Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder characterized by inflammatory cell infiltration, leading to persistent synovitis and joint destruction. The pathogenesis of RA remains unclear. This study aims to explore the immune molecular mechanism of RA through bioinformatics analysis.Five microarray datasets and a high throughput sequencing dataset were downloaded. CIBERSORT algorithm was performed to evaluate immune cell infiltration in synovial tissues between RA and healthy control (HC). Wilcoxon test and Least Absolute Shrinkage and Selection Operator (LASSO) regression were conducted to identify the significantly different infiltrates of immune cells. Differentially expressed genes (DEGs) were screened by “Batch correction” and “RobustRankAggreg” methods. Functional correlation of DEGs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Candidate biomarkers were identified by cytoHubba of Cytoscape, and their diagnostic effectiveness was predicted by Receiver Operator Characteristic Curve (ROC) analysis. The association of the identified biomarkers with infiltrating immune cells was explored using Spearman’s rank correlation analysis in R software.Ten significantly different types of immune cells between RA and HC were identified. A total of 202 DEGs were obtained by intersection of DEGs screened by two methods. The function of DEGs were significantly associated with immune cells. Five hub genes  were screened by R package “UpSet”. CCL5+CXCR4 and GZMA+CD8A were verified to have the capability to diagnose RA and early RA with the most excellent specificity and sensitivity, respectively. The correlation between immune cells and biomarkers showed that CCL5 was positively correlated with M1 macrophages, CXCR4 was positively correlated with memory activated CD4+ T cells and follicular helper T (Tfh) cells, and GZMA was positively correlated with Tfh cells.CCL5, CXCR4, GZMA, and CD8A can be used as diagnostic biomarker for RA. GZMA-Tfh cells, CCL5-M1 macrophages, and CXCR4- memory activated CD4+ T cells/Tfh cells may participate in the occurrence and development of RA, especially GZMA-Tfh cells for the early pathogenesis of RA. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease . The maiImmune cells in synovial membrane, including resident and infiltrating immune cells, play a vital role in the occurrence and development of RA, an autoimmune disorder . StudiesWith the development and widespread use of microarray and high-throughput sequencing technology, bioinformatics analysis can be used to identify novel genes and biomarkers for many diseases, including autoimmune disease –14. A prIn this study, CIBERSORT was performed to analyze immune cell infiltration in RA by using five microarray datasets. Wilcoxon test and LASSO regression were conducted to identify the significantly different infiltrates of immune cells in RA and HC. After DEGs were screened, functional correlation was analyzed by GO and KEGG, and hub genes were identified by R package “UpSet.” ROC logistic regression was conducted to analyze the predictive of biomarkers. Spearman’s rank correlation in R software was also used to analyze the correlation between biomarkers and significantly different infiltrates of immune cells. This work not only systematically analyzed the infiltration of immune cells in the synovial membrane of RA but also screened novel and effective diagnostic biomarkers for RA. Results provide a new method for the diagnosis and treatment of RA.http://www.ncbi.nlm.nih.gov/geo) database. The inclusion criteria were as follows: (1) expression profiling by array or high-throughput sequencing of mRNA; (2) availability of the synovial tissues of patients with RA or HC from joint in the datasets; and (3) ten or more synovial specimens in the dataset. Six eligible datasets were selected, including GSE1919, GSE12021, GSE55235, GSE55457, GSE77298, and GSE89408. The details of all data are shown in Gene expression profiling of RA was screened using the GEO  based on R package “affy.” Platform annotation file was used to convert the probe expression matrix into a gene expression matrix. For the case of multiple probes corresponding to one gene, the average value was obtained. After five gene matrixes were merged by Perl script, R package “sva” was applied to eliminate heterogeneity caused by different experimental batches and platforms. Finally, we obtained one merging normalized gene expression matrix and used R package “limma” for analysis. For series matrix files, Perl script was used to extract each probe expression matrix one by one and then convert them into gene expression matrices, respectively. R package “limma” was used to analyze each gene expression matrix. The validation RNA-Seq dataset, GSE89408, was downloaded by the form of gene expression matrix. R package “edgeR” was employed for subsequent analysis. The flow diagram of this study is shown in http://CIBERSORT.stanford.edu/), LM22 was used as a reference expression signature with 1000 permutations. The LM22 signature matrix defined 22 infiltrating immune cell components, including subsets of macrophages , T cells , natural killer (NK) cells (resting NK cells and activated NK cells), mast cells (resting mast cells and activated mast cells), B cells (naïve B cells and memory B cells), dendritic cells (resting dendritic cells and activated dendritic cells), monocytes, plasma cells, neutrophils, and eosinophils. The p-values and root mean squared errors were determined for each expression file in CIBERSORT. Only data with a CIBERSORT p value < 0.05 was filtered and reserved for the following analysis. The output was directly integrated to generate an entire matrix of immune cell fractions. The results from CIBERSORT were visualized using the R packages “corplot”, “vioplot”, “ggplot2”, and “glment”.CIBERSORT algorithm was performed to evaluate immune cell infiltration in synovial tissues between RA and HC. This algorithm can transform the normalized gene expression matrix into the composition of infiltrating immune cells. After data were submitted to the CIBERSORT web portal  merged the downloaded five raw datasets into an expression matrix and then analyzed the DEGs with R package “limma” after batch correction and normalization. The second method  used the R package “limma” to analyze the DGEs of the downloaded gene expression matrices. The DEGs of each dataset were integrated with R package “RobustRankAggreg.” DEGs obtained by the two methods were intersected by Venn diagram to extract final DEGs. The threshold points for DEGs were adj.P.Val < 0.05 and |log fold change (FC)| > 1.The gene names of DEGs were converted to gene ID by R package “org.Hs.eg.db”. Analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for DEGs were performed by R package “clusterProfiler.” The significantly different GO terms and signal pathways were screened by the threshold p value <0.05 and q value <0.05. The results were visualized by R package “enrichplot” and “ggplot2”.https://string-db.org) was performed to analyze the PPI network of DEGs with a highly reliable filtering condition (score>0.7). The interaction file (string_interactions.tsv) was downloaded. Perl was conducted to obtain the network file. The cytoHubba of Cytoscape (v 3.7.2) was conducted to score each node gene by top 10 algorithms, namely, MCC , DMNC (Density of Maximum Neighborhood Component), MNC (Maximum Neighborhood Component), Degree, EPC (Edge Percolated Component), BottleNeck, EcCentricity, Closeness, Radiality, and Betweenness. The top 60 node genes scored by each algorithm were used to screen hub genes by R package “UpSet”. The RNA-Seq dataset GSE89408 was used to validate hub genes.STRING  value was utilized to determine the diagnostic effectiveness in discriminating RA from control samples in the GSE89408 dataset.The association of the identified gene biomarkers with the levels of infiltrating immune cells was explored using Spearman’s rank correlation analysis in R software. The resulting associations were visualized using the chart technique with the “ggplot2” package.Five microarray raw datasets, including 56 RA and 41 normal synovial tissues, were selected for the study of immune cell infiltration. The data before (A and B) and after (C and D) batch correction was presented in Five microarray datasets, including 57 RA and 41 normal samples, were used by the two methods to identify DEGs. A total of 360 DEGs were obtained by “Batch correction” and included 335 upregulated and 25 downregulated genes. A total of 461 DEGs were obtained by “RRA” and included 298 upregulated and 163 downregulated genes. Some of DEGs are shown in After being converted into gene ID, DEGs were analyzed by GO and KEGG. The GO annotations of DEGs consisted of three parts including CC (cellular component), BP , and MF (Molecular function), which were used to analyze the functional enrichment of DEGs. The DEGs were mainly related to the biological activity of immune cells, such as lymphocyte differentiation, T cell activation, and leukocyte  and 10 significantly differential immune cells  were analyzed in RA synovial tissues. The correlation results are presented in RA is a systemic autoimmune disease characterized by synovitis of joints. Innate and adaptive immune responses play an indispensable role in the pathogenesis of RA. Increasing number of studies have shown that the complex interaction and activation of infiltrating immune cells are key factors in the formation of synovitis and persistent joint damage. Effective biomarkers, especially for the early stage, have not been established due to the significant heterogeneity of RA. Early diagnosis and treatment of RA could effectively prevent the disease progression of 90% patients . TherefoCompared with normal control M1 macrophages, the numbers of Tfh cells, memory activated CD4+ T cells, and plasma cells were significantly higher in RA synovial tissues, while those of regulatory T cells, activated dendritic cells, activated NK cells, memory resting CD4+ T cells, resting mast cells, and activated mast cells were significantly lower in RA synovial tissues. Macrophages can be polarized into M1 and M2 macrophages under different conditions due to their high degree of heterogeneity and plasticity. In the synovial environment of RA, macrophages mainly differentiate into type M1, which plays a pro-inflammatory role . The patTo improve the accuracy of the results, the DEGs were screened by two independent methods. Further analysis indicated that the function of these DEGs were significantly associated with immune cells. Finally, five hub genes  were screened, which were further verified by the validation dataset (GSE89408). Compared with osteoarthritis, all the five hub genes were also significantly higher expressed in RA, which made our result more convincing. Surprisingly, the expression levels of GZMA and CD8A were significantly higher in early RA than in established RA. Therefore, GZMA and CD8A might play an important role in the pathogenesis of early RA, while CXCR4, CCL5, and CD247 might play a vital role in the overall pathogenesis of RA. ROC regression analysis further found that CCL5+CXCR4 and GZMA+CD8A had the capability to diagnose RA and early RA with excellent specificity and sensitivity, respectively.GZMA is a member of the granzyme family and is mainly secreted by cytotoxic cells . In addiGiven the important role of immune infiltrating cells and hub genes in RA, the correlation among four effective biomarkers  and the top 10 significantly differential immune cells was further investigated in RA. CCL5 was positively correlated with M1 macrophages , and CXCR4 was positively correlated with memory activated CD4+ T cells  and Tfh cells . Previous research revealed that CCL5 could directly activate M1 polarization, and CXCR4 had the ability to active memory activated CD4+ T cells and Tfh cells –53. Therin vivo and in vitro are needed.This research has some limitations. First, the study lacks clinically relevant information, including the activity of the disease, and the use of drugs for the disease. Second, this study was conducted only from the perspective of gene transcriptome and lacks multi-group trials. Last, only bioinformatics methods were used for data analysis, and subsequent confirmatory experiments In summary, our study not only offers insights into the landscape of immune cells associated with RA but also identifies effective diagnostic biomarkers for RA. GZMA-Tfh cells, CCL5-M1 macrophages, and CXCR4- memory activated CD4+ T cells/Tfh cells may participate in the occurrence and development of RA; in particular, GZMA-Tfh cells may be involved in the early pathogenesis of RA. Therefore, this study may provide a new perspective for the diagnosis and immune cellular-molecular mechanism of RA.http://www.ncbi.nlm.nih.gov/geo) database.Publicly available datasets  were analyzed in this study. All the datasets were obtained from the GEO (SZ analyzed and wrote the manuscript. HL designed the experiments and analyzed the data. MX devised the concept and supervised 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.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.


