Per- and polyfluoroalkyl substances (PFAS) are widespread chemicals. Legacy PFAS have been phased out of production in most developed countries and emerging PFAS (short-chain PFAS and polyfluorinated compounds) are used as legacy PFAS alternatives. The effect of legacy and emerging PFAS on cytokine homeostasis in human remains poorly understood. This study aimed to evaluate the associations between legacy and emerging PFAS and cytokine profiles, and identify the main contributors to the disturbance of cytokine homeostasis. We quantified 21 PFAS in 198 Chinese women of childbearing age from 2015 to 2016. 13 cytokines were measured using the Meso Scale Discovery U-PLEX and V-PLEX platforms. The associations between PFAS exposure and cytokine levels were assessed using multiple linear regression (single-exposure), and Bayesian kernel machine regression (BKMR) models (PFAS mixture exposure). In single PFAS models, legacy and alternative PFAS were positively associated with Th1 and Treg cytokines, and negatively associated with Th2 and Th17 cytokines. For instance, each ln-unit increase in 6:2 chlorinated perfluoroalkyl ether sulfonic acid (6:2 Cl-PFESA), perfluorooctanoic acid (PFOA), and perfluorooctane sulfonate (PFOS) was associated with a decrease in IL-10 by − 0.228 , − 0.153 , and − 0.174 , respectively. The BKMR model showed a significantly positive association of PFAS mixture with TGF-β and a negative association with IL-10. Overall, these results indicate that both legacy and emerging PFAS may affect the homeostasis of cytokines. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two of the most studied long-chain PFAS, and are reported to induce adverse effects on human, including immunotoxicity2, carcinogenicity3, and reproductive and developmental toxicity4. Due to their persistence in the environment and potential health implications in humans, PFOS and PFOA production and applications have been restricted in Europe and North America6. Consequently, PFAS alternatives with either a short chain (perfluorosulfonic acid with carbon number < 6 and perfluorocarboxylic acid with carbon number < 81) or a slightly modified structure have emerged to meet market demand7. These alternatives are increasingly found in environmental and human samples at relatively high concentration due to the unrestricted use. For example, 6:2 chlorinated perfluoroalkyl ether sulfonic acid (6:2 Cl-PFESA) concentrations were as high as 2.13 ng/mL in Shenyang population, China8, and have become one of the main PFAS in Chinese local surface waters9. And PFAS alternatives showed comparable or even greater toxicities in both epidemiological and toxicological studies11. However, literature on immunotoxicity of PFAS alternatives is still scarce and inconsistent.Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds that have been used in many industrial and consumer applications for over 70 years because of their high thermal and chemical stability12. The balance between pro- and anti-inflammatory cytokines in female immune system is essential for embryo implantation and pregnancy maintenance. T-helper 1 (Th1) and Th2 cells are the major subsets of Th cells with different patterns of cytokine production and different roles in immune responses13. In female, the deviation of the immune system from Th2 toward Th1 has been implicated in pregnancy complications, such as recurrent miscarriage14, preeclampsia15 and fetal growth restriction16. Th1 cells secrete pro-inflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), IL-8, and IL-12p70, that can activate macrophages and cell-mediated reactions that play critical roles in resistance to infection by intracellular pathogens and in cytotoxic and rejection reactions. Th2 cells secrete IL-4, IL-6, IL-10, and IL-13, which induce antibody production and are associated with strong humoral immunity17. IL-17 and IL-22 are the main Th17 effector cytokines, which are related to protective immunity against extracellular microbes during pregnancy18. Transforming growth factor-β (TGF-β) is secreted by regulatory T (Treg) cells and has the function of local inflammatory immune responses, potentially detrimental to the fetus13. Epidemiological findings regarding the possible relationship of PFAS and immune cytokines are limited and inconsistent. A recent study in 87 middle-aged women found that PFHpS was positively associated with Th1 cytokine (IL-8)19. But the C8 Health Project found that PFHxS, PFOA, and PFOS were inversely associated with TNF-α in 200 women aged 40–70 years. Similarly, PFOS and PFNA were negatively associated with IL-820. The inconsistency of previous findings warrants further research. We speculated that PFAS might affect pregnancy by disrupting immune balance19.The immune system plays a critical role in human reproductionWe conducted a cross-sectional study to examine the association between legacy and emerging PFAS exposure and cytokine levels and their balances.A total of 198 women were included in our study. Table Table Table P for trend < 0.001).Tables Figure In the PFAS mixture analysis, Th1 cytokines have inconsistent trends Fig. a–d, and Our study found that both legacy and emerging PFAS alternatives were associated with cytokine levels in women of childbearing age. Certain PFAS alternatives and isomers were positively associated with Th1 and Treg cytokines and negatively associated with Th2 and Th17 cytokines.21 in the general population and 1.54 ng/mL by Chen et al.22 in pregnant women in China. Moreover, 6:2 Cl-PFESA, 8:2 Cl-PFESA and HFPO-DA were detected in more than 95% of our population, suggesting that humans are also widely exposed to the PFAS alternatives.Consistent with our study, previous studies also reported high concentrations of PFAS alternatives. For example, the plasma concentration of 6:2 Cl-PFESA concentration was 1.4 ng/mL in our study, which is comparable to 1.75 ng/mL reported by Yu et al.14, preeclampsia15, and fetal growth restriction16. In addition, this study found a positive association between PFAS and TGF-β. As the principal function of TGF-β is to regulate cellular functions including cell adhesion, invasion and angiogenesis, which is essential for endometriosis development23, elevated TGF-β may have effects on endometriosis.Our results reported that PFAS were positively associated with Th1 cytokines and negatively with Th2 cytokines. Th1 cytokines tend to induce proinflammatory responses while Th2 cells produce anti-inflammatory cytokines. Th1/Th2 balance is beneficial to the maternal–fetal immune tolerance and maintenance of pregnancy. A shift in the Th1/Th2 balance toward Th1 dominance may cause miscarriages24. A significantly inverse association with PFOA was also found for IL-10 (β = − 0.22) among 101 healthy 1-year-old children25. Additionally, prenatal PFOS and PFNA in children aged 8 years were inversely related to Th2 cytokines (IL-4 and IL-13) in children from 1,101 mothers-child pairs in the Human Early Life Exposome project (HELIX)26. These evidences seem to suggest that PFAS may induce the production of pro-inflammatory cytokines. However, Zhu et al.27 showed that serum PFAS were positively associated with Th2 cytokines (IL-4 and IL-5) and negatively associated with Th1 cytokines (IFN-γ and IL-2) among male asthmatics. Positive associations were also observed for PFAS and Th2 cytokine (IL-6)  in 103 overweight or obese pregnant women in the U.S.28. Thus, evidence so far has been inconsistent.A recent study found that PFHpS was positively associated with IL-8  in 87 middle-aged women35. Dong et al. (2013) reported positive associations between PFOA, PFOS, PFBS, PFDA, PFDoA, PFHxS as well as PFNA exposure and asthma among 231 Taiwanese Children (10–15 years old). They also showed that PFAS were positively associated with serum IgE concentrations, absolute eosinophil counts (AEC), eosinophilic cationic protein (ECP) concentrations, and asthma severity scores among asthmatics31. However, PFAS were not statistically associated with the asthma diagnosis, or eosinophil count in 118 World Trade Center (WTC) participants33. As for immune globulin, no association between PFOS, PFOA and immunoglobulins IgA-IgG-IgM was found among 53 American male workers in PFOA production department29. No significant relationships between serum PFOA and white cell blood counts was discovered in 371 American residents30.A number of epidemiological studies in humans focused on the associations between legacy PFAS and immune outcomes such as asthma, allergies, and propensity to infections32. Moreover, a longitudinal study found that the PFAS concentrations in 5-year-old children were associated