To observe and determine the effect and mechanism of psoralen on tumor necrosis factor-α (TNF-α)-induced muscle atrophy.Three sets of C2C12 cells, including blank control, TNF-α (10 or 20 ng/ml) treatment and a TNF-α (10 or 20 ng/ml) plus psoralen (80 μM) administration were investigated. Cell viability was assessed using Cell Counting Kit-8 (CCK-8) assay. Western blot analysis was used to detect protein expression of atrophic markers. Flowcytometry was used to observe the effect of psoralen on apoptosis. A quantitative real-time PCR (qRT-PCR) assay was performed to detect the mRNA level of miR-675-5P.P<0.001), while 24 hr of psoralen administration increased the viability, and lowered TNF-α cytotoxicity (P<0.001). MURF1, MAFbx, TRIM62 and GDF15 expressions were significantly increased in TNF-α (10 ng/ml or 20 ng/ml)-treated group (P<0.001), and psoralen could significantly decrease the expression of these proteins (P<0.001). Apoptotic rate of C2C12 myoblasts was increased after TNF-α (10 ng/ml and 20 ng/ml) treatment, and was significantly decreased after psoralen treatment (P<0.001). miR-675-5P was increased in TNF-α-treated C2C12 myoblasts compared to control group, and it was significantly decreased after psoralen treatment.TNF-α  treatment inhibited C2C12 myoblast viability (Psoralen could reduce TNF-α-induced cytotoxicity, atrophy and apoptosis in C2C12 myoblasts. The therapeutic effect of psoralen may be achieved by down-regulating miR-675-5P. Muscle wasting is a life-threatening consequence of many critical disease -3 includPrior research has shown that microRNAs, a class of small non-coding RNAs of ~22 nucleotides, act as key regulators in the process of metabolic homoeostasis by promoting degradation or inhibiting the translation of specific mRNAs . It playInflammatory cytokines have been shown to play a central role in the loss of skeletal muscle mass. For example, TNF-like weak apoptosis-inducing factor (TWEAK) is a major cytokine that causes muscle atrophy -20. In tCell culture 2 and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal calf serum  and 1% penicillin-streptomycin G . The myoblast (C2C12) cell line was supplied by the Cell Bank of Type Culture Collection of Chinese Academy of Sciences . Cells were incubated at 37°C in a humidified atmosphere containing 5% COCCK-8 assay3 in a 96-well plate  for 24 hr. After washed by phosphate-buffered saline (PBS), we added 100 μl RPMI-1640 medium and 10 μl CCK-8 to each well. After 2 hr, the 96-well plate was measured at 450 nm using a standard micro plate reader . The cell viability was calculated according to the following formula: cell viability =  × 100%. Cell viability was determined by Cell Counting Kit (CCK-8) assay according to the manufacturer’s instructions. Briefly, C2C12 myoblasts were seeded at a density of 6×10Western blot assayCells were harvested and washed with PBS and then lysed in radioimmunoprecipitation assay buffer . The protein concentrations were determined using a BCA Protein Assay kit . Proteins were separated using SDS-PAGE gels based on the molecular weight of the target protein and was then transferred to an PVDF membranes , followed by incubation with primary antibodies overnight. The membrane was then incubated with HRP-conjugated secondary antibodies and developed using an ECL detection kit . The primary antibodies were anti-GAPDH , anti-MURF1 , anti-MAFbx , anti- tripartite motif containing 62 (TRIM62) , and anti- growth differentiation factor-15 (GDF15) .Flow cytometric analysisC2C12 myoblasts with different treatments were added into 6-wells plate and incubated overnight. Apoptosis rate was measured by flowcytometry using Annexin-V/FITC double staining  and the results were analyzed using the FlowJo software. Apoptosis of C2C12 myoblasts were analyzed in triplicates and repeated three times independently.RNA extraction and quantitative real-time PCR (qRT-PCR) assayThe mRNA expression levels were detected using the SYBR-Green PCR Master Mix kit (Takara). Total RNA was isolated using TRIzol  following the manufacturer’s instructions and reversely transcribed to cDNA by RT-PCR assay using a miScript II RT Kit . The target genes and controls were analyzed by qRT-PCR and the test was performed on ABI 7500 system  with primers specific for miR-675-5P . All data are expressed as the mean±SD of three independent experiments. The sequences of primers used were as follows:U6 primers: 5’-CTCGCTTCGGCAGCACATATACT-3’ (forward primer) 5’-ACGCTTCACGAATTTGCGTGTC-3’ (reverse primer); miR-675-5P primers: 5’-ACACTCCAGCTGGGTGGTGCGGAGAGGGCC-3’ (forward primer) 5’-CTCAACTGGTGTCGTGGA-3’ (reverse primer)Statistical analyses P<0.05 was considered statistically significant. Data were analyzed by Student’s t-test and variance (ANOVA) using SPSS 19.0 software . Each experiment was repeated at least three times. Data were summarized and presented as means±SD of three or more biological replicates. Psoralen reduced TNF-α-induced cytotoxicity in C2C12 myoblastsP<0.001), indicating that TNF-α has cytotoxic effect on C2C12 myoblasts. To examine the effect of psoralen on C2C12 myoblasts growth viability, we used different psoralen concentrations  to examine on normal cultured, as well as TNF-α -treated C2C12 myoblasts. As shown in CCK-8 assay was carried out to evaluate cell viability. As illustrated in Psoralen attenuated TNF-α-induced muscle atrophyP<0.001), and psoralen could significantly decrease the expression of these proteins (P<0.001).The protein expressions of MURF1, MAFbx, TRIM62 and GDF15 atrophy markers were measured by western blot. As showed in Psoralen inhibited TNF-α-induced apoptosis in C2C12 myoblastsP<0.001). However, it was significantly decreased after psoralen treatment (P<0.001). We suggested that psoralen suppressed TNF-α-induced apoptosis in C2C12 myoblasts.Flowcytometry assay was used to explore whether cell apoptosis could be affected by psoralen. As showed in Psoralen inhibited miR-675-5P expression in C2C12 myoblasts induced by TNF-αThe results of qRT-PCR indicated that expression of miR-675-5P was increased in TNF-α-treated C2C12 myoblasts compared to control group. However, the miR-675-5P expression was decreased after psoralen treatment . Muscle atrophy is caused by the imbalance between anabolic and catabolic processes. In the course of its development, the protein breakdown exceeds protein synthesis and ultimately results in a loss of muscle mass. Sepsis, cancer, diabetes mellitus, heart failure and surgery are all the reason of muscle atrophy -24. Exeret al. and Cai et al. demonstrated that the proinflammatory transcription factor nuclear factor-kappa B (NF-kB) regulated the expression of large number of genes associated with the ubiquitin-proteasome system  up-regulated the expression of MURF1, MAFbx, TRIM62 and GDF15 proteins in C2C12 myoblasts. In order to observe the effect of psoralen on muscle cells, we designed a cell group using psoralen alone and found that psoralen does not induce muscle atrophy in C2C12 myoblasts. However, when we used psoralen together with TNF-α in C2C12 cells, we found that psoralen could attenuate TNF-α-induced muscle atrophy. We speculate that psoralen has a protective effect on C2C12 myoblasts.Researches show that miR-675 is overexpressed in many human cancers; however, the function of miR-675-5P is largely unknown . In our Overall, our current study shows that, by down-regulating miR-675-5P, psoralen can inhibit cytotoxic and pro-apoptotic effects of TNF-α-treated C2C12 myoblasts, thereby exerting its protective effect on muscle atrophy. Our research laid a clinical foundation for psoralen on the treatment of muscle atrophy, and we also provided a theoretical basis for the future research on the mechanism of muscle atrophy.