Subcutaneous fat and intramuscular fat (IMF) deposition are closely related to meat production and pork quality. Dingyuan pig, as a native pig breed in China, low selection leads to obvious genetic and phenotypic differences in the population. Individuals with extreme fat content in the population are ideal models for studying the mechanism of fat deposition. In this study, we used RNA-Seq and tandem mass tags-based (TMT) proteomics to analyze the key pathways and genes that specifically regulate subcutaneous fat and IMF deposition in Dingyuan pigs. We identified 191 differentially expressed genes (DEGs) and 61 differentially abundant proteins (DAPs) in the high and low back fat thickness  groups, 85 DEGs and 12 DAPs were obtained in the high and low intramuscular fat  groups. The functional analysis showed that the DEGs and DAPs in the backfat groups were mainly involved in carbohydrates, amino acids, and fatty acids metabolism, whereas the IMF groups were involved in the insulin pathway, longevity, and some disease-related pathways. We found 40 candidate genes that might tissue-specifically lipids deposition for subcutaneous and intramuscular fat. Our research provides theoretical reference materials for the improvement of fat deposition traits of local pig breeds in my country. Commercial pig breeds increase the proportion of lean meat and reduce the deposition of subcutaneous fat, while the reduced intramuscular fat content at the same time results in poor flavor5. Porcine subcutaneous fat is quantified by backfat thickness, which is negatively correlated with lean meat percentage and positively correlated with intramuscular fat6. The IMF content plays a decisive role in the quality and flavor of pork7. Due to the positive genetic correlation between the two fat traits8, increasing the intramuscular fat content while reducing the deposition of subcutaneous fat has always been a challenge in pig breeding.China's pig breeds are rich in genetic resources and have good meat quality. However, they also suffer from problems such as a slow growth rate, high fat deposits, and low lean meat percentagesThe Dingyuan pig is an indigenous pig breed distributed in Dingyuan County, Anhui Province, China. It has the advantages of high intramuscular fat, and good meat quality, but also has a low lean meat percentage and relatively slow growth rate. A large population of Dingyuan pigs was phenotyped using multi-production trait indexes, and found that the growth rate and IMF content of some individuals were similar to those of others, but their subcutaneous fat was thinner. Therefore, we believe that this population may harbor key genes that specifically regulate subcutaneous and intramuscular fat deposition, providing a good study system to investigate the molecular mechanisms regulating tissue-level differences in fat deposition.9. Sequencing of subcutaneous fat and IMF of 100 pigs identified 215 subcutaneous fat genes and 90 IMF genes10. The mechanism of subcutaneous fat and IMF deposition has not only been studied in different breeds of pigs, but also in Italian Great White pigs or Duroc pigs11 of the same breed with different phenotypes6. Dingyuan pig is an excellent indigenous pig breed, but there is no relevant report yet.RNA-Seq has been widely used to analyze the transcriptome profile of pig subcutaneous and intramuscular fat. Jinhua pig and Landrace pig intramuscular fat transcriptome sequencing revealed the differences in fat and protein metabolism at the transcriptional level of the two pigslongissimus dorsi (LD) muscle from pigs of similar age and body weight with divergent backfat thickness and intramuscular fat content. We used RNA-Seq to study gene expression in subcutaneous and intramuscular fat simultaneously. We also used proteome analysis technology based on TMT because it has improved proteome coverage and more reliable peptide identification and quantification, which is suitable for analyzing thousands of proteins in complex biological samples12. Such integrated transcriptome and proteome analyses can provide insights into key functional genes and their regulatory mechanisms between different types of fat deposits in pigs.In this study, we collected tissues of subcutaneous fat and the P = 0.001). However, the body weight, intramuscular fat content, and lean meat percentage were not significantly different between the two groups. The IMF content was significantly higher in the HIMF group than in the LIMF group (P = 0.006).The phenotypic traits of the pigs used in this study are listed in Table The total number of raw reads obtained by sequencing the 12 samples was 125.21 Gb and the number of clean reads was 120.87 Gb, and the average ratio of clean reads was 96.11%  analysis, we compared the expression patterns of protein-coding genes in different samples. A total of 17,302 genes (FPKM > 0.05) were obtained in the LD muscle and back subcutaneous fat tissue of Dingyuan pigs, and 13,081 genes were commonly expressed in the four groups , protein kinase AMP-activated non-catalytic subunit gamma 3 (PRKAG3), and solute carrier family 7 member 5 (SLC7A5).To identify the genes that specifically regulate subcutaneous and intramuscular fat deposition, we compared the DEGs from the two tissues and found 3 overlapping DEGs—keratin 80 , amino acid metabolism , propanoate metabolism (ECHDC1 and ACSS3), and carbon metabolism (ALDH1L1 and ALDH6A1), as well as 11 genes  associated with adipocytes differentiation, butanoate metabolism, the PPAR signaling pathway, regulation of gluconeogenesis, and glucose metabolism , the transcription factor PPARA , the adipocyte secretory product LEP (leptin), genes related to fatty acid β-oxidation, the lipid metabolism-related gene RETREG1 (reticulophagy regulator 1), GK (glycerol kinase), BDH1 (3-hydroxybutyrate dehydrogenase 1), SMPDL3A (sphingomyelin phosphodiesterase acid like 3A), and PRKAG2 . The expression levels of these candidate genes in the two tissues are shown in Fig. In the LD muscle, following GO and KEGG analysis, we selected 8 genes mainly involved in fat metabolism-related pathways as candidate genes for the specific regulation of intramuscular fat deposition. These included the fat synthesis gene A total of 29,519 peptides were obtained from the MS analysis of the subcutaneous fat tissues, and 4,099 proteins were identified. In contrast, 14,619 peptides and 2,178 proteins were obtained from the LD muscle. Among the identified proteins, 78.3% of those in the subcutaneous fat tissue . Moreover, all 8 genes showed the same expression trends in terms of mRNA and protein. The PRG4 gene was highly expressed in the HBF group, whereas the remaining 7 genes were low in HBF. Based on the DEGs and DAPs, a protein–protein interaction (PPI) network was established for the Sus scrofa database using the STRING v.10.0 online software —was found. This gene is mainly involved in the processes of myofibril assembly and muscle structure development. The PPI network diagram shows that the DEGs and DAPs in the LD muscle group were mainly centered on the INS  to test the validity of the RNA-Seq and TMT-based proteomic results. For this, we performed RT-qPCR using RNA samples from fat and muscle tissues of Dingyuan pigs. The expression levels of these genes in each group are shown in Supplementary Fig. We selected ten DEGs , propionate metabolism , ketone body metabolism (HMGCL), and the citric acid cycle (IDH3A); the genes coding these proteins may be candidate genes for regulating subcutaneous fat in pigs. Our transcriptome proteome had very few overlapping genes, and the results indicate that there is post-transcriptional regulation of fat deposition in Dingyuan pigs. In addition, due to the limitations of sequencing technology, the number of proteins identified by the proteome was far lower than that in the transcriptome. Moreover, the number of proteins detected in the two tissues was very different, and many proteins were not detected. Therefore, we believe that DEGs that exist only in the transcriptome can also be used as important candidate genes.Among the 8 genes identified in the subcutaneous fat group, PRKAG3 was the only DEG found to participate in fat metabolism in the transcriptome of both tissues. The PRKAG3 gene encodes the AMPK regulatory subunit γ327. AMPK activity is associated with increased glucose uptake and fatty acid oxidation, as well as inhibition of glycogen synthase activity and fatty acid synthesis28. Two missense mutations in the pig PRKAG3 gene—A595G  and G154A (Gly52Ser)—are related to the IMF content, water-holding, and meat quality of pigs29, and the AMPKγ3 (R200Q) mutation results in a lack of AMP dependence and elevated basal activity of AMPK28. The current research on pig PRKAG3 is focused on IMF. Based on the FPKM value of the transcriptome, we found that PRKAG3 expression is high in muscle tissue and very low in fat tissue; therefore, the role of this gene in pig backfat deposition remains to be verified.In our study, 31. Fatty acid synthase (FASN) is a key enzyme in the de novo fatty acid synthesis pathway and cell substrate energy metabolism32. Acetoacetyl-CoA synthetase (AACS) is an enzyme that provides acetyl units for the biosynthesis of cholesterol and fatty acids33. AACS gene expression in white adipose tissue is lower in Zucker fatty rats than in lean rats; however, in high-fat diet induced obese rats, the expression of this gene is increased34. ALDH1L1 and ALDH6A1 belong to the aldehyde dehydrogenases family. The ALDH6A1 gene is involved in the metabolism of a variety of aliphatic and aromatic aldehydes produced by various endogenous and exogenous precursors, and is also an important candidate gene that affects drip loss35. Moreover, ALDH1L1 is an enzyme that metabolizes