Malnutrition (MN) in nursing home (NH) residents is associated with poor outcome. In order to identify those with a high risk of incident MN, the knowledge of predictors is crucial. Therefore, we investigated predictors of incident MN in older NH-residents.n = 3), function (n = 4), nutrition (n = 1), diseases (n = 5) and medication (n = 4)) with incident MN (weight loss ≥ 10% between nD and follow-up (FU) or BMI (kg/m2) < 20 at FU) was analyzed in univariate generalized estimated equation (GEE) models. Significant (p < 0.1) variables were selected for multivariate GEE-analyses. Effect estimates are presented as odds ratios and their respective 99.5%-confidence intervals.NH-residents participating in the nutritionDay-project (nD) between 2007 and 2018, aged ≥65 years, with complete data on nutritional status at nD and after 6 months and without MN at nD. The association of 17 variables , a quarter (2.15 [1.56–2.97]) or half of the meal eaten (1.72 [1.40–2.11]) (vs. three-quarter to complete intake), the lowest BMI-quartile (20.0–23.0) (1.86 [1.44–2.40]) (vs. highest (≥29.1)), being between the ages of 85 and 94 years  (vs. the youngest age-group 65–74 years)), severe cognitive impairment  (vs. none) and being immobile (1.28 [1.00–1.62]) (vs. mobile) predicted incident MN in the final model.10.5% of non-malnourished NH-residents develop MN within 6 months. Attention should be paid to high-risk groups, namely residents with poor meal intake, low BMI, severe cognitive impairment, immobility, and older age. The number of nursing home (NH) residents was estimated by the number of beds at about 1.6 million in the US in 2015 and 4.0 million in Europe in 2017 . TherefoMany factors are associated with malnutrition in cross-sectional studies. In a systematic review, including studies with at least 100 NH-residents each, depression, poor oral intake, swallowing disorders, chewing problems, and eating dependency were associated with weight loss (WL) in 6 studies, and immobility and dependence in activities of daily living (ADL), poor oral intake, chewing problems, dysphagia and female sex were associated with low Body Mass Index (BMI) in 7 studies . Since tBased on repeated measurements data, a decline in nutritional status  was observed in 23% of NH-residents within 1 year . A 2-yeaIdentification of factors increasing the risk and predicting the development of malnutrition is of utmost importance to help prevent malnutrition and its negative consequences. Thus, our aims were to examine the incidence of malnutrition and to identify predictors of incident malnutrition in a large sample of NH-residents.This manuscript adheres to the reporting standards for observational studies . The nD-project was approved by the ethical committee of the Medical University of Vienna, Austria, by the ethical committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany and by local ethical committees as required by national rules. All residents or their legal representatives gave written or oral consent prior to participation and all information was assessed completely anonymised.This analysis is part of the Joint Action Malnutrition in the Elderly Knowledge Hub (MaNuEL) of the European Joint Programming Initiative A Healthy Diet for a Healthy Life (JPI‐HDHL) that investigated determinants and risk factors for (incident) malnutrition in older people in different settings –24 and i2 and/or WL of >5 kg in the past year (assessed via questionnaire)) were excluded to gain data on real incidence.We included NH-residents aged 65 years or older participating in the nD-project between 2007 and 2018. Residents without data on body weight at FU were excluded as well as residents from Japan due to major differences in the nursing home system  and a co2. Height at nD was used for calculation of BMI at nD and FU.Questionnaires were completed by local NH staff. Body weight in kilogram (kg) was measured using scales from the NH wards at nD and FU, or the most recent weight documented in the nursing records was used for both dates. NH staff was asked to measure body height using a stadiometer for residents who were able to stand. If this was not possible, height was extrapolated based on the measurement of knee height or was recorded from the information in nursing records or identification cards. BMI was calculated according to body weight (kg)/height (m)2 and/or WL ≥ 10% between nD and FU, which was calculated as body weight difference between the two points in time.Incident  malnutriSeventeen variables in 5 categories were included as potential predictors of incident malnutrition:2).General characteristics (3 variables): age , sex , BMI : mobility chair and unable to stand up without any help by other persons), cognitive impairment , dysphagia (yes/no), chewing problems (yes/no).Nutrition (1 variable): intake at lunch .Diseases (5 variables): diagnosis of cancer (yes/no), neurologic diseases  (yes/no), musculoskeletal diseases  (yes/no), cardiovascular/pneumological diseases (yes/no), other diseases (yes/no).Medication (4 variables): intake of antibiotics (yes/no), opiates (yes/no), psychoactive substances (yes/no) and number of drugs (<5/≥5).Potential predictors are described by median with minimum and maximum for continuous variables (age and BMI) and by absolute numbers, percentages for categorical variables.For categorical variables with more than 0.1% missing values, we added a missing category. Cases with missing values in a variable with less than 0.1% missing values  and cases with missing values for age or sex were excluded.p-value p < 0.1 were tested for multicollinearity (variation influence factor (VIF)) and those with a VIF < 5 were added to the multivariate model. A p-value of p < 0.005 was defined as significant in the final model ) (vs. highest BMI-quartile (29.1–64.8 kg/m2)), the age-group 85–94 years  (vs. 65–74 years)), severe cognitive impairment  (vs. none) and being immobile (OR 1.28 [1.00–1.62]) (vs. mobile) predicted incident malnutrition in the final model , associations and effect sizes were similar, only immobility could not retain significance  global consensus definition , are widThe incidence of malnutrition at 6 months according to our definition was 10.5%, with 8.0% according to the WL-criterion and 4.9% according to the low BMI-criterion. Data on incident malnutrition in NH-residents is scarce and hardly comparable due to different definitions of malnutrition, different methods to assess malnutrition (e.g. measurement vs. self-report), and different FU-periods. Beck reported a WL of ≥10% within 6 months in only 2.1% of NH-residents  and MamhResidents aged 85–94 years were more likely to develop malnutrition compared to residents aged 65–74 years. While chances for the other age groups were also higher compared to the reference group, they did not show significance which could be due to the statistical power: nearly half of all participants were in the age group 85–94 years  had a 1.86 times higher chance of incident malnutrition  to develop malnutrition compared to mobile residents. This is in line with results from a systematic review of cross-sectional studies which reported an association between low BMI and immobility in several studies . Two lonDepending on the malnutrition screening tool used in the NH-setting , our ideOur study has several strengths. First, we could include a very large sample from different countries and different care systems. The questionnaires were standardized and available in different languages. Second, we present data on incident malnutrition which are very rare so far by excluding residents with malnutrition at baseline to reduce the risk of wrong causation. Third, we were able to consider a large set of variables from different domains to investigate the potential prediction of incident malnutrition. Moreover, we report our analyses according to a standardized reporting guideline (STROBE-statement) to ensure transparent and thorough reporting.However, when interpreting our results, several limitations must be considered. First, data were assessed by local staff and not by trained scientific personnel. Data presented here are mostly assessed as part of daily routine with simple and quick methods and not via validated scales (such as Katz or Barthel Index for ADL). Our analyses are limited to the variables available in the nD dataset, and eventually relevant potential predictors, e.g. ADL, multimorbidity, or hospitalisation, could not be taken into account. Food intake on nD was assessed estimating the consumed amount of food via a plate diagram and not via dietary records or other more valid—but also more time-consuming—dietary assessment methods . ThirdlyIn this large nD data set, 6-month incidence of malnutrition in nursing-home residents was 10.5%. A low intake at lunch, a low BMI, immobility, severe cognitive impairment and older age were identified as predictors for the development of malnutrition.are on physical and cognitive status are well known, which might also affect the nutritional status positively. In case of severe functional or cognitive impairment, at least special attention should be given to these residents. Food intake also represents a modifiable risk factor, and if food intake (at lunch) is low, effective nutritional care strategies with a potential to prevent malnutrition  are available and should be offered.While older age represents a non-modifiable factor, it is also of importance since special attention should be given to older residents to timely start with respective preventive interventions. In contrast, functional and cognitive impairments represent at least partly modifiable risk factors. Beneficial effects of physical exercise and cognitive training interventions Further prospective, large-scale cohort studies in nursing homes with longer follow-up periods and repeated measurements of nutritional status including reasons for WL (e.g. voluntary vs involuntary) and reasons for low intake should be conducted to complement and extent our results. In addition, food intake should be assessed more detailed, and reasons for drop-out should be recorded. Finally, it should be investigated whether inclusion of our identified risk factors in malnutrition screening tools will improve nutritional care and health outcomes.Supplemental material


