized as a phage host, was tested for the detection of prophage, adapting the method of If not specified, all chemical compounds were purchased from Sigma–Aldrich .W) was performed, as well as a nonparametric test  analyzing the day of collection as the independent variable and the microbial plate counts as the dependent variable. All the tests on plate counts were performed using the STATISTICA data analysis software system, version 9.1 .A normality test  by means of plug-in implemented in QIIME2. For the differential abundance test, the taxonomy information for each OTU sequence was provided using ANCOM method (Differences in diversity indices (OTUs number and Shannon diversity index) of different samples were tested by Kruskal–Wallis test by a plug-in implemented in QIIME2. The overall structural changes of bacterial community were visualized by principal coordinate analysis (PCoA) based on both weighted and unweighted UniFrac distance matrices. The statistical significance was assessed M method  by meansThe WAM counts of thermophilic bacteria in NWS and cNAW samples during the monitoring from February to December are shown in The NWS pH mean values ranged between 3.3 and 3.6 and the titratable acidity between 28°SH and 32°SH / 50 mL without any significant difference for month or cheese factory (data not shown).As the final acidification activity in NWS cultures is directly associated with a slow temperature decrease from 55°C to 40°C during the overnight process of NWS production , the NWSAt dairy plants A, B, and C, the first thermophilic was at least 2 h 25 min shorter than the second mesophilic stage, conversely at dairy plant D, the first was approximately 7 h longer than the second stage. At cheese factories E and F, there was no relevant difference between the first and second stage of NWS culture production cycle.A total of 576 isolates were picked up from WAM agar plates. A RAPD-PCR fingerprinting had been performed (i) to preliminarily recognize the bacterial isolates purified from colonies onto the same plate originated from genetically alike single mother cells and (ii) to investigate the microbial diversity beyond the species level, establishing the number of different biotypes inside cheese factory and over time. One isolate for each biotype was selected for further identification by 16S rRNA gene sequencing.L. helveticus, 120 as Levilactobacillus brevis, 13 as Lacticaseibacillus paracasei, eight as Lactiplantibacillus plantarum, and the other seven as Limosilactobacillus fermentum  was calculated, using both the number of observed OTUs and Shannon diversity indexes. Both α-diversity indexes were not significantly different between cNAW and NWS samples , and no significant difference was found for month or dairy of sampling (data not shown).To address the hypothesis that species richness and biodiversity vary with sample source (cNAW or NWS sample), month , and cheese factory , the intragroup diversity estimation ; the month of sampling was slightly significant in cNAW and NSW samples, and in particular, the samples collected in November showed a significantly different bacterial community. Cheese factory was the main factor affecting microbial population diversity in all the samples, and the cheese factory D showed a significantly higher effect on cNAW and NSW samples  and not in all the samples.Considering only the OTUs whose relative abundance in each sample was greater than 0.01% , Firmicu samples . OTUs idLactobacillus and Streptococcus spp. were informative for characterizing the microbial community of cheese factories A and B.To identify taxonomic groups driving differences among the bacterial community, a differential abundance test by ANCOM method had been performed. No difference was found considering the month of sampling. Some taxa had been found differentially abundant among the cheese factories; in particular, both All the 72 NWS samples collected from the six local cheese factories had been phage screened. The bacterial biotypes isolated from the different NWS cultures  represenL. helveticus host isolate, with the exception of two phages lytic for L. fermentum. To detect the presence of prophage in their genomes, the 120 host bacterial isolates were treated with sublethal concentrations of MytC+. In none of the isolates, the MytC+ induced a sharp decrease in plate counts, confirming the lytic nature of all the phages.One hundred twenty phages were readily detected in 41 of the 72 NWS samples  and wereL. helveticus phages, titers ranged between 2 × 106 and 9 × 108 pfu/mL with the exception of all the 12 phages from dairy factory C and six phages from dairy factory F, whose titers were in a range between 2 × 102 and 4 × 105 pfu/mL.Phage titers were determined in all the 41 positive NWS samples. For The NWS cultures used in Grana-like cheese production originate from artisanal back-slopping