folate36. At present, there are almost no reports on the relationship between these two genes and fat deposition. However, based on the various metabolic pathways they participate in and their differential expression in the HBF and LBF groups, we speculate that they may play a role in the subcutaneous fat deposition process in pigs.Our results showed that in the HBF and HIMF groups, most of the candidate genes that regulate the biological process of adipogenesis were downregulated. Lipoprotein lipase (LPL) is one of the key enzymes involved in lipid metabolism. It is the rate-limiting enzyme for the catabolism of triglycerides and can remove TG-rich, very low-density lipoproteins and chylomicrons from the bloodADHFE1 expression is highly enriched in adipose tissue and other highly metabolically active tissues, and its expression is closely related to the phenotype of mature adipocytes in vivo and in vitro37. The candidate genes related to subcutaneous adipogenesis that were upregulated in the HBF group included KLF2, ACP5, SERPINE1, PPARD, UBD, and UCP2. KLF2 inhibits fat formation by negatively regulating adipocyte differentiation, but does not affect the commitment of multipotent stem cells to the preadipocytic lineage38. ACP5, which is associated with excessive fat deposition and the development of atherosclerosis in pigs39. SERPINE1  is mainly produced in white adipose tissue and increases the cardiovascular risk in obese and diabetic patients, whereas its deficiency can prevent obesity and metabolic dysfunction41. The peroxisome proliferator-activated receptor delta gene (PPARD) is a key regulator of lipid metabolism, and its haplotype is associated with pig backfat thickness42. Studies in C2C12 myoblasts have shown that overexpression of PPARD inhibits myotube formation and enhances adipocyte differentiation43. In mice, knockout of the UBD gene prevents the development of age-related obesity while prolonging lifespan and vitality44. UCP2 participates in the oxidation of mitochondrial substrates and plays an important role in metabolic pathways46. Studies have reported that some SNPs in UCP2 are related to obesity and type 2 diabetes49. Among these differential genes, genes that are positively related to adipogenesis were down-regulated , and some were upregulated , indicating that fat metabolism in the body is a dynamic and balanced process.RETREG1, also known as pFAM134B, promotes the accumulation of subcutaneous fat in pigs by increasing the mRNA levels of PPARγ, FAS, and ACC, and reducing the levels of ATGL and HSL50. PRKAG2 is an important regulator of cardiac metabolism51; an SNP of PRKAG2 is associated with insulin sensitivity and is essential for obesity, triglycerides, and HDL cholesterol52. SMPDL3A—a recently identified phosphodiesterase—plays a role in cholesterol and fat metabolism by regulating the liver X-receptor53. The insulin receptor substrate 2 (IRS2) is the first downstream effector of the insulin receptor. Studies have found that the absence of IRS2 in myeloid cells improves glucose homeostasis and enhances resistance to metabolic dysfunction induced by a high-fat diet54. In addition, the proteins LEP55, GK56, and PPARA57—reported to be associated with obesity or adipose-related biological processes—were also included in our results.In the transcriptome of the IMF group, we found many enriched pathways related to lipid metabolism. Based on these pathways, we identified genes that may regulate intramuscular fat deposition. UBD expression was upregulated in our HBF transcriptome and in the proteome of the HIMF group. Studies have shown that UBD has a positive regulatory effect on intramuscular and subcutaneous fat, and that inhibition of UBD in intramuscular and subcutaneous adipocytes results in the inhibition of cell proliferation and lipid droplet production58.The purpose of our experiment was to identify genes that specifically regulate subcutaneous and intramuscular fat in pig with extreme backfat and IMF. Interestingly, some of the DEGs and DAPs identified in the backfat groups in this study. For instance, RETREG1 gene was downregulated in the HIMF group. Although it has been suggested that this gene has an inhibitory effect on intramuscular fat deposition in Dingyuan pigs, studies have shown that it also promotes subcutaneous fat deposition. The opposing roles of genes in the two types of adipose tissue are beneficial for the selection of high-quality pigs, and their functions can be explored through further studies.From our results, it is worth mentioning that the All animal work was conducted following the guidelines for the care and use of experimental animals. The Animal Welfare Committee of the State Key Laboratory for Agro-Biotechnology of the China Agricultural University approved all procedures for animal care . The study was carried out in compliance with the ARRIVE guidelines.The Dingyuan pigs were raised in the Ankang Agriculture and Animal Husbandry Company, Dingyuan County, Anhui Province, with consistent feeding conditions. Over 200 unmated sows that were 10 months old and showed normal growth were selected and their body weight and live backfat thickness were measured using a platform scale and a B-ultrasound instrument . Based on the measurements, 12 individuals with similar body weights were selected such that six had extremely high live backfat thickness and six had extremely low live backfat thickness. The pigs were slaughtered to measure the carcass weight, backfat thickness and lean meat percentage. The LD muscle and back subcutaneous fat tissues were collected, immediately frozen in liquid nitrogen, and stored at − 80 °C.A fresh sample of the LD muscle tissue was cut into pieces, the connective tissue and fascia were removed, and the remaining tissue was homogenized. The LD muscle samples were then dried to a constant weight in an oven and the dried samples were ground into powder. The Soxhlet extraction method was used to determine IMF content in LD muscle tissue.Based on backfat thickness and IMF content, subcutaneous fat samples were divided into high backfat thickness  and low backfat thickness , and LD muscle samples were divided into the high intramuscular fat  and low intramuscular fat  groups. was used for transcriptome sequencing, and the sequencing read length was paired-end 150 bp. The obtained raw data were filtered to clean data with fastp (version 0.12.3) by removing reads containing adapters, low-quality reads , and reads with an N content > 5%. Clean reads were mapped to the reference pig (Sus scrofa) genome version scrofa.Sscrofa11.1.9959 (http://ftp.ensembl.org/pub/release-99/fasta/sus_scrofa/dna/Sus_scrofa.Sscrofa11.1.dna.toplevel.fa.gz) using the HISAT2 (V2.0.5) software for alignment. We used the Cufflinks (V2.2.1) software to calculate FPKM values to represent gene expression levels. The DESeq2 (V1.25.9) was used to analyze the differences between groups, and the P-values were adjusted using the Benjamini–Hochberg method. Differentially expressed genes (DEGs) were defined as those with false discovery rate (FDR) < 0.01 and |log2 fold change|≥ 1.Total RNA was extracted from the LD muscle and back subcutaneous fat tissues using the TRIzol  method. Isolated total RNA was quantified  and quality controlled with typical curves . The RNA integrity number (RIN) values of all samples were above 7, and 2.5 µg of total RNA was used to construct the cDNA library . The Illumina NovaSeq6000 platform The samples used for the proteomic analyses were the same as those used for RNA-Seq. An appropriate amount of sample tissue was added to the protein lysis solution . After sonication, the samples were placed on ice for 20 min, centrifuged at 10,000× g at 4 °C for 30 min, transferred to a new tube, and stored at − 80 °C. Protein concentrations of the samples were determined using a BCA kit (Thermo Fisher Scientific), and protein quality was detected by SDS-PAGE. The peptides were labeled with TMT isobaric tags (Thermo Fisher Scientific) at room temperature for 1 h.2O, pH10), loaded onto the mass spectrometer pre-column using the autosampler, and then separated by an analytical column. The separated samples were analyzed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific).The TMT‐labeled samples were fractionated using a high-pH peptide fractionation protocol. Each sample was fractionated into 60 fractions using a step gradient of 0–95% acetonitrile. Next, the 60 fractions were pooled into 10 fractions in a non-contiguous manner. Each sample was separated using a high-performance liquid chromatography (HPLC) system. The chromatographic column was balanced with 95% solution A  for peptide filtering. Quantification of experimental bias was set to normalize the total peptide amount. FC ≥ 1.2, FC ≤ 0.833, and P-value ≤ 0.05 were set as thresholds for identifying differentially abundant proteins (DAPs).After mass spectrometry analysis, the Proteome Discoverer 2.4 software (Thermo Fisher Scientific) was used to process all the original files against the Sus_scrofa.Sscrofa11.1.pep.all.fa database. The peptide confidence was set to a high level (https://kobas.cbi.pku.edu.cn/anno_iden.php). Functional analysis of DAPs was conducted using the UniProt ftp database and the KEGG PATHWAY database60. The Pathway Maps tool was used to enrich the pathways, and P-values were calculated using R software based on a hypergeometric distribution, with the default database being used as the background.GO and KEGG pathway enrichment analysis of the DEGs was performed using the KOBAS 3.0 enrichment analysis tool . Graphs were prepared using GraphPad Prism (version 7), and data are presented as the mean ± standard error.Supplementary Figures.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Table S8.