Xanthomonas citri subsp. citri, which affects all commercial sweet orange (Citrus sinensis [L.] Osbeck) cultivars. Salicylic acid (SA) and systemic-acquired resistance (SAR) have been demonstrated to have a crucial role in mediating plant defense responses against this phytopathogen. To induce SAR, SA is converted to methyl salicylate (MeSA) by an SA-dependent methyltransferase (SAMT) and translocated systemically to prime noninfected distal tissues. Here, we generated sweet orange transgenic plants  overexpressing the SAMT gene from Citrus (CsSAMT) and evaluated their resistance to citrus canker. We obtained four independent transgenic lines and confirmed their significantly higher MeSA volatilization compared to wild-type controls. Plants overexpressing CsSAMT showed reduced symptoms of citrus canker and bacterial populations in all transgenic lines without compromising plant development. One representative transgenic line (V44SAMT) was used to evaluate resistance response in primary and secondary sites. Without inoculation, V44SAMT modulated CsSAMT, CsNPR1, CsNPR3, and CsWRKY22 expression, indicating that this plant is in a primed defense status. The results demonstrate that MeSA signaling prompts the plant to respond more efficiently to pathogen attacks and induces immune responses in transgenic plants at both primary and secondary infection sites.Citrus canker is a destructive disease caused by  Xanthomonas citri subsp. citri, which affects all commercial citrus cultivars and causes extensive economic damage worldwide  pathway in plastids and the phenylalanine ammonia-lyase (PAL) pathway in the cytosol  is a plant hormone that plays a crucial role in both local immune responses and systemic-acquired resistance (SAR) . In plan cytosol . SA is eX. citri . Citrus nce rate . Similarnce rate .NPR1 (Non-expressor of pathogenesis-related genes 1) is a key gene required for SA defense responses and SAR activation  and its homolog from Citrus maxima (CtNH1) increased resistance to citrus canker in Duncan grapefruit (Citrus paradisi Macfadyen), Hamlin sweet orange (C. sinensis), and pummelo (Citrus maxima), respectively  proteins  in tobacco (Nicotiana tabacum) showed a significant symptom reduction in response to Xylella fastidiosa subsp. pauca  in Wanjincheng oranges (C. sinensis) was demonstrated to enhance the tolerance to Huanglongbing caused by Candidatus Liberibacter asiaticus (LAS) in citrus species  . MeSA isesponses . In distesponses . Tomato nfection . Similarde races , 2016. Wp. pauca . In addie plants . Recentl species .C. reticulata CrSAMT belongs to the SABATH family of methyltransferases containing SAM- and SA-binding residues (C. sinensis (CsSAMT)  ] . Thus, c (NCBI)] , we geneCrSAMT from C. reticulata was produced as described by CrSAMT sequence is identical to CsSAMT used in other studies  as previously described (Citrus x limonia Osbeck) and transferred to the greenhouse at an average temperature of 28°C.The binary vector pCambia2201 containing the target gene  studies , we willescribed . Shoots escribed  supplemeescribed  when leaCsSAMT gene  using a specific primer pair designed to anneal to the FMV promoter and the CaMV35S terminator to avoid amplification of the endogenous AMT gene . GenomicAMT gene . The intCsSAMT gene expression analysis by qRT-PCR. Total RNA was extracted using the PureLink™ RNA Mini Kit  according to the manufacturer’s instructions. The RNA quality and concentration were determined by spectrophotometry NanoDrop ND-8000  and electrophoresis. The absence of cross-contamination with genomic DNA was verified by conventional PCR using RNA as template and endogenous primers  following the manufacturer’s instructions. Specific primers for qRT-PCR were designed using Primer Express® Software v3.0.1 . The reactions were performed using 6.5 μl of GoTaq® qPCR Master Mix , 120 nm of each gene-specific primer pair (ΔΔCt method (ubiquitin (UBI) gene was used as an internal control for transcript normalization (t-test (*p < 0.05) in GraphPad Prism 9 for Windows .Plants with amplicons of the expected size were subjected to  primers . One micmer pair , and 3 μmer pair , and relt method . The ubilization . ExperimCsSAMT lines and WT plants were placed into glass flasks arranged in parallel directly connected to a glass tube packed with Super-Q absorbent . Leaf volatiles were continuously captured under vacuum (640 mm Hg) for 24 h  mode . The identification of MeSA in plants was performed by comparison with the mass spectra of the GC/MS system database (NIST 62 Library) and commercial standard . In addition, the retention times of MeSA were compared with the retention times of the methyl salicylate standard. The quantification of MeSA in the samples was carried out by the external standard method in GC/MS . This procedure was performed based on the peak area of the ion at m/z 120, which was used as the target ion, followed by the ions at m/z 92 and m/z 152 as reference ions to confirm the compound specificity. The calibration curve was prepared with a commercial methyl salicylate standard diluted in dichloromethane (chromatographic grade) at the following concentrations: 1.2 μg/ml; 1.0 μg/ml; 0.8 μg/ml; 0.6 μg/ml; 0.4 μg/ml; and 0.2 μg/ml. Aliquots of 1 μl of each solution were injected and analyzed under the same chromatographic conditions as the samples in triplicate.The volatilized MeSA was detected and quantified using the dynamic headspace method  with modfor 24 h . The volt-test (p < 0.05 ** < 0.01).The concentration of MeSA was calculated by measuring the areas of each MeSA peak and plotting in a standard curve , accordiX. citri strain 306 carrying a GFP marker gene  containing ampicillin at 100 μg/ml and gentamicin at 5 μg/ml. Three regions on each leaf surface were pierced with a needle (inoculation point) and infiltrated with an X. citri suspension (104 CFU/ml) in phosphate saline buffer (PBS) using a needleless syringe. Symptoms and bacterial populations (CFU/mL) were evaluated at 7 and 14 days after inoculation (DAI). Phenotypic analysis of the canker lesions was performed using an Olympus MVX10 stereomicroscope with a U-MGFPHQ filter to detect GFP. Leaf disks were excised exactly at the inoculation point and out of the inoculation point within each inoculated region (t-test (p < 0.05) in GraphPad Prism 9 for Windows .Three fully expanded leaves of similar age and size per line were infiltrated with ker gene . X. citrd region . Disks wC. sinensis plants, primary inoculation of X. citri was carried out on a leaf surface (0 h), and then, after 24 and 48 h, secondary inoculations were performed on distal leaves  stereomicroscope. Four areas were analyzed for canker lesions by measuring the pixels using ImageJ software (t-test (p < 0.05) in GraphPad Prism 9 for Windows . In addition, noninfected leaves were evaluated from a representative transgenic plant and the wild type for expression analysis of defense-related genes  by Agrobacterium tumefaciens-mediated transformation using the CsSAMT_Cambia2201 vector  technique , which ef 0.9912 . The anan the WT ,B. The i HSAMT49 . These rreported .CsSAMT could promote resistance to X. citri, canker symptoms and the bacterial population were evaluated at 7 and 14 days after inoculation (DAI) of a green fluorescent protein (GFP)-expressing strain of X. citri. Although no difference in symptoms was observed at 7 DAI (CsSAMT lines showed reduced canker symptoms at 14 DAI compared to the WT (CsSAMT lines were restricted to the inoculation wound region (IWR), while WT developed typical canker symptoms, such as water soaking and erupted pustule lesions, over the infiltrated leaf area in both Valencia and Hamlin cultivars , and then, after 24 and 48 h, secondary inoculations were performed on other leaves of the same plants  infections compared to the WT ,B, but ted to WT , which ied to WT . The deced to WT .CsSAMT in VSAMT44 was significantly higher in all the leaves . Curiously, CsWRKY22, a transcription factor associated with susceptibility to citrus canker and SA  and secondary (SI) inoculations (24 and 48 h after PI), named T0, T24, and T48, respectively . As expee leaves . The PALthan VWT . Moreover and SA , showed nfection .CrSAMT in tobacco increases MeSA production and leads to reduced symptom development of the citrus bacterial pathogen X. fastidiosa  overexpressing The original contributions presented in the study are included in the article/AS conceived and designed the research. AS, MM, and MT provided reagents and analytical tools and discussed the data. CN and RC performed the experiments. CN and NT-S analyzed data. CN, NT-S, and AS wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a research grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—2013/10957-0). CN, a Ph.D. student from the Graduate Program in Genetics and Molecular biology (UNICAMP), was supported by a fellowship from (CAPES-Grant 001). NT-S and RC are postdoctoral fellows supported by FAPESP (2019/01447-5 and 2017/16142-0). MT, MM, and AS are recipients of research fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).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.


Patient satisfaction is identified as an indicator of the right to health . TraditiAn observational, cross-sectional, exploratory-descriptive study with a non-probabilistic and accidental sample was performed. Data were collected through the “Children Care Quality at Hospital” instrument, after translation and validation to Portuguese. The instrument includes three domains: nurse characteristics, nursing activities and nursing environment. Also, children were asked to rate global satisfaction with nursing care from 1 (less satisfied) to 5 (more satisfied). Statistical analysis was performed using SPSS statistical tool (version 24.0). Authorisation was obtained from National Data Protection Commission as well as ethics committees in each of the 6 health institutions were the study was applied.n = 252) includes mainly boys  with 8.9 years (SD = 1.4) as mean age and most children had unscheduled admissions . Global nursing care (1–5) was rated with a score of 4.51 (SD = 0.645). There was no significant difference between sex , age  or scheduled/unscheduled admissions  and the score attributed by children.The sample (In this sample, school-aged children are satisfied with nursing care provided during hospitalisation. Sociodemographic factors seem to have effect on overall satisfaction in previous studies with better scores of satisfaction in: older patients , male pa


This work provided a novel and reliable approach to recover gut microbiota genomes, facilitating the discovery of new bacteria species and furthering our understanding of the microbiome that underlies human health and diseases.Human gut microbiota modulates normal physiological functions, such as maintenance of barrier homeostasis and modulation of metabolism, as well as various chronic diseases including type 2 diabetes and gastrointestinal cancer. Despite decades of research, the composition of the gut microbiota remains poorly understood. Here, we established an effective extraction method to obtain high quality gut microbiota genomes, and analyzed them with third-generation sequencing technology. We acquired a large quantity of data from each sample and assembled large numbers of reliable contigs. With this approach, we constructed tens of completed bacterial genomes in which there were several new bacteria species. We also identified a new conditional pathogen Further, disorder of the gut microbiota is a potential risk factor for various chronic diseases such as type 2 diabetes, gastrointestinal cancer, and brain disorders6. Furthermore, the gut microbiota influences drug effectiveness because of its effects on drug metabolism8. Despite decades of research, the composition of the gut microbiota remains unidentified. For example, through analysis of more than 150,000 human microbial genomes, Edoardo et al. recapitulated nearly 5000 species-level genome bins, in which 77% are not in public repositories9. One of the main difficulties is that many microbes are uncultivable and require isolation9. In addition, culture-dependent genomic research is rare in large cohorts10. Bypassing the need to cultivate microbes, high-throughput sequencing overcomes these shortages and is a powerful method for bacterial metagenomics.There is increasing evidence that the gut microbiota, the human commensals, influences normal physiological functions, such as maintenance of barrier homeostasis and modulation of metabolism, inflammation, immunity, and development12. However, as only a single region of 16s rRNA in the genome is detected, it provides limited information about the microbial community14. Furthermore, it underestimates the diversity of microbes. Compared with 16s rRNA sequencing, WGS technology examines whole bacterial genomes, and provides more accurate detection at the species and diversity levels15. Through WGS technology, it is feasible to assemble the entire bacterial metagenome. However, there are several drawbacks in WGS technology owing to short sequencing reads (mostly <200 bp). For example, many microbiota genomes are fragmented into numbers of contigs16. Besides, it is difficult to assemble the de novo genome because there is too little sequence information17. Moreover, great phenotypic differences exist even between highly related strains of the same species19, but the differences between