practices and face several selective pressures during the production process such as heat treatments and strong lactic acid fermentation. The bacterial consortia are characterized by a lower species complexity but high degree of strains diversity , and bacThis study was carried out to investigate the bacterial and phage dynamics of cNAW and NWS production analyzing samples collected in 72 different days of Trentingrana production in six dairy plants, over a year period, by means of a combined approach, using plate counts, bacterial isolation, metataxonomic analysis, and phage isolation.The NWS culture thermophilic counts, acidity, and pH were not significantly different for month (with exception of cheese factory C) or cheese factory, and their values were according to previously studied NWS cultures for Trentingrana  and ParmL. helveticus and L. brevis were the two dominant ones . L. paracasei, L. plantarum, and L. fermentum were present in few NWS samples collected only in February and May. L. helveticus is well known as dominant in NWS cultures for Grana-like cheeses  and during the overall year was surprising. The presence of 51 biotypes of L. brevis could not be explained as a contamination. L. brevis is one of the most common nonstarter LAB species found in the dairy industry , confirming the importance of NWS technology of production in modulating the microbial community of the NWS cultures. The analysis of both metataxonomic and quantitative data obtained by plate counts and isolation suggested that L. helveticus decreased in relative abundance but increased in amount from cNAW to NWS. This was in agreement with L. helveticus growth was mainly favored, in the acidic conditions occurring during the overnight fermentation of NWS. In Trentingrana dairy plants A, B, and C, the NWS cultures showed a high abundance of L. brevis isolates adapted to survive in thermophilic environment. Their dominance was also confirmed by metataxonomic data. Lactobacillus spp. relative abundance was very high (29–39%) when compared to previous metataxonomic studies on NWS for Grana-like cheeses  can be found below: EF and AMe devised the study. EF and AMa drafted the manuscript. EF, AMa, NC, AG, IC, and MR performed sampling plane and the experiments. MZ contributed to the study with her know-how on phages. EF, AMe, and KT provided resources and intellectual input that supported the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Citrus aurantium L.) is one of the biological sources of polymethoxyflavones (PMFs), which are often used to deal with gastrointestinal diseases. The intestine is highly sensitive to irradiation damage. However, limited certain cures have been released for irradiation-induced gastrointestinal injury, and the potentials of sour orange PMFs as radio-resistance agents have not been fully discussed yet. The present study aims to (1) investigate the PMF components in 12 sour orange cultivars, (2) determine the protective effects of PMFs on irradiation-induced intestinal injury by treating mice that received 12 Gy abdominal irradiation with different doses of PMFs and observing the changes in organ indexes and pathological sections and (3) test cytotoxicity of PMFs by CCK-8 method. The results showed that sour orange PMFs appeared to have high intraspecies similarity. Besides, PMFs protected mice from irradiation-induced injury by alleviating body weight loss, reliving organ index changing and maintaining the intestinal structure. Finally, IC50 concentrations to cell line CCD 841 CoN of PMFs and nobiletin were calculated as 42.23 μg/mL and 51.58 μg/mL, respectively. Our study uncovered PMF contents in 12 sour orange materials and determined the protective effects on irradiation-induced intestinal injuries, providing guidance for the utilization of sour orange resources.Sour orange ( The intestinal tract is one of the most important immune organs and undertakes functions of digestion. The intestinal epithelium draws a clear distinction between internal and external environments of organisms and is composed of different cell types, including Paneth cells, intestinal stem cells (ISCs), endocrine cells, goblet cells, tuft cells, M cells and intestinal epithelial cells (IECs) ,2. The hThe gastrointestinal (GI) form of acute radiation sickness (ARS) is a common disease often observed among patients who received intraperitoneal radiotherapy. Symptoms may appear in any section of the intestine and may be featured as abdominal pain, diarrhea (or obstruction), ulcer and apostasies ,6. The sCitrus aurantium L.), regarded as the crude materials of ‘zhiqiao’ and ‘zhishi’, has often been used in herbal medicine to deal with gastrointestinal diseases. A variety of bioactive compounds have been detected in sour oranges, such as flavonoids, volatile components  and alkaloids [Sour orange . The PMF extracts were given through irrigation