Although H–B grade improvement occurred in both groups, the degree of improvement was not significantly different between groups. (4) Conclusions: Facial nerve decompression surgery in severe Bell’s palsy patients did not significantly improve prognosis beyond that offered by conservative treatment alone. Additional surgical decompression may not be necessary in patients with severe Bell’s palsy if they receive sufficient conservative treatment.(1) Background: The effectiveness of decompression surgery for Bell’s palsy is controversial. This study investigated the effects of facial nerve decompression in patients with severe Bell’s palsy who were expected to have a poor prognosis. (2) Methods: We retrospectively reviewed 1721 patients with Bell’s palsy who visited the Kyung Hee University Hospital between January 2005 and December 2021. Of these, 45 patients with severe Bell’s palsy were divided into two groups; 30 patients were treated conservatively with steroids and antiviral agents alone, while 15 patients underwent additional decompressive surgery after the conservative treatment. Outcomes were measured using House–Brackmann (H–B) grade for least 6 months after treatment was finished and conducted until full recovery was achieved. (3) Results: There was no significant difference in the rate of favorable recovery (H–B grade 1 or 2) between the surgery group and the conservative treatment group (75% vs. 70.0%,  Bell’s palsy is an acute, unilateral, peripheral facial nerve paresis or paralysis of unknown cause. It has an annual incidence of 20–30 per 100,000 population . Facial There is strong evidence supporting the use of steroids as the initial medical treatment in Bell’s palsy to reduce facial nerve inflammation ,7. TreatElectroneuronography (ENoG) and electromyography (EMG) are objective electrical tests that are used to estimate the degree of paralysis and prognosis in Bell’s palsy patients. Patients with Bell’s palsy are at a high risk of poor recovery if they demonstrate the following clinical findings: a complete lack of facial movement on clinical examination; ENoG findings of >90% degeneration; and EMG findings of no voluntary motor unit potentials ,10,11,12For patients at risk of poor recovery, surgical decompression of the facial nerve has been proposed as an additional treatment option to release nerve entrapment in the facial canal and improve outcomes ,14,15. HThe purpose of this study was to investigate the effects of surgical decompression in patients with severe Bell’s palsy who were expected to have a poor prognosis based on clinical and electrodiagnostic tests. This study also uses the House–Brackmann (H–B) grade to compare the outcomes of decompression surgery with those of conservative treatment alone.We conducted a retrospective case review of 1721 patients with Bell’s palsy who visited the outpatient clinic in the Department of Otolaryngology at Kyung Hee University Hospital between January 2005 and December 2018. A total of 775 patients, who were treated with high-dose steroids and antiviral agents within one week of diagnosis, were enrolled. The steroid treatment schedule consisted of prednisolone 80 mg/day for the first 4 days, 60 mg/day for 2 days, 40 mg/day for 2 days, 20 mg/day for 2 days, and 10 mg/day for 2 days. Patients were also treated with antiviral agents, including 1000–2400 mg/day acyclovir for 5 days, or 750 mg/day famciclovir for 7 days.Blood samples were obtained from all of the patients within 5 days after the onset of facial paralysis. Complete blood count (CBC), differential count (DC), and HbA1c were measured. The neutrophil to lymphocyte ratio (NLR) was defined as the absolute neutrophil count divided by the absolute lymphocyte count. The platelet-to-lymphocyte ratio (PLR) was defined as the platelet count divided by the absolute lymphocyte count. The upper limit of NLR for adults was set to 3.53 based on previous studies ,19. The Out of the 775 patients with Bell’s palsy treated with high-dose steroids and antiviral agents, 45 met the following inclusion criteria: (1) adult >16 years of age; (2) severe facial palsy on early examination (based on an H–B grade of 5 or 6); (3) degree of denervation >90% on electroneurography (ENoG) performed between 5 and 14 days from the onset of facial weakness; (4) no voluntary electromyographic (EMG) compound motor action potentials (CMAP) performed 14 days or later after the onset of facial palsy; and (5) follow-up for at least 6 months after onset. Patients with recurrent facial palsy, traumatic facial palsy, suspected Ramsay–Hunt syndrome, previous history of otologic surgery, or those who were pregnant/breastfeeding were excluded.Ultimately, 45 patients with severe Bell’s palsy were divided into two groups according to the treatment regimen. The conservative treatment group included 30 patients who were treated with prednisolone and antiviral agents. The surgery group included 15 patients who underwent nerve decompression surgery.In the surgery group, transmastoid decompression was performed 21–70 days after the symptom onset of Bell’s palsy by a single experienced surgeon. The facial nerve was decompressed from the distal portion of the labyrinthine segment to the stylomastoid foramen using the transmastoid approach. The incus was temporarily removed to obtain wide access to the geniculate ganglion. The geniculate ganglion and the distal part of the labyrinthine segment were exposed by removing the supralabyrinthine cells anterior to the bony superior semicircular canal. More than 50% of the surface of the facial canal could be exposed. Finally, the incus, which had been temporarily removed, was returned to its original position.The grade of facial palsy was followed using the H–B grade for at least 6 months until full recovery was achieved. A favorable recovery was defined by a final H–B grade of 1 or 2. An H–B grade ≥3 was defined as an unfavorable recovery. The study protocol was approved by the Institutional Review Board of Kyung Hee University Hospital. Informed consent was not required, as this was retrospective study (IRB No 2019-07-065).p-value was < 0.05. All values were presented as means ± standard deviations, or as frequencies and percentages.All statistical analyses were performed using IBM SPSS version 20.0 . Categorical variables were analyzed using the chi-square test and Fisher’s exact test. Continuous variables were compared using the independent t-test or Mann–Whitney U-test, as appropriate. Univariate analysis was performed to assess whether the treatment methods and comorbidities influenced the outcome in severe Bell’s palsy patients. Statistical significance was considered when the p < 0.001), ENoG score , white blood cell (WBC) count , neutrophil percentage , lymphocyte percentage , NLR , and the percentage with a favorable recovery (90.7% (524/54) vs. 75.8% (147/47), p < 0.001). In contrast, there were no differences between the groups with regard to age, BMI, or HbA1c. The prevalence of patients with hypertension and diabetes mellitus also did not differ significantly across the groups  had mild to moderate palsy , and 197 (25.1%) had severe palsy . These two groups differed significantly in the final H–B grade , but the difference was not statistically significant.p = 0.633), those with and without hypertension , with and without diabetes mellitus , with normal weight or obesity , or with normal or high NLR . The presence of each risk factor tended to show less improvement in the H–B grade, but this increase was not statistically significant   .We performed univariate analysis, using age, sex, HTN, DM, BMI, and blood test variables in addition to the treatment methods. None of these factors was statistically significant in predicting unfavorable recovery in patients with severe Bell’s palsy .In this study, we selected the patients with severe Bell’s palsy with poor prognosis based on their ENoG, EMG, and physical examination findings. We also evaluated the effect of surgical decompression of the facial nerve.Previous studies have revealed that most Bell’s palsy patients are not surgical candidates, because of the excellent overall rates of recovery. Spontaneous recovery ranges from approximately 70% with no treatment, to 94% with steroids. However, long-term sequelae, including facial paresis, paralysis, and synkinesis, are still significant consequences in a small portion of Bell’s palsy patients. Electrodiagnostic testing in patients with severe paralysis can identify a subset of patients who have an increased likelihood of poor prognosis ,21. ENoGIn an experimental animal model, improved nerve regeneration occurred in cats with facial nerve palsy when nerve decompression was performed within 12 days of the injury . The samThere have been multiple studies of surgical decompression of the facial nerve in Bell’s palsy. However, there is no consensus regarding its utility in this clinical setting, especially with regard to surgical indications, the optimal timing of surgery and the appropriate surgical approach. In general, surgical decompression is performed in patients with complete paralysis after medical treatment has failed. Still, the optimal timing of surgery remains unclear.Some studies have reported favorable outcomes after delayed surgical decompression in severe Bell’s palsy. Bodenez et al., described favorable outcomes after delayed decompression surgery (30–120 days after disease onset) in 13 patients with severe facial palsy . AnotherAlthough we found that surgical decompression (performed 21–70 days after symptom onset) in severe Bell’s palsy improved functional outcomes, the functional improvement was not statistically different from that of the control group at the 6-month follow-up. Contrary to our findings, Yanagihara et al., found that delayed transmastoid decompression performed 15–120 days after symptom onset may be more beneficial than steroid treatment alone in patients with severe Bell’s palsy . There aSome studies have suggested that decompression surgery results in better facial nerve outcomes if it is performed within 14 days of symptom onset than it does if performed after 14 days. Gantz et al., conducted a multi-centered, prospective, case-control study that enrolled 70 patients treated for severe Bell’s palsy. The results show that 31 of 34 (91%) patients who underwent middle fossa decompression within 14 days of disease onset achieved H–B grades 1 or 2 compared with only 15 of 36 (42%) patients treated with steroids alone . DespiteThe optimal approach of decompression surgery for Bell’s palsy is still debated. Several studies recommend the middle fossa approach to reach the bottle neck of the fallopian canal from the internal auditory canal . One preAlthough the use of surgical facial nerve decompression is still a topic of debate. it is used in patients of complete paralysis with evidence of extensive neurodegeneration who, on nerve conduction test, show conduction velocities on the lesion side that are less than 10% of those on the unaffected side. When the voluntary motor unit potential is not observed in the needle electromyography test, a therapeutic effect can be expected if the surgical facial neve decompression is performed early.Patients who underwent decompression surgery 21–70 days after symptom onset of severe Bell’s palsy did not show better prognosis than did those who were treated with conservative treatment alone. These findings are consistent with the previous guideline and systematic reviews, which have not recommended surgical decompression for patients with Bell’s palsy . Our res


Bifidobacterium breve, Saccharomyces boulardii, Lactobacillus acidophilus, L. rhamnosus, and amylase, was developed to rebalance the bacterial–fungal gut microbiome, with the goal of reducing inflammation and maintaining a healthy gut population. To test the effect of BIOHM on human subjects, we enrolled a cohort of 49 volunteers in collaboration with the Fermentation Festival group . The profiles of gut bacterial and fungal communities were assessed via stool samples collected at baseline and following 4 weeks of once-a-day BIOHM consumption. Mycobiome analysis following probiotic consumption revealed an increase in Ascomycota levels in enrolled individuals and a reduction in Zygomycota levels . No statistically significant difference in Basidiomycota was detected between pre- and post-BIOHM samples and control abundance profiles (p > 0.05). BIOHM consumption led to a significant reduction in the abundance of Candida genus in tested subjects , while the abundance of C. albicans also trended lower than before BIOHM use, albeit not reaching statistical significance. A reduction in the abundance of Firmicutes at the phylum level was observed following BIOHM use, which approached levels reported for control individuals reported in the Human Microbiome Project data. The preliminary results from this clinical study suggest that BIOHM is capable of significantly rebalancing the bacteriome and mycobiome in the gut of healthy individuals, suggesting that further trials examining the utility of the BIOHM probiotic in individuals with gastrointestinal symptoms, where dysbiosis is considered a source driving pathogenesis, are warranted.Gastrointestinal microbiome dysbiosis may result in harmful effects on the host, including those caused by inflammatory bowel diseases (IBD). The novel probiotic BIOHM, consisting of  Escherichia coli and Serratia marcescens, and the fungus, Candida tropicalis, demonstrated increased abundance in the GI tract of CD patients when compared with their non-Crohn healthy relatives [C. tropicalis and the two bacterial species cooperate in a strategic way to form in vitro pathogenic biofilms capable of causing damage to the epithelial cell lining of the gut and initiating an inflammatory response [Human gastrointestinal (GI) microbiome research has primarily focused on resident bacteria and their associated bacterial–host interactions, both beneficial and detrimental. However, solely