Some of these mechanisms, such as RBP-J and its mediated regulatory network, uniquely and predominantly limit osteoclastogenesis mediated by TNF but not by RANKL. As a consequence, targeting RBP-J activities suppresses inflammatory bone destruction but does not significantly impact normal bone remodeling or inflammation. Hence, discovery of these intrinsic inhibitory mechanisms addresses why TNF has a weak osteoclastogenic potential, explains a significant difference between RANKL and TNF signaling, and provides potentially new or complementary therapeutic strategies to selectively treat inflammatory bone resorption, without undesirable effects on normal bone remodeling or immune response in disease settings.TNF (Tumor necrosis factor) is a pleiotropic cytokine that plays an important role in immunity and inflammatory bone destruction. Homeostatic osteoclastogenesis is effectively induced by RANKL (Receptor activator of nuclear factor kappa-B ligand). In contrast, TNF often acts on cell types other than osteoclasts, or synergically with RANKL to indirectly promote osteoclastogenesis and bone resorption. TNF and RANKL are members of the TNF superfamily. However, the direct osteoclastogenic capacity of TNF is much weaker than that of RANKL. Recent studies have uncovered key intrinsic mechanisms by which TNF acts on osteoclast precursors to restrain osteoclastogenesis, including the mechanisms mediated by RBP-J signaling, RBP-J and ITAM (Immunoreceptor tyrosine-based activation motif) crosstalk, RBP-J mediated regulatory network, NF- Under inflammatory conditions, abnormal osteoclast differentiation and function often results in excessive bone resorption, which is a common characteristic of many diseases, such as osteoporosis, rheumatoid arthritis (RA), psoriatic arthritis and periodontitis  has been a medical breakthrough that successfully ameliorates the quality of life of patients suffering from TNF-mediated diseases. TNFi includes monoclonal antibodies to TNF, such as Infliximab, Adalimumab, Certolizumab Pegol, and Golimumab, as well as soluble TNF receptor(s), such as Enbrel. Using TNFi has shown to help treat inflammation and joint erosion that occur in RA. However, immunosuppression from long-term utilization of TNFi is a side effect that can result in patients being susceptible to opportunistic infections. Understanding the mechanistic difference between RANKL-mediated physiological and TNF-mediated inflammatory osteoclastogenesis, and especially TNF-induced intrinsic inhibitory mechanisms, will strengthen the development of therapeutic approaches to treat pathological bone destruction in disease settings and prevent negative side effects on bone remodeling and immunity.Inflammatory conditions have complex impacts on osteoclastogenesis and bone remodeling , 5, 6. Cvia multiple mechanisms, such as increasing osteoclast precursor cells, acting on other cell types and in synergy with additional cytokines, mostly with RANKL (Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that is important for inflammation, immunity, and disease pathogenesis. TNF is known to play a key role in driving chronic inflammation as well as in pathological bone erosion associated with multiple inflammatory bone diseases, such as RA, periodontitis and periprosthetic osteolysis , 2, 7, 8th RANKL , 8. SincRecent studies have provided evidence that uncover TNF-mediated intrinsic inhibitory mechanisms during osteoclastogenesis. In this review, we will highlight these discoveries and discuss their potential clinical relevance in treating inflammatory bone destruction.in vivo  translocate to the nucleus and bind to RBP-J, which induces Notch target gene transcription. RBP-J has also been shown to function as a transcriptional activator or repressor for other signaling pathways, such as TNF (β-catenin (κB (Recombinant recognition sequence binding protein at the Jκ site (RBP-J) is a nuclear DNA-binding protein that is expressed in a wide range of cell types and was originally identified as a key transcription factor in the canonical Notch signaling . Upon ach as TNF , TLR , deletion of Notch 1/2/3, or constitutively-active NICD1 expression in the myeloid compartment (NICD1M) in mice did not exhibit significant bone defects under physiological conditions , which are osteoclast precursors, and dramatically suppresses TNF-induced osteoclastogenesis and bone resorption  in vivo , 32, whi in vivo , 32, 40.nditions , 41. Howignaling . Thus, RThe mechanism by which RBP-J restrains TNF-induced osteoclastogenesis is by suppressing induction of NFATc1 through attenuation of c-Fos activation and suppression of Blimp1 induction . These eγ subunit (FcRγ). These adaptors associate with various receptors in myeloid cells to mediate signaling, including DAP12-associated triggering receptor expressed in myeloid cells 2 (TREM2), signal-regulatory protein β 1 (SIRPβ1), FcRγ-associated osteoclast-associated receptor (OSCAR), paired immunoglobulin-like receptor-A (PIR-A) and FcRs. Osteoclasts require co-stimulation of RANK via ITAM-mediated signaling pathways to drive osteoclastogenesis  is a highly conserved signaling motif contained in the cytoplasmic domain of transmembrane receptors and adaptors that are crucial mediators of various cellular activities, particularly immune response and cancer activation. ITAM-mediating signaling regulates hematopoietic cells including myeloid osteoclast precursor cells. The main ITAM-containing adaptors expressed by myeloid osteoclast precursors that play crucial roles in the osteoclast program include DNAX-activating protein 12 (DAP12) and Fc receptor common ogenesis . Deficie defects . This inntiation   and protein kinase double-stranded RNA-dependent (PKR) compared to healthy donors , 47. TheκB family is involved in a number of cellular functions and plays a key role in immunity and proinflammatory signaling pathway. This family includes the transcription factors p65/RelA, RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). NF-κB activation can be induced via canonical or classical signaling pathway involving TNFR activation or TLR ligand binding and dependent on IKKβ-induced IκBα degradation, which leads to RelA/p50 activation and downstream gene transcription. The non-canonical or alternative pathway involves NIK-induced phosphorylation of NF-κB2/p100, which is crucial for p100 processing to p52 and RelB/p52 activation for downstream gene transcription. RelB was found to be essential for RANKL-induced osteoclast maturation and TNF-induced bone resorption , limiting TNF-induced osteoclastogenesis  , 57, Eosvia interplay of DNA methylation and histone post-translational modifications. DNA methylations via de novo DNA methyltransferases (DNMTs), such as DNMT3a and DNMT3b, and histone post-translational modifications (histone tail modifications), such as acetylation, methylation, phosphorylation, ubiquitylation, and sumoylation of histones, are common types of epigenetic modifications. Epigenetic repressors generally include DNMTs, histone deacetylases (HDACs), and polycomb group proteins (PcG). It has been shown that epigenetic repression of Irf8 by Dnmt3a-mediated DNA hypermethylation leads to decreased IRF-8 expression and enhanced osteoclastogenesis and bone resorption , also known as IRF4-binding protein (IBP) or SWAP-70-like adaptor protein of T cells (SLAT), is a type of guanine nucleotide exchange factor (GEF) expressed predominantly in T cells and regulates T cell development, activation, and function , 66. Expogenesis . Def6 deogenesis , leadingRBP-J was validated as an RA risk allele in a genome-wide association study (GWAS) meta-analysis  and The Tow Foundation . The content of this manuscript is solely the responsibilities of the author and does not necessarily represent the official views of the NIH.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epichloë coenophiala represent two of the most prevalent stressors to beef cattle in the United States and cost the beef industry millions of dollars each year. The rate at which a beef cow sheds her winter coat early in the summer is an indicator of adaptation to heat and an economically relevant trait in temperate or subtropical parts of the world. Furthermore, research suggests that early-summer hair shedding may reflect tolerance to fescue toxicosis, since vasoconstriction induced by fescue toxicosis limits the ability of an animal to shed its winter coat. Both heat stress and fescue toxicosis reduce profitability partly via indirect maternal effects on calf weaning weight. Here, we developed parameters for routine genetic evaluation of hair shedding score in American Angus cattle, and identified genomic loci associated with variation in hair shedding score via genome-wide association analysis (GWAA).Heat stress and fescue toxicosis caused by ingesting tall fescue infected with the endophytic fungus h2 = 0.34 to 0.40), with different repeatability estimates between cattle grazing versus not grazing endophyte-infected tall fescue. Our results suggest modestly negative genetic and phenotypic correlations between a dam’s hair shedding score (lower score is earlier shedding) and the weaning weight of her calf, which is one metric of performance. Together, these results indicate that economic gains can be made by using hair shedding score breeding values to select for heat-tolerant cattle. GWAA identified 176 variants significant at FDR < 0.05. Functional enrichment analyses using genes that were located within 50 kb of these variants identified pathways involved in keratin formation, prolactin signalling, host-virus interaction, and other biological processes.Hair shedding score was moderately heritable (This work contributes to a continuing trend in the development of genetic evaluations for environmental adaptation. Our results will aid beef cattle producers in selecting more sustainable and climate-adapted cattle, as well as enable the development of similar routine genetic evaluations in other breeds. Lolium arundinaceum) is the most widely available forage in the United States [Epichloë coenophiala. E. coenophiala produces ergot alkaloids that benefit the forage by increasing drought tolerance and pathogen resistance [At the beginning of the summer, many mammalian species molt thick winter coats in response to changing day length in order to prepare for warmer temperatures –6. Thered States , thanks sistance , but negsistance . Therefosistance . Howeversistance –19, partsistance . Here, wAll data originated from purebred cattle registered in the American Angus Association (AAA) and commercial cattle enrolled in the AAA Breed Improvement Record program. Phenotypic