these strains are difficult to distinguish by WGS. Therefore, there is a dire need to detect the bacterial metagenome through more powerful sequencing methods.There are two main methods for bacteria metagenome sequencing: 16S rRNA-based sequencing and whole-metagenome shotgun sequencing (WGS). The 16S rRNA-based sequencing is widely used to assess microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community20. Compared with the second-generation sequencing technology of 16s rRNA and WGS, it can detect much longer fragments, so TGS produces genome assemblies of unprecedented quality20. For example, Hui et al. de novo assembled a chromosome-level reference genome of the red-spotted grouper with TGS21. Though TGS has been used in eukaryotic genome detection extensively, but unfortunately its application in microbiota genomes have not been of sufficient scale. For instance, Johanna et al. assembled a certain specific microbial species living in the vagina with an abundance of more than 75%22. Thidathip et al. compared the taxonomic abundance of gut microbiota of head and neck cancer patients; while the mean length is approximately 1 kb, which is only a tenth of that of the buffalo genome (11.5 kb)24. One difficulty in the detection of gut microbiota genomes is that it is challenging to obtain high quality genomic DNA, because of complex fecal features. Therefore, large numbers of bacterial species in the gut microbiota have not yet been identified. In this study, we successfully extracted high quality gut microbial genomic DNA. By detecting the gut microbiota metagenome via Single Molecule Real-Time (SMRT) Sequencing of Pacbio, we assembled the bacteria genomes efficiently and discovered new species of bacteria.Third-generation sequencing (TGS) technology, also known as long-read sequencing, detects the isolated genomic DNA without amplification and produces surprising long reads (average 10–20 kb)g for 5 min at room temperature and decanted the supernatant. Subsequently, we added 978 μL sodium phosphate buffer, shook the mix, and vibrated the mixture for 15 s, then added 122 μL MT Buffer and shook up and down gently for 5 min. Then, we placed the samples in the shaker at 4 °C for 30 min followed by centrifugation at 14,000 × g for 5 min and transferred the supernatant to a clean EP tube. Then, 250 μL of PPS solution was added and the samples were vigorously mixed and incubated at 4 °C for 10 min followed by centrifugation at 14,000 × g for 2 min. The supernatant was transferred to the binding matrix solution in a 15 mL conical tube and shook gently for 5 min. Then, the samples were centrifuged at 14,000 × g for 2 min and the supernatant was decanted. Afterwards, we washed the binding mixture pellet with 1 mL wash buffer #1 and transferred the binding mixture to a SPIN filter tube and centrifuged at 14,000 × g for 1 min. We emptied the catch tube and added 500 μL of prepared wash buffer #2 to the SPIN filter tube and gently resuspended the pellet. Afterwards, we centrifuged the samples at 14,000 × g twice to extract residual ethanol. Finally, we transferred the SPIN filter bucket to a clean 1.9 mL catch tube and added 100 μL TES to resuspend the genomic DNA. DNA was detected using agarose gel electrophoresis, and the top bands were isolated and purified with a DNA Purification Kit . The DNA concentration and integrity were assessed using a NanoDrop2000 spectrophotometer .All methods in this study were approved by the Research Medical Ethics Committee of Shanghai University of Medicine & Health Sciences affiliated Zhoupu Hospital. Fecal samples were collected from a 34-year-old male and a 10-month-old baby and stored in an ultra-low temperature freezer . Genomic DNA extraction was performed with a FastDNA Spin Kit for Feces . To acquire sufficient high-quality gut microbiota genomic DNA, we improved the experimental procedure as follows: we added 500 mg feces in a 2 mL Lysing Matrix E tube, mixed the feces with 825 μL sodium phosphate buffer and 275 μL PLS solution, and shook the mix and vibrated for 15 s. Then, we centrifuged the samples at 14,000 × For PacBio sequencing library preparation and SMRT sequencing, DNA was fragmented by a Covaris g-TUBE device (10 kb) and was concentrated with AMPure PB beads according to the manufacturer’s instructions . DNA damage and ends were repaired in a LoBind microcentrifuge tube. Blunt ligation reaction was performed by adding 1 μL of blunt adapter (20 μM) and 1 μL of ligase to 30 μL DNA followed by incubation at room temperature for 15 min. SMRTbell™ templates were purified with AMPure PB beads and the concentration was measured by Qubit. Sequencing was performed on a PacBio Sequel instrument by OE Biotech Co., Ltd. .−5. The taxonomy of the species was obtained as a result of the corresponding taxonomy database of the NR library.Metagenome assembly was performed with flye software after obtaining valid reads. Open reading frame prediction of assembled scaffolds using prodigal was performed and translated into amino acid sequences. Non-redundant gene sets were built for all predicted genes using CD-HIT. The clustering parameters were 95% identity and 90% coverage. The longest gene was selected as the representative sequence of each gene set. The gene set representative sequence (amino acid sequence) was annotated with NR, KEGG, COG, SWISSPROT, and GO databases with an e-value of 1ehttps://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/). A total of 266,319 prokaryote genomes have been identified until now  . Then, we screened similar sequences in the order of identity, and downloaded the relative sequences. We identified the bacterial species of the assembled contigs and analyzed their evolutionary relationship.We acquired the full-length 16s rRNAs from the assembled contigs and blasted the 16s rRNAs using the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) and selected the most related full-length 16s rRNAs by identity. With these 16s rRNAs, we analyzed the phylogenetic tree with Mega725. The average nucleotide identity (ANI) were used to analyze whether two genomes belong to the same species26.To examine the differences between full-length 16s rRNAs, we compared the 16s rRNAs and visualized the differences using BioEdit software . For phylogenetic tree analysis, we blasted the full-length 16s rRNAs in the NCBI database , and GC% (gene region). The ncRNA predictions were harnessed using tRNAscan-SE (v1.3.1)28 (tRNA), RNAmmer (v1.2)29 (rRNA), and Rfam(v10.0)30 (sRNA). Repeat sequence prediction was analyzed with RepeatMasker (v4.0.7)31. Common function potential analyses were performed using the Non-Redundant (https://www.ncbi.nlm.nih.gov), Swissprot (http://www.uniprot.org), KEGG (http://www.genome.jp/kegg/pathway.htmL), Cluster of Orthologous Groups of proteins (https://www.ncbi.nlm.nih.gov/COG/), comprehensive antibiotic resistance database (CARD) (https://card.mcmaster.ca)32, and carbohydrate-active enzymes (http://www.cazy.org) databases33.Single bacteria analyses included gene prediction, non-coding RNA (ncRNA) prediction, repeat sequence prediction, nonredundant analysis, and common function potential analyses. We performed gene prediction with the Prokaryotic Dynamic Programming Genefinding Algorithm [prodigal (v2.6.3)]t-test. Data are presented as mean ± SD (standard deviation) or mean ± SEM (standard error of the mean) as indicated in the figure legends. P-values were considered statistically significant at P < 0.05.R programming language version 3.4.3 was used for statistical analysis. Statistical significance between two groups was determined using an unpaired two-tailed Student’s https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/)  Fig. , consistEscherichia coli (U00096.3) . Four contigs  had high identities (>99%), while the identity of contig_638 was less than 97%  was a conditional pathogen; therefore we sought to evaluate whether healthy people also carry other conditional or common pathogens. We selected 27 common and conditional pathogens, including E. coli, Staphylococcus aureus, Shigella flexneri, and Helicobacter pylori. Only S. flexneri was found in the gut of the adult and baby samples, while other pathogenic bacteria were not found  for further analysis. Contig_82 belongs to ies Fig. . Similarlis Fig. . Analysilis Fig. –d. We thlis Fig. . The moslis Fig. . Interesund Fig. .Fig. 4PhEnterococcus faecalis showed only 8% similarity , which is also consistent with other species of Enterococci  might be new human species or subspecies. Using large-scale metagenomic assembling, Edoardo et al. and Alexandre et al. uncovered thousands of new species of bacteria36. These results, together with our work, indicated that further investigation of gut microbiota is worthwhile. Furthermore, the approach used in this study will help assemble the genome of unknown bacteria, and thus might facilitate the Human Microbiome Project and the Genomic Encyclopedia of Bacteria and Archaea38. Finally, bacteria are widely distributed in various environments, such as natural lakes, oceans, and soils. Therefore, it is of great interest to investigate bacteria living in different environments with our methods.Extensive studies of the human gut microbiota have been reported in past decades. However, large numbers of species in the gut microbiota remain unknown. By improving the extraction method, we successfully acquired high-quality genomic DNA of the gut microbiota, and obtained approximately 85 Gb TGS data from each sample, which was much greater than previous studies39. Intriguingly, our work discovered more than 10,000 unknown genes without known domain . We found that the contig lengths were closely correlated with the coverage (Supplementary Fig. Enterococcus tongjius, with multiple traditional microbiological means, including different medium and antibiotics; but we have not isolated Enterococcus tongjius clone successfully until now. Our inability to culture this bacterium has limited our understanding of its ecological role in the intestinal environment and its relation to human’s health.Despite the promise that this study holds for gut microbiota, it is important to note its limitations. First, the lengths of bacterial genomes acquired were between 1.5 and 3.5 Mb, much smaller than that of In summary, we established an effective extraction method to obtain high quality gut microbiota genomic DNA and detected the samples with TGS technology. We identified not only a large number of unknown genes, but also several new subspecies and species with our methods. This work provided a novel and reliable framework for exploring gut microbiota genomes, improving our understanding of the mechanisms that underlie the role of the microbiome in health and disease.Supplementary legendsSupplemental Fig. 1Supplemental Fig. 2Supplemental Fig. 3Supplemental Fig. 4Supplemental Fig. 5Supplementary Tab. 1Supplementary Tab. 2Supplementary Tab. 3