focusing on bacteria has neglected the potential influence of the host’s fungal community (mycobiome) on health and disease. In a previous study, we characterized the gut bacterial microbiota (bacteriome) and the mycobiome in family members with Crohn’s disease (CD) and their healthy relatives in an attempt to define the interactions leading to dysbiosis in CD. We identified a positive correlation between bacteria and fungi, wherein the bacteria, elatives . Subsequresponse . Not onlOne approach to combat IBD symptoms by preventing and treating microbiome dysbiosis includes the use of probiotics, which the World Health Organization (WHO) has defined as live microorganisms that confer health benefits on the host when administered in adequate amounts . The impCandida vaginitis [Since the cooperative interaction of fungi and bacteria in the dysbiotic state has been shown to produce harmful effects on the host, it is logical to suggest that the introduction of different combinations of microbes in the form of probiotics to restore overall balance may help to counteract these detrimental effects. Probiotics have been shown to be effective in preventing and ameliorating various medical conditions, particularly those involving the GI tract in children. Recently, certain probiotic bacteria have been studied as a potential method to prevent opportunistic infectious diseases by stimulating the host immune system ,7,8. Preaginitis  and vulvaginitis ,11, oralaginitis , GI infeaginitis , colon caginitis , and recaginitis ,20,21,22Bifidobacterium breve 19bx, Saccharomyces boulardii 16mxg, Lactobacillus acidophilus 16axg, and L. rhamnosus 18fx, combined with the enzyme amylase based on its anti-biofilm activity [Since it has been demonstrated that microbial dysbiosis is implicated in GI diseases such as IBD, ulcerative colitis, and CD, developing probiotics that can rebalance and maintain the gut microbiota is a reasonable approach to counteract the effect of dysbiosis. The development of the BIOHM probiotic was guided by microbiome analysis based on a large cohort of individuals who were analyzed through the BIOHM gut testing platform to design a probiotic that would affect organisms increased in individuals with intestinal dysbiosis. Our aim was to select appropriate microbes that target pathogenic bacterial and fungal strains while supporting beneficial ones. To achieve this, we conducted correlation analyses of bacterial–bacterial and bacterial–fungal interactions to identify appropriate probiotic strains. This work led to the development of a new probiotic, BIOHM, consisting of activity ,24,25.In order to determine the effect of BIOHM on the comprehensive intestinal microbiome  of human subjects, in this study, we enrolled a cohort of 49 volunteers in collaboration with the Fermentation Festival group . The CIM profiles of bacterial and fungal communities were assessed at baseline and following 4 weeks of BIOHM use. We then compared the bacteriome of our subjects with those reported by the Human Microbiome Project (HMP) for healthy subjects as a control for bacterial abundance. For fungal controls, we used cumulative fungal abundance data generated through the BIOHM Gut Test data repository of healthy individuals.Candida (Lactobacillus rhamnosus 18fx (2.38 × 1010 CFU/g), Saccharomyces boulardii 16mxg (5.6 × 109 CFU/g), and Lactobacillus acidophilus 16axg (2.38 × 1010 CFU/g)), as well as a bacterium that antagonizes both S. marcescens and E. coli (Bifidobacterium breve 19bx (2.38 × 109 CFU/g)) [Appropriate probiotic strain selection is critical to the probiotic design process. To select optimal probiotic strains that antagonize (inhibit the growth of) harmful microorganisms while supporting beneficial ones, we conducted correlation analyses of bacterial–bacterial and bacterial–fungal interactions. Based on our results, we identified individual bacterial and yeast strains that antagonize  CFU/g)) .Based on our data, which showed that fungi and bacteria cooperate in strategic ways to form pathogenic, inflammation-inducing biofilms, we included the enzyme amylase in our formulation, which has been shown to inhibit biofilms and can be safely incorporated into a probiotic mixture .S. boulardii and L. rhamnosus can survive at a pH of 1.5, while L. acidophilus and B. breve are able to survive the acidified stomach environment if ingested within 30 min of a meal [Prior to reaching the small intestine, probiotics must first pass through the harsh acidic environment of the stomach. The pH of the stomach can increase to a range of 4.0–6.0 after ingestion of a meal but normally returns to the baseline acidic range of 1.5–3.5 within approximately 2 h . It has f a meal .n = 49) who signed informed consent at baseline and following 4 weeks of once-a-day BIOHM consumption, these individuals are represented as “before” and “after” in all figures. In addition, a “normal” population was generated by comparing the bacteriome of our subjects to those reported by the Human Microbiome Project (HMP) for healthy subjects as a control for bacterial abundance (see below). For fungal “normal” controls, we used cumulative fungal abundance data generated through the BIOHM Gut Test data repository of healthy individuals.To evaluate the effect of BIOHM on the microbiome structure of healthy individuals, we collaborated with the slow-food movement Fermentation Festival, Santa Barbara group  to enroll in the present study . Fecal s2, as well as subjects on medications . This resulted in selecting 950 individuals considered healthy , with a BMI of 18.6–34.9 kg/m2, in our analysis.To select the healthy normal subjects, we followed the inclusion and exclusion guidelines of the Human Microbiome Project . SpecifiFecal samples were analyzed for their bacterial and fungal communities using Ion Torrent sequencing technology. Samples were transferred to tubes containing glass beads with the lysis solution included in the QiaAmpFast DNA Extraction Kit . Bacterial and fungal DNAs were isolated and purified following the manufacturer’s instructions with minor modifications: In this regard, we incorporated an additional bead-beating step , with the MP FastPrep-24 speed setting of 6 M/s and 2 × 40 s cycles. The quality and purity of the isolated genomic DNA were confirmed using a NanoDrop 2000 . DNA concentration was quantified using a Qubit 2.0 instrument applying the Qubit dsDNA HS Assay  and adjusted to 100 ng per sample. Extracted DNA samples were stored at −20 °C.For bacteria, the V3-V4 region of the 16S rRNA gene was amplified using 16S-515F: GTGCCAGCMGCCGCGGTAA and 16s-806R: GGACTACHVGGGTWTCTAAT primers, while the fungal ITS region was amplified using ITS1 (CTTGGTCATTTAGAGGAAGTAA) and ITS 2 (GCTGCGTTCTTCATCGATGC) primers. The reactions were carried out on a 100 ng template DNA, in a 50 µL  reaction mixture consisting of Q5 PCR Master Mix , for a final primer concentration of 400 nM. Initial denaturation at 94 °C for 3 min was followed by 30 cycles of denaturation for 30 s each at 94 °C, annealing at 57 °C (16 s) or 59 °C (ITS) for 30 s, and extension at 72 °C for 10 s. Following the 30-cycle amplification, there was a final extension time of 15 s at 72 °C. The size and quality of amplicons were screened on a 1.5% TAE agarose gel, separated using 100v, and electrophoresed for 45 min then stained with ethidium bromide. The PCR products were sheared for 20 min, using Ion Shear Plus Fragment Library Kit . The amplicon library was generated with sheared PCR products using Ion Plus Fragment Library Kit (<350 bp) according to the manufacturer’s instructions. The library was barcoded with Ion Xpress™ Barcode Adapter and ligated with the A and P1 adaptors.The adapted barcoded libraries were concentrated 4–6× in a speed-vac  and the concentrated pooled libraries were then quantified using a TaqMan Quantitation Kit . The libraries were adjusted to 100 pM and attached to the surface of Ion Sphere particles (ISPs) using an Ion PGM Template OT2 400 bp Hi-Q View Kit  according to the manufacturer’s instructions, via emulsion PCR. The quality of ISP templates was checked using Ion Sphere™ Quality Control Kit (Part no. 4468656) with the Qubit 2.0 device. Sequencing of the pooled libraries was carried out on an Ion Torrent PGM System using the Ion Sequencing 400 bp Hi-Q View Kit  for 150 cycles (600 flows) with a 318 v2 chip, following the manufacturer’s instructions. De-multiplexing and classification were performed using the Qiime Platform (ver. 1.8). The resulting sequence data were trimmed to remove adapters, barcodes, and primers during the de-multiplexing process. In addition, the sequence data were filtered for the removal of low-quality reads below the Q25 Phred score and de-noised to exclude sequences with a read length below 100 bp . De novot-test for a given genus, species, or phylum. A p value < 0.05 was considered significant.Pre- and post-BIOHM consumption data were analyzed for each sample. Statistical significance levels were calculated, comparing the changes across groups by p value < 0.01) with a subsequent decrease in phylum abundance also matched healthy control profiles. No statistically significant difference in Basidiomycota was detected between pre- and post-BIOHM samples and control abundance profiles (p > 0.05).Mycobiome analysis following probiotic consumption (“after”) showed an increase in Ascomycota levels in enrolled individuals, and the abundance of this phylum increased to levels observed in healthy control profiles, a reduction in Zygomycota levels , while the abundance of C. albicans also tended to be lower than before BIOHM use, albeit not reaching statistical significance, compared with healthy control profiles . Subjects in the enrolled cohort had significantly higher phylum levels of Proteobacteria (known to be a red flag for inflammation) at baseline, compared with the HMP healthy control values . The phyla Actinobacteria, Tenericutes, and Verrucomicrobia were detected at low abundance in all subjects irrespective of the time of collection relative to BIOHM use  disease, acute diarrhea in adults and children induced by a variety of enteric pathogens, traveler’s diarrhea, and relapses of CD or UC. Benefits of S. boulardii are believed to be related to direct enzymatic effects, modulation of the gut endogenous flora, and enhancement of the immune response. Samonis et al. evaluated the virulence of S. boulardii when used as a probiotic, and its role in preventing GI colonization by Candida in a murine model [ne model . They shLactobacillus spp., Bifidobacterium spp., and S. boulardii have shown efficacy against intestinal disorders, especially if treatment is introduced early. Orally administered L. acidophilus and L. rhamnosus (as cheese ingredients) have also been shown to reduce oral Candida colonization in denture wearers [ wearers .An in vitro study by Ribeiro et al. showed that both cells and supernatant of L. rhamnosus reduced C. albicans biofilm formation, filamentation, gene expression of adhesins (ALS3 and HWP1), and transcriptional regulatory genes (BCR1 and CPH1) [L. paracasei, L. rhamnosus, and L. fermentum were used in a Galleria mellonella larvae model to evaluate whether clinical isolates of Lactobacillus spp. are able to provide protection against C. albicans infection [L. paracasei strain 28.4 had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans, demonstrating that Lactobacillus can modulate the immune system.nd CPH1) . Furthernfection . Their dThus, a probiotic that will restore fungal and bacterial balance in the gut should be of enormous benefit to individuals suffering from IBD, as well as to the health of the general population. The ability of BIOHM to reduce polymicrobial biofilm formation may be an outcome of particular importance considering the pathogenesis associated with biofilms and the refractory nature of organisms incorporated in biofilms to traditional therapeutics . The abiCandida suggests that this probiotic should be further examined using expanded clinical trials including IBD patients, where we know imbalance in polymicrobial interactions is a key to dysbiosis and pathogenesis [Our preliminary results show that BIOHM consumption results in the regulation of both bacterial and fungal abundance in the gut within 4 weeks of daily consumption. Importantly, the ability to significantly decrease the pathogenic genus ogenesis .Limitations of the current study include the modest number of participants in the study as well as the lack of matched controls, although each participant did serve as their own control at baseline. Further limits include subject demographics and knowledge regarding potential dietary differences or the use of other potential probiotic regimens prior to participation in the current study. A more longitudinal sampling approach in future studies would provide more insight regarding the natural variability of the microbiome and how it reacts to external factors, such as changes in diet or the intake of probiotics.Given our early success in demonstrating the ability of BIOHM to modulate the gut microbiome structure, more extensive placebo-controlled clinical trials are warranted to determine whether this novel probiotic could ameliorate or prevent symptoms in persons with IBD or gut dysbiosis. Further clinical implications regarding BIOHM consumption to consider are the face validity of being able to modulate both bacterial and fungal gut constituents. Modulation of the gut microbiome suggests that in addition to clinical approaches such as fecal microbiome transplant, it may be possible one day to tailor probiotics that would augment host microbial composition and may show efficacy as primary or adjuvant therapies for the treatment of diseases such as irritable bowel syndrome (IBS) or obesity. Indeed, the ability to modulate the microbiome through the rational design of probiotic would be useful in any number of clinical outcomes influenced by the gut microbiome, including potential immune modulation.