once per day, and mice were sacrificed on day 14 to evaluate the protective effects of sour orange PMFs on mice ARS. The results of our study uncover the composition of PMFs in sour orange cultivars and determine the potential of PMFs being a radio-resistant agent, providing practical guidance for the utilization of sour orange resources.Citrus reticulata Blanco cv. ‘dahongpao’, abbreviated as ‘DHP’), sweet orange (Citrus sinensis (L.) Osbeck. var. brasliliensis, abbreviated as ‘NHE’) and pomelo (Citrus maxima (Burm) Merr. cv. ‘liangpingyou’, abbreviated as ‘LPY’) as outgroups . CitrusCitrus materials, we chose the type species (cultivar ‘daidai’), also known as a common crude material of Chinese traditional medicine ‘zhiqiao’ and ‘zhishi’, out of 12 sour orange cultivars for the following extraction and purification. The main flavonoids of the mixture after enrichment include two flavones, eight flavanones and eight polymethoxyflavones  mice to mine the differentially expressed genes and pathways as the candidates of the potential target pool. Considering that estrogen might contribute to the radio-resistance, only male mice samples were extracted for the following analysis. Ionizing irradiation significantly changed the gene expression pattern in abdominal-irradiated mice compared to mice that received no irradiation A. The diThe potential targets of PMFs were downloaded from online databases SWISS and SEA. The probability of PMF monomer acting as an antiradiation component was estimated preliminarily by calculating the counts of enriched pathways which met with those obtained from the public dataset. The potential pathways of PMFs matched well to pathways enriched by public sequencing data. PMFs showed the potential to have an impact on mice ARS as the targets downloaded were significantly enriched in all top three KEGG pathways  in the candidate pool C. ProteiTo verify the radio-protective effects of PMFs, male KM mice pretreated with or without PMFs were anesthetized and received abdominal γ-ray irradiation at a single dose of 12 Gy to establish a radiation-induced intestinal injury mice model A. The miAs injury might occur in multiple organs when experimental animals are exposed to abdominal irradiation, organ indexes of the liver and spleen were also calculated to determine the protective effects of PMFs on multiple organs. Both the liver index and spleen index of mice in the VEH group significantly changed after irradiation, while intake of 200 mg/kg sour orange PMFs per day, to some extent, reversed such trends of organ index changes. Interestingly, oral administration of a high dose of PMFs showed opposite effects on liver and spleen indexes, while the low dose of PMFs intake did not show any influence on organ indexes D. Mice eAs oral administration of 200 mg/kg sour orange PMFs positively promoted irradiation-induced injury repair, different section staining methods were used for further analysis of the protective effects of PMFs from a pathological perspective. H&E staining showed that high-dose abdominal irradiation shortened intestinal villus and caused infiltration in crypts and lamina propria in the ileum. Medium-dose PMF pretreatment, to some extent, maintained relatively normal small intestinal histological structures by keeping the integrity of villus and crypts. In the region of the proximal and distal colon, administration of 200 mg/kg PMFs mitigated loss of crypts and infiltration A. To det50 values of PMFs in CCD 841 CoN and HCT-116 were 42.23 μg/mL and 36.00 μg/mL, respectively. The cytotoxicity of three major monomers in sour orange PMFs was also tested using cell line CCD 841 CoN. Nobiletin was shown to crystalize in complete medium at 198 μM (80 μg/mL), and the IC50 was calculated as 128.18 μM (51.58 μg/mL). The crystallization concentrations of tangeretin and sinensetin were 53 μM (20 μg/mL) and 214 μM (80 μg/mL). The inhibition of cell viability brought by tangeretin and sinensetin was no more than 50% under their saturated concentration  and HCT-116 , were used to determine the influences of PMFs on cell viability. As PMFs started to crystalize in complete medium at a concentration of 100–120 μg/mL, concentration gradient of PMFs was set as 0, 5, 10, 20, 40, 60, 80, 100 and 120 μg/mL. The ICntration .50 concentrations of PMFs and nobiletin were calculated as 42.23 μg/mL and 51.58 μg/mL, respectively.The gastrointestinal form of acute radiation sickness (ARS) or acute radiation enteritis (RE) is a common complication of intraperitoneal radiotherapy which affects the postoperative life quality of patients and sometimes causes mortality. Studies have been carried out to explore approaches to alleviate the symptoms of ARS ,18, inclCitrus aurantium L.) is important germplasm often used as rootstock and crude material of herbal medicine with a long history of application. It has been proven that sour