data comprised hair shedding scores recorded by beef cattle producers enrolled in the Mizzou Hair Shedding Project (MU data) between 2016 and 2019 in combination with hair shedding scores collected by technicians in 2011, 2012, 2018, and 2019 as part of Angus Foundation-funded projects at Mississippi State University and North Carolina State University (AGI data). Across all years and both datasets, scores were recorded on 1 day between April 17th and June 30th in the late spring or early summer, with most scores recorded in mid- to late-May. Hair shedding was evaluated using a 1 to 5 visual appraisal scale, where 1 was 0% dead winter coat remaining and 5 was 100% winter coat remaining based on the systems developed by Turner and Schleger  and GrayRecords were removed when the breeder-reported sex of an animal did not match the sex recorded in the AAA pedigree. Hair shedding scores that originated from male animals comprised less than 1% of the dataset and only female records were retained. Age classifications were assigned to each record based on age in days determined by the AAA-recorded birth date and the date the hair shedding score was recorded. Similar to the system used in the Beef Improvement Federation (BIF) Guidelines for age-of-dam classification , age claentclass1pt{minimaGenotypes for 3898 of the 8619 animals were imputed to a union marker set (te v.3.0 . TherefoIn single-step genomic best linear unbiased prediction (BLUP) as used in the AAA National Cattle Evaluation (NCE), relationships between individuals are represented in the matrix matrices , allowinn method  and was  program . In all Understanding how and which environmental factors shape phenotypic variation enables the development of more appropriate contemporary group definitions during genetic evaluation. In order to quantify the effect of animal age on hair shedding score, we fitted age as a categorical fixed effect in a repeated records animal model. Age categories were defined in three ways. First, age in years was fit . Second, ages were grouped as 1, 2, 3, or other (“four-class model”). Third, age groups were defined according to the guidelines set forth by the BIF for age-of-dam effects on birth weight and weaning weight  plus yeCattle reared in heat-stressed regions but not exposed to endophyte-infected fescue demonstrate similar benefits from early summer hair shedding, but it is unclear if the biological mechanisms that govern hair shedding under fescue toxicosis and heat stress alone are the same. This could have implications for routine genetic evaluation, as it might require that some hair shedding score observations be treated as a separate trait. In order to clarify the relationship between hair shedding score while grazing toxic fescue versus while not grazing toxic fescue, we calculated the covariance and genetic correlation between hair shedding score grazing toxic fescue and not grazing toxic fescue using the bivariate repeated records animal model below:In addition, we fitted a univariate model with toxic fescue grazing status included as a categorical fixed effect. The goal of this model was to quantify the effect of reported toxic fescue grazing status on hair shedding score:In both models, only females with known toxic fescue grazing status were retained for analysis. Contemporary groups with fewer than five animals or no variation were discarded, resulting in 5832 observations from cattle grazing toxic fescue and 4197 observations from cattle not grazing toxic fescue. Three hundred ninety-six animals had observations over multiple years both grazing and not grazing toxic fescue.Variance components, heritability, repeatability, and breeding values were estimated using the univariate repeated records animal model below implemented in AIREMLF90 .The definition of contemporary groups used in this final prediction model was informed by the results of the age classification and toxic fescue grazing status analyses above. It included a combination of farm, year scored, calving season , toxic fescue grazing status (yes or no), age group , and score group. In herds where cattle were scored for hair shedding over more than 1 day, the score group was determined using a 7-day sliding window to maximize the number of animals per contemporary group. In the future, it will be recommended that producers score all cattle for hair shedding within a week of one another to maximize the size of contemporary groups. Although yearling heifers have not yet experienced the stress of pregnancy, calving season/birth season is a good proxy for management group in the absence of breeder-reported codes. Therefore, “calving season” was included in the contemporary group definition for all animals regardless of reproductive status. Contemporary groups with fewer than five animals or no variation were dropped. This resulted in 14,438 total scores from 8449 animals in 395 contemporary groups.In order to evaluate model bias, we estimated breeding values in ten separate iterations, excluding all phenotypes for a randomly selected 25% of animals. These “partial” breeding values were then compared to breeding values obtained via the “whole” model including all possible information using the “LR method” parameters proposed by Legarra and Reverter . First, The effects of heat stress on pre-weaning growth are well characterized in cattle. Heat stress impacts calf performance most severely via reduced milk production in the dam . Fescue Weaning weight phenotypes and contemporary group designations came from the weekly growth run of the AAA national cattle evaluation (NCE). Prior to entering the NCE, phenotypes were adjusted for age-of-dam effects as used in the AAA weekly NCE and to 205 days-of-age. Weaning weight data were retrieved for: (1) own weaning weight of cows with at least one hair shedding score recorded, (2) all of cow’s recorded calves, (3) cow’s weaning weight contemporary group peers, and (4) all of their recorded calves’ weaning weight contemporary group peers. Weaning weights from animals born via embryo transfer and contemporary groups with fewer than five animals or no variation were excluded, resulting in 40,794 total weaning weights and 14,039 total hair shedding score records. Of the 45,420 phenotyped animals retained for analysis, 3850 had both a recorded weaning weight and at least one hair shedding score. Furthermore, 6448 dams had both hair shedding scores and calf weights recorded in at least 1 year (n = 9092 score/weight pairs).Conceivably, environmental factors that affect a dam’s hair shedding performance could also affect the direct weaning weight of her calf and her maternal effect on the calf’s growth, creating a residual covariance between the two traits. In order to reflect this covariance, a bivariate model was fitted in which a direct hair shedding score effect was modeled for the cow, a direct weaning weight effect was modeled for the calf, and a maternal weaning weight effect was modeled for the cow. In practice, this model was implemented by fitting a maternal genetic effect for hair shedding, no direct genetic effect of hair shedding , and direct and maternal genetic effects for weaning weight. This model created a direct tie between a dam’s hair shedding score and the calf she weaned that year, which reflects more accurately the relationship of interest and is similar to models used to assess the correlations between weaning weight and actual milk yield . For cowWe also evaluated the phenotypic relationship between dam hair shedding score and calf weaning weight using the subset of 6448 dams with both hair shedding scores and calf weights recorded in at least 1 year. We did this by calculating the estimated change in calf weaning weight as a function of dam hair shedding score using four separate simple linear regression models. In the first two models, unadjusted calf weaning weight was regressed on unadjusted dam hair shedding score. Using weaning weight unadjusted for age in days captures increased gain from an earlier birth date (older when weighed), which might be an indicator of increased fertility for earlier shedding cows. In the other two models, 205-day, age-of-dam, and contemporary group solution adjusted calf weaning weight was regressed on unadjusted dam hair shedding score. Both the unadjusted weaning weight and adjusted weaning models were fitted separately for all available data, dams explicitly recorded as grazing toxic fescue, and dams explicitly recorded as not grazing toxic fescue.In order to evaluate the genetic architecture of hair shedding and identify variants that contribute to hair shedding score breeding values, we performed a single-SNP genome-wide association analysis using the SNP1101 v.1 software . The bren method .q-value lower than 0.05. The size of our search space was determined based on the density of our marker set, and the resulting gene list was used as input for cluster enrichment analysis within ClueGO v.2.5.6 [Using the UMD 3.1 bovine genome assembly  coordina v.2.5.6 . KEGG pa v.2.5.6 , consideThe results of the age-in-years model suggest a non-linear effect of age with larger effect sizes in 2-year-old, 3-year-old cows, yearlings, and old cows relative to mature cows . Furthermore, the Pearson correlation between breeding values grazing and not grazing toxic fescue was 0.99. The total phenotypic variation in hair shedding grazing toxic fescue was slightly higher than hair shedding not grazing toxic fescue, which suggests that reduced peripheral blood flow caused by fescue toxicosis is more detrimental to hair shedding than heat stress alone (Table r) was much higher for hair shedding while grazing toxic fescue . For dams not grazing toxic fescue, the rg between the direct and maternal effect of weaning weight fell near commonly reported estimates  and for all possible dams (rg = − 0.43) (Table rg between the direct effect of weaning weight and hair shedding ranged from − 0.10 (dams not grazing toxic fescue) to − 0.03  to 0 (dams grazing toxic fescue).All three bivariate models suggest a moderately negative genetic correlation between weaning weight and hair shedding score. In the model using all available data, the estimated 19 Table . When th − 0.29; ) but wasIn the simple linear models predicting unadjusted weaning weight from dam hair shedding score, unadjusted calf weaning weight was estimated to decrease by 1.30 kg with every unit increase in hair shedding score using all available data, by 3.22 kg for dams grazing toxic fescue and by 5.08 kg for dams not grazing toxic fescue. Slope estimates from the simple linear models predicting adjusted weaning weight from dam hair shedding score