with higher basophil counts , but no significant relationship for neutrophils, eosinophils, lymphocytes, monocytes, and white cell counts. Furthermore, no association was found between PFAS from mothers as well as children at 18 months with aforementioned cell counts34.Knudsen et al. (2018) reported significant negative associations between PFAS and lymphocyte , neutrophil , monocyte , and white cell counts  in 192 Greenlandic pregnant women37. For example, 10 mg/kg PFOS exposure was positively associated with the expression levels of IL-1β and TNF-α in rats after 14-day exposure36. PFAS can also induce a markable upregulation of proinflammatory cytokines TNF-α expression in the jejunum of the PFOS 1 mg/kg and 10 mg/kg groups compared with the control in rats37. Collectively, these results indicated that PFOS exposure induces an upregulated pro-inflammatory infiltration. But inconsistent results also reported that PFOS exposure decreased TNF-α and increased IL-10 in the mice colon tissues38. Similarly, another study found that exposure to PFOS and F-53B significantly downregulated the expression of pro-inflammatory cytokine genes, including IL-8 and TNF-α, in the liver of adult zebrafish11.Many in vivo and in vitro experiments have demonstrated that dysregulation of cytokine caused by environmental pollutants induces the inflammatory responseThe main strength of our study is that we measured various types of PFAS, including PFAS alternatives and isomers. We also measured an array of cytokines including Th1, Th2, Th17, and Treg cytokines. We further used more advanced statistical methods to take into account of PFAS mixture exposure. Thus, our study has given a comprehensive and robust assessment of the association between PFAS, especially the emerging alternatives, and cytokine homeostasis in humans.39. It remains to be tested whether our finding is due to such unmeasured confounding.Our study also has several limitations. Firstly, the cross-sectional study design does not allow us to draw causal conclusions. Secondly, previous studies showed that other persistent chemicals like polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs) and bisphenols are also associated with cytokinesIn conclusion, our study suggests that both legacy PFAS and emerging alternatives may affect the homeostasis of cytokines in humans.A total of 198 healthy females were recruited in the Center for Reproductive Medicine of Shandong University, China, between 2015 and 2016. Non-pregnant women who had no chronic diseases, endocrine disorders, autoimmune diseases, or active infection were eligible. Blood specimens were collected at the recruitment and centrifuged at 1500 rpm for 20 min to collect serum and plasma, and subsequently stored at − 80 °C until analysis. The study was performed in accordance with the Declaration of Helsinki, the ethics committee of Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine granted ethical clearance for this study (XHEC-C-2015-046), and all participants provided a written informed consent.A total of 21 PFAS analytes were detected in 0.1 mL of plasma by ultra-performance liquid chromatography Agilent 1290 Infinity II coupled with an Agilent 6495C triple-quadrupole tandem mass spectrometry  in the negative electrospray ionization (ESI-) mode in all subjects. They included PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorododecanoic acid (PFUdA), perfluorododecanoic acid (PFDoA), perfluorohexane sulfonate (PFHpS), 6:2 Cl-PFESA, 8:2 chlorinated perfluoroalkyl ether sulfonic acid (8:2 Cl-PFESA), hexafluoropropylene oxide dimer acid (HFPO-DA)], perfluorobutane sulfonate (PFBS), perfluorobutanoic acid (PFBA), perfluoroheptanoic acid (PFHpA), perfluorohexanoic acid (PFHxA), linear perfluorohexane sulfonate (n-PFHxS), br-PFHxS, linear PFOS (n-PFOS), 1 m-PFOS, 3 m-PFOS, 4 m-PFOS, 5 m-PFOS and 6 m-PFOS. In addition, ∑3,4,5 m-PFOS was calculated as the sum of 3 m-PFOS, 4 m-PFOS, and 5 m-PFOS due to limited separation capacity. Br-PFOS was computed as the sum of 1 m-PFOS, ∑3,4,5 m-PFOS and 6 m-PFOS, and total PFOS (PFOS) was the sum of n-PFOS and br-PFOS. Similarly, total PFHxS (PFHxS) was the sum of n-PFHxS and br-PFHxS. Internal quality control samples and procedure blanks were included along with each batch of samples for quality assurance throughout the project.40 and Benskin et al.41. Briefly, the Oasis HLB extraction cartridges (3 cc)  were pretreated with 1 mL methanol and 1 mL formic acid (0.1 M). 100 μL plasma samples were spiked with 2 ng of mass-labelled standard, and 1 mL formic acid (0.1 M). After shaking, the sample was loaded onto the cartridge. Then the cartridge was washed with 1 mL 0.1 M formic acid, 3 mL of 50% 0.1 M formic acid/50% methanol, and 0.5 mL of 1% ammonium hydroxide in succession. The PFAS were eluted with 1.8 mL of 1% ammonium hydroxide in acetonitrile (ACN). The eluate was evaporated and reconstituted to 0.1 mL in a mixture of methanol and 10 mM ammonium acetate. Finally, the mixture was transferred to UPLC-MS/MS for analysis. The limit of detection (LOD) was from 0.002 to 0.0083 ng/mL for all PFAS examined. More detailed information on LOD and limits of quantification (LOQ) can be found in Supplementary Table 42.Extraction methods were modified by Kuklenyik et al.43. Briefly, cytokines including Th1 cytokines , Th2 cytokines , and Th17 cytokines (IL-17 and IL-22) were quantified by a multiplex assay U-PLEX platform with electrochemiluminescent enzyme-linked immunosorbent assays . And Treg cytokines (TGF-β) were measured by a single-plex assay V-PLEX assay platform following the manufacturer’s instructions. MESO QuickPlex SQ 120 plate reader  was used to determine the analyte concentration.The method for measuring cytokines in serum has been detailed in a previous study2, continuous), and education .Potential confounding factors included age , age at menarche , body mass index  or number (percentage) for general characteristics among the participants. The plasma PFAS concentrations and serum cytokines were presented as median (interquartile range) and mean (SD). PFAS and cytokines concentrations were natural log transformed to reduce skewness in further analyses. Spearman’s rank correlation was used to assess correlations between different plasma PFAS.Multiple linear regression models were used to assess the associations between plasma PFAS concentrations and cytokines. In adjusted single-PFAS models, age, age at menarche, BMI, and education were used as covariates. Subsequently, PFAS levels were categorized into quartiles and the β coefficients (95% CI) of cytokine were estimated using the first quartile as the reference.44. Individual exposure-outcome relationships in the mixture can be inconsistent, and this method produces unbiased estimates of overall mixture effects in small sample sizes with an acceptable CI coverage44. It has the advantage to allow chemicals to interact with the outcome in either a positive or negative direction and is performed in the following three steps. First, all PFAS were categorized into quantiles. Second, PFAS, covariates, and cytokines were fit in a linear model. Finally, an index weight was calculated between 0 and 1 for each exposure, corresponding to the contributing individual component of the mixture effect on the outcome. The overall mixture effect can be considered as the change in outcome per one quantile of change in all PFAS exposures after adjusting for covariates.Quantile g-computation (gqcomp) is an improved modeling method based on weighted quantile sum (WQS) regression that can assess the joint effect of all mixture exposures by one quartile at a time45 was conducted as a semi-parametric machine learning method to investigate non-linear dose–response associations, interactions among PFAS, and overall cumulative effects of the PFAS mixture. In this study, the BKMR models were applied as Yi = h (PFAS) + ßT Zi + ei, where Yi is the change of cytokines; h  is an exposure–response kernel function; Zi contains a set of potential confounders; β is the regression coefficient; and ei represents residuals. We identified the most important PFAS contributing to cytokine changes within the mixture using the hierarchical variable selection and presented the posterior inclusion probability (PIP) for each PFAS.Bayesian kernel machine regression (BKMR)P-values were two-tailed, and statistical significance was defined as p value less than 0.05.Most statistical analyses were performed using the survey analysis procedures in SAS . In addition, we used R software  with “qgcomp” and “bkmr” packages to conduct PFAS mixture analyses. All the Supplementary Information.