Benign prostatic hyperplasia (BPH) is a common disease in aging males. It has been proven that the Hedgehog (HH) is implied as an effective and fundamental regulatory growth factor signal for organogenesis, homeostasis, and regeneration. Smoothened (SMO), as the major control point of HH signals, activates aberrantly in most human solid tumors. However, the specific function of SMO and its downstream glioma-associated oncogene (GLI) family in BPH has not been well understood. Here, we first revealed that the SMO cascade was upregulated in BPH tissues and was localized in both the stromal and the epithelium compartments of human prostate tissues. Cyclopamine, as a specific SMO inhibitor, was incubated with BPH-1 and WPMY-1, and intraperitoneally injected into a BPH rat model established by castration with testosterone supplementation. SMO inhibition could induce cell apoptosis, cell cycle arrest at the G0/G1 phase, and a reduction of tissue fibrosis markers, both in vitro and in vivo. Finally, a tissue microarray, containing 104 BPH specimens, was constructed to analyze the correlations between the expression of SMO cascade and clinical parameters. The GLI2 was correlated positively with nocturia and negatively with fPSA. The GLI3 was in a positive relationship with International Prostate Symptom Score and nocturia. In conclusion, our study suggested that SMO cascade could play important roles in the development of BPH and it might be rediscovered as a promising therapeutic target for BPH. Bothersome LUTS can interfere with activities of daily living, causing significant impairment of disease-specific quality of life. The presence of histological BPH at autopsy is ~50% in those aged 51–60 years and 70% in those aged 61–70 years2. Androgen levels and aging are two indispensable driving factors of BPH3. Despite the high prevalence, the causative factors of the initial event leading to the development of BPH remain unclear. Consequently, it is of great significance to identify new markers and new therapeutic targets for BPH.Benign prostatic hyperplasia (BPH) refers to the nonmalignant growth of the prostate that occurred commonly in aging men and can lead to lower urinary tract symptoms (LUTS)4, playing an instrumental role during diverse processes of cell differentiation and organ development . New glands, which can only be seen in the embryonic period, are often found in the human hyperplastic prostate. It has been proposed that the occurrence of BPH is the “reawakening” of the embryonic process that prostate mesenchyme induced epithelial differentiation8. Several HH target genes have been identified in the mesenchyme of developing prostate, including the cytokine Cxcl14, the insulin-like growth factor-binding protein Igfbp3, and the delta/notch-like epidermal growth factor-related receptor Dner9. Indeed, HH pathway target genes have been implicated in the placement of prostate epithelial buds and in subsequent ductal branching and outgrowth10.It has been documented that robust Hedgehog (HH) signals occur frequently during embryonic development and infancy11.The activation of HH signaling cascade is initiated by binding HH ligands to the membrane receptor Patched1 (PTCH1), thus releasing smoothened (SMO) to be activated by phosphorylation. SMO ultimately decreases the interaction between suppressor of fused homolog and glioma-associated oncogenes (GLIs). GLI proteins belong to zinc-finger transcription factors and are the terminal effectors of SMO cascade. This family consists of three major regulators in mammals, i.e., GLI1–3. GLI1 acts only as a transcriptional activator (GLI-A), whereas GLI2 and GLI3 can be processed into both transcriptional activators and repressors (GLI-R), depending on the specific cell context and on the activation state of HH signalingGLI1, PTCH1, HHIP1), proliferation and differentiation , apoptosis , epithelial-to-mesenchymal transition , and stem cell self-renewal 13. Contrarily, in the absence of HH ligand, PTCH1 represses the HH signals by inhibiting the activity of SMO. Therefore, SMO is the central transductor in HH signaling and is of major academic and pharmaceutical interest. In addition, the actions of HH signaling, especially SMO and its downstream GLI family, on growth, epithelial–mesenchymal interactions, and fibrosis in BPH are not well documented.The transmembrane protein SMO, a member of the Frizzled class of G-protein-coupled receptor superfamily, is a critical control point of the HH signaling pathway. The accumulation of activated SMO suppresses the generation of GLI-R forms and allows GLI-A proteins to translocate into the nucleus and transcriptionally activate their target genes that include HH-GLI pathway autoregulation  from our institute. In the hyperplastic prostate, the mRNA level of SMO and GLI1–3 was significantly increased over 2-fold and the protein level of these molecules was consistently increased  datasets  and the Oncomine database were explored to analyze the differentially expressed SMO cascade genes between BPH specimens and normal prostate (NP) specimens. As shown in Fig. ase Fig. . Normal sed Fig. . Meanwhised Fig. . Consistsed Fig. . Hence, Human cell lines were treated with the SMO inhibitor CYC. The Cell Counting Kit-8 (CCK-8) assay was applied to detect BPH-1 and WPMY-1 cell viability after CYC treatment and calculated the half-maximal inhibitory concentration (IC50) of CYC for the cytotoxic effect. We found that the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells was 29.8 and 32.6 μM, respectively Fig. . Based oAs shown in Fig. p < 0.01), which may be due to the physiological effect of T. In addition, the ventral prostate weight and prostate index [prostate wet weight (mg)/body weight (g)] were decreased by 22% and 13% in CYC-treated rats . However, the increased weight of the body and seminal vesicles in CYC-treated rats did not reach statistical significance. Histologically, in BPH rats, the epithelium component was relatively increased, large glands lined with tall columnar epithelium, which are stratified, pseudostratified, or papillary fronds protruding into the lumen. Nevertheless, SMO blockade by CYC effectively prevented the progression of BPH induced by castration with TP supplementation. The shrunk glands were lined with a single layer of columnar epithelium to low cuboidal cells, along with slight edema ) in BPH rats. Interestingly, the stromal component was relatively decreased (a 41% loss of SM and a 50% loss of collagen fibers) and the epithelium component was also reduced by 26% in the CYC-treated BPH rat model  was obviously increased for castrated rats with 4-week T supplementation (BPH model) Fig. . Interesats Fig. , respectema Fig. . Masson’del Fig. . In addidel Fig. .Fig. 6CYThe BPH rats had a nearly 2.5-fold increase in Ki-67-positive rate Fig.  and a 50Bcl-2, BAX, caspase-3, CDK2/4, cyclin D1, α-SMA, and collagen I, which subsequently modulates apoptosis, cell proliferation, cell cycle, and fibrosis through the GLI-dependent transcription network in BPH12.As shown in Fig. Finally, we calculated the percentage of the positive area of these proteins on tissue microarray (TMA) of 104 BPH patients and analyzed the correlation between the expression of these proteins and clinical parameters. As shown in Supplementary Figs. Our novel data demonstrated that SMO and GLI1–3 were localized in the stroma and the epithelium compartments of human prostate tissues and upregulated in hyperplastic prostate tissues. We also showed that SMO cascade inhibition could induce cell apoptosis, cell cycle arrest at the G0/G1 phase, and a reduction of tissue fibrosis markers both in vitro and in vivo. Our study suggested that SMO system could play a vital role in the development of BPH.15 showed that SMO and GLI1–3 were broadly expressed in the human ovary, endometrium, and other tissues, including prostate. In our current study, SMO cascade was abundantly expressed in human prostate tissues, rat ventral prostates, and cultured prostate cell lines. There are adequate experimental evidences demonstrating that prostatic stromal and epithelial cells maintain a sophisticated autocrine/paracrine type of communication17. Therefore, it is necessary for our subsequent study to determine that SMO cascade proteins can either be expressed solely in the epithelia or in both epithelia and stroma. Zhu et al.18 have reported that SMO was mainly detected in the ductal epithelia with minimal to moderate expression in the stromal cells of human fetal prostates. A previous TMA analysis19 also showed that SMO cascade was localized in both the stromal and the epithelium compartments of adult prostate tissues, which was consistent with our current observation. The high expression of SMO cascade indicated that it could play a role in established BPH by modulating this autocrine/paracrine signaling pathway. Indeed, SMO, GLI2, and GLI3 were all significantly upregulated in BPH when compared with normal ones analyzed through two public databases (GEO and Oncomine). However, GLI1 was found to be significantly increased in BPH in our current study, which may attribute to a limited sample size of BPH or NP accessed by a public database. Besides, the sample size (eight BPH samples and eight control samples) in our current study was much larger than those public datasets of BPH. The altered expression of SMO cascade was further demonstrated via RT-PCR and western blotting. The expression levels of SMO cascade were upregulated in the human hyperplastic prostate and BPH rat prostate when compared with normal controls.An analysis of the human tissue-specific expression by genome-wide integration of transcriptomics21. We observed that CYC could induce delayed cell proliferation, cycle arrest at the G0/G1 phase, and apoptosis for prostate cells. The percentage of apoptotic epithelial cells (45.81%) was higher than that in stromal cells (29.00%) at 20 μM of CYC, which could be explained by the fact that prostatic epithelial cells might be more sensitive to a high dosage of CYC than stromal cells in vitro. Proteins involved in apoptosis were strongly altered, especially a high BAX/Bcl-2 ratio, which is involved in mitochondrial-dependent apoptosis22, and induce a subsequent activation of caspase- 3 and -7 down in the intrinsic apoptotic pathway24. Also, it was found that proteins related to cell cycle regulation, such as cyclin D1 and CDK2/4, were all reduced after CYC treatment.To explore the potential functional activities of SMO cascade involved in the progression of BPH, two common human prostate cell lines BPH-1 and WPMY-1 were selected. SMO downregulated prostate cell model was generated using CYC treatment. CYC, a natural plant-derived alkaloid, acts by inhibiting cellular response to SMO cascade28. During the past decades, several studies have demonstrated that fibrosis may act as a risk factor contributing to BPH/LUTS etiology32. Mechanistically, in response to exposure to the canonical profibrotic protein TGF-β1 and other inflammatory proteins33, prostate stromal fibroblasts seem to be able to differentiate into myofibroblasts and can be triggered to express fibrosis-associated collagen 1 and 3 and α-SMA34. As CYC has a cytotoxic effect, we calculated the IC50 of CYC for the cytotoxic effect of BPH-1 and WPMY-1 cells . Therefore, in this current study, we chose 5, 10, and 20 μM dosages for human prostate cell lines and 10 mg/kg of CYC for in vivo intraperitoneal (i.p.) injection. Thus, CYC induced apoptosis, delayed cell growth, and reduced fibrosis markers in the prostate independent of SMO inhibition, rather than an off-target effect or a cytotoxic effect.Interestingly, CYC incubation lowered the mRNA and protein levels of fibrosis markers α-SMA and collagen I in cultured immortalized prostate stromal cells WPMY-1. Fibrosis, as a hallmark of pathologic remodeling in many tissues, has adverse effects in many organs and leads to numerous clinical diseases35. However, there is no animal model that could totally mimic all characteristics of human BPH. In our current study, BPH was induced in rats by castration with TP supplementation, which was a widely used, convenient, time-saving, and inexpensive model. Moreover, our TP-induced BPH model was verified with the significantly increased weight of the androgen-sensitive organs  when compared with sham rats. In line with previous observations38, T injection mainly led to a notable involution of acinar epithelium hyperplasia, such as increased acinus amount, papillary fronds protruding into the glandular cavities, and thickening of the epithelial layer with no change in the percentage of the stromal component. Interestingly, it was noticed that the loss of body weight in BPH rats seems to be due to the exogenous T supplementation inducing an improvement of metabolic characteristics and