were more modest but also negative. Adjusted calf weaning weight was estimated to decrease by 1.45 kg using all possible data, by 2.47 kg among dams grazing toxic fescue, and by 1.11 kg among dams not grazing toxic fescue with every unit increase in hair shedding score , which are involved in creating structural epithelial cells like hair.We found 176 variants that passed the genome-wide false discovery rate threshold of 0.05 and 56 variants that passed the false discovery rate threshold of 0.01  for pathways involved in virus-host interaction, fat cell differentiation, prolactin signalling, cellular response to starvation, vasopressin-regulated water reabsorption, and other biological processes Table . We alsoThe expression of a phenotype is not always consistent across lifespan . We founAlthough our results suggest a high correlation between hair shedding score breeding value across toxic fescue grazing status, we found a slightly higher heritability estimate and much larger effect of permanent environment among cattle grazing toxic fescue than those not grazing toxic fescue. Stress can sometimes increase phenotypic variation , which crg lower than 0.80 are considered “very different” [rg of 0.93, which suggests minimal re-ranking of breeding values. However, the magnitude of the difference in permanent environment effects found here may justify treating hair shedding grazing and not grazing toxic fescue as separate traits in research studies that examine physiological or non-additive genetic effects. For the implementation of the American Angus NCE, we have chosen to minimize the effect of toxic grazing status by including it in the definition of contemporary groups. Many biotic and abiotic factors affect the prevalence of toxicity-inducing ergot alkaloids within forage, including moisture, reproductive status, soil nitrogen, and most notably, temperature [Typically, measurements of the same trait across different environments that result in genetic correlations fferent” . Hair shperature . Previouperature . HoweverPRL) and its receptor (PRLR) that cause abnormal pelage, milk production, and thermoregulation phenotypes in cattle [rg = − 0.58), which was identical to the correlation reported by Turner and Schleger [rg between hair shedding score and the maternal effect of weaning weight increases relative to the rg estimated using all data. This is similar to the results reported in Hoff et al. [Our enrichment results identified pathways associated with prolactin signalling, which is a well-known modulator of seasonal hair shedding and hair growth as well as milk production . In 2014n cattle . Furthern cattle . Gray etn cattle  suggesten cattle   proteins [BBS1 was associated with local adaptation in Red Angus cattle [The two most significant associations were both detected in eceptors , 60. Phoeceptors , and chaproteins . Recentls cattle . Furthers cattle  affectinMany strategies have been proposed to phenotype heat stress in cattle. These methods often require the use of specialized equipment and training , or at Cattle produced in sub-tropical environments account for nearly 80% of the global beef herd . HoweverWe developed a prototype genetic evaluation for early-summer hair shedding in American Angus cattle in order to enable genetic selection for heat tolerance. In agreement with previous research , 22, we


The fecal viral load was positively associated with disease severity and negatively associated with serum anti-rotavirus immunoglobin A. High and persistent rotavirus shedding among vaccinated children with breakthrough disease may contribute to ongoing transmission in this setting.Quantifying rotavirus shedding among vaccinated individuals will aid understanding of vaccine indirect effects. Serial stool samples were collected from 196 children who presented with rotavirus gastroenteritis to health facilities in Blantyre, Malawi, and were tested for rotavirus using a VP6 semi-quantitative, real-time polymerase chain reaction. The median duration of fecal shedding was 28 days . The median copy numbers for peak shedding were 1.99 × 10 Serial stool samples from Malawian children with rotavirus gastroenteritis tested for rotavirus using polymerase chain reaction had high levels of viral shedding despite vaccination. High and persistent rotavirus shedding among vaccinated children with breakthrough disease may contribute to ongoing transmission. Rotavirus vaccine has been introduced in over 90 countries worldwide, including 45 low-income or GAVI Alliance-eligible countries . HoweverThe dynamics of fecal shedding of rotavirus in unvaccinated populations has been examined in children with both asymptomatic rotavirus infection and with clinical rotavirus disease using electron microscopy, enzyme-immune assay, and polymerase chain reaction (PCR) . Fecal sThere is a lack of shedding data from vaccinated infants in any setting, including infants from sub-Saharan Africa, in whom differences in intestinal integrity, nutritional state, comorbidities, and immune responses may lead to variations in shedding, with concomitant impacts on transmission. We aimed to describe patterns of wild-type rotavirus shedding over time and identify the factors associated with fecal viral load in children age-eligible for vaccination with symptomatic rotavirus disease in Malawi, an LIC in southern Africa. The live oral monovalent rotavirus vaccine was introduced into Malawi’s national immunization program on 29 October 2012, with doses at 6 and 10 weeks of age.Children age-eligible for vaccination (born on or after 12 September 2012 and >6 weeks old) presenting with acute gastroenteritis to Queen Elizabeth Central Hospital and to 3 government health centers in Blantyre, Malawi, between 16 February 2015 and 11 November 2016 were tested for rotavirus using immunochromatographic tests on a fecal sample. Following informed consent, rotavirus-positive children had demographic data and 2 mls of serum collected at recruitment to measure anti-rotavirus immunoglobin (Ig) A titres; a second bulk stool sample was obtained 48 hours after presentation (primary cohort). A subset of children had more intensive stool sampling carried out for 28 days (intensive cohort); samples were collected daily from the time of presentation for the first 7 days after symptom onset, twice weekly from 7 until 14 days, and weekly from day 14 until day 28.Disease severity was defined using the 20-point Vesikari score . Severe acute malnutrition was defined by either a weight-for-height Z-score (WHZ) less than -3 standard deviations, a mid–upper arm circumference <115 mm, or nutritional edema . WeightsStool samples were tested for wild-type rotavirus using a real-time semi-quantitative reverse transcription PCR . An addiSerum anti-rotavirus IgA titres were measured using a semi-quantitative sandwich enzyme-linked immunosorbent assay . ResultsP value of ≤.1 on a univariate analysis were selected for evaluation in the multivariable model. Nested models were compared using F tests. Variables which improved the model fit (P ≤ .05) were retained in the final model.The viral load data did not follow a normal distribution, so were log-transformed for analysis. Risk factors for fecal viral shedding density were investigated using multivariable linear regression, where the outcome variable was the peak log-viral load  in children from the primary cohort. Variables achieving a Wald test Data from the intensive cohort were used to evaluate changes in viral load over time using linear mixed models with a random intercept to account for within-child clustering; for this analysis, all data were included, regardless of viral load. Polynomial terms (quadratic and cubic) were included to account for the non-linear relationship between fecal viral load and time.A time-to-event analysis was used to estimate the duration of viral shedding in children from the intensive cohort. The event of interest was defined as the cessation of shedding and the start time for analysis was the onset of symptoms. Cessation of shedding was defined as the last time point from which rotavirus could be detected until censoring. Thus, an individual with no detectable rotavirus at a given analytical time point but who was shedding rotavirus in subsequent samples was classified as having ongoing shedding at the time of analysis. The follow-up was limited to 28 days.We recruited 196 index children, from whom 374 fecal samples were collected . The 2 unvaccinated children had peak viral loads comparable to the vaccinated children . In a multivariable analysis, a positive association between peak shedding density and Vesikari score and a negative association between peak shedding density and IgA titres were identified  protection appears limited despite high vaccine coverage . HoweverIncreasing anti-rotavirus IgA titres at presentation were negatively associated with fecal viral load, independent of disease severity. Serum anti-rotavirus IgA titres are known to correlate with intestinal IgA titres , and it We found that rotavirus viral shedding density is negatively associated with WHZ, implying that increasingly malnourished children shed more of the virus. Careful interpretation is required, as the standard deviations for our anthropometric measurements are outside the World Health Organization range for data quality assessment purposes . NeverthChildren in Malawi shed large quantities of rotavirus for an extended period following an episode of moderate-to-severe diarrhea, despite prior vaccination. Persistently high fecal virus shedding may contribute to the high prevalence of asymptomatic infections in young children in the community and to ongoing rotavirus transmission. While reduced disease severity in Malawian children was associated with lower viral shedding density, the potential impact of this on population-level rotavirus transmission remains to be determined.Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at The jiz612_suppl_TableS1Click here for additional data file.jiz612_suppl_TableS2Click here for additional data file.jiz612_suppl_TableS3Click here for additional data file.jiz612_suppl_TableS4Click here for additional data file.