Purpose: Patients with syndromic 46, XY disorders/differences of sex development (DSD) are characterized by gonadal and phenotypic genders inconsistent with their chromosomal sexes as well as abnormalities of multiple extragonadal organs. They are caused by mutations in specific genes, which are expressed in the affected organs and regulate their development, and over fourteen genes have been identified. In this study, we aimed to determine the underlying cause of a patient with syndromic 46, XY DSD and review the clinical presentations and genetic findings of all reported similar cases.Methods: Whole-exome sequencing (WES) was performed to find a molecular cause of the patient. In silico tools were used to analyze the pathogenicity of the variants. Reports of cases with similar clinical features and involved genes were summarized by searching through PubMed/MEDLINE using keywords “PPP2R3C” or “G5PR” and “46,XY disorders of sex development”.Results: Compound heterozygous variants (p.F229del/p.G417E) in PPP2R3C were identified in the 24-year-old female by WES and verified by Sanger sequencing. The patient presents complete testicular dysgenesis, low birth weight, facial deformity, cubitus valgus, and decreasing number of CD19+ B lymphocytes and CD4+ T lymphocytes. A total of thirteen 46, XY DSD cases with four homozygous PPP2R3C mutations  have been reported previously, and their clinical manifestations are roughly similar to those of our patient.Conclusion: Novel compound heterozygous variants in PPP2R3C cause specific syndromic 46, XY gonadal dysgenesis, which broadened the pathogenic variants spectrum of PPP2R3C. The typical phenotype of PPP2R3C mutation is complete 46, XY gonadal dysgenesis with multiple extragonadal anomalies, including facial deformities, skeletal system abnormalities, muscle abnormalities, impaired nervous system, impaired hearing and vision, heart and kidney anomalies, and gastrointestinal dysfunction. The disease may present a dysgenetic gonadal, testicular, ovotesticular, or ovarian tissue and variable degrees of virilization of the external genitalia, along with structures derived from the Wolffian or Müllerian duct. According to the different pathogenesis, 46, XY DSD is mainly divided into gonadal development abnormalities, insufficiency of androgen synthesis, insensitivity to androgens, or Müllerian duct persistent syndrome . The etiWT1, NR5A1, SOX9, FGFR2, HOXA13, DHCR7, and XH2, most of which are genes encoding transcript factors  is located on 14q13.2, which has 13 exons encoding a 453-amino acid protein. Recently, it was found that the PPP2R3C gene regulates the balance of phosphorylation and dephosphorylation of Sox9 protein by forming a protein phosphatase complex. The mutation of the PPP2R3C gene is identified as a novel genetic etiology for syndromic 46, XY DSDs -box 9 (SOX9) protein, a very important transcript factor, regulates the onset, development, and maintenance of the testis formation, and it is also essential for the early chondrogenesis in humans. Mutations or deletions of li cells . Human P XY DSDs . HomozygPPP2R3C variants in a Chinese patient diagnosed with 46, XY gonadal dysgenesis. The clinical presentations and genetic findings of similar cases reported previously were also reviewed to improve our understanding of the phenotypes and genotypes in patients with PPP2R3C mutations.In this study, we described the clinical, laboratory characteristics, and novel compound heterozygous Patient’s genomic DNA was extracted from 200 μl peripheral blood leukocytes using the Qiagen DNA Blood kit . First, 50 ng DNA was broken into a size of about 200bp by fragmentation enzymes. The DNA fragments were then end-repaired, and the 3’end was added with one A base. Second, the DNA fragments were ligated with barcoded sequencing adaptors, and fragments of about 320bp were collected by XP beads. After PCR amplification, the DNA fragments were hybridized and captured by NanoWES according to the manufacturer’s protocol. The hybrid products were eluted and collected and then subjected to PCR amplification and purification. Next, the libraries were quantified by qPCR, and size distribution was determined using an Agilent Bioanalyzer 2100 . Finally, the Novaseq6000 platform , with 150 bp pair-end sequencing mode, was used for sequencing the genomic DNA of the patient. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data. The sequencing reads were aligned to the human reference genome (hg19/GRCh37) using the Burrows–Wheeler Aligner tool. GATK software was employed for variant calling. Variant annotation and interpretation were conducted by ANNOVAR.PPP2R3C gene were amplified by PCR, and then the PCR products of exon 7 and exon 13 were purified and underwent Sanger sequencing. Primers were designed using Primer3 software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Primer sequences were as follows: PPP2R3C-E7-F: 5′-ACTACATATTGGAACTTATCCCTACG-3′, PPP2R3C-E7-R: 5′-GGCAG TGAC AGGAAAATAAATGATA-3′; PPP2R3C-E13-F: 5′-ATTTTATTCCTCGTACTTCAG TGGA-3′, PPP2R3C-E13-R: 5′-GGTCCTATT GCTTCTAAACTATT GC -3′.Exon 7 and exon 13 of the TA clone of exon 7 was performed for a small deletion existing in one allele of the patient. The Taq polymerase-amplified PCR products of exon 7 were inserted into the pMD19T vector, and then the constructs were transformed into DH5α-competent cells and grew in the X-Gal and IPTG plates. The white clones were selected and amplified, and their DNA was extracted and sequenced.https://blast.ncbi.nlm.nih.gov/Blast.cgi) taking the following sequence as references: cDNA: NM_017917.4; protein: NP_060387.2.Sequencing of PCR products and TA clone was performed using a Taq big dye terminator sequencing kit and an ABI-3730 automated sequencer . DNAs from both parents were sequenced to ascertain the inheritance. We read the sequencing results by 4Peaks  and then blasted each in NCBI (https://swissmodel.expasy.org). Then, we downloaded the PDB file and uploaded it into UCSF ChimeraX (version: 1.2.5), a molecular visualization program. By comparing the changes in the contacts and hydrogen bonds with other amino acids in a spatial model, we can predict the potential pathogenic effects the specific variant brings to the protein.Structural models of wild PPP2R3C protein and mutant protein were built by inputting protein sequences in the SWISS-MODEL server , luteinizing hormone (LH), and testosterone (T), which were measured by chemiluminescent immunoassays .PPP2R3C variations in PubMed and MEDLINE, and the keywords for searching included “PPP2R3C” or “G5PR” in combination with “46,XY disorders of sex development”. Available data on clinical evaluations and genetic findings were extracted and summarized.We searched for reports about PPP2R3C and confirmed by Sanger sequencing  of quencing  in the pquencing . The delquencing .A 24-year-old female of Chinese Han descent was diagnosed with 46, XY gonadal dysgenesis at age 18, when she was presented with poor development of secondary sexual characteristics and primary amenorrhea. She was born of a non-consanguineous marriage at term by vaginal delivery without any perinatal complications and was raised female gender. She was born with a birth weight of around 1 kg and normal body length. There was no history of feeding difficulties or developmental retardation in childhood. At ten years of age, she presented retardation of bone age about two years, and the closure of bone epiphysis did not appear till over 20 years old. The patient was 148 cm tall (-3SD) at 18 years old and was diagnosed with short stature. She received growth hormone therapy for three months and gained the height increase of about 2.5 cm in the following two years. The patient did not receive other therapy till presentation.On physical examination, she had a height of 155 cm, a weight of 48 kg, and a blood pressure of 124/75 mmHg. Tanner staging was B1P2. External genitalia revealed the clitoris of normal and puerile female genitalia. There were two distinct openings in the urethra and vagina. No gonads or lumps were found in the inguinal region. The patient did not have facial hair, acne, male torso, body hairs, and deepening of voice. Systemic examination revealed remarkable facial features including unruly scalp hair, thin eyebrows, thin lips, and long and smooth philtrum. The patient had hypoplastic ala nasi and beaked nose, low-set ears, and facial pigmented nevus which were non-ignorable facial characteristics as well. The systemic examination also revealed abnormalities of the musculoskeletal system including a short fifth phalanx, webbing of the neck, and cubitus valgus .PPP2R3C gene is highly expressed in lymphocytes and plays a role in the survival of CD19+ B cells. Gene