Temperature, motor Activity and body Position) in Sleep Disordered Breathing (SDB), its relation to SDB severity and the effect of continuous positive airway pressure (CPAP) on these circadian rhythms. To do this, we monitored motor activity and body position rhythms of 78 SDB patients  and 32 healthy subjects  for 1 week. On the last day of that week, SDB patients underwent a polysomnography followed by a Maintenance of Wakefulness Test, Multiple Sleep Latency Test and Sustained Attention to Response Task protocol. A subgroup of 18 moderate to severe SDB patients was treated with CPAP and monitored again after 3 months under treatment. A non-parametrical analysis was performed to characterize the circadian patterns to assess differences between groups and associations between sleep and circadian parameters. Circadian variables were altered in SDB, exhibiting a direct relationship to SDB severity. The motor activity pattern showed a clear improvement with CPAP treatment. Thus, circadian ambulatory monitoring, including the integrated variable TAP, could be used to evaluate the circadian alterations caused by SDB and activity pattern to monitor the effect of CPAP treatment.Our aim was to evaluate the circadian rhythm of motor activity, body position and integrated variable TAP (composed by wrist  Among these processes, the sleep–wake cycle is regulated by an homeostatic sleepiness accumulation, a circadian process and sleep inertia3.The circadian system is the main responsible for the temporal regulation of most physiological processes, including thermoregulation, metabolism or the sleep–wake cycle4. The chronodisruptive effect of SDB is supported by both the circadian system alteration at several levels, such as thermoregulation or the sleep–wake cycle itself8, and the circadian restoration of sleep, blood pressure, thermoregulation, immune response or haemostatic system by Continuous Positive Airway Pressure (CPAP)13. Also, this relationship seems bidirectional since the circadian system modulates the incidence and length of apnoea events14.The increasing prevalence of SDB through the world has become a major health concern, due to its multiple consequences including hypertension, diabetes, cardiovascular and cerebrovascular disease, cognitive impairment and even cancer15, used as choice method for circadian sleep disorders assessment16 and has been validated against polysomnography in SDB patients17. However, isolated marker rhythms are submitted to masking, thus multivariable recordings19 and variable integration have been developed15. In this sense, the integrated variable TAP  developed for sleep detection and successfully validated against polysomnography20, can improve the accuracy provided by a single marker rhythm21.The rest-activity marker rhythm is inherently associated to the sleep–wake cycleThus, the main purpose of this study was to evaluate the circadian rhythm of motor activity, body position and the integrated TAP variable in order to determine how these rhythms are affected by sleep disordered breathing severity and continuous positive airway pressure treatment, and in consequence Ambulatory Circadian Monitoring (ACM) could constitute a screening tool for this disorder as well as CPAP efficacy under free-living conditions.5 for clinical and polysomnographic characteristics). With regard to the 30 patients requiring CPAP, one refused to complete the protocol and 11 presented non valid recordings (removing the sensors or insufficient monitoring time); therefore, 18 moderate-to-severe SDB patients receiving CPAP were revaluated at least 6 weeks after the baseline study. PSG revealed a complete resolution of SDB in all patients and improved excessive daytime sleepiness measured by ESS, BSI sleepiness index, MSLT-SL, MWT-SL and MWT-E. However, CPAP treatment did not improve SART errors .From the consecutive SDB patients originally studied (n = 98), twenty were excluded due to the following reasons: irregular sleep–wake rhythms (n: 3), acute sleep deprivation prior to the sleep study (n: 1), REM sleep without atonia (n: 1), severe depressive symptoms (n: 1), migraines during the nap protocol (n: 1) and removing the sensors  either at home or during the PSG procedure (n: 13). Then, the group finally comprised 78 adults with a wide spectrum of disease  and healthy subjects (n: 32) are shown in Fig. The decision tree to discern between healthy subjects n: 32) and SDB patients (n: 78) selected WT stability (IS) with a maximum agreement rate (89.2%) at 0.53 . The ROC curve shows an area under the curve of 0.89 (p < 0.001) as shown in Fig.  and SDB 5.The SDB severity effect on the circadian rhythmic parameters Fig.  was addr5, the best WT parameter for SBD classification was also assessed. Thus, WT robustness, as measured by CFI, with a cut-off point of 0.44, yielded an agreement rate of 76.7%  and the ROC area under the curve was 0.76 (p < 0.01) as it is shown in Fig. The best criteria to discriminate, by means of a decision tree, from mild (n: 31) to severe (n: 28) SDB patients, was TAP robustness (CFI) with a cut-off point of 0.62 yielding an agreement rate of 83.3% . The ROC for TAP robustness Fig.  reached Regression analysis was performed to determine the association between sleep and circadian parameters (n: 78). It was controlled for age, sex and BMI. For motor activity (Supplementary Table The influence of CPAP treatment on circadian parameters was addressed by a Mixed Effects Model (n: 18). For the motor activity pattern, CPAP reduced fragmentation (IV), nocturnal activity L5) and increased day-night contrast (RA) as it is shown in Fig.  and incrFinally, the decision tree to discern between pre-treatment and CPAP treated severe patients (n: 15) selected a value of 10.4°/min for nocturnal activity (L5) with an agreement rate of 86.7% . The Fig. Our results highlight that SDB impairs rest-activity, body position and TAP variable circadian rhythms in accordance to SDB severity, whereas CPAP treatment improves motor activity pattern. Besides circadian parameters allow quantitative classification of populations providing a useful clinical screening tool for SDB and CPAP efficacy.20, ageing22 and cancer chemotherapy circadian impairment23. In the case of body position, SDB patients experienced a nocturnal phase advance, a characteristic associated with an aged circadian system25, and a more standing position during daytime, probably due to the less pronounced nap period in our sample.SDB patients showed lower stability and robustness for motor activity and the integrated variable TAP than healthy subjects, conditions related to other sleep disorders26 and the subsequent excessive daytime sleepiness5 which can contribute to frequent and unexpected or arrhythmic WT fluctuations.The above-mentioned differences between healthy and SDB subjects´ circadian rhythms can be used as a screening tool for SBD prone population. In a previous paper we showed that SDB patients exhibited lower wrist temperature stability values (IS) with respect to healthy subjects with an agreement rate of 89%, probably due to apnoea-dependent sympathetic activation at night27, seems to impair circadian indexes , reinforcing the hypothesis of circadian disruption caused by sleep apnoea5, as also occurs with other pathological conditions such as obesity, metabolic syndrome, diabetes, cardiovascular disease or, even, mortality risk31. Nocturnal values were increased for activity, body position and TAP variable as SDB severity progressed, which indicates a less deep sleep32, confirmed by the increase in N1 duration and the decrease of N3 and REM duration. Besides, and according to regression analyses, TAP stability, fragmentation, amplitude, robustness and nocturnal values impairment were related to higher daytime sleepiness as measured by MWT and MSLT as it was previously suggested for WT5, and could be a reflect of the sleep structure impairment15. Thus, these circadian alterations can be used as a screening procedure to detect individuals prone to suffer SDB before their definitive diagnosis in a sleep unit. Moreover, low TAP robustness, assessed by CFI, allowed distinction in SBD severity reaching an agreement rate of 83%. Given that the CFI variable can be used as an indicator of circadian system status15, SDB worsening could implicate a progressive deterioration of the circadian system.The severity of SDB, as measured by the Apnea–Hypopnea Index5 and sleep itself34, but also for other alterations such as core body temperature rhythm6, cognitive impairment4, metabolic syndrome35, fibrinolytic10 and inflammatory markers13, coagulation factors11, blood pressure12 or autonomic function36. Although the main limitation of this study is the absence of the individual adherence and compliance CPAP data, all monitored patients accomplished at least five hours per night during six consecutive weeks37 of CPAP treatment, an threshold value for an objective CPAP use that has demonstrated its efficacy for improving nocturnal sleep34. In this sense, the activity pattern a great improvement occurred, probably because CPAP treatment reduces nocturnal awakenings38 and, hence, would reduce nocturnal activity. Previous studies reported no effect of CPAP treatment40, probably due to differences in treatment compliance or monitoring device sensitivity. Activity nocturnal values diminution, and the subsequent increase in amplitude, were compatible with a less fragmented41 and deeper sleep due to the reduced