Listeria monocytogenes, Mycobacterium tuberculosis, and Leishmania major. Furthermore, we discuss the role of TNF in the generation of proinflammatory macrophages in mouse models of infection and summarize briefly the potential consequences of anti-TNF treatment for infectious diseases.An interesting puzzle is the fact that an infection of a tumor necrosis factor α (TNF)-deficient host with pathogens such as bacteria or parasites that reside intracellularly inevitably ends fatally. Is this due to one specific role of TNF in the immune defense or are different functions responsible for this outcome? In this review we provide an update of the functions of TNF in the defense against the intracellular pathogens  Listeria monocytogenes, Mycobacterium tuberculosis, and Leishmania spp. Since macrophages are the predominant host cells of many of those infections, this cell type and the role of TNF in its differentiation will be discussed in particular.The increased susceptibility of a host organism to pathogens has many underlying factors. These range from environmental causes such as the effects of malnourishment on the immune system through to intrinsic differences resulting from genetics. Some susceptibility genes have been relatively easy to identify since the functions of their encoded proteins are directly associated with pathogenic resistance. For example, the natural resistance-associated macrophage protein (Nramp) has been shown to be crucial in the fight of macrophages against intracellular pathogens including mycobacteria, salmonella, and leishmania . SusceptListeria monocytogenes is an intracellular, food-borne, gram-positive pathogen that causes listeriosis, which is characterized by gastroenteritis, septicaemia, and meningitis. Infections have high hospitalization and fatality rates in immunocompromised patients and can result in spontaneous abortions during pregnancy  and a functional MyD88 pathway which leads to the expression of proinflammatory cytokines, including TNF and IL-12  is caused by an infection with orldwide . With muorldwide . Predomirculosis . In the rculosis . This earculosis .M. tuberculosis infection with increased bacterial loads  has also been produced as an important effector in host defense against nfection . Moreovetibility . Interes of iNOS . The vieype mice . A caveaM. tuberculosis strains in resistant and iNOS–/– mouse strains showed that the role of iNOS in protection depended to a large extent on the virulence of the infecting strain. The attenuated strain was controlled by both mouse strains while the iNOS–/– mice showed exacerbated infection with the virulent strain  and attenuated (H37Ra) t strain .+ T cell depletion caused reactivation despite unimpaired iNOS expression implying other macrophage factors in host defense  and to reactive nitrogen species, and to citrulline which can be reused via the citrulline-NO cycle . High leIn mouse models, M2 macrophages are characterized by expression of arginase (Arg1), an enzyme which hydrolyzes L-arginine to ornithine and urea. The arginase pathway facilitates the downstream synthesis pathways for polyamine and proline via ornithine, which M2 macrophages can utilize for tissue repair. Importantly, since Arg1 utilizes the same substrate as iNOS, Arg1 naturally limits L-arginine availability for NO synthesis by iNOS . M2 macrWhile TNF is a major product of activated macrophages it also shows a strong influence on the activation and development of these cells. IFN-γ-induced macrophages express TNF in autocrine or paracrine manner  which syA single pulse of TNF produces an extended yet transient activation of the NF-κB pathway in macrophages  which, cL. monocytogenes infection (L. monocytogenes infection. After infection monocytes are recruited to the spleen via CCR2 (int/CD11cint dendritic cells (TipDCs). These cells are not required for in vivo T-cell priming but represent an effector arm of the innate immune system producing iNOS and TNF that elicit protective immune responses against L. monocytogenes  succumbed to L. monocytogenes infection, demonstrating that other effector mechanisms can compensate for an absence of NO (Nitric oxide is one of the central effector molecules for defense against nfection . For exanfection . Macrophvia CCR2  and diffytogenes . Interesce of NO . In the ce of NO .M. tuberculosis. However, a common signature is the expression of various amounts of iNOS, IL-1β, TNF, and IL-12 which corresponds to an M1 program (M. tuberculosis has been shown to significantly reduce the innate immune response significantly. Binding of ESAT-6 to TLR-2 prevented the downstream activation of NF-κB (Macrophages respond differently to infection by different genotypes of  program . The con program . This ea program  and also program . Howeverof NF-κB .M. tuberculosis infection (M. tuberculosis (M. tuberculosis burden and smaller cellular infiltrates in the lungs after aerosol infection (M. tuberculosis by macrophages (A different line of research has shown Wnt6 signaling in granulomas induces arginase and downregulates TNF expression, driving a M2 polarization in a mouse model of nfection . This parculosis . The altrculosis . Howeverrculosis . Notablynfection . Alternanfection . Likewisrophages .L. major infections is driven by a strong Th1 response and an aligned M1 polarization of macrophages (L. major (L. major infected TNF–/– mice (+iNOS+ cells than did WT lesions but additionally almost all iNOS+ cells co-expressed Arg1. This occurred only in around 50% of WT CD11b+iNOS+ cells. This overexpression of Arg1 in the absence of TNF resulted in a lack of tyrosine nitration in the lesion and the draining lymph nodes, which caused by a strongly reduced activity of NO in the tissue despite a presence of iNOS (Resistance to experimental rophages . The expL. major . However–/– mice . An anal of iNOS . Indepen of iNOS .in vivo even under so called steady state conditions (While there is now broad agreement about the development and activation pathways of monocytes and macrophages, it is increasingly clear that we are not looking at one relatively homogenous population but a continuum of very different developmental states . Macrophnditions . Under inditions  and thisnditions .in vivo context has been a major goal, but it is not fully supported by the transcriptional analysis of signature molecules (in vivo models at a single cell level represents the ultimate challenge and the understanding of the regulation of transcriptional changes in response to a dynamic and complex environment is still in its infancy.As a consequence, the notion to define myeloid cell populations by only a few marker molecules has been challenged. Identifying combinations of key marker molecules that accurately characterize macrophage subpopulations and related dendritic cells in the olecules . Rather,olecules , 2014 anolecules  or the colecules . TherefoFor many years, TNF has been acknowledged as an important pro-inflammatory cytokine that plays a clearly important yet still only vaguely defined role in the immune response. A puzzling observation has been the dominant part it plays in response to intracellular infections by bacteria and parasites where the humoral response is subservient. Recently, a new role of TNF in the biology of macrophages was demonstrated  and the XLi: original draft preparation. HK and XLiu: review, editing, and supervision. XLi and XLiu: funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sodium–glucose linked transporter type 2 (SGLT-2) inhibition has been shown to reduce cardiovascular mortality in heart failure independently of glycemic control and prevents the onset of atrial arrhythmias, a common co-morbidity in heart failure with preserved ejection fraction (HFpEF). The mechanism behind these effects is not fully understood, and it remains unclear if they could be further enhanced by additional SGLT-1 inhibition. We investigated the effects of chronic treatment with the dual SGLT-1&2 inhibitor sotagliflozin on left atrial (LA) remodeling and cellular arrhythmogenesis  in a metabolic syndrome-related rat model of HFpEF.2+ transients  and spontaneous Ca2+ release were recorded by ratiometric microscopy using Ca2+-sensitive fluorescent dyes (Fura-2) during various experimental protocols. Mitochondrial structure (dye: Mitotracker), Ca2+ buffer capacity (dye: Rhod-2), mitochondrial depolarization (dye: TMRE) and production of reactive oxygen species (dye: H2DCF) were visualized by confocal microscopy. Statistical analysis was performed with 2-way analysis of variance followed by post-hoc Bonferroni and student’s t-test, as applicable.17 week-old ZSF-1 obese rats, a metabolic syndrome-related model of HFpEF, and wild type rats (Wistar Kyoto), were fed 30 mg/kg/d sotagliflozin for 6 weeks. At 23 weeks, LA were imaged in-vivo by echocardiography. In-vitro, Ca, LA cardiomyocytes in HFpEF showed an increased incidence and amplitude of arrhythmic spontaneous Ca2+ release events (SCaEs). Sotagliflozin significantly reduced the magnitude of SCaEs, while their frequency was unaffected. Sotagliflozin lowered diastolic [Ca2+] of CaT at baseline and in response to glucose influx, possibly related to a ~ 50% increase of sodium sodium–calcium exchanger (NCX) forward-mode activity. Sotagliflozin prevented mitochondrial swelling and enhanced mitochondrial Ca2+ buffer capacity in HFpEF. Sotagliflozin improved mitochondrial fission and reactive oxygen species (ROS) production during glucose starvation and averted Ca2+ accumulation upon glycolytic inhibition.Sotagliflozin ameliorated LA enlargement in HFpEF in-vivo. In-vitro2+-mediated cellular arrhythmogenesis in-vitro . The safety and efficacy of combined SGLT-1&2 inhibition for the treatment and/or prevention of atrial cardiomyopathy associated arrhythmias should be further evaluated in clinical trials.The SGLT-1&2 inhibitor sotagliflozin ameliorated LA remodeling in metabolic HFpEF. It also improved distinct features of Ca Heart failure (HF) with preserved ejection fraction (HFpEF) is an increasingly prevalent disease. Left atrial (LA) cardiomyopathy and remodeling are hallmark features of HFpEF and commonly associated with LA enlargement and (precursors of) atrial fibrillation (AF) –3. Cathe+] of cardiomyocytes, mitochondrial dysfunction and oxidative stress, endothelial inflammation, fibrosis and cardiac bioenergetics  after exposure to 2 µM Ca2+ expressed as ΔF = F—F0. The change of mitochondrial density (Δ%) was quantified as a measure of mitochondrial swelling.Mitochondrial Can detail . LA card2+ and 10 nM TMRE. Two-dimensional images of TMRE  and MitoTracker green fluorescence  were acquired for a duration of 6 min . A binary mask of both channels was derived using a Bernsen thresholding algorithm (ImageJ). Positive pixels of the MitoTracker green image were defined as mitochondria and a positive overlay of the TMRE image assumed to indicate a polarized state. The standard deviation of polarized mitochondria over time was taken as a measure of spatiotemporal oscillation.LA cardiomyocytes were loaded with TMRE and MitoTracker green, transferred to an LSM 800 laser scanning microscope