knockout (PPP2R3C-/-) mice by conditional targeting in CD19+ B cells showed a deficit in B-cell survival and a reduced number of mature B cells. After we identified the PPP2R3C variants of our patient, we tested her T/B cell subsets. The analysis of T/B cell subsets revealed a decreased number of CD19+ B cells  and CD4+ T cells  , FSH 70.88IU/L , T 0.34 ng/ml , E2 11 pg/ml , 17-OHP 1.19 ng/ml (-), FT4 1.19 ng/dl , FT3 2.95 pg/ml , and TSH1.2μIU/ml  . Lab eva0–46.0%) . The kar0–46.0%) .Considering the risk of gonadal malignancy, she underwent bilateral gonadectomy at twenty years of age, and streak gonads and bilateral oviducts were found during the operation. Histopathology of gonadectomy material showed an oviduct-like structure bilaterally. After gonadectomy, she was treated with Femoston (estradiol/dydrogesterone: 1/10 mg), with a dose of one tablet daily for almost four years.PPP2R3C variants. Eleven out of thirteen 46, XY DSD patients were presented with complete gonadal dysgenesis, complete female vulva with hypoplastic labium major, and hypoplastic uterus. Sex hormone levels were compatible with complete gonadal dysgenesis. One patient presented with partial testicular dysgenesis, and he had ambiguous genitalia, micropenis, and left cryptorchidism. PPP2R3C variants are also reported to be associated with syndromic 46, XX DSD. Three such patients were presented with complete gonadal dysgenesis, recognizable facial features, and multiple extragenital malformations. All patients with PPP2R3C mutations had some similar extragonadal system manifestations, such as facial deformity , retardation of bone age , and delayed development of the nervous system . The typical facial deformity includes abnormal eyebrows, low small-ears, and dysplasia of the alar nose. Among sixteen patients, more than half of the cases had special clinical manifestations, such as impaired vision , low birth weight , and myopathy . Symptoms including, renal agenesis , gastrointestinal dysfunction , sensorineural hearing loss , and cardiac defect  also appeared in some patients  and gnomAD (0.000194452) databases. These two variants were predicted to be harmful to protein function by REVEL, an ensemble method for predicting the pathogenicity of variants. So far, four other variants  in PPP2R3C have been reported in cases with similar clinical characteristics. Three missense mutations  were predicted to be pathogenic according to in silico tools, conserved among species, and not found in gnomAD, ExAC, or 1000 Genomes databases. Therefore, the pathogenic variants of PPP2R3C cause a syndromic 46, XY complete gonadal dysgenesis in an autosomal recessive inheritance pattern.In the present study, we reported a syndromic 46, XY DSD due to the compound heterozygous variants in the PPP2R3C variants manifested gonadal dysgenesis and complete female external genitalia along with the hypoplastic uterus seen in ultrasound or laparoscopy. Extragonadal multisystem abnormalities were also shared in these patients. They were presented with flat faces, poorly developed nasal alars, thin lips, and low ears. The facial abnormalities might be related to dysplasia of cartilage in ears and nose in the early chondrogenesis. The SOX9 protein played a significant role in early chondrogenesis, and it was suggested that pathogenic variants in PPP2R3C can impair SOX9 signaling by upregulating the catalytic function of PP2A  mice by conditional targeting in CD19+ B cells showed a deficit in B-cell survival and low level of mature B cells (+ T lymphocytes and increased NK cells, and this condition suggested that PPP2R3C plays a role in the survival of multiple lymphocytes and identified PPP2R3C variants as a potential cause of the disorder of immune cells.46, XY DSD patients with  of PP2A . In addi B cells . It is s B cells . SignifiPPP2R3C cause specific syndromic 46, XY gonadal dysgenesis, which broadened the pathogenic variant spectrum of PPP2R3C. The typical phenotype of PPP2R3C mutation is complete gonadal dysgenesis with multiple extragonadal anomalies, including facial deformities, skeletal system abnormalities, muscle abnormalities, impaired nervous system, impaired hearing and vision, heart and kidney anomalies, and gastrointestinal dysfunction. In addition, we found a decreased number of CD19+ B cells and CD4+ T cells in our patient, which is a new phenotype in syndromic 46, XY gonadal dysgenesis.In conclusion, novel compound heterozygous variants in


Ceanothus velutinus, commonly known as snowbrush ceanothus, from the natural habitat and the greenhouse-grown native soil-treated snowbrush ceanothus plants. We also conducted a microbial diversity analysis of the rhizosphere of greenhouse-grown native soil-treated and untreated plants (control). Twenty-seven of the 64 isolates were from the rhizosphere of the native region, and 36 were from the greenhouse-grown native soil-treated plants. These isolates were also tested for plant growth-promoting (PGP) traits such as their ability to produce catalase, siderophore, and indole acetic acid, fix atmospheric nitrogen and solubilize phosphate. Thirteen bacterial isolates tested positive for all five plant growth-promoting abilities and belonged to the genera Pantoea, Pseudomonas, Bacillus, and Ancylobacter. Besides, there are isolates belonging to the genus Streptomyces, Bacillus, Peribacillus, Variovorax, Xenophilus, Brevundimonas, and Priestia, which exhibit at least one of the plant growth-promoting activities. This initial screen provided a list of potential PGPR to test for plant health improvement on model and crop plants. Most of the bacterial isolates in this study have a great potential to become biofertilizers and bio-stimulants.Continuous demand for an increase in food production due to climate change and a steady rise in world population requires stress-resilient, sustainable agriculture. Overuse of chemical fertilizers and monoculture farming to achieve this goal deteriorated soil health and negatively affected its microbiome. The rhizosphere microbiome of a plant plays a significant role in its growth and development and promotes the plant’s overall health through nutrient uptake/availability, stress tolerance, and biocontrol activity. The Intermountain West (IW) region of the US is rich in native plants recommended for low water use landscaping because of their drought tolerance. The rhizosphere microbiome of these native plants is an excellent resource for plant growth-promoting rhizobacteria (PGPR) to use these microbes as biofertilizers and biostimulants to enhance food production, mitigate environmental stresses and an alternative for chemical fertilizer, and improve soil health. Here, we isolated, purified, identified, and characterized 64 bacterial isolates from a native plant,  Sample collected from the five plants from the elevation 1950m AMSL and three plants from the elevation 2289m AMSL. Snowbrush ceanothus is a big shrub with profound roots in the national forest. The methodology described elsewhere was used with slight modifications, where whole plant was not uprooted as described in protocol . The tubes with samples were shaken on a rotary shaker to separate the rhizosphere soil. The roots were removed, and the rhizosphere soil was collected by centrifuging tubes at 3000g for five minutes. The rhizosphere soil pellets were washed with phosphate buffer without surfactant and stored at 4°C.Rhizosphere soil samples from protocol . The pri2.2-3 gypsum , 1.57 kg m-3 dolomitic lime , and 0.65 kg m-3 wetting agent , and controlled-release fertilizer (3-N) content (3-N) content (ppm) by a NO3-N meter . Five replicates were taken from each pot’s leachate to measure the NO3-N content. The rhizosphere soil was collected from the treated plants for PGPR isolation after six months of native soil treatments and stored at 4°C. Rhizosphere samples were also saved for metagenomics analysis and stored at -80°C. Sample collection was similar to the one followed for the native samples , 25% vermiculite , 0.89 kg.mrtilizer . To see rtilizer . Cutting content  were mea samples . An anal2.3https://www.novogene.com/us-en/services/research-services/metagenome-sequencing/16s-18s-its-amplicon-metagenomic-sequencing/). The amplification reaction mixture (25 µl) contained 13 µl of water, 10 µl of Platinum Hot Start PCR Master Mix (MM) (Thermo Fisher), 0.5 µl of 10 mM forward and reverse primer each, and 1 µl of 5 ng/µl of the DNA. The polymerase chain reaction (PCR) conditions were as follows: an initial denaturation of three minutes at 94°C followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 60 seconds, an extension at 72°C for 60 seconds, and a final extension at 72°C for 10 minutes, and ending with an infinite