number of arousals5, as also supported by lesser time in N1 and the AI and SDB indexes improvement. In accordance with the nocturnal decrease in activity level, L5 values can be used to assess CPAP efficacy with an agreement rate of 86%.The restorative effect of CPAP treatment has been thoroughly demonstrated for sleepinessIn summary, although WT robustness allow us to discern between SDB patients and healthy subjects, when motor activity, body position and TAP robustness are considered reliability for discriminating between mild and severe SDB as well as CPAP usage increases with respect to wrist temperature alone .Considering jointly motor activity, body position and WT in the integrated variable TAP reveals that Sleep Disordered Breathing patients present circadian disruption, that increases with the severity of the disease and improves with Continuous Positive Airway Pressure treatment. Thus, TAP variable could constitute, a useful screening tool for SBD prevalence and severity as well as treatment efficacy adherence.5, we analysed the wrist temperature (WT) circadian pattern in the same group of subjects, using the information from the ACM devices. In the current study we will focus on the information provided by the circadian pattern of motor activity, body position and integrated variable TAP as well as data provided by the Maintenance of Wakefulness Test (MWT), and the Multiple Sleep Latency Test (MSLT).In a previous study5. The patients were informed about the objectives of the study, the willingness of their participation and that whatever their decision, it would not affect their treatment. Participants who accepted to participate in the study signed an informed consent. Exclusion criteria were being under 18 years of age, use of medications affecting wakefulness or sleep, shift work or irregular sleep–wake schedules during the four weeks before the sleep study, and major medical or psychiatric disorders. Patients with any concomitant sleep disorder other than SDB were excluded by nocturnal polysomnography (PSG).Ninety-eight consecutive patients with suspected SDB Table  were eva42.A total of 32 healthy subjects were recruited  at the Chronobiology Lab of the University of Murcia Table . They arThe study follows the bioethical principles set out by the Declaration of Helsinki. Data from the volunteers were protected according to Spanish Law 15/1999 from 13 September. The study was approved by the Hospital Clinic of Barcelona and the University of Murcia ethics committees for the SDB patients and healthy subjects, respectively.43, both test were performed at the end of the monitoring period, allowing at least 6 days of continuous recordings .Healthy volunteers were monitored for an entire week whereas SDB patients were monitored 6 days under free-living conditions and an additional day, the last one, under controlled conditions in hospital. During this week, the subjects were encouraged to maintain their habitual life style. Since PSG and nap-protocols could interfere with the normal sleep pattern of the patients32, placed on the wrist of the non-dominant hand over the radial artery as already described47, and isolated from the environment by a double-sided cotton sport wrist band32. In addition, every subject was monitored with a HOBO Pendant G Acceleration Data Logger UA- 004–64 actimeter  programmed every 30 s, positioned on the non-dominant arm by means of a sport band for motor activity and body position monitoring as already described45. Briefly, motor activity was measured as the rate of change in degrees per minute and position represents the angle between the X-axis of the accelerometer  and the horizontal plane. Data from activity and body position were averaged for 10-min intervals to facilitate WT comparisons. Wrist Temperature, motor Activity and body Position were combined in order to obtain the integrated variable TAP, according to the algorithms previously described15. In summary, and as previously described22, each variable was normalized between 0 and 1 and averaged to obtain the integrated variable (TAP). Thus, a TAP value of 1 indicates the lowest values of WT, the highest values of activity and a standing position (compatible with wake periods), whereas a score of 0 corresponds to the highest WT values, the lowest activity values and a horizontal position (compatible with sleep periods)15.For ambulatory circadian monitoring, all subjects wore a Thermochron iButton DS1921H  programmed to sample every 10 min over the whole week to measure WT5. After admission, the Epworth Sleepiness Scale (ESS) and Barcelona Sleepiness Index (BSI) were used to assess subjective daytime sleepiness48. Nocturnal PSG was performed according to standard practice parameters and diagnostic criteria50 with sleep stages independently and manually double scored according to the American Academy of Sleep Medicine (AASM) criteria, using 30 s epochs51. The nap protocol was initiated the morning after PSG in order to objectively measure daytime sleepiness throughout the day . In brief, we used the research version protocol comprising 5 sets of the Maintenance of Wakefulness Test (MWT), the Multiple Sleep Latency Test (MSLT) that started at 08:30 and every 2 h onwards52. Each nap set was preceded by a measurement of vigilance with Sustained Attention to Response Task (SART), with a duration of 4 min53 and using commission errors, missed errors and total errors54. Due to their skewed distribution, MWT and MSLT were log transformed previously to statistical analysis.On the last day of the week, patients with suspected SDB underwent a 24-h sleep study, including questionnaires and neurophysiological tests as previously described55. CPAP compliance was measured objectively using a built-in CPAP meter. For the analysis, five hours per night, measured by the CPAP, during six consecutive weeks were considered the strict minimum use of CPAP37.From the cohort of 78 patients, eighteen moderate or severe SDB patients with resistant hypertension or excessive daytime sleepiness were treated with CPAP and evaluated again following the same protocol . CPAP titration was performed following the recommendations of the Spanish Sleep Society5. The mean daily patterns for motor activity, body position and TAP variable were calculated per individual, and then averaged per group.The WT data were obtained, filtered and processed from data previously reported5. This analysis determines the following parameters: interdaily stability , intradaily variability , relative amplitude (RA) and circadian function index (CFI) calculated by the integration of IS, IV and RA oscillating between 0 (absence of circadian rhythmicity) and 1 (robust circadian rhythm)56. RA was calculated as the difference between M10  and L5  divided by the sum of M10 and L5 for variables with acrophase during daytime , as previously published56. The timing for L5 and M10 were used as nocturnal and diurnal phase markers .In order to characterize the aforementioned circadian rhythms a nonparametric analysis was performed as previously describedCircadian variables parameters in healthy subjects and SDB patients were compared using a General Linear Model controlled by age, gender and body mass index (BMI). Differences between snorers/mild SDB (AHI < 15 events/hours), moderate SDB (AHI between 15 and 30 events/hour) and severe SDB (AHI > 30 events/hour) were assessed by a General Linear Model controlled by age, gender and BMI (followed by post hoc pairwise comparisons and a Bonferroni test). In addition, a regression analysis was performed between sleep and circadian parameters controlling again for age, gender and BMI (with Bonferroni correction for multiple comparisons). A Mixed Effects Model was performed to determine the effect of CPAP treatment on SDB patients.5), activity, body position and TAP were entered in WEKA version 3.8.3 57. An independent classification was then performed to discern between i) healthy subjects and SDB patients, ii) mild and severe SDB patients and iii) pre-treatment and CPAP therapy groups by means of a J.4.8 decision tree that uses the C 4.5 algorithm for decision making58, which selects the decision that maximizes information gain at each step. In order to simplify the algorithm, all decision trees were required to use only one decision. Each individual decision tree corresponds to the best of 100 iterations performed with 66% of the data randomly selected and checked against the other 33%. Then, specificity (test’s ability to detect correctly (i) SDB patients, (ii) severe SDB patients and (iii) pre-treatment group), sensitivity (test’s ability to detect correctly (i) healthy subjects, (ii) mild SDB patients and (iii) CPAP therapy group), positive predictive value  SDB, (ii) severe SDB patients and (iii) pre-treatment patients), negative predictive value  healthy subjects, (ii) mild SDB patients and (iii) CPAP therapy group), agreement rate and ROC curve for every decision rule were calculated, which were compared by a Wilcoxon test. Data were processed using Microsoft Office Excel 2016, and all statistical analyses were performed using SPSS version 23.0 software . Values of p < 0.05 were considered to be statistically significant for General Linear Model and Wilcoxon test and p < 0.00625 for Regression analysis.Circadian parameters for WT (showed in a previous workThe raw data supporting the conclusions of this manuscript are available on request to the corresponding author.Supplementary Information.