and kept in Tyrode’s solution containing 2 mM Ca0, reactive oxygen species (ROS) accumulation calculated as ΔF = F − F0 per image, averaged per image sequence and reported as the respective rate ΔF/(F0 * t).LA cardiomyocytes were starved of glucose for 1 h at 37 °C, loaded with H2-DCF and transferred to an LSM 800 laser scanning microscope. Two-dimensional images  were acquired for a duration of 30 s . Cells were exposed to 30 mM glucose and another set of images acquired for a duration of 90 s. Image sequences acquired between 0–30 s (glucose starved) and 90–120 s (glucose saturated) were individually assessed. H2-DCF signal intensity (F) of the initial image was defined as FLA tissue homogenate was run on a 4–12% Bis–Tris gel and transferred to a 0.45 µm nitrocellulose membrane for 120 min. The total protein on the membrane was stained with Ponceau S. Non-specific binding was blocked with 5% dried milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween-20. Membranes were probed with anti-SGLT-1 , anti-SGLT-2 . Anti-mouse IgG linked with IRDye 680RD or anti-rabbit IgG linked with 800CW (LI-COR) were used as a secondary antibody. The signal was recorded with an Odyssey CLx System. Band intensities and total protein were determined by Image Studio software (LI-COR).p-values are supplied for each graph in the figure legend. A p-value of < 0.05 was considered to be of statistical significance.Results are shown as mean ± standard error. Individual data points are shown where spatially feasible. Statistical tests and In-vivo LA volume obtained via echocardiography showed severely enlarged atria in the HFpEF group  and regarding (the closely related) diastolic sarcomere length (R2 = 0.63), indicative of LV/LA interaction in this HFpEF atrial cardiomyopathy model  of LA cardiomyocytes were more frequent and their Ca2+ release amplitude increased in HFpEF  to be a potential modulator of arrhythmic SR Ca2+ release in this model . When challenged with 3 Hz and 5 mM extracellular [Ca2+] however, Sota lead to a significant reduction of diastolic [Ca2+] in HFpEF, while leaving CaT amplitudes unchanged. Interestingly, Sota also prevented glucose-mediated influx of diastolic Ca2+ in HFpEF.First, we examined CaT of LA cardiomyocytes at 1 Hz stimulation and 2 mM extracellular  was not determined in the current study, a probable driver might be a reduction of  cytosolic [Na+]. Different mechanisms of [Na+] lowering seem plausible: SGLT-2 inhibitors have been demonstrated to inhibit the Na+/H+ exchanger in murine cardiomyocytes [+] and [Ca2+] by its inhibition. Work by Lambert et al. indicates a contribution of the SGLT-1 transporter to cytosolic [Na+] in failing hearts, in particular in the presence of metabolic dysfunction , making it another plausible site-of-action  of LV cardiomyocytes at rest has been associated with increased cytosolic [Ca2+], mitochondrial swelling and reduced mitochondrial respiration [2+ buffer capacity . Indeed, Sota prevented cytosolic Ca2+ accumulation upon glucose influx and glycolytic inhibition in HFpEF. Dual SGLT-1&2 inhibition lowered ROS production during glucose starvation. Interestingly, ROS production normalized upon reintroduction of glucose only in HFpEF, indicating an increased glucose-dependency to meet cellular energetic demand while maintaining an adequate degree of pro-arrhythmogenic ROS production.A reduced cardiac energy reserve and metabolic disorders are hallmark features of severe HF. In addition, almost 50% of HFpEF patients suffer from T2DM and are at particular high risk for HF hospitalization . SGLT inergetics . Moreoveergetics . We therThe dual SGLT-1&2 inhibitor sotagliflozin ameliorated LA remodeling in HFpEF and exerted an anti-arrhythmic effect on LA cardiomyocytes. The safety and efficacy of dual SGLT-1&2 inhibition for the treatment and/or prevention of AF in HFpEF should be further evaluated in clinical trials.Additional file 1: Figure S1. (A) Longitudinal and (B) transversal LV fractional shortening as observed in echocardiography . (C) Correlation of longitudinal and (D) transversal LV fractional shortening with LA volume. (E) Exemplary electrocardiogram to monitor atrial fibrillation (excerpt of 30 min continuous recording). (F) Total occurrence of atrial fibrillation during screened time intervals. (G) Serum concentration of creatinine and (H) urea. Statistical analysis: Two-way ANOVA followed by post-hoc Bonferroni . n = animals. 10.016, 2 < 0.001, 3 < 0.001, 40.014, 5 < 0.001. Figure S2. (A) Correlation of diastolic Ca2+, (B) CaT amplitudes, (C) CaT time-to-peak (TTP), (D) CaT tau of decay, (E) diastolic sarcomere length, (F) sarcomere shortening, (G) sarcomere time-to-peak (TTP) and sarcomere RT50 (H) of LV and LA cardiomyocytes. p-values (deviation from zero): 1 < 0.0001, 20.005, 30.01. n = average per animal. Figure S3. (A) Western Blot of LA SGLT-1 expression (above) and respective total protein visualized by Ponceau S staining (below). (B) Corresponding statistical analysis of SGLT-1 signal intensity after normalization to total protein. (C) Western Blot of LA SGLT-2 expression (above). and respective total protein (below), kidney (K) shown as positive control. (D) Corresponding SGLT-2 signal intensity after normalization to total protein. Statistical analysis: Two-way ANOVA followed by Fisher’s LSD test. n = animals. 10.003, 20.048, 30.03. Figure S4. (A) Example of polarized mitochondria  at baseline (left) and after spontaneous depolarization (right) and corresponding signal trace (below). (B) Standard deviation (SD) of signal trace over the course of 6 min. (C) Maximum depolarization per cell. Statistical analysis: Two-way ANOVA followed by post-hoc Bonferroni. Figure S5. (A) Example of SCaEs during a non-stimulated interval, following 3 Hz stimulation for 10 s at baseline (above) and after 10 min incubation with acetylcysteine . (B) Related occurrence of SCaEs . Statistical analysis: Two-way ANOVA followed by post-hoc Bonferroni. p-values: 10.069, 20.018.


This case-control study explores de novo candidate gene variants in 18 multiplex families with bipolar disorder. We present a DNV study in 18 multiplex bipolar families, combining 32 individuals previously reported4 with 29 additional participants.The detection of de novo variants (DNVs) by next-generation sequencing has facilitated the identification of candidate genes in psychiatric disorders.STREGA) reporting guideline.This study was approved by the University of New South Wales Ethics Committee. Participants provided written consent. This study follows the Strengthening the Reporting of Genetic Association Studies . The DNA samples were prepared and whole-genome sequencing was performed on the HiSeq X platform at The Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, Australia. Whole-exome sequencing was previously performed at the Lottery State Biomedical Genomics Facility, University of Western Australia.We performed whole-genome sequencing of 9 multiplex families  in this case-control study. Identification of DNVs used 2 variant calling software packages, GATK version 3.4.0 (Broad Institute) and RTG-Core sequencing software version 3.7.1 . All variants were validated by Sanger sequencing and were combined with variants previously identified by whole-exome sequencing in 32 offspring,multi snp-wise model in MAGMA statistical software version 1.06b  using a threshold of P < .05 and genome-wide association study summary statistics from the Psychiatric Genomics Consortium. For BD, there were 20 352 cases and 31 358 controls3; for schizophrenia, there were 40 675 cases and 64 643 controls; and for major depression, there were 170 756 cases and 329 443 controls. Protein interaction network analysis was performed using IPA statistical software release date February 8, 2019 . Data were analyzed from February 2017 to June 2019.For each gene harboring DNVs, a gene-based association study was performed using the 4 for a combined analysis of 63 coding DNVs from 61 white participants . In total, 42 DNVs were identified in cases . However, missense and gene-truncating DNVs were statistically significantly more frequent in cases compared with unaffected relatives . Interestingly, the rate for potentially disrupting DNVs in brain-expressed genes 4 was higher in participants with BD compared with unaffected relatives (14 of 42 [33%] vs 3 of 21 [14%]), which is comparable to the previous BD report (30%)5 and rates observed in autism (36%) and schizophrenia (34%).2Thirty-two DNVs from 12 female and 17 male offspring were validated and combined with 31 DNVs from our previous report,in cases  and 21 vHP) and pyruvate carboxylase (PC), 2 brain-expressed genes associated across 3 psychiatric disorders that carry potentially pathogenic DNVs. The PC gene encodes a mitochondrial enzyme involved in glucose metabolism and neurotransmitter synthesis, which lies within one of 30 BD-associated loci recently reported,3 and it had the strongest gene-based association  (Gene-based association tests for the 42 DNV-bearing genes implicated haptoglobin (= 0.042) .3 were combined with 112 genes carrying DNVs from the current and previous BD studies.5 Protein-protein interaction network analysis implicated 4 genes: microtubule associated protein 4 (MAP4), which promotes microtubule assembly and is reported to carry DNV in autism; WD repeat and HMG-box DNA binding protein 1 (WDHD1), a DNA replication initiation factor; eukaryotic translation initiation factor 4E (EIF4E), which interacts with cytoplasmic Fragile X Mental Retardation interaction protein and is implicated in autism; and striatin (STRN), a largely unknown calmodulin-binding protein with a potential role in dendritic Ca2+ signaling and striatal neuron maturation (MAP4 and WDHD1 harbored predicted-pathogenic variants with nominal gene-based association in schizophrenia (To identify relevant networks, 218 genes from BD-significant locituration . Of thesophrenia .6 Although this study is limited by the small sample size, the overall de novo mutation rate was comparable in cases and unaffected offspring, whereas deleterious DNVs were observed more frequently in participants with BD, which is consistent with previous reports in autism and schizophrenia.2 This study highlighted HP, PC, MAP4, and WDHD1 as potential susceptibility genes for BD. Additional sequencing studies in larger cohorts are needed to further delineate the impact of DNVs in BD.Examination of DNVs in psychiatric disorders has traditionally focused on singleton families. These findings suggest that DNVs may also contribute to mutational load in multiplex BD families, as previously observed for multiplex autism families.