hold at 4°C using the DNA engine dyad Peltier thermal cycler. The PCR products were diluted 50 times, and a secondary PCR reaction attached the indexes. The second reaction mixture consisted of 5 µl of MM, 2 µl of i5 index, 3 µl of i7 index, and 1 µl of the diluted PCR product. The PCR conditions are as follows: an initial denaturation step at 94°C for one minute followed by 15 cycles of denaturation at 94°C for 15 seconds, annealing at 64°C for 15 seconds, and an extension at 72°C for one minute, and a final extension at 72°C for three minutes using a DNA engine dyad Peltier thermal cycler (BIO-RAD). Once the indexes were attached, the samples were cleaned up with AMPureXP beads, using a 1:1 ratio. The PCR products were quantified by fluorometry, and quality was analyzed on the TapeStation. The samples were then pooled and sequenced on the MiSeq using a 2×250 paired-end sequencing chip size (Illumina).Microbial DNA was isolated from the rhizosphere soil of snowbrush ceanothus plants (control and treatment) from the greenhouse using Qiagen DNeasy PowerSoil Pro Kit. The isolated DNA was quantified by nanodrop 2000 (Thermo Scientific). The V4 variable region of 16S rRNA was sequenced. The 16S rRNA gene was amplified using the V4 variable region-specific primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVHHHTWTCTAAT-3’) (R ver 4.0 (dada2 (phyloseq (phyloseq package. Heat maps of the relative abundance of bacteria in different samples were generated using the “heatmap.2” function of gplots (version 3.1.1)The sequenced data were analyzed using various packages available in  ver 4.0 . Analysi0 , Yeast Mannitol Agar (SIGMA-Life Science), Minimal M9 Media (BD Difco), and Actinomycete Isolation Agar (SIGMA-ALDRICH)  as the forward primer and 1492R (V9 region- 5′-TACGGYTACCTTGTTACGACTT-3′) as the reverse primer using DreamTaq DNA polymerase. The PCR program was as follows: 95°C initial denaturation for two minutes, 35 cycles of 95°C denaturation for 30 seconds, 54.3°C annealing for 30 seconds, and 72°C extension for one and a half minutes, followed by a final extension at 72°C for 10 minutes (Applied Biosystems- ProFlex PCR system). The PCR products were sequenced and results were run on a BLAST  against a 16SrRNA database on NCBI to identify the bacterial species. The sequences were submitted to the GenBank on NCBI, and accession numbers are given in 2.6Bacillus megaterium ATCC14581 was used as a positive control. The siderophore-producing bacteria were screened on CAS (chrome azurol S) agar (Millipore SIGMA) (Pseudomonas chlororaphis O6 (produce pyoverdine siderophore) and positive control for the siderophore test. Nitrogen-fixing (NF) bacterial isolates were screened on Norris Glucose Nitrogen-Free Medium (NGNF) (HIMEDIA) for their ability to fix atmospheric nitrogen. The bacterial isolates were streaked on the plates and incubated at 28 °C for three days or until a clear zone around the colony appeared. The appearance of a clear zone indicates a positive result for nitrogen fixation  . The bace SIGMA) . We usedfixation   was mixed with 1 ml of the supernatant and incubated for 25 minutes. IAA producing isolate developed pink color , PolR- (5′ ATS GCC ATC ATY TCR CCG GA 3′), universal primers for the presence of Nitrogenase gene . The bac2.7The fresh weight of 40 grams of bulk soil from each sample was taken and dried in a hot air oven at 60 °C for 72 hours, and dry weight was measured. Soil moisture percentage was calculated using the following equationThe pH and EC of the snowbrush ceanothus native soil were estimated by adding one part of the soil to two-part distilled water to make a soil slurry. A few drops of clear solutions from this slurry were taken to measure the pH and EC by an EC and pH meter (Thermo Scientific Orion Star A112 2011) .3 (0.5 mol/L), and ammonium molybdate ascorbic acid was added. The available P was measured calorimetrically at 880 nm . A blank was used without soil. A standard curve for inorganic phosphate was plotted using KH2PO4 (potassium dihydrogen phosphate), and the amount of available phosphorus (in µg/g) was determined. An analysis of variance (ANOVA) and Tukey-Kramer method for multiplicity at α = 0.05 was done to depict the statistical differences among different samples on SAS Studio .The total carbon (TC) and available phosphorus were measured in the bulk soil samples collected from the snowbrush ceanothus plants in Tony Grove. Triplicates were used per sample for each test. Five grams of each soil sample were air-dried for 72 hours. Dried samples were crushed using a mortar and pestle and sieved through a 250 µm sieve. Total carbon content was measured in dried soil samples of 60 to 230 mg using the SKALAR Carbon Analyzer 2008. The amount of phosphorus was calculated using Olsen’s sodium bicarbonate extraction method . One gra3 Method (2 extract (The available potassium and nitrogen were measured in the native and greenhouse soil samples (control and treatment) at the Utah State University Analytical Laboratories (USUAL) using Olsen NaHCO3 Method  and the  extract , respect extract .33.1The native soil-treated snowbrush ceanothus plants showed a visual difference in growth compared to the control plants , 22 from ¼ Tryptic Soy Agar (TSA), 21 from ¼ Nutrient Agar (NA), six from Actinomycete Isolation Agar (AIA), and 11 were from Yeast Mannitol Agar (YMA) Table S1Streptomyces, Pseudomonas, Variovorax, Priestia, Bacillus, Xenophilus, Acidovorax, Ancylobacter, and Pedobacter. Most isolates belonged to Pseudomonas (41.7%) and Streptomyces (19%)   from the greenhouse-treated plants’ rhizosphere showed nitrogen fixation ability using Norris Glucose Nitrogen Free media but 7 of them amplified the 393bp se gene) Figure 33.5.5Pantoea, Brevibacterium, Leifsonia, and Streptomyces. Two of them, GK_NR_133 (Pantoea sp.) and GK_NR_149 (Brevibacterium sp), produced the highest amounts of IAA, 33.52 ± 0.15 µg/ml and 14.88 ± 0.11 µg/ml, respectively  produced more than 1 µg/ml of IAA Figure 3Pseudomonas, Agrobacterium, Priestia, Acidovorax, Xenophilus, Streptomyces, Ancylobacter, Bacillus, and Brevundimonas.Out of all the bacteria isolated from the greenhouse snowbrush ceanothus rhizosphere, 32 out of 36 or 89% of the isolates from snowbrush ceanothus treated plants produced more than 1 µg/ml of IAA Figure 3Pseudomonas, Pantoea, Bacillus, and Ancylobacter  plants revealed the significant dominance of the bacteria belonging to phyla ugarcane . Many phPseudomonas and Streptomyces. In this study, bacteria from genus Streptomyces (41%) dominated the isolates from the rhizosphere of the natural habitat  reported to have various species of the Priestia possess plant growth-promoting activities and help plants grow and develop. Priestia aryabhattai promotes plant growth in soybean by increasing phytohormones, such as ABA and GA, and is also tolerant to oxidative stress  and Brevibacterium (14.88 ± 0.11 µg/ml) .The data presented in the study are deposited in the BioProject: PRJNA853068 at NCBI repository, BioSample accessions SAMN29359218, SAMN29359219, SAMN29359220, SAMN29359221, SAMN29359222, SAMN29359223, SAMN29359224 and available at (nifH+ gene. AK, JG, and VS analyzed and interpreted the data. The manuscript was written jointly with contributions from all the authors. All authors have read and approved the manuscript.AK conceptualized the idea and designed it and supervised the experiments. JG isolated DNA and prepared the 16S rRNA library, isolated, identified, and characterized bacteria, and carried out the plant growth assays. VS carried out the metagenomic analysis. KH assisted JG with experiments. AK and KH isolated bacterial DNA and amplified