probably exerts its effects by a direct action on lean body mass. Meanwhile, in the CYC-treated group, BPH rats were treated with 10 mg/kg CYC (i.p.) once a day for 14 days by observing the reduction of ventral prostate weight and shrunk glands lined with a single layer of columnar epithelium to low cuboidal cells. Furthermore, both the stromal and the epithelium components were significantly reduced in the CYC-treated BPH rat model compared with BPH rats. In addition, SMO cascade proteins were upregulated in BPH rat prostate and downregulated with CYC administration. The Ki-67-positive cell rates were attenuated, whereas apoptosis rates were enhanced. The alteration of proteins involved in apoptosis and cell cycle were detected by western blotting by observing an increasing expression of BAX and a decreased expression of CDK2/4, cyclin D1, bcl-2, and cleaved caspase-3. Moreover, downregulation of SMO resulted in a suppressed status for the fibrosis markers α-SMA and collagen I in vivo. All these changes are in parallel with our in vitro findings.Over the past few decades, various types of animal models of BPH have been established to provide useful tools for the study of BPH, including spontaneous BPH models, T-induced BPH models, xenograft models, etc.39. In their study, only primary cultured rat prostate cells and rat models were used. No human data were available. Moreover, SMO was not determined in their study. Importantly, they lacked a more exact and scientific process of creating an age-related BPH rat model. Their BPH rats are only 6–8 weeks old, which is hard to find in enlarged prostate.Recently, Yuan and colleagues had reported that CYC administration suppressed BPH by inhibiting epithelial and stromal cell proliferation40, which includes the assessment of nocturia. Nocturia, common among older individuals with insomnia, affects people of different ages, races, and genders all over the world. The prevalence of nocturia in both men and women increased with age41. In fact, men with BPH more commonly present with irritative than obstructive symptoms, and the most common presenting symptom is nocturia. Our findings suggested that SMO cascade, especially GLI2 and GLI3, was in positive correlation with the progression of BPH. However, the correlation of the SMO system with clinical data is worth further investigation.Finally, a TMA of 104 BPH tissues was constructed to analyze the correlation between the expression of SMO cascade proteins and corresponding clinical parameters. The GLI2 was correlated positively with nocturia and negatively with fPSA and the GLI3 was in a positive relationship with IPSS score and nocturia via a Pearson correlation analysis. The IPSS is recommended as the symptom scoring instrument that is used for the baseline assessment of symptom severity in men with LUTSCollectively, our present study demonstrated the expression and functional activities of SMO cascade in the prostate. SMO cascade is localized in both the stroma and the epithelium compartments of human prostate tissues and it is upregulated in hyperplastic prostate tissues. Moreover, SMO cascade appears to be involved in the development of BPH via modulation of imbalance of cell apoptosis, cell proliferation, and cell cycle progression, as well as the fibrosis process in vivo and in vitro. Besides, GLI2 and GLI3 are positively correlated with the progression of BPH. Thus, our data suggest that SMO cascade may represent a potential therapeutic target for the treatment of BPH in the future.14, GSE386842, and GSE11919543) were obtained from GEO (https://www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information and the mRNA expression profiles of SMO cascades between human BPH specimens and human NP specimens were analyzed. Meanwhile, the Oncomine database (https://www.oncomine.org/) was used for the transcriptional validation of our concerned genes.Three gene expression profiling datasets (GSE6099n = 12 per group): sham group, perineal incision + corn oil  injection (subcutaneously (s.c.)) + dimethyl sulfoxide (DMSO)  administration ; C + T group, bilateral orchiectomy + T  (2 mg/day)/corn oil injection (s.c.) + DMSO administration (i.p.); C + T + CYC group, bilateral orchiectomy + T (2 mg/day)/corn oil injection (s.c.) + CYC  administration . Rats were euthanized under anesthesia using a euthanasia solution containing 10% [w/v] chloral hydrate on day 37, and ventral prostates and seminal vesicles were harvested and weighed. The tissues were snap frozen in liquid nitrogen and stored at −80 °C for subsequent molecular analyses or put into 10% neutral-buffered formalin for histological examination. All surgical procedures were performed under anesthesia by i.p. injection of pentobarbital sodium . We are blinded to the group allocation when assessing the in vivo effect of CYC on rat prostates. Animal experiments were conducted at the Animal Center of Zhongnan Hospital of Wuhan University and all animal protocols were approved by the Medical Ethics Committee for Experimental animals of Zhongnan Hospital of Wuhan University.The design of this experiment is outlined in Supplementary Fig. Eight prostate samples from young brain-dead men  undergoing organ donation were obtained as controls. A total of 104 specimens of BPH samples with clinical data were obtained from the patients who underwent transurethral resection prostate at the Department of Urology, Zhongnan Hospital of Wuhan University. Postoperative prostate pathology examination revealed BPH. Prostate tissues were divided into two strips and were, respectively, stored in liquid nitrogen for PCR and western blotting analysis and stored in 10% neutral-buffered formalin for immunofluorescence microscopy and the construction of TMA. Collection and treatment of all human specimens were carried out in accordance with the approved guidelines of the Ethics Committee at Zhongnan Hospital of Wuhan University.2O2. Subsequently, all slides were incubated with primary and secondary antibodies  was purchased from the Procell Co., Ltd. in Wuhan, China. Identification of the cell lines was performed at the China Center for Type Culture Collection in Wuhan, China. SV40 large-T antigen-immortalized stromal cell line WPMY-1 (Cat. #GNHu36) was purchased from the Stem Cell Bank, Chinese Academy of Sciences in Shanghai, China. The BPH-1 cells were cultured in RPMI-1640 medium  containing 10% fetal bovine serum (FBS) . The WPMY-1 cells were cultured in Dulbecco’s modified Eagle’s medium  containing 1% penicillin G sodium/streptomycin sulfate and 5% FBS. All the cell lines were recently authenticated and were cultured in a humidified atmosphere consisting of 95% air and 5% CO6 cells) were harvested, washed with phosphate-buffered saline (PBS), and then centrifuged. Pellets were resuspended with 1 ml DNA staining solution, which contained 50 μg/ml propidium iodide and 0.1 mg/ml RNaseA, and 10 μl permeabilization solution . The DNA content distribution was analyzed by flow cytometry analysis  after incubation in the dark at 37 °C for 30 min. For cell apoptosis analysis, fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I  was used. BPH-1 and WPMY-1 cells (1 × 106 cells) were harvested and then stained with FITC Annexin V Apoptosis Detection Kit I according to the manufacturer’s instruction.For cell cycle analysis, BPH-1 and WPMY-1 cells (1 × 10l-lysine-coated 96-well plates and subjected to various treatments as described above. CCK-8 solution (10 μl/100 μl) was added to each culture well, and neurons were incubated for 2 h at 37 °C. Finally, we measured the absorbance at 450 nm with a microplate reader .The viability of cells was examined by CCK-8  assay. Briefly, the neurons (~5000 cells/well) were seeded in poly-For cell immunofluorescence microscopy, cells were cultured as aforementioned, followed by seeding on 12 mm coverslips and washing by ice-cold (PBS (pH = 7.4). The coverslips were then fixed with 4% paraformaldehyde (PFA) for 30 min, followed by 0.1% Triton X-100 incubation, and then blocked in goat serum for 30 min at room temperature. Afterward, they were incubated with primary antibody  according to the manufacturer’s instructions and quantitated at 260/280 nm using a NanoPhotometer spectrophotometer . Two micrograms of total RNA was reverse transcribed to complementary DNA via the SuperScript II First-Strand Synthesis System according to the manufacturer . QRT-PCR was performed to determine the level of mRNA expression of a gene of interest based on SYBR Green using a Bio-Rad  CFX96 system. The expression levels of genes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and compared by 2g for 10 min at 4 °C. Then, the protein concentration was measured by bicinchoninic acid assay. Protein extracts were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane  using a Bio-Rad wet transfer system. The membrane was then blocked in Tris-buffered saline with 0.05% Tween-20 buffer containing 5% skim milk, and incubated sequentially with primary and secondary antibodies  on ice for 30 min. The supernatant was collected after centrifugation at 14,000 × 2O. The sections were then stained in 10% hematoxylin  for 7 min, followed by washing under the tap water for 10 min to reveal the nuclei. Afterward, the sections were stained in 1% eosin  containing 0.2% glacial acetic acid for 5 min. After staining, the sections were washed with tap water, dehydrated in increasing grades of ethanol , and cleared in xylene for 3 × 10 min. The sections were imaged by an Olympus-DP72 light microscope .Prostate paraffin sections (5 μm) were deparaffinized in xylene for 3 × 10 min, rehydrated in descending concentrations of ethanol , and H46, prostate tissues were embedded into paraffin after being fixed in 10% formalin for 24–36 h and cut into 5 μm sections. Then, the sections were stained using Masson’s trichrome staining. Staining was detected by light microscopy. Prostatic smooth muscle (SM) cells, collagen fibers, and epithelial cells were stained red, blue, and orange, respectively. In each sample, we analyzed three areas randomly under magnification (×200). The area percentage of SM, collagen fibers, and glandular epithelium were quantitated with Image Pro Plus 5.0.As previously describedRat prostate tissues were fixed in 4% PFA, embedded in paraffin, and then digested with proteinase K for 20 min. The sections were then incubated with a fluorometric DNA Fragmentation Detection Kit  according to the manufacturer’s instructions. Nuclei were labeled with DAPI. Visualization was done with a Laser Scanning Confocal Microscope . DAPI and fluorescence-labeled images were merged and TUNEL-positive apoptotic cells in the merged images were quantified by the counting of positively stained cells.Tissues were sectioned in 10-μm-thick slices and thawed, mounted onto glass slides using a cryostat , air-dried, and fixed for 10 min in ice-cold acetone. Slides were washed in PBS and incubated for 2 h in a mixture of PBS supplemented with 0.2% Triton X-100 and 0.1% bovine serum albumin, followed by incubation overnight with the primary antibodies (listed in Supplementary Table t test (two groups compared) or one-way analysis of variance and Tukey’s post tests with SPSS 20.0 (multiple means compared). Statistical significance was considered as a p value < 0.05.All analyses were performed at least three times and represented data from three individual experiments. The data values were expressed as the means ± standard deviation (SD). Statistical analysis was performed using either the Student’s Supplementary table S1Supplementary table S2Supplementary table S3Supplementary table S4Supplementary table S5Supplementary table S6Supplementary figure legendsSupplementary figure S1Supplementary figure S2Supplementary figure S3