TNF downregulates IFN-α and TNF production by human pDCs.TNF downregulates IRF7 and NF-κB pathways and upregulates Ag processing in pDCs.TNF enhances Ag presentation and T cell activation properties in pDCs. + T cell activation. Previous studies showed a cross-regulation between TNF-α and IFN-α, but many questions remain about the effect of TNF-α in regulating human pDCs. In this study, we showed that TNF-α significantly inhibited the secretion of IFN-α and TNF-α of TLR-stimulated pDCs. Instead, exogenous TNF-α promoted pDC maturation by upregulating costimulatory molecules and chemokine receptors such as CD80, CD86, HLA-DR, and CCR7. Additionally, RNA sequencing analysis showed that TNF-α inhibited IFN-α and TNF-α production by downregulating IRF7 and NF-κB pathways, while it promoted Ag processing and presentation pathways as well as T cell activation and differentiation. Indeed, TNF-α–treated pDCs induced in vitro higher CD4+ T cell proliferation and activation, enhancing the production of Th1 and Th17 cytokines. In conclusion, TNF-α favors pDC maturation by switching their main role as IFN-α–producing cells to a more conventional dendritic cell phenotype. The functional status of pDCs might therefore be strongly influenced by their overall inflammatory environment, and TNF-α might regulate IFN-α–mediated aspects of a range of autoimmune and inflammatory diseases.Human plasmacytoid dendritic cells (pDCs) play a vital role in modulating immune responses. They can produce massive amounts of type I IFNs in response to nucleic acids via TLRs, but they are also known to possess weak Ag-presenting properties inducing CD4 A common DC progenitor in the bone marrow can generate both pDCs and conventional DCs (cDCs), but pDCs are unique in their ability to produce type I IFNs in response to viral infection . Upon lioduction –7.Apart from type I IFN production, other cytokines such as TNF-α can also be produced by pDCs upon viral stimulation . Early s+ T cells, inducing their activation  molecules and are able to capture, process, and present Ags to CD4tivation , 11. Rectivation , 13. TLRtivation –16. Despesponses , 17, 18.ntiation –22.+ hematopoietic progenitors clearly demonstrated a cross-regulation between TNF-α and type I IFNs . Cell sorting was carried out at the St. James Campus Infrastructure and Facilities Cytometry and Imaging Facility of the Wellcome Trust Brenner Building of the University of Leeds, with a BD Influx Six-Way Cell Sorter (BD Biosciences).Human PBMCs were separated from whole blood by density gradient centrifugation using Leucosep tubes (Greiner Bio-One). A pDC-enriched population was isolated from PBMCs by negative selection using the Diamond pDC Isolation Kit II (Miltenyi Biotec). pDCs were further sorted using an Ab to BDCA-4 (Miltenyi Biotec). Naive CD4+ T cells at 1:5 ratio for 5 d. For cytokine detection, cells were cultured in the last 4 h with GolgiPlug (BD Biosciences). Cell proliferation was measured using the CellTrace Violet Cell Proliferation kit (Thermo Fisher Scientific), according to the manufacturer’s instructions.Cells were cultured in RPMI medium 1640 with GlutaMAX supplement (Thermo Fisher Scientific) containing 10% (vol/vol) FBS and 100 U/ml penicillin/streptomycin. For cytokine production, cells were stimulated with 2 μM class A CpG  or ORN R-2336 (Miltenyi Biotec). For pDC/T cell coculture, pre-enriched pDCs were treated with 10 ng/ml recombinant human TNF-α (R&D Systems) for 24 h and then washed twice to remove residual TNF-α before use in subsequent culture. Untreated or TNF-α–treated pDCs were cultured with allogeneic naive CD4Pre-enriched pDCs were stimulated with ODN 2216 (1 ng/ml) or ORN R-2336 (1 ng/ml) in the presence or absence of human TNF-α Ab (R&D Systems). After 24 h, the plates were centrifuged to collect the supernatants, and the cells were washed twice before restimulation for an additional 24 h. Supernatants collected at 24 and 48 h were analyzed by Human IFN-α Platinum ELISA Kit (eBioscience), according to the manufacturer’s protocol.For cell surface staining, fluorochrome-conjugated mAbs against human CD3, CD4, CD19, CD14, CD56, CD11c, HLA-DR, CD123, CD303, CD317, CD80, CD85g, isotype controls (Miltenyi Biotec), CD304, CD69 (BioLegend), CD86, and CCR7 (BD Biosciences) were used. For intracellular staining, cells were first stained for surface markers and then fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) before incubation with fluorochrome-conjugated mAbs against human IFN-α and TNF-α (Miltenyi Biotec) or TNF-α, IFN-γ, and IL-17A (BioLegend). Flow cytometry was performed on LSRII (BD Biosciences) and Cytoflex S (Beckman Coulter) cytometers, and the data were analyzed using FACSDiva (BD Biosciences) and CytExpert (Beckman Coulter) software.pDCs were isolated from PBMCs (Miltenyi Biotec) and were then cultured in RPMI medium 1640 with GlutaMAX supplement (Thermo Fisher Scientific) containing 10% (vol/vol) FBS and 100 U/ml penicillin/streptomycin in a 96-well plate. Purified pDCs were cultured with or without 10 μg/ml DQ OVA (Molecular Probes) in the presence or absence of 50 ng/ml TNF-α. After 18 h, the cells were collected and then washed twice before data were analyzed using flow cytometry.RNA from sorted pDCs was extracted using PicoPure RNA Isolation Kit (Thermo Fisher Scientific) and quantified using Qubit RNA HS Assay Kit (Thermo Fisher Scientific). RNA libraries were made by using SMART-Seq V4 Ultra Low Input RNA Kit (Takara Bio) and Nextera XT DNA Library Preparation Kit (Illumina) for next-generation sequencing. Indexed sequencing libraries were pooled and sequenced on a single lane on HiSeq 3000 instrument as 151 bp paired-end reads. Pooled sequence data were then demultiplexed using Illumina bcl2fastq software, allowing no mismatches in the read index sequences.Raw paired-end sequence data in Fastq format was initially analyzed using FastQC software to identify potential issues with data quality. Cutadapt software  was thenReads trimmed to fewer than 30 nt and orphaned mate-pair reads were discarded to minimize alignment errors downstream.Reads were aligned to human hg38 analysis set reference sequences, obtained from University of California Santa Cruz (UCSC) database  using sphttps://www.ncbi.nlm.nih.gov/bioproject, accession number PRJNA645253.Bioconductor R package RSubread  was used+SIGLEC6+ DCs (AS-DCs), we analyzed key transcripts associated with each cell type. This confirmed a high expression of the pDC marker TCF4 and a low expression of the AS-DC markers SIGLEC 1, 2, 3, and 6, KLF4, and AXL . Continuous variables were compared using either Student Although pDCs are mostly recognized for their IFN-α–producing capacity, they are also capable of producing other proinflammatory cytokines. To evaluate this, peripheral blood pDCs were analyzed by flow cytometry for the production of both IFN-α and TNF-α upon stimulation with TLR9 (ODN 2216) or TLR7 (ORN R-2336) agonists. Circulating pDCs produced no IFN-α and/or TNF-α without external stimulation . After eTo understand the role of TNF-α on pDC function, we first investigated the effect of TNF-α on cytokine production in the presence or absence of TLR stimulation. Freshly isolated PBMCs were cultured in the absence or presence of different concentrations of recombinant human TNF-α (1–50 ng/ml) for 24 h. We observed no induction of IFN-α and/or TNF-α production in TNF-treated pDCs without exogenous stimulation . Howeverp < 0.05). This effect could not be seen in pDCs restimulated with ORN R-2336 (TLR7 agonist) as the levels of IFN-α secreted were similar in both anti-TNF– and control-treated pDCs.We next investigated whether neutralization of endogenous TNF-α had an impact on IFN-α production. We isolated a pDC-enriched population from PBMCs by negative selection using magnetic beads (purity >92%), and the cells were stimulated with ODN 2216 or ORN R-2336 in the absence or presence of anti-TNF Ab or isotype control. After 24 h, the supernatants were collected, and IFN-α production was measured by ELISA . The celn = 3) were purified by negative selection using magnetic beads. The pre-enriched pDCs (purity >85%) from each donor were divided into two aliquots before they were cultured in the presence or absence of human recombinant TNF-α (10 μl/ml) for 18 h. After incubation, untreated and TNF-treated pre-enriched pDCs from all three donors (n = 6) were washed and sorted based on CD304 (BDCA-4) expression to achieve purity >99%.We sought to investigate how TNF-α regulates TLR-mediated cytokine production and induces further transcriptional changes in human pDCs. pDCs from healthy donors  as well as MHC-II .Among the upregulated genes in TNF-treated pDCs compared with untreated pDCs were HLAs corresponding to MHC class I , molecules promoting endocytosis (CD59) and cell adhesion (ADAM8), ICOS ligand (ICOSLG), and IL-27 subunit β (EBI3). In contrast, downregulated genes included CCR2, PTPRC, SYK, IL1B, LILRB2, and SOCS1, among others. TNF-α also upregulated genes associated with T cell differentiation , particularly toward Th17, Th1, and Th2 pathways , phagosomal maturation , and regulation of trafficking and processing of endosomal TLRs .TLR9, MyD88, IRAK2, IRAK4, and IRF7. In contrast, there was upregulation of the NF-κB inhibitor (NFKBIA) known to block the translocation of NF-κB to the nucleus  for 24 h. After washing thoroughly, pDCs were cocultured with allogeneic naive CD4+ T cells for 5 d. T cell proliferation was assessed based on CellTrace Violet dilution upon cell division using flow cytometry. Both sets of pDCs could induce T cell proliferation without exogenous stimulation  sharing features of both cDCs and pDCs (“AS-DCs”)  , 50. AS-AS-DCs”) . In conthiCD5+CD81+ was also shown to induce strong T and B cell activation but not to be able to secrete type I IFNs  and secretion  as well as TCF4 alongside its binding targets . Hence, TNF-α strongly influenced the transcriptional profile of pDCs by downregulating their classical pathways and upregulating genes  mostly related to the cDC phenotype. Our data suggest that TNF-α can alter intracellular pathways and may interfere with phosphorylation of transcription factors such as IRF7 and IκB involved in cytokine production and other known functions of pDCs.Another single-cell RNA sequencing study revealed the diversification of human pDCs in response to influenza virus into three phenotypes  with distinct transcriptional profiles and functions . The P3-e I IFNs . In our TNFRSF1A  but not TNFRSF1B  and TNFRSF6B , whereas it upregulated TNFRSF4 , FAS , and CD40 . Interestingly, TNF-α promoted the downregulation of IFNAR1 and IFNAR2 and the upregulation of IFNGR1 and IFNGR2, receptors for IFN-γ. This may suggest that changes in TNFR1 and TNFR2 receptor expression accompany the functional changes in pDCs that we observed. Although TNFR1 can mediate apoptosis, such an effect did not appear consistent with our results, with an increase in T cell activation by pDCs after TNF exposure. Changes in receptor expression at a protein level and studying their functional significance require further work. In future work, we will also study what signaling mechanisms explain the differential responses to TLR7 and TLR9 ligands.Other cytokines such as IL-21 have been shown to have a regulatory impact on the function of pDCs promoting the secretion granzyme B, which impairs their capacity to induce T cell proliferation . In contLinking these data to human disease, anti-TNF treatment has been associated with lupus-like symptoms as well as an induction of IFN signature in peripheral blood , 54, 55.In summary, our data elucidate the plasticity of pDC function according to their inflammatory environment. Although pDCs possess weak Ag-presenting properties, TNF-α can enhance these functions by switching their main role as IFN-α–producing cells to a more cDC phenotype. This cytokine therefore plays a key role in modulating the functional status of pDCs and may regulate IFN-α–mediated aspects of a range of autoimmune and inflammatory diseases.Data Supplement