Cow-calf operations rely mostly in forage-based systems. Different supplementation strategies are used by beef producers in order to overcome nutrient deficiencies and achieve targeted growth or reproductive performances. This study provides information on the impacts of feeding pregnant replacement heifers with vitamin/mineral and protein/energy supplements on heifer performance and fetal outcomes. Our study shows that moderate rates of gain (achieved via protein/energy supplementation) resulted in fetuses with heavier femurs and reduced liver mass relative to fetal body weight. Moreover, vitamin and mineral supplementation increased fetal liver mass, and vitamin and mineral supplementation combined with moderate gain treatments resulted in greater fetal intestinal weights. These findings indicate that replacement heifer nutrition during early gestation can alter the development of organs in the fetus that are relevant for future offspring performance. Liver and intestines are key organs related to energy metabolism; therefore, this study shows that compensatory mechanisms are in place in the developing conceptus that can alter the growth rate of key metabolic organs possibly in an attempt to increase or decrease energy utilization.p < 0.01). Concentrations of NEFA were greater for LG than MG (p = 0.04) and were affected by a VMSUP × day interaction (p < 0.01), with greater concentrations for NoVTM on d 83. Insulin was greater for NoVTM than VTM (p = 0.01). A GAIN × day interaction (p < 0.01) was observed for IGF-1, with greater concentrations for MG on d 83. At d 83, P4 concentrations were greater for MG than LG , and MG had greater (p < 0.01) corpus luteum weights versus LG. Even though fetal BW was not affected (p ≥ 0.27), MG fetuses had heavier (p = 0.01) femurs than LG, and VTM fetuses had heavier (p = 0.05) livers than those from NoVTM. Additionally, fetal liver as a percentage of BW was greater in fetuses from VTM  than NoVTM (3.79 ± 0.06% BW), and from LG  than MG (3.78 ± 0.06% BW). A VMSUP × GAIN interaction was observed for fetal small intestinal weight (p = 0.03), with VTM-MG being heavier than VTM-LG. Therefore, replacement heifer nutrition during early gestation can alter the development of organs that are relevant for future offspring performance. These data imply that compensatory mechanisms are in place in the developing conceptus that can alter the growth rate of key metabolic organs possibly in an attempt to increase or decrease energy utilization.Thirty-five crossbred Angus heifers  were randomly assigned to a 2 × 2 factorial design to evaluate effects of vitamin and mineral supplementation  and different rates of gain  during the first 83 d of gestation on dam hormone and metabolic status, fetal tissue and organ mass, and concentration of glucose and fructose in fetal fluids. The VMSUP was initiated 71 to 148 d before artificial insemination (AI), allowing time for mineral status of heifers to be altered in advance of breeding. At AI heifers were assigned their GAIN treatment. Heifers received treatments until the time of ovariohysterectomy (d 83 ± 0.27 after AI). Throughout the experiment, serum samples were collected and analyzed for non-esterified fatty acids (NEFA), progesterone (P4), insulin, and insulin-like growth factor 1 (IGF-1). At ovariohysterectomy, gravid reproductive tracts were collected, measurements were taken, samples of allantoic (ALF) and amniotic (AMF) fluids were collected, and fetuses were dissected. By design, MG had greater ADG compared to LG (0.85 vs. 0.34 ± 0.04 kg/d, respectively;  Dam nutrition during the early stages of pregnancy may have metabolic and physiological effects on the fetus ,2,3, witTo continue investigating the effects of maternal nutrition in fetal development outcomes, we developed a research model where beef heifers were subjected to a vitamin and mineral supplementation and two rates of gain achieved via protein/energy supplements  during the first 83 d of gestation. We speculate that strategic supplementation may modulate rates of gain and by doing so potentially affect offspring growth in utero and postnatally. Currently, there is a large variation in vitamin/mineral  and protPreviously , we repoDiniz et al.  observedAll animal procedures were approved by the North Dakota State University Institutional Animal Care and Use Committee (#A19012).n = 72; 11 to 12 months of age, initial BW = 359.5 ± 7.1 kg) were initially housed at the Central Grasslands Research Extension Center , where they were randomly assigned to one of 2 vitamin and mineral supplementation treatments , thus allowing time for mineral status of heifers to be altered in advance of breeding. Therefore, NoVTM heifers were submitted to a mineral depletion period of at least 71 days, as explained further in the text. Heifers were group-fed and diets were delivered once daily via total mixed ration (TMR) and consisted of triticale hay, corn silage, modified distillers grains plus solubles, ground corn, and if indicated by treatment, vitamin and mineral premix (delivered at a 0.45 kg•heifer−1•day−1 to target 113 g of product). The vitamin and mineral premix provided vitamins A, D, and E and macro and trace minerals to meet 110% of the requirements specified by the NASEM [−1•day−1.Crossbred Angus heifers (he NASEM  and cons2) with 6 heifers per pen. Therefore, a same pen housed VTM and NoVTM heifers. Heifers were individually fed daily in an electronic head gate facility , and continued to receive their respective VMSUP treatments until the time of artificial insemination (AI). At this facility, the VTM heifers received a pelleted vitamin and mineral supplement fed at a 0.45 kg•heifer−1•day−1, consisting of 113 g of the vitamin and mineral supplement and formulated to deliver similar levels of vitamins and minerals that were fed pre-breeding, while the NoVTM heifers received a pelleted carrier product fed at a 0.45 kg•heifer−1•day−1 with no added vitamins or minerals. The duration of time VTM and NoVTM heifers received their treatments varied according to the AI group they were assigned . At ANPC, heifers were randomly allocated in 12 group-pens (23.7 mAt AI (d 0 of our experiment), heifers were assigned randomly to either LG or MG treatments—within their respective VMSUP factor—completing the factorial arrangement of treatments. Heifers that received a protein/energy supplementation  were targeted to gain 0.79 kg/d (MG), while heifers that did not receive the protein/energy supplementation were targeted to gain 0.28 kg/d (LG). All heifers received a basal diet made up by prairie grass hay, corn silage, and dried distillers grains plus solubles . If indin = 9); (2) No vitamin and mineral supplement, moderate gain ; (3) Vitamin and mineral supplement, low gain ; (4) Vitamin and mineral supplement, moderate gain . Heifers were weighed at weekly intervals and individual feed intake adjusted during the course of the study to achieve targeted BW gains. The targeted daily gains proposed in our research model were both within the range observed in normal production scenarios, reflecting what has been observed in unsupplemented and supplemented grazing beef cattle [Pregnancy diagnosis was performed 35 days after AI, and fetal sex was determined on d 65 after AI using transrectal ultrasonography . After pf cattle ,23,24. ADiet TMR samples were collected weekly throughout the experiment and composited over the feeding period. The composited sample was dried in a 55 °C oven and ground to pass through a 2 mm screen. Samples were analyzed for dry matter, ash, N (Kjehldahl method), Ca, P, and ether extract by standard procedures . Crude protein was determined by multiplying N by 6.25. Neutral detergent fiber and acid detergent fiber concentrations were determined by the modified method of Van Soest et al. [g at 4 °C for 20 min, and stored in plastic vials at −20 °C until further analysis.Serum samples from the jugular vein were collected into 10 mL serum vacutainer tubes  at the time of VMSUP factor initiation , controlled internal drug release (CIDR) insertion (9 days before AI), and on d 14, 28, 42, 56, 70, and 83 ± 0.27 after AI to determine concentrations of glucose, non-esterified fatty acids (NEFA), and P4. Serum samples collected at VMSUP factor initiation, at the time of CIDR insertion, and at the time of ovariohysterectomy (d 83 ± 0.27 after breeding) were further analyzed to determine concentrations of insulin and insulin-like growth factor 1 (IGF-1). All blood samples were collected prior to the morning feeding, placed on ice, centrifuged at 1500× Samples were analyzed for glucose and NEFA using the Synergy H1 Microplate Reader  with the Infinity Glucose Hexokinase Kit  and NEFA-C Kit . The intra- and inter-assay CV were 2.87 and 6.80%, respectively, for glucose and 2.96 and 17.46%, respectively, for NEFA.Concentrations of P4, insulin ,27, and Ovariohysterectomy procedures were conducted at d 83 ± 0.27 of gestation to collect utero-placental tissues as previously described by McLean et al. . ImmediaSubsequently, the fetus was dissected, and the fetal liver, heart, intestine, pancreas, hindlimb, brain, and ovaries were collected. Fetal organs were individually weighed, processed, and stored at −80 °C for further analysis.g and 18 °C to remove any debris that would interfere with the assay. The thawed, pre-cleared ALF supernatants were diluted 1:80 and AMF samples diluted 1:10 with 18 mΩ water. The EnzyChrom Fructose Assay Kit  was utilized on a Synergy H1 Microplate Reader (BioTek) at 565 nm and 25 °C. Final results were reported in µmol of fructose. Pooled samples of ALF and AMF were used as controls .Allantoic (ALF) and amniotic (AMF) fluids were collected using a 22-gauge needle  to penetrate the respective fetal membranes, and fluids were