These results suggest that the UPR operates as a modifier of an individual's propensity for body weight gain in a manner that, at least in part, involves the regulation of an inflammatory response by skin fibroblasts.The unfolded protein response (UPR) is involved in the pathogenesis of metabolic disorders, yet whether variations in the UPR among individuals influence the propensity for metabolic disease remains unexplored. Using outbred deer mice as a model, we show that the intensity of UPR in fibroblasts isolated early in life predicts the extent of body weight gain after high-fat diet (HFD) administration. Contrary to those with intense UPR, animals with moderate UPR in fibroblasts and therefore displaying compromised stress resolution did not gain body weight but developed inflammation, especially in the skin, after HFD administration. Fibroblasts emerged as potent modifiers of this differential responsiveness to HFD, as indicated by the comparison of the UPR profiles of fibroblasts responding to fatty acids This article has an associated First Person interview with the first author of the paper. Highlighted Article: Using outbred deer mice, we show that the unfolded protein response operates as a modifier of propensity for body weight gain in a manner involving the regulation of an inflammatory response by skin fibroblasts. The regulation of body weight gain is an extremely complex process that involves the neurochemical and hormonal regulation of appetite, the absorption of nutrients and calorie-rich compounds, and their processing, distribution and, ultimately, storage or utilization in the periphery . ExtremePeromyscus maniculatus) that exhibit highly variable, yet tightly coordinated, UPR among individuals , which constitutes a conserved homeostatic response developing in cells to resolve endoplasmic reticulum (ER) stress . Numerouividuals . AnimalsP. maniculatus at weaning, exposed to tunicamycin, which induces canonical UPR, and the levels of the chaperones BiP , GRP94  and calnexin were evaluated. As expected, highly variable UPR among different individuals was detected that followed a positive linear correlation in the pairwise comparisons of all chaperones. This in turn identified some animals as high (HR) and others as low (LR) UPR responders , which, depending on the specific strain, may show an increase in body weight after HFD consumption that can exceed 50% .When body weight gain data were analyzed with respect to the UPR responsiveness of fibroblasts, it was revealed that the HR group gained significantly more body weight than the LR group D,E, and Fig. S2).The fact that inflammation is inherently linked to the consumption of the HFD, obesity and appetite led us to explore whether and to which extent distinct proinflammatory responses differentiate LR and HR animals following HFD consumption. To that end, we utilized a mouse cytokine array to simultaneously assess the levels of several proinflammatory cytokines in the animal sera. Surprisingly, we unveiled a clear trend, applicable to almost all cytokines evaluated, for induction, specifically in the group of LR animals and only after the administration of the HFD A. Thus, It is known that, depending on the context and the severity of the response, inflammation can produce contrasting effects in appetite and obesity. Inflammation is associated with anorexia and appetite loss , yet chrin vitro, recapitulate the response of certain peripheral organs to the HFD, operating as surrogates. To that end, the induction of inflammation was assessed histologically in brain, liver and adipose tissue, which represent established target tissues of the HFD in mice. As shown in Fig. S3). Hepatic steatosis did develop occasionally, in ∼30% of the animals, but was not associated with body weight gain, indicating that liver dysfunction proceeds independently of the body weight gain in this model  (P<0.05) and marginally with oleate (P=0.06). These observations imply the existence of an inverse relationship between UPR gene expression and the levels of proinflammatory cytokines in primary, unchallenged fibroblasts from genetically diverse animals.To test whether fibroblasts induce a proinflammatory response following fatty acid administration  NFKBIA) A. As shoP. maniculatus fibroblasts exhibiting a diverse UPR response were analyzed at the whole-transcriptome level for the mode of expression of the major chaperone BiP and several proinflammatory cytokines. Pairwise scatterplots were then developed, depicting the correlation of each transcript in the transcriptome between BiP and a roster of proinflammatory cytokines , were closed-colony bred in captivity since 1948 and descended from 40 ancestors wild caught near Ann Arbor, MI, USA. Sonoran deer mice, P. maniculatus sonoriensis (SM2 Stock), were derived from ∼50 animals wild caught by Jack Hayes in 1995 near White Mountain Research Center, Bishop, CA, USA  and the Department of Health and Human Services, Office of Laboratory Animal Welfare, University of South Carolina .Deer mice,  CA, USA . In this CA, USA . Blood w2. Cells were passed when adherent layers reached 90% confluence and for no more than seven passages. For tunicamycin treatment, cells were split into six-well plates, at 300,000 cells/well, and cultivated for 24 h. Then cells were treated with tunicamycin (5 μg/ml) for 5 h, immediately followed by RNA extraction.Fresh ear punches were collected during routine weaning and marking procedures at ∼3 weeks of age. After the ear punch tissue was bathed in 70% ethanol for 2 min, it was placed in RPMI 1640 medium (HyClone) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.292 mg/ml L-glutamine (HyClone), as previously described . Ear punGapdh.RNA was isolated with a Qiagen RNeasy Plus Mini Kit according to the manufacturer's recommendations. RNA was eluted using 30 μl nuclease-free water. Complementary DNA (cDNA) synthesis was performed using an iScript cDNA synthesis kit (Bio-Rad) according to the supplied protocol. RT-qPCR was performed on a T100 thermocycler (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). Specific oligonucleotide primers for target gene sequences are listed in Primary fibroblasts were split into six-well plates, at 300,000 cells/well, and cultivated for 24 h. Then, cells were treated with ThT (4 μM) and tunicamycin (5 μg/ml) for 5 h. ThT is known to be able to detect ER stress by exhibiting fluorescence when it binds to protein aggregates . After wAt sacrifice, the brain, liver, skin and adipose tissue were excised from each animal, fixed for 3-5 days in 4% formaldehyde, dehydrated with 100% ethanol and embedded in wax. Paraffin sections were stained with Hematoxylin and Eosin (H&E). Histological examination of the specimens was performed blindly by a certified pathologist. Images shown were obtained by an ICC50 HD (Leica Microsystems).The serum samples of deer mice were pooled for each group, and the relative levels of serum cytokines and chemokines were assessed with a Mouse Cytokine Antibody Array C3 (RayBio C-Series). Blocking, biotinylated antibody cocktail and HRP-streptavidin incubation, washing conditions and chemiluminescent detection were performed according to the manufacturer's instructions.Blood samples of deer mice for leptin level detection were collected before HFD consumption and at 4 and 28 weeks after HFD administration. The concentration of serum leptin for each animal was measured using an Ultra Sensitive Mouse Insulin ELISA Kit  according to the manufacturer's instructions.Paraffin-embedded skin tissues underwent antigen retrieval in 0.01 M citrate buffer (pH 6.0), permeabilization with 0.1% Triton X-100 and blocking with 10% goat serum. The sections were then incubated in phosphate-buffered saline with 0.1% Tween 20 and 1% bovine serum albumin at 4°C overnight with anti-vimentin . After washing, the sections were incubated with goat anti-rabbit IgG H&L Alexa Fluor 488  for 1 h at 25°C and then incubated with anti-BiP  conjugated to Alexa Fluor 647  at 4°C overnight. Images were obtained with a confocal microscope (Carl Zeiss LSM 700) and analyzed with ImageJ.Palmitic acid  and oleic acid  were dissolved in 100% ethanol and conjugated to 10% fatty acid-free bovine serum albumin (BSA)  in RPMI 1640 medium (HyClone). The molar ratio of PA/OA:BSA is 6:1. The primary fibroblasts were treated with PA or OA at a concentration of 500 μM for 24 h.P. maniculatus genome [HU_Pman_2.1 (GCA_003704035.1)] in ensembl.org using STAR v2.7.2 (GSE179058). The flowchart of the transcriptome coordination analyses was described previously  COBRE Center for Targeted Therapeutics Functional Genomics Core. RNAs were isolated with a Qiagen RNeasy Plus Mini Kit. RNA integrity was evaluated using an Agilent Bioanalyzer, and samples had a quality score ≥8.6. RNA libraries were prepared using established protocol with Next Ultra II Directional RNA Library Prep Kit for Illumina . Each library was made with one of the TruSeq barcode index sequences and pooled together into one sample to be sequenced on the HiSeq 2×150 bp pair-ended sequencing platform (Genewiz). Sequences were aligned to the R v2.7.2 . Reads wR v2.7.2  using Eneviously . BrieflyThe levels of total, free, HDL and LDL/VLDL cholesterol in sera of HR and LR deer mice after 14 weeks of HFD administration were measured with a cholesterol assay kit  according to the manufacturer's instructions. The cholesterol concentrations were detected on a microplate reader at an optical density of 570 nm.t-test, one-way ANOVA with Tukey's multiple comparisons test or Pearson's correlation as indicated. Statistical analyses were conducted using Prism software . P<0.05 was considered statistically significant.Data were expressed as mean±s.e.m, unless specified otherwise. Results were analyzed using paired or unpaired two-tailed Student's


Before measurements, blood samples were collected to determine the immune  and hormonal  profiles. Participants (n = 37) completed the study within the controlled experimental conditions. Protein intake increased in the EX+DM  and DM (1.3 ± 0.5 to 1.8 ± 0.6 g kg−1 BM day−1) groups during the intervention. Absolute fat-free mass increased in the EX+DM [mean  = 0.65 (0.25–1.0) kg] and EX [0.49 (−0.44 to 1.40) kg] groups (P < 0.001) compared to DM [−0.54 (−1.6 to 0.05) kg]. Relative fat mass decreased  in DM [−1.8% (−3.3 to −0.35%)] and EX+DM [−1.3% (−2.3 to −0.31%)], which was a greater reduction than that in the CON [0.10% (−0.80 to 1.0%)] group (P < 0.01). Relative maximal strength increased in both the EX and EX+DM  groups, but not in the DM and CON groups. The change in 1RM strength outcomes was higher in EX+DM compared to all other groups . There was an increase in resting plasma IL-10 concentration in EX+DM (88%), compared to all the other groups (P = 0.016). No other differences in systemic inflammatory cytokines were observed. There were no significant changes in all hormone concentrations measured among all groups. In conclusion, a high-protein dairy milk beverage providing additional protein did not further enhance the effects of PRT on outcomes of fat-free mass, power, or physical performance. However, there was a significant augmentative effect for high-protein dairy milk consumption on changes to maximal strength outcomes during PRT in healthy active older adults.The study aimed to investigate the independent and combined effects of consuming a high-protein dairy milk beverage, twice daily, with or without a progressive resistance training (PRT) program on outcomes of age-related sarcopenia, in healthy active older (≥50 years) adults. In this 12-week, 2 × 2 factorial study, participants were randomly allocated into one of four groups: dairy milk beverage (DM), exercise and dairy milk beverage (EX+DM), exercise alone (EX), and control (CON). The EX group underwent a 12-week whole-body PRT schedule (three sessions/week) and a high-protein dairy milk beverage (DM) was consumed twice daily (30 g protein/day). At weeks 0, 6, and 