The inflammation that occurs at stem cell transplant sites (such as that resulting from TNF-α) is a contributing factor for stem cell treatment failure. Currently, there is little evidence regarding the protective role of melatonin with regard to the negative effects of TNF-α on the stemness of MSCs. In this study, we report a melatonin-based method to reduce the inflammatory effects on the stemness of bone marrow mesenchymal stem cells (BMMSCs). The results of colony formation assays, Alizarin red staining, western blotting, and reverse transcription-polymerase chain reactions suggest that melatonin can reverse the inflammatory damage caused by TNF-α treatment in the third, seventh, and tenth generations of primary BMMSCs (vs. control and the TNF-α-treated group). Meanwhile, a detailed analysis of the molecular mechanisms showed that the melatonin receptor and YAP signaling pathway are closely related to the role that melatonin plays in negative inflammatory effects against BMMSCs. In addition, in vivo experiments showed that melatonin could reverse the damage caused by TNF-α on bone regeneration by BMMSCs in nude mice. Overall, our results suggest that melatonin can reverse the loss of stemness caused by inflammatory factor TNF-α in BMMSCs. Our results also provide a practical strategy for the application of BMMSCs in tissue engineering and cell therapy.Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term  In addition, MSCs that experience oxidative stress may undergo premature aging, which can significantly affect their ability to differentiate into different types of cells /[number of cells inoculated]. Melatonin, TNF-α, Luz, and VP were obtained from Sigma-Aldrich (USA).BMMSCs at P3, P7, and P10 were induced toward osteogenic differentiation and adipogenic differentiation. For osteogenic induction, BMMSCs were plated in DMEM supplement with osteogenic differentiation assay . After 2 weeks, differentiated BMMSCs were stained with Alizarin red S (ARS) solution . The detailed experimental processes of the tests were conducted as described previously . For adiμL final volume from 400 ng total RNA using the TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit  according to manufacturer's instructions. GAPDH, RUNX2, OPN, OCN, HTERT, SOX2, C-MYC, NANOG, PPARγ-2, LPL, ADIPONECTIN, and YAP mRNA levels were determined by RT-PCR, and primers were utilized as described in μL volumes containing 2x SYBR® Premix Ex Taq™ , PCR Forward Primer (10 μM), PCR Reverse Primer (10 μM), cDNA template, and ddH2O. RT-PCR was performed using a Bio-Rad CFX96 Real-Time PCR System. Thermal cycling conditions were as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s. Data were collected and calculated using the Bio-Rad CFX Manager Software1.6. RNA expression was calculated based on a relative standard curve with the 2ΔΔct− method.RNAiso Plus reagent  was used to exact total RNA from BMMSCs. Reverse transcription-PCR (RT-PCR) analysis was carried out in 20 γ-2, Lipoprotein lipase (LPL), ADIPONECTIN, and GAPDH. Before detection with an ECL chemiluminescence detection kit , proteins were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (H&L) and HRP-conjugated goat anti-mouse IgG (H&L) secondary antibodies (1 : 5000 dilution) for 1 h at room temperature . The bands were examined with ImageJ software .Total cellular protein from differently treated BMMSCs was isolated by the addition of 1% PMSF and RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS). After boiling in SDS-PAGE sample buffer for 5 min, the samples underwent sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes . After being blocked for 1 h at room temperature, the membranes were incubated with a 1 : 1000 dilution of the following antibodies (Abs) from Abcam  overnight: Abs against HTERT, SOX2, C-MYC, NANOG, OPN, OCN, RUNX2, PPARn = 10), osteoporosis simulation group , OVX+P3 BMSC treatment , OVX+P3 BMSCs+20 ng/mL TNF-α treatment , OVX+P3 BMSCs+TNF-α+100 μM Mel treatment , and OVX+P3 BMSCs+TNF-α+Mel+5 μM verteporfin . After the model of osteoporosis was established , BMMSCs treated with Mel, TNF-α, and VP were injected into the tail veins. Eight weeks after modeling, femur tissues were collected for further experimentation.Eight-week-old Balb/c nude mice were obtained from Charles River . Sixty nude mice were randomly divided into six groups, including control , trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular pattern factor, were measured automatically using the Siemens Preclinical Imaging System in a multislice in standard resolution mode [To observe the bone microstructure, micro-CT examination was carried out with SCANCO Medical AG (Switzerland) before sacrificing, as previously described . The parion mode , 28.t-test. P values < 0.05 were considered to be a significant difference between two measurements.Statistical differences in this study were assessed using SPSS 18.0 software. The mean values of the statistical results for the measured data are presented as the means ± standard error. The values in this study were analyzed by Student's P < 0.01)  in P3, P7, and P10 BMMSCs   . For wes Figures . The res Figures . In addi Figures . In summα  to simulate an inflammatory environment. RT-PCR analysis of biomarkers of BMMSC stemness  showed that mRNA abundances of these biomarkers were significantly lower in the three treated groups than in the control group (P < 0.01) . However, colony formation in the TNF-α-treated group could be partially recovered with melatonin treatment (P < 0.01 vs. TNF-α-treated group) , osteogenic differentiation , and adipose differentiation  in P3, P7, and P10 cells with different treatments were examined with RT-PCR . However, the expression levels of these genes were recovered with additional melatonin treatment (P < 0.01 vs. TNF-α-treated group). Similar results were found in western blot analysis. Protein abundances of these biomarkers in TNF-α-treated P3, P7, and P10 cells were also significantly lower than those in the control group (P < 0.01). Meanwhile, melatonin treatment could partially recover the protein expression of these genes (P < 0.01) . Melatonin treatment was able to increase mineral deposition in the TNF-α-treated group (P < 0.01 vs. TNF-α-treated group)  . Based od group) . mRNA ab Figures . The res Figures . Alizarid group) . Therefoα, TNF-α+Mel, and TNF-α+Mel+Luz. For colony formation analyses, colony formation ratio in the TNF-α group was significantly lower than that in the control (P < 0.01). Colony formation ratio in the TNF-α+Mel group was significantly higher than that in the TNF-α group (P < 0.01). However, this value in the TNF-α+Mel+Luz group was significantly lower than that in the TNF-α+Mel group (P < 0.01)  in P3 cells with different treatments were examined . Therefore, we speculated that melatonin could reverse the inflammatory effects on BMMSC stemness via its receptor.In this study, we have employed luzindole (a melatonin receptor inhibitor) to study the molecular mechanism of melatonin in BMMSC P3 generation. BMMSCs were divided into four groups: control, TNF- < 0.01) . RT-PCR  Figures . The resμM verteporfin (a YAP inhibitor) to study the molecular mechanisms of melatonin on BMMSC stemness. BMMSCs were divided into four groups: control, TNF-α, TNF-α+Mel, and TNF-α+Mel+VP. RT-PCR and western blot analyses of YAP expression showed that TNF-α treatment could significantly decrease YAP mRNA and protein abundance in P3, P7, and P10 BMMSCs. However, melatonin treatment could reverse YAP mRNA and protein expression in TNF-α-treated BMMSCs (P < 0.01)  (α+Mel+VP group was significantly lower than that in the TNF-α+Mel-treated group (P < 0.01)  in P3 cells with different treatments . In summary, YAP is a potential target of melatonin in reversing the impacts of inflammation on BMMSC stemness.In this study, we have employed 5  Figures . Moreove < 0.01) . For col < 0.01) . In addi Figures . The mRNα treatment aggravated osteoporosis. However, melatonin was able to reverse the damage of osteoporosis. VP, one of the YAP inhibitors, was shown to weaken the therapeutic effect of melatonin in osteoporosis. Similar results were also observed in H&E staining analysis (P < 0.05). Meanwhile, the above indices in the OVX+P3 BMSC+TNF-α+Mel group were significantly higher than those in the OVX+P3 BMSC+TNF-α group (P < 0.05). However, VP could reverse the expression in the OVX+P3 BMSC+TNF-α+Mel+VP group . Meanwhile, both indices in the OVX+P3 BMSC+TNF-α+Mel group were significantly lower than those in the OVX+P3 BMSC+TNF-α group (P < 0.01). However, they were upregulated in the OVX+P3 BMSC+TNF-α+Mel+VP group compared with the OVX+P3 BMSC+TNF-α+Mel group , and basic fibroblast growth factor (bFGF) signals can promote the differentiation of MSCs into muscle [β-catenin [β-catenin and thus increase nuclear β-catenin-mediated osteogenesis [α inhibited BMMSC stemness. However, melatonin was able to restore YAP expression. Our results confirm that melatonin treatment can reverse the loss of BMMSC stemness caused by TNF-α via YAP signaling.Maintenance of stemness in MSCs is regulated by a variety of cellular signals, such as BMP signaling and Wnt signaling, which can promote the differentiation of MSCs into osteoblasts. However, a lack of BMP signal or Wnt signal will inhibit the differentiation of MSCs into osteoblasts and promote the differentiation of MSCs into fat cells –53. Plato muscle –56. Receo muscle , 58. YAPo muscle , 60. Reco muscle , signal o muscle , and β-c-catenin . RUNX2 i-catenin . Pan et ogenesis . In thisin vitro culture. TNF-α could result in the inflammatory-based reduction in HMSC stemness. However, melatonin could reverse the inflammatory reduction of HMSC stemness via the melatonin receptor and YAP signaling. In osteoporotic nude mice, melatonin improved the effects of HMSC treatment. This work provides a detailed understanding of the effects of melatonin treatment of HMSCs.In summary, we demonstrated that HMSC stemness decreases with increased generations of