suctioned with a 10 mL syringe . Aliquotp-values ≤ 0.05 were considered significant.Data were analyzed using the MIXED procedure of SAS 9.4 . Body weight, blood hormones and metabolites were analyzed as repeated measures with VMSUP, GAIN, day, and their interactions as fixed effects and heifer as a random effect. The covariate structures were tested, and the structure with the lowest Akaike information criterion/Bayesian information criterion was used for all variables analyzed. Maternal performance, uterine and fetal measurements, and glucose and fructose concentrations in ALF and AMF were analyzed using the MIXED procedures of SAS with VMSUP, GAIN, and a VMSUP × GAIN interaction as fixed effects and heifer as a random effect. Means were separated using the PDIFF procedure of SAS with a Tukey–Kramer adjustment. Results are presented as least squares means (LSMEANS) with their standard errors. For all analyses, heifer was considered the experimental unit and p = 0.72) or by a VMSUP × GAIN interaction  for MG heifers (0.85 ± 0.04 kg/d) than LG heifers  was not affected by VMSUP (p < 0.01) for MG (7.72 ± 0.21 kg/d) than LG (4.98 ± 0.19 kg/d) heifers (p < 0.01) for MG than LG heifers (p ≥ 0.49) or the VMSUP × GAIN interaction (p ≥ 0.08).To achieve targeted gains, total dry matter intake (DMI) was greater . Concentrations of glucose averaged 3.96 ± 0.15, 4.18 ± 0.14, 3.86 ± 0.19, and 4.25 ± 0.17 mmol/L for NoVTM-LG, NoVTM-MG, VTM-LG, and VTM-MG, respectively.There were no differences in plasma glucose concentrations due to main effects or interactions (p = 0.04), with lower concentrations for MG (262.54 ± 15.28 µmol/L) than LG (309.57 ± 17.03 µmol/L) heifers throughout the experiment. Further, there was a VMSUP × day interaction (p = 0.002) for concentrations of NEFA, with greater concentrations for NoVTM than VTM heifers (p = 0.02) at d 83 of gestation  for MG than LG heifers on d 83 of gestation . Concentrations of P4 were not affected by the main effects of VMSUP (p = 0.86) or GAIN (p = 0.11) nor other interactions (p ≥ 0.18).A GAIN × day interaction . Concentrations of insulin increased throughout the experiment (p < 0.01), with greater concentrations for all groups of heifers at d 83 of gestation (51.07 ± 2.76 pmol/L) compared to samples collected before VMSUP initiation (26.11 ± 2.76 pmol/L) and at the time of CIDR insertion (34.29 ± 2.76 pmol/L). Further, a VMSUP effect was observed (p = 0.01), with greater concentrations for NoVTM (43.16 ± 2.97 pmol/L) than VTM (31.15 ± 3.12 pmol/L) heifers.Concentrations of insulin in maternal serum were not affected by any of the  or GAIN (p = 0.25) nor any of the other interactions tested (p ≥ 0.30).A GAIN × day interaction (p ≥ 0.35), GAIN (p ≥ 0.43), or their interaction , both being greater for MG than LG heifers.Gravid and empty uterine weight and mean diameter of the 3 largest placentomes were not affected by VMSUP (p ≥ 0.27) or their interaction , where intestine from VTM-MG fetuses were heavier than those from VTM-LG (p = 0.04), with all other treatments being similar (p ≥ 0.08). This effect was not observed (p ≥ 0.15) when fetal intestinal mass was evaluated as a percentage of fetal BW. No other fetal organ weights  were affected by a VMSUP × GAIN interaction (p ≥ 0.15). However, femur weight was affected by GAIN, where fetuses from MG had heavier (p = 0.01) femurs than those from LG. Fetal liver weight was heavier (p = 0.05) for fetuses from VTM than NoVTM when analyzed as absolute weight, and when analyzed as a percentage of fetal BW. Interestingly, fetal liver weight as a percentage of fetal BW was greater (p = 0.04) for LG than MG heifers. Fetal heart, pancreas, hindlimb, brain, and ovaries  were not affected by main effects of VMSUP (p ≥ 0.14), GAIN (p ≥ 0.07), or their interaction (p ≥ 0.08). Fetal body measurements , GAIN (p ≥ 0.20), or their interaction (p ≥ 0.06).Fetal BW was not affected by main effects of VMSUP or GAIN  by VMSUP, GAIN, or their interaction. Concentrations of glucose averaged 2.61 ± 0.11 and 2.13 ± 0.04 mmol/L in ALF and AMF, respectively; while concentrations of fructose averaged 28.61 ± 1.8 and 4.15 ± 0.14 mmol/L in ALF and AMF, respectively.Concentrations of glucose and fructose in ALF and AMF were not affected  were targeted to gain 0.79 kg/d and had an actual ADG of 0.85 kg/d, whereas unsupplemented heifers (LG) were targeted to gain 0.28 kg/d and had an actual ADG of 0.34 kg/d. We reported that gestating heifers grown at a moderate rate of gain had increased concentrations of IGF-1 and P4, lower concentrations of NEFA throughout the experiment, greater CL weight at d 83 of gestation, and their fetuses had greater fetal femur weight and reduced liver weight (as a percentage of BW) compared with heifers grown at a lower rate of gain. We did not observe differences in concentrations of glucose and fructose in fetal fluids in response to maternal treatments; however, this study demonstrated that providing a vitamin and mineral supplement during the first trimester of gestation resulted in greater fetal liver weight, whereas the interaction between vitamin/mineral and protein/energy supplementation resulted in greater fetal intestinal weight. Findings from this study indicate that response variables that diverged in the dam were also experienced in some way by the fetus. Our results may help to understand how maternal nutritional strategies in early pregnancy can alter the development of organs that are relevant for future offspring performance.It is well known that levels of metabolic hormones and metabolic factors are regulated by nutritional status. For example, key responses such as follicular development, oocyte quality, maternal-embryo recognition, embryo and fetal development, and pregnancy maintenance have been shown to be affected by nutrition, body condition, and metabolic status of beef heifers and cows ,33. In tAdditionally, nutritional plane and rates of gain are associated with IGF-1 levels. Similar to our study, Rodríguez-Sánchez et al.  reportedThe fact that fetal liver mass (as a % of BW) was increased in fetuses from LG heifers indicates that the liver grew at a greater rate than that of the overall body during the first 83 d of gestation. Studies investigating fetal hepatic and small intestinal in vitro oxygen consumption in pregnant ewes, cows, and their fetuses ,41,42 haAn interesting observation was that heifers receiving VTM had reduced concentrations of NEFA on d 83 compared with heifers not receiving VTM. Leading up to d 83, heifers were handled 2 additional days for surgical preparation and were fasted for 24 h, and water was deprived for 12 h before the d 83 sample collection; therefore, the dramatic increase in NEFA for all heifers on this day was expected. Such stressors may increase the levels of catecholamines and glucocorticoids that stimulate lipolysis . PerhapsOther novel finding of the present study was the greater fetal liver weight (absolute weight and as a percentage of BW) of fetuses from VTM heifers, indicating that a vitamin and mineral supplementation at prebreeding and early gestation play a role in fetal liver development. Further, we observed that heifers supplemented with vitamins/minerals and protein/energy presented greater fetal intestinal mass than those receiving a VTM supplement but not supplemented with protein/energy, implying a greater intestinal functional area. However, small intestine growth and development is affected not only during organogenesis, but also during the perinatal and neonatal periods . TherefoIn a previous study  we reporIn summary, vitamin/mineral and protein/energy supplementation of pregnant beef heifers are advantageous as it overcomes potential mineral deficiencies in forage-based diets and are a solid strategy to achieve targeted production goals for growth and reproductive performance of commercial beef herds. In addition, results of this study demonstrate that (1) moderate rates of gain result in heavier fetal femur weight and reduced fetal liver proportional mass; (2) vitamin and mineral supplementation results in greater fetal liver absolute and proportional mass; and (3) vitamin and mineral supplementation combined with moderate rates of gain resulted in greater fetal intestinal mass than VTM supplementation combined with low rates of gain. These preliminary data are interpreted to imply that fetuses have the ability to alter the growth rate of key metabolic organs in an attempt to increase or decrease energy utilization. Further research is necessary to study energy expenditure of fetal tissues, which could result in the development of nutritional strategies to improve mitochondrial function and the efficiency of energy utilization in the future offspring.