12, body composition (iDXA), strength [one-repetition maximum (1RM): leg press, chest press, lateral ( There has been considerable research exploring the age-related decline in skeletal muscle mass and function , collectively known as sarcopenia , 2. The −1 BM day−1), to overcome the increased requirements of amino acid utilization from the exercise stimulus and the blunted response to muscle protein synthesis (MPS) known as “anabolic resistance”  per day] have been suggested for active older adults, exceeding the current recommended dietary allowance (RDA) of protein ] and a state of chronic low-grade inflammation, a term known as “inflammaging” , 32. RaiThe current study aimed to determine the independent and combined effects of a high-protein dairy milk beverage provided at breakfast and lunch (or after resistance exercise), with or without PRT, on outcomes of FFM, skeletal muscle strength and power, and physical performance in a cohort of healthy active older adults. We sought to evaluate the study hypothesis that providing a high-protein milk on its own would maintain outcomes of FFM, skeletal muscle strength and power, and physical performance compared to those that receive no intervention . In comparison, we hypothesized that a high-protein dairy milk beverage in conjunction with PRT would further enhance the effects of PRT, leading to augmented gains in FFM and skeletal muscle strength and power and an improved physical performance compared to PRT alone.The study protocol obtained approval from the Monash University Human Research Ethics Committee (project number 12812), in accordance with the Helsinki Declaration for human research ethics. Informed written consent was obtained from all participants before they were enrolled in the trial. The study was registered with the Australian and New Zealand Clinical Trial Registry as ANZCT12618001088235.2; and (16) had participated in a structured resistance training program in the past 12 months. Once participants were deemed eligible, data were collected during the period from September 2018 to January 2020.Older adult males and females  performing exercise training for recreational fitness and/or sports competitions  three or more structured exercise sessions per week, totaling ≥90 min/week of structured exercise duration, plus additional unstructured physical activity that accounted for meeting the Australian physical activity guidelines , were reA total of 65 participants expressed interest in participating; of these, 51 were eligible to participate and were randomly assigned into one of four groups: high-protein dairy milk beverage alone (DM), exercise and high-protein dairy milk beverage (EX+DM), exercise alone (EX), and control (CON) . ParticiPrior to commencing any physical activity, participants filled out a physical activity readiness questionnaire (PAR-Q), in which they self-reported their level of activity including exercise volume and type. The participants were asked to complete a 3-day food–fluid diary prior to their baseline visit, and again at 6 and 12 weeks during the intervention, as previously described . Food–fl−1 BM day−1) over the entire duration of the experimental procedure, facilitated by an accredited practicing dietitian. Energy requirements were calculated using the participants' resting metabolic rate (RMR) scaled by an activity factor based on reported exercise (1.37 ± 0.09). RMR was determined by indirect calorimeter  in temperate ambient conditions (22.2 ± 1.4°C) and in accordance with best practice guidelines (Participants assigned to DM and EX+DM were asked to consume 500 ml/day (2 × 250 ml) of reduced fat (1.5%) fresh dairy milk . Participants were provided with a measuring cup and asked to consume 250 ml of dairy milk in the morning (with breakfast) and another at lunchtime . Each 250-ml cup of dairy milk contained: 535 kJ energy, 15.0 g protein (1.57 g leucine), 8.3 g carbohydrates (8.3 g lactose), 3.8 g fat, and 435 mg calcium. The participants received food provisions to deliver 100% of the total daily estimated energy requirements and 100% of the total daily estimated protein requirements (~1.2 g kgidelines . Intake latissimus dorsi (lat) pull-down, and chest press . Additional exercises including biceps curls, triceps extensions, shoulder raises, calf raises, cable deadlifts, leg curls, back rows, and abdominal exercises were used on a cable machine  and rotated throughout the program to ensure the development of muscle balance. During the first 2 weeks of the PRT program, the participants completed three sets of 10–15 repetitions of 50–60% of their one-repetition maximum (1RM) with 2-min rest intervals. For the following 4 weeks, the training volume was set at three sets of 8–12 repetitions at an intensity of 69–75% of 1RM, which increased to 80–95% for six to eight repetitions for the remaining 6 weeks. Each week, weight progressively increased by 5–10%. For all exercises, the participants were instructed to perform each repetition in a slow, controlled manner, with a rest of 2 min between sets. Testing for participants' 1RM occurred at baseline and week 6; weights were adjusted according to their new 1RM to account for strength gains throughout the protocol. Participants in the exercise groups  had 2 ± 5 days between their last exercise session and their mid- and post-assessment to minimize a carryover effect. Exercise compliance was determined by the number of sessions attended. All participants in the exercise groups  were instructed to continue their normal physical activity outside the PRT program.Participants allocated to EX+DM and EX were required to attend supervised PRT sessions, on three non-consecutive days per week, for 12 weeks at the research laboratory. These sessions were conducted either in a morning session (7:00 a.m. to 10:00 a.m.) or afternoon session (1:00 p.m. to 3:00 p.m.), to account for participants' work–life schedule. During the course of the trial, all exercise sessions were instructed by a strength and conditioning qualified investigator to ensure correct lifting, to monitor the appropriate amount of exercise and rest intervals, and to check compliance. Each training session (30 ± 15 min) consisted of full-body resistance training, which included leg press, All participants were asked to resume their usual lifestyle activity levels and were required to wear an activity monitor  on their non-dominant wrist. Participants were instructed to wear the activity monitor during the course of the intervention trial from waking to bedtime and to take the monitor off only when engaged in aquatic activities. A new activity monitor was provided every 2 ± 1 weeks due to the limited (25 days) battery life. Data were uploaded to analytical software . A valid day was defined as ≥80% wear time.2).The participants arrived at the laboratory between 7:00 a.m. and 9:00 a.m. in a fasted state [plasma osmolality = 296 ± 5.6 mOsmol/kg  and total body water = 53.3 ± 6.4% ]. All participants were required to avoid strenuous exercise for a 24-h period prior to all laboratory assessments. Height was assessed using a fixed stadiometer . BM was measured (Seca 515 MBCA) to the nearest 0.1 kg using standardized anthropometrical procedures. Total (in kilograms) and relative (in percent) fat mass (FM) and FFM and bone mineral content (BMC) were assessed by a trained radiographer using a dual-energy X-ray absorptiometry . Appendicular lean mass (ALM) was determined by adding the total arm and leg mass, and then it was adjusted for height  and a metabolic cart . Procedures were adjusted from standard fitness testing protocols . The iniA force plate  was used to measure relative muscle power (in watts per kilogram), jump height (in centimeters), and velocity (in meters per second) during a countermovement jump (CMJ) test. The participants were asked to start in a full erect standing position in the middle of the force plate and then instructed to dip to a self-selected depth and to perform a static squat jump. Hands were kept on the hips to minimize any influence of arm swing . The parTo assess gait speed, a walking course of 4 m length was marked on the floor. The participant was instructed to walk from one end of the course to the other at their usual walking pace. The timer began as the participant started walking and the timer stopped with the first footfall after the 4-m line. The test was repeated twice and the fastest time of the two scores was recorded .lat pull-down, and bench press exercises were measured. The 1RMs were normalized by body weight (1RM/BM). Hand grip strength (HGS) was measured using a digital hand dynamometer . HGS was measured in a standing position with the participants elbow by their side and flexed to 90° and a neutral wrist position. The participants were asked to apply the maximum grip strength by squeezing the dynamometer with as much force as possible using their dominant hand. This was repeated three times with a 1-min rest in between attempts. HGS was defined as the highest value for their dominant hand  were determined by the HemoCue system  in duplicate from heparin whole blood samples. The coefficients of variation (CVs) for blood glucose concentration, hemoglobin, and total leukocyte counts were 3.0, 1.5, and 4.6%, respectively. Hematocrit was determined using the capillary method in triplicate (CV = 1.1%) from heparin whole blood samples and using a microhematocrit reader (ThermoFisher Scientific). Hemoglobin and hematocrit values were used to estimate changes in plasma volume relative to baseline and used to correct plasma variables. The remaining heparin whole blood samples were centrifuged at 4,000 rpm  for 10 min within 15 min of sample collection. Aliquots of heparin plasma were placed in 1.5-ml microstorage tubes and frozen at −80°C until analysis, except 2 × μl plasma was used to determine plasma osmolality in duplicate (CV = 1.1%).Circulating concentrations of cortisol , insulin , IGF-1 , testosterone , and estradiol  were measured by enzyme-linked immunosorbent assay (ELISA). The plasma concentrations of TNF-α, IL-6, IL-8, IL-2, and IL-10 were determined by high-sensitivity multiplex ELISA . All assays were performed as per the manufacturer's specifications, with standards and controls on each plate. The CV for the analyzed circulating biomarkers was ≤ 7.2% and for the systemic inflammatory cytokine profile was ≤ 13.5%. Systemic cytokine profile was established, as previously described .n = 36 (n = 8 per group), using a randomized controlled design as reported in Hanach et al. . Data in the text and tables are presented as either mean ± SD or mean and 95% confidence interval (CI), as indicated. For clarity, data in figures are presented as mean ± standard error of the mean (SEM). Only participants who completed the experimental design, adhered to the controlled intervention conditions, and with full datasets within each specific variable were included in the data analysis, as indicated in the table and figure legends. All data were checked for distribution using the Shapiro–Wilk test of normality. Variables with singular data points were examined using a one-way ANOVA or non-parametric Kruskal–Wallis test, when appropriate. Variables with multiple data points were examined using a two-way repeated-measures ANOVA with a matrix including group  and time . Assumptions of homogeneity and sphericity were checked, and when appropriate, adjustments to the degrees of freedom were made using the Greenhouse–Geisser correction method. Significant main effects were analyzed using a post-hoc Tukey's HSD test. Statistics were analyzed using SPSS statistical software  with significance accepted at P ≤ 0.05.Only participants that attended ≥80% of the PRT sessions and consumed ≥80% of the DM beverage over the 12-week intervention were included in the data analysis. Based on the statistical test, mean, standard