S. aureus from silicone menstrual cups. Methods: In this in vitro study, we tested four cleaning techniques: (1) cold water; (2) cold water and liquid soap; (3) cold water followed by steeping the cup in boiled water for 5 min in a ceramic mug covered with a small plate; and (4) cold water and soap followed by steeping the cup in boiled water as in (3). Human blood was coated to the inner and outer surface of each cup, dried, and incubated with 106S. aureus colony-forming units (CFU/mL). All tests were performed in triplicate. Viable bacterial abundance was measured with decadic dilution and drop plate or surface plating. Results: Bacteria were most effectively eliminated by cleaning cups with soap and water and then steeping in boiled water . This was not statistically significantly different from washing cups with water only and steeping 5 min in boiled water (14 CFU/cup). Raised lettering on the outer surface of the menstrual cups resulted in more bacterial recovery from pieces with lettering than without lettering. Conclusions: These results advance knowledge of between-period menstrual cup cleaning recommendations, suggesting that the logistical challenges of continuous boiling may be eliminated with steeping at least 5 min.Background: We sought to determine the effectiveness of common cleaning procedures in eliminating  Healthy management of menstruation requires access to water, sanitation, puberty and hygiene health education, both for individuals and communities, as well as effective, safe and affordable menstrual products with access to disposal facilities. Accessibility and affordability are particularly relevant in impoverished areas, where the lack of resources affects work and school attendance and productivity during menstruation. Demand for environmentally friendly products that produce less waste is increasing ,2,3. TheStaphylococcus aureus [S. aureus in the typically anaerobic vaginal environment [Toxic shock syndrome (TSS) was first identified in 1978 and in the early 1980s, when cases of menstrual TSS (mTSS) were reported, associated with tampons. mTSS is caused by the production of the toxin TSST-1 by ironment . As withironment , among tironment . There is concern that a buildup of menstrual effluent creates a microbial biofilm, which could increase the risk of mTSS . Thus, mS. aureus from the surface of menstrual cups. The study aims to address the knowledge gap related to appropriate cleaning of menstrual cups and to provide consumers of these cups information on the best cleaning practice. Specifically, the effectiveness of various cleaning procedures in eliminating pathogenic, biofilm forming bacteria from menstrual cups was assessed using the einhorn brand menstrual cups and four different cleaning methods.The goal of this study is to compare different cleaning techniques for their effectiveness in removing biofilms of This study was determined as exempt from human subject ethical review by the University of Illinois Chicago Institutional Review Board.S. aureus DSM 25630 from Jeonom University Hospital (South Korea) was used . This strain can produce the toxic shock syndrome toxin 1 (TSST-1), is resistant to multiple antibiotics, including methicillin, and is considered a methicillin resistant S. aureus (MRSA). The strain contains a gene for enterotoxin G and has been typed as Spa type t002. According to the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH), the strain grows optimally at a human body temperature of 37 °C. The material tested was the “Papperlacup” einhorn brand menstrual cup . There aBiofilm development on the different surfaces of the Papperlacup was analyzed. The surface of the inside of the cup is shiny and smooth, while the outside is matte and contains raised lettering, as can be seen in S. aureus. The tested cleaning techniques reflect between-period cleaning instructions commonly included on menstrual cup products as well as real-life cleaning as practiced by menstrual cup users. We tested four cleaning techniques, including cleaning with (1) cold water; (2) cold water and liquid soap ; (3) cold water followed by steeping the menstrual cup in boiled water (250 mL) for 5 min (poured immediately after reaching boiling) in a mug covered with a small plate; and (4) cold water and soap followed by steeping the menstrual cup in boiled water (250 mL) for 5 min (poured immediately after reaching boiling) in a mug covered with a small plate. The room temperature in the lab where experiments were conducted was maintained at 25 °C ± 1 °C. For all four cleaning methods, the cups were rubbed and turned between fingers under running water for 30 s without using a brush or scrub. This study was conducted to test a range of cleaning techniques on their ability to remove biofilms formed by In our study, a ceramic mug was used for steeping the cups in the boiled water. A new mug was used for each individual cup and cup piece test. As tin cans are more commonly available in low-resource settings, we performed an initial comparison of mugs and tin cans to observe the temperature profiles and to assess whether the mugs could approximate conditions in tin cans. Water was boiled before pouring into mugs and tin cans and measured with a thermometer . Temperature decline curves were plotted over 10 min , and the® incubator). Control cups without blood were similarly incubated. Subsequently, each cup was placed in 125 mL tryptic soy broth (TSB) medium (CASO-Bouillon) containing S. aureus (106 colony forming units (CFU)/mL) or TSB without S. aureus. The cups in the TSB medium were incubated for 12 h at 37 °C to match the general maximum recommended wearing time for such cups [For complete cup assays, human blood from a lab worker was applied to the entire inner and outer surface of each menstrual cup using a sterile cotton swab (Carlroth). The coated cups were dried in an incubator at 37 °C ± 1 °C for a minimum of 30 min  containing 10 mL sterile NaCl-Tween . A decadic dilution series was performed in volumes of 1 mL of the sample in the stomacher® bag plus 9 mL 0.9% sterile NaCl, at each step vortexed for 30 s and then shaken by hand for 20 s. Agar plates  were inoculated with 10 µL from each dilution in triplicate by the drop plate method, and plates were incubated at 37 °C for 12 h. After growth, plates with 25–250 colonies were used for counting, and CFU were determined applying the appropriate dilution factor to the number of colonies counted [® bag solution on an agar plate. If no bacteria were recovered, then the entire volume of each stomacher® bag was filtered through a 0.45 µm filter , using a filter funnel and vacuum system. The cup and the stomacher® bag were rinsed again with sterile NaCl (20–80 mL) to ensure that no bacteria remained. Filters were then placed directly on the tryptic soy agar plates and incubated for 12 h at 37 °C prior to colony counting  pieces of the menstrual cup, with and without raised indentation from the lettering. Only the outer surface was evaluated as a prior test showed that the inner, smooth surface retained substantially fewer bacteria than the outer cup surface (data not shown). Two pieces, each with and without the raised indentation from the lettering , were cut out of a complete cup. The outer surface of each piece of cup was coated with human blood with a cotton swab and then dried, as described above. Individual pieces of cup were placed in 6-micro-well plates  and covered with TSB medium (1.8 mL/well). After 12 h of incubation at 37 °C ± 1 °C (Memmert® incubator), the pieces of cup were cleaned using four different cleaning methods similar to the above, but modified for small silicone pieces. Specifically, each piece of cup was rubbed between the fingers under running water for 4 s. In two cleaning scenarios, after handwashing the cup pieces, they were dropped in the ceramic mug, boiling water poured over it and left to steep for 5 min. CFU were counted as described for the complete cups, applying the stomacher® test method followed by the drop plate, surface plating and, finally, the filter method.To determine the effect of the raised indentations (lettering) on bacterial growth and removal, viable bacterial counts were determined on 2 × 2 cm . The statistical significance of differences in bacterial counts for each cleaning method compared to the reference (without cleaning) was determined using non-parametric testing. Because of the multiple paired comparisons between cleaning methods, we applied Dunn’s multiple comparison test with the Benjamini–Hochberg adjustment. For comparison of bacterial recovery from pieces with and without lettering, we applied the non-parametric Wilcoxon rank sum test as each of these were independent tests. For comparison of cleaning methods for pieces with and without lettering, Dunn’s test was applied, and 8 CFU of S. aureus recovered per cup (p < 0.05) reductions were observed for Method 3 (rubbing with cold tap water for 30 s followed by steeping the cup in boiling water in a mug for 5 min) and Method 4 (rubbing with soap and cold water for 30 s followed by immersion in boiling water in a mug for 5 min), resulting in recovery of an average of 14 (Method 3) and 0 CFU (Method 4) per cup.Without any cleaning (reference), there were on average 2.075 × 10 per cup . StatistAcross all test scenarios, the CFU recovered were higher for cup pieces with lettering than without lettering, although this did not reach statistical significance in any comparison . SimilarEvidence-informed cleaning recommendations are required to minimize the risk of menstrual cup-associated infection. A recent white paper report by PATH, a global public health non-profit organization, summarized qualitative analyses of menstrual cup products and cleaning instructions, and suggested a need for more evidence and consensus on effective cup cleaning to inform users and bolster support for the recommended cleaning practices . BoilingS. aureus, we found that cleaning cups with soap and water and then steeping with boiled water for 5 min effectively eliminated bacterial recovery. The use of soap and boiling water reduced bacterial recovery to zero, though this was not significantly different to recovery from cups that were only cleaned through steeping in boiled water for 5 min without soap. However, while cleaning cups with soap and then steeping in boiled water had the greatest reduction in bacteria, using soap to wash the cups has not been recommended because soap can irritate the vagina. If soap can be adequately removed prior to use, this is the most effective sterilization method tested. Future observational studies of women using menstrual cups should assess actual cleaning practices, and whether these cleaning practices are associated with differences in vaginal microflora or vaginal symptoms.In our study, after seeding cups with a high bacterial load of S. aureus growth and TSST-1 production using the tampon sac method [S. aureus and TSST-1 production associated with cups in their study may have been a result of their experimental procedures, and could reflect the greater volume of air inserted into the experimental apparatus with cups relative to tampons, due to their shape. Thus, the Nonfoux et al. study does not fully quantitate the impact of menstrual cups on S. aureus growth and TSST-1 production in comparison to tampons. Schlievert performed a similar analysis of S. aureus growth with three methods: stationary incubation, incubation with shaking, or the tampon sac method (designed to mimic the vagina) [S. aureus inoculum. Schlievert found no effect on S. aureus growth or production of TSST-1 on the menstrual cup pieces (2.5 cm2) as compared to controls without menstrual cup pieces. Similarly, in an older study, Tierno et al. found no production of TSST-1 on menstrual cups, though the Tassaway cups used were non-absorbent elastic polymer rather than silicone [Three previous in vitro studies have compared bacterial growth in menstrual products ,22,23. Nsilicone . S. aureus growth, none systematically evaluated the effects of cleaning on bacterial recovery, which was the goal of our study. There are only a few studies that have examined how cup users clean their cups. Some data relate to cleaning the cup during a period , while other data relate to cleaning the cup between periods. In South Africa, 93% of the cup users reported emptying and cleaning their menstrual cups at home with tap water when at home, though this decreased to 32–44% of women when having to empty outside the home [S. aureus, G. vaginalis or Bacteroides spp. In addition, the abundance of Lactobacillus spp. was maintained at normal levels. Among 604 Kenyan schoolgirls aged 14–16 years using menstrual cups over a median of 10.9 months, the S. aureus vaginal colonization prevalence did not statistically or meaningfully differ by MHM sanitary product/material, and was 9.4% among cups users versus 10.7% for pads and 10.5% controls [While these studies reflect the relevance of examining the association between menstrual cups and the home . In situthe home ,26. Systthe home , cases othe home . Among 4controls . TSST-foS. aureus and if it is TSST-1 producing; initial/first use cleaning, how long users wear their cup, cup characteristics , how users clean the cup during a cycle, dropping or other adverse events, storage conditions and hand washing; and pathological conditions that may impair or make menstrual cup use more difficult [Staphylococcus growth but which are unlikely in vivo. Different bacteria strains  can inhibit each other in the vagina, a mechanism that helps the host suppress growth of pathogenic bacteria [S. aureus strain means that there was no competition between different bacterial strains for nutrients and cell adhesion, further contributing to the ‘worst-case’ scenario we constructed in these experiments. While the comparison between cleaning methods and surface type (with and without lettering) indicates that some cleaning methods are more effective, the concentration of S. aureus grown in these experiments cannot be ‘scaled-up’ to an entire cup due to (1) the complete cup has a different surface structure; and (2) we used a shorter cleaning time (4 s) for the cup pieces as in reality one would not spend 30 s cleaning a 4 cm2 area. We conducted our study with liquid soap, as with bar soap it would have been more difficult to control the amount of soap used. However, this could be a limitation as many menstrual cup users in LMIC use bar soap. We did not test the fourth cleaning technique on the cup pieces, and this should be incorporated in future studies. Lastly, the effectiveness of the cleaning methods needs to be verified across different brands of menstrual cups, which have a varying texture, size, shape, and firmness, which could affect usage and safety [The strengths of this in vitro study include novel assessment of steeping as a cleaning technique, and controlled conditions of biofilm formation and cleaning techniques, allowing replicability across labs and menstrual cups. However, population-based studies are needed to encompass the variability across individuals and how this is related to infection risk. For example, important mediating factors may include whether users have ifficult . We creabacteria . The used safety .S. aureus from silicone menstrual cups. We observed that cleaning with tap water and soap and steeping in boiled water for 5 min was most effective at reducing viable bacteria, but that cleaning with tap water and steeping in boiled water for 5 min was not statistically significantly different. These results present an advancement in the knowledge of menstrual cups between-period cleaning recommendations, suggesting that the logistical challenges of continuous boiling may be eliminated with steeping. We advise that menstrual cup manufacturers, menstrual health programmers and donors advocate for and conduct studies across an increasing variety of cup types, along with evaluation in vivo, as understanding the real-world conditions would be of value.In conclusion, we conducted an in vitro study to systematically evaluate the different cleaning methods for removing