orange peel contains various bioactive compounds, such as synephrine and rutinum [3) which mainly exist in citrus fruit and vegetative tissues [Citrus. Our results demonstrated that the content and component of PMFs showed significant variation in different sour orange cultivars. The differences observed may be caused by the natural selection that occurred during the process of evolution. As all sour orange materials were collected at the same time and on the same field, which limited the environmental factor changes, variation of PMF content was possibly brought by genetic factors rather than epigenetic factors. Since Scora (1975) [Citrus based on components of essential oil, phytochemicals have been considered as parameters for plant taxonomic classification. In other words, closely related varieties should have similar content or component of phytochemicals.Sour orange   and Barra (1975)  describeCitrus, we used the hierarchical clustering method for further analysis of the diversity and similarity of PMFs in sour orange. Pomelo, sweet orange and mandarin were introduced as outgroups. Based on molecular biological evidence, sour orange is considered to be a hybrid origin taxon, and pomelo is proven to be one of the crossing ancestors . While pomelo and mandarin are regarded as two basic independent origin species in the genus Citrus, sweet orange may be the backcross progeny of (pomelo × wild orange A) × wild orange B. Our clustering results agreed with the phylogenetic relationships among four citrus types demonstrating that PMFs have high homogeneity within taxa Citrus aurantium L. and differ from other species in the genus Citrus.To determine whether the variation of PMFs intraspecies matched the phylogenetic relationships of Natural components play important roles in human health, but it challenges researchers that a great variety of chemicals with different structures lead to extremely complex bioactivities. With the development of computer algorithms and databases, network pharmacology has been released for target prediction and drug discovery. In the present study, the potential of PMFs as radio-resistance agents was predicted by calculating targets enriched in pathways influenced by irradiation, which was similar to a method known as CIPHER developed previously . NetworkThe intestine, regarded as an important digestive and immune organ, has complete strategies of cell differentiation and proliferation to meet the requirement of digestion and resistance to external threats. Various intestinal cells and complex intestine microecology guarantee the homeostasis of the gut and, at the same time, provide support to intestinal barrier function . Acute rSecretory cells, such as goblet cells, produce mucus containing lysozymes, mucopolysaccharides, glycolipids and digestive enzymes which make up the intestinal chemical barrier. Changes in the number and function of goblet cells may, to some extent, reveal the changes in the chemical barrier . ColonicIt was inferred that small intestinal goblet cells did not show differences between VEH- and medium-dose-treated groups because the fast-pass characteristic of contents in the ileum and relatively low microbial abundance limited the interaction between PMFs and intestine. Evidence is mounting that the gut microbiota is essential for PMF metabolism and PMFs can shape the microorganism characteristics. Modification of PMFs might be carried out by gut microorganisms, including demethylation, hydroxylation, glycosylation and ring opening . MeanwhiPMFs have been confirmed to have multiple bioactivities and, for a long time, have been considered as nontoxic agents . BecauseThere are some limitations of this study. First, PMFs used in the experiment were purified from a single growth stage, and PMFs in other tissues were ignored. Further and processive analysis of the diversity of PMFs in different sour orange cultivars, the variation of PMFs during the growth stages of sour orange and the polymorphism of PMFs in different tissues of sour orange should be carried out to provide theoretical and practical guidance for the utilization of sour orange materials. Second, because the metabolites of PMFs in vivo remain unclear, only major monomers were adopted for network pharmacological analysis. Given the development of MS and computing technologies, the metabolic process of PMFs could be uncovered by new pharmacology methods, such as Poly PK analysis. Finally, the strains of mice and cell lines covered are relatively limited. Omics and genetically modified animals should be used to explore the mechanism of PMFs as radio-resistant agents.Sour orange fruits of 12 cultivars were collected from National Citrus Germplasm Repository, Citrus Research Institute, SWU/CAAS, China, in December 2020. The fruits were collected following a five-point sampling method to reduce errors brought by