deviation, and effect size  for outcomes of FFM, skeletal muscle strength, and physical performance and applying standard alpha (0.05) and beta (0.80) values, a sample size of h et al. , is estiP ≤ 0.05. Additionally, Cohen's d was applied to determine the magnitude of effect size for significant differences, with d ≥ 0.20 for small, d ≥ 0.50 for medium and d ≥ 0.80 for large effect size.Furthermore, correlations between changes in the primary variables  and the inflammatory and hormone markers were conducted at 6 and 12 weeks. This was carried out with a Pearson's or Spearman's correlation test, based on the data distribution. Significance was accepted at n = 8; EX+DM, n = 9; EX, n = 10; and CON, n = 10. Participants' baseline variables, based on group allocation, are summarized in The participants from this study came from a variety of sporting backgrounds, including endurance runners/race walkers (61%), cyclists (9%), aerobic gym goers (16%), or a combination of multiple activities (14%). The dropout rate for the current study was 20% between all four groups, with the dropout reasoning depicted in P = 0.538). In DM and EX+DM, the average compliance for the high-protein dairy milk beverage provisions, according to the food diaries and milk bottle returns, was 93% (88–97%) and did not differ between groups (P = 0.969). Average adherence to the standardized meal plan and food provisions, based on food diaries, was 81% (76–85%) and did not differ between groups (P = 0.821).In EX+DM and EX, the PRT was well-tolerated, with the average compliance for participants in the exercise program being 89% (95% CI = 85–93%), and did not differ between groups (*time interaction (P = 0.048) was observed for energy intake, indicating a significant increase in the DM (19%) and EX+DM (22%) groups at 12 weeks compared to baseline (P = 0.001) and relative protein intake  in the DM and EX+DM groups at 6 weeks  and 12 weeks . Similarly, there was a group*time interaction for calcium intake (P = 0.007). Further analysis indicated that DM and EX+DM had a significant increase in calcium intake at 6 weeks  and 12 weeks  compared to baseline (*time interaction effect was observed for protein (in grams per kilogram BM) at breakfast (P = 0.005) and dinner (P = 0.012) and toward significance at lunch (P = 0.055). Further analysis indicated that, compared to baseline, protein at breakfast significantly increased at 6 and 12 weeks in DM (≥44%) and EX+DM (≥55%) compared to EX and CON. Whereas, there was a significant decrease of relative protein intake (in grams per kilogram) at dinner in the EX+DM (50%) and EX (10%) groups at 6 and 12 weeks compared to baseline.A groupbaseline . Consumpbaseline . Based oP = 0.754). Based on the analysis of the accelerometer over the intervention period, there were no differences between groups for the amount of hourly kilocalories, time in sedentary, or time in light physical activity , absolute FFM (P = 0.051), and absolute and relative FM , or resting metabolic rate , chest press (P < 0.001 and P < 0.001), and lat pull-down .There was a significant groupVO2), and physical performance , there were no significant main effects or interactions from baseline to week 12 , cardiorespiratory fitness . There were no main effects or interactions observed for any other immune biomarkers measured. There was no significant correlation between the changes in any of the primary outcomes  and any of the systemic hormonal and inflammatory cytokine markers.There were no main effects or interactions observed for any of the hormonal biomarkers measured . There wlat pull-down) compared to PRT alone, but did not further augment changes in FFM, power, or physical performance. Moreover, the consumption of a high-protein dairy milk beverage during the PRT period resulted in significant increased levels of cytokine IL-10, suggesting an anti-inflammatory effect at this intervention, but it did not result in any anabolic hormone enhancements compared to other interventions or the control. Overall, these results suggest that the consumption of a high-protein dairy milk beverage, in combination with PRT, elicits greater effects on skeletal muscle strength outcomes than consuming the dairy milk beverage or PRT in isolation. This suggests that the DM does not seem to impact FFM, power, or physical performance any more than the CON in healthy active older adults. Therefore, high-protein dairy milk in combination with PRT may be an effective strategy in the prevention and management of age-related sarcopenia in the active aging population.This study aimed to determine the independent and combined effects of a high-protein dairy milk beverage provided at breakfast and lunch (or after resistance exercise), with or without PRT, on outcomes of FFM, skeletal muscle strength and power, and physical performance in active older adults. In conflict with the hypotheses, a high-protein dairy milk beverage did not influence gains in FFM, skeletal muscle strength, power, or performance compared to the control. Whereas, in accordance with the hypotheses, a high-protein dairy milk beverage provided and consumed twice daily, in conjunction with PRT, resulted in significant increases in strength  was significantly higher in EX+DM (53–78%) compared to EX (35–36%), DM (4–7%), and CON (7–11%), indicating an interaction effect. These findings align with a recent meta-analysis which reported that protein supplementation (20 ± 18 g protein/day) further augments strength (33%), as measured by 1RM leg press, in community-dwelling older adults (≥45 years) (−1 BM day−1 of protein at baseline (−1 BM day−1) to treat sarcopenia, it is below (1.6 g kg−1 BM day−1) the threshold recommended to support significant changes in muscle size and strength during prolonged resistance training in healthy active adults that are novice to weight training , the lack of a further significant effect may be influenced by the CON and EX groups that were consuming high habitual protein intakes throughout the study intervention  (−1 BM day−1), the EX+DM group still showed a significantly greater increase in maximal strength than the EX group. This may suggest that increasing protein intake by this magnitude with PRT can lead to additional adaptations in skeletal muscle strength, as previously reported in younger adults (−1 BM day−1) intakes than those currently recommended for active older adults (≥1.2 g kg−1 BM day−1) may be required to see optimal strength adaptations. This requirement is in accordance with the nutrition guidelines for strength and power athletes for adaptations in skeletal muscle strength following resistance training (The progressive decline in strength and FMM begins to be detectable from the age of ≥50 years . The rat5 years) . However5 years) , 50. One5 years) . Secondlbaseline , 50. Alttraining –55. FurtM day−1) . Althougr adults . The fintraining , 55.−1 BM day−1). Resistance training acutely sensitizes skeletal muscle mass to anabolic effects of ingested protein (The current study provided participants with a 15-g protein (1.57 g leucine) dose at breakfast and lunch (or after resistance exercise). This significantly increased the relative protein intakes at those meal times (≥25%) in DM and EX+DM compared to baseline. The provision of this protein dose at these time points was based on previous reports suggesting that the distribution of protein is often inadequate at those times in older adults . Additio protein . When co protein , 57. TheThere was a significant increase in absolute FFM in the EX+DM (1.2%) and EX 0.85%) groups at 12 weeks from baseline, whereas the DM group had a significant decrease in FFM (−1%) over the course of the intervention trial. The absence of any greater increase in FFM from the consumption of additional protein in DM aligns and conflicts with previous findings in studies that investigated PRT and protein intake in healthy community dwellers , 27, 51 5% groupsAging is associated with the redistribution of FM, characterized by an increase in the abdominal region  and a decrease in the appendicular (mostly subcutaneous fat) . This stn = 108) within the study by Daly et al.  is consistent with two meta-analyses that have reported mixed findings with regard to the benefit of additional dairy milk protein or protein supplementation on outcomes of physical performance , 67. In y et al. . Overallin vitro and in vivo models and could be possibly explained by the addition of the high-protein milk beverage. In particular, the addition of branched-chained amino acids (BCAAs) found in dairy milk acts as a substrate for the synthesis of short-chained fatty acids (SCFAs) such as butyrate , which has a direct correlation with the onset of sarcopenia , 84. In −1 BM day−1). These participants were not provided a control diet based on previous studies that have suggested that up to 50% of active older adults do not meet the protein requirement of 1.2 g kg−1 BM day−1  and compliance rate (≥80%) to the intervention, and in the comprehensive outcomes measured, including FFM, strength, power, and physical performance, accounting for outcomes that may be more relevant in an active aging cohort . This study also controlled for variables such as dietary intake through using food provisions and monitoring food intake through diaries. Physical activity was monitored over the course of the clinical trial to ensure that changes in outcomes were due to the exercise intervention and not due to the participants increasing habitual exercise outside the trial, which other studies have failed to implement. The measurement of blood markers  provided an extensive insight into the pathophysiology and potential mechanisms of the interventions. Previous nutrition and exercise intervention studies have shown significant interaction effects on sarcopenia outcomes  with a study size of 6–196 participants per group, in two to four group studies 0% and co. HoweverBM day−1 . TherefoBM day−1 , which m−1 BM day−1). There was a significant increase in FFM following the PRT, but no augmented effect with the high-protein dairy milk beverage. There was a significant decrease in FM in those consuming the high-protein milk beverage. Potentially, the influence from the protein or other constituents of the high-protein milk beverage  may have contributed to the significant reduction in FM observed. Additionally, EX+DM led to a significant increase in resting anti-inflammatory cytokine , which all may play a significant role in improving skeletal muscle mass and strength outcomes in active older adults . Finally, these results suggest that consuming a high-protein milk beverage at times that are usually inadequate in protein with PRT can facilitate gains in muscle strength, FFM, and reductions in FM and is well-tolerated by active older adults. Overall, the findings of this study are novel and define opportunities for future interventional studies examining age-related sarcopenia in the healthy active aging population.This study showed that a daily consumption of two high-protein milk beverages at breakfast and lunch (or after PRT) significantly enhanced the effects of PRT on skeletal muscle strength outcomes in active older adults who already have high levels of protein intake (≥1.2 g kgThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Monash University Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.ZH and RC contributed toward the original research idea. ZH, RC, and JP contributed toward development of the experimental design. ZH, RC, and AP contributed toward various aspects of data collection, sample collection, and analysis. ZH and RC contributed toward the analysis of the raw data. ZH was responsible for the initial manuscript draft. All authors contributed to the critical review of the manuscript and approved the final manuscript for submission.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.