environmental factors. The materials used are listed in v/v) (A) and methanol (B) at the rate of 0.4 mL/min, and the gradient profile was as a previous study reported. All the prepared samples were tested in triplicate [https://www.originlab.com) (accessed on 15 December 2021).The peels of sour orange were separated from flesh and immediately frozen in liquid nitrogen. The peels were then porphyrized and sieved to obtain materials for extraction. The powder-like materials were kept at −80 °C until extraction. The powders were rewarmed to room temperature 1 h before extraction. One gram of each powder was moved into a 15 mL centrifugal tube and mixed with 8 mL methanol. The mixtures were then treated with ultrasound . The supernatants were removed and placed into 50 mL centrifugal tubes after centrifugation . Sediments were supplemented with 8 mL methanol and repetitively extracted twice. The supernatants were combined and diluted with methanol to a final volume of 25 mL. The extracts were filtered with 0.22 μm hydrophilic polytetrafluoroethylene (PTFE) syringe filters to obtain a sample solution for ultrahigh-performance liquid chromatography  equipped with a photodiode array detector, a quaternary solvent delivery system and a column temperature controller) detection. Chromatographic separations were performed on a 2.1 × 100 mm, 1.7 µm ACQUITY UPLC HSS C18 column . The mobile phase consisted of water/formic acid . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). The differentially expressed genes were obtained by Deseq2 package. The heatmap was plotted by the ComplexHeatmap package. The enrichment of differentially expressed genes was carried out using the ggplot2 package and GOplot package.The potential targets of PMFs were downloaded from online databases SWISS and SEA. The downloaded targets were merged and duplicates were removed, followed by GO/KEGG enrichment using ggplot2 and GOplot packages. The protein–protein interaction (PPI) network was established by STRING and modified by Cytoscape Version 3.8.2 .n = 5): Blank, mice received no PMFs or irradiation; VEH, mice received no PMFs but were injured by irradiation; Low, mice received 50 mg/kg PMFs per day and were injured by irradiation; Medium, mice received 200 mg/kg PMFs per day and were injured by irradiation; High, mice received 800 mg/kg PMFs per day and were injured by irradiation; Drug, mice received 200 mg/kg PMFs per day but were not injured by irradiation. After a 7-day adaption, mice were irrigated with different doses of purified PMFs  dispersed in sterile 0.8% CMC-Na solution once per day for consecutive 14 days. On the 7th day after the first irrigation, mice were anesthetized by a combination of 0.5% Zoletil-50 (Virbac) and 0.5% xylazine hydrochloride  and received abdominal γ-ray irradiation at a total dose of 12 Gy. The mice were transported to the laboratory animal room and fed normally. Mice were sacrificed on the 14th day after the first irrigation. The small intestines were divided into 5 segments numbered 1 to 5 and colons were also divided into 5 segments numbered 6 to 10. Segments 1, 3, 5, 7 and 9 were fixed with 10% formaldehyde–PBS solution while segments 2, 4, 6, 8 and 10 were treated with RNA later  and kept at −80 °C. Livers and spleens were separated and weighed for the calculation of organ indexes. The formula of organ index calculation was as follows:Eight-week-old KM male mice weighing 28−30 g were purchased from Hunan SJA Laboratory Animal Co., Ltd.  (production certificate No. SCXK (Xiang) 2019-0004), and kept in the laboratory animal room of the Institute of Combined Injury, AMU. Mice were randomly divided into 6 groups , AB-PAS staining kit , OLFM4 (D1E4M) XP Rabbit mAb  and Ki-67 (D3B5) Rabbit mAb  .2 for one generation and then seeded into 96-well plates (1 × 105 cells per well). PMFs and monomers were dissolved in DMSO and then diluted with DMSO  to obtain secondary mother liquor of different concentrations . The liquor was added into the medium (1 μL per well) and incubated with cells overnight. Cell viability was detected by CCK8 kit  following the recommended protocols.Cell lines of CCD 841 CoN and HCT-116 were provided by State Key Laboratory of Trauma, Burns and Combined Injury . Cells were resuscitated and cultured in DEME  containing 10% FBS  under the conditions of 37 °C and 5% COt-test (p < 0.05) and visualized by GraphPad Prism Version 9.0.0 for Windows ) and OriginPro 2022.Data were expressed as the means ± standard deviation of replicates. Significant differences among groups were analyzed by In summary, the present study analyzed PMF contents in 12 sour orange materials and determined the protective effects on ARS. The in vivo and in vitro toxicities of PMFs were analyzed to provide information for PMF development. This study provides theoretical and practical guidance for the utilization of sour orange resources.