To investigate the safety and efficacy of chemoradiotherapy or radiotherapy combined with nimotuzumab in the treatment of unresectable oesophageal squamous cell carcinoma (ESCC) in elderly patients.This study retrospectively analysed 54 cases of elderly patients (aged over 70 years) with unresectable ESCC in our centre between December 2016 and November 2019. The patients were treated with a radiation dose of 50–61.6 Gy (25–30 fractions) combined with nimotuzumab for targeted therapy with or without chemotherapy according to each patient’s condition. The patients were observed for quality of life, safety, side effects and survival before and after the treatment.Among the 54 patients, 26 were treated with nimotuzumab combined with chemoradiotherapy and 28 were treated with nimotuzumab combined with radiotherapy. Toxicities were mainly oesophagitis , myelosuppression  and hypoproteinaemia . The rates of complete response, partial response, disease stability and disease progression were 11.1% (6/54), 81.5% (44/54), 3.7% (2/54) and 3.7% (2/54), respectively, and the overall objective response rate was 92.6% (50/54). The median follow-up time was 35.1 months, and the 1- and 2-year overall survival (OS) and progression-free survival (PFS) rates were 61.1% (1 year OS) and 35.2% (2 year OS), 42.6% (1 year PFS) and 16.7% (2 year PFS), respectively. The median OS and PFS rates were 16.0 and 10.0 months, respectively.Nimotuzumab combined with chemoradiotherapy or radiotherapy was well tolerated in elderly patients with unresectable ESCC. This combination can achieve a good treatment response and enhance survival. Oesophageal carcinoma is one of the most common gastrointestinal cancers and ranks seventh in malignant tumours and sixth in mortality worldwide. Almost 1 in every 20 cancer deaths occurs in China .Epidermal growth factor receptor (EGFR) is overexpressed in ESCC tissues and has been significantly associated with high local recurrence rates and low overall survival (OS) rates in multivariate analyses . In receAt present, chemoradiotherapy is the primary treatment for patients with local advanced oesophageal carcinoma . For eldA retrospective study showed that radiotherapy combined with nimotuzumab in elderly patients with oesophageal carcinoma was preliminarily proven to be safe and effective . The comIn this study, the safety and efficacy of nimotuzumab combined with chemoradiotherapy or radiotherapy in elderly patients with ESCC were investigated.Elderly patients with ESCC who were treated with radiotherapy/chemoradiotherapy + nimotuzumab at our hospital from December 2016 to November 2019 were analysed retrospectively.The inclusion criteria were as follows: (1) patients aged 70 years or older, (2) ESCC confirmed by pathology, (3) patients who were unresectable or unwilling to have surgery or endoscopic treatment, (4) Stage I–IVA (based on imaging and the American Joint Committee on Cancer’s staging [8th edition]), (5) estimated survival time ≥ 3 months, (6) Karnofsky Performance Status ≥ 70, (7) largely normal bone marrow function and (8) patients who signed an informed consent form.Unresectable ESCC in this study was defined as follows: (1) The tumour was T4b, involving the heart, large blood vessels, trachea, vertebral body or adjacent abdominal organs, including the liver, pancreas, lung and spleen, (2) cervical or thoracic oesophageal cancer, with a lesion < 5 cm from the cricopharyngeal muscle, (3) the tumour was stage IVA at the lower oesophagus, but the regional lymph node involved blood vessels or other organs, making resection impossible or (4) unresectable because of comorbidities. Each case was discussed by a multidisciplinary team to determine whether surgery could be performed.The exclusion criteria were as follows: (1) oesophageal adenocarcinoma or other pathological types, (2) distant metastases, (3) tumour recurrence, (4) severe infectious disease, (5) a combination of other tumours and (6) history of surgery, chemotherapy or radiotherapy.The rejection criteria were: (1) refusal of treatment for various reasons (most of the patients who refused treatment in this study were unable to afford the cost of treatment) and (2) incomplete or missing information.All patients in this study signed an informed consent form, and the study protocol was approved by the institutional review board of our hospital.This study retrospectively analysed 54 elderly patients with ESCC. Among them, 28 patients received nimotuzumab combined with radiotherapy and were enrolled in an NRT group. The other 26 patients received nimotuzumab combined with chemoradiotherapy and were enrolled in an NCRT group.International Commission on Radiological Units and Measurements 62. The gross tumour volume (GTV), including the primary lesions and the involved lymph nodes, was determined according to CT or positron emission tomography (PET), endoscopy and oesophagus barium meal. Elective node irradiation was applied to the clinical target volume (CTV), which was 3 cm superior and inferior to the GTV, 1 cm horizontal to the GTV and included the corresponding lymphatic drainage areas. The planning target volume (PTV) was defined as the CTV plus 5 mm three-dimensionally. The PTV included at least 95% of the isodose lines, and 95% of the PTV dose was 50–61.6 Gy in 28–30 radiations. The radiotherapy was delivered by a Varian linear accelerator, once a day, five times a week.All patients received intensity-modulated radiation therapy. Patients were positioned in the supine position and underwent a computed tomography (CT) scan with a 5-mm slice after the position was fixed using thermoplastic film. The target area was outlined according to the definition in the The organ-at-risk dose limits were V20 ≤ 25%, V30 ≤ 20% and V5 ≤ 60% for both lungs and V40 ≤ 30% and V30 ≤ 40% for the heart. The maximum dose for the spinal cord was 45 Gy.2 d1 + cisplatin 20 mg/m2 d1, once a week (3/54) [All patients received (1) tegafur–gimeracil–oteracil potassium capsules , 40–60 mk (3/54) .Nimotuzumab was administered at a dose of 400 mg weekly .Eligible patients were required to complete a history interview, physical examination, blood examination, electrocardiogram, oesophagoscopy and biopsy, oesophagus barium meal, chest and abdomen CT, neck ultrasound, radionuclide bone scan and brain magnetic resonance imaging or PET/CT.The evaluation of treatment efficacy was performed according to the Response Evaluation Criteria in Solid Tumours scale (version 1.1) , includiAdverse reactions of radiotherapy  were evaluated according to the Radiation Therapy Oncology Group’s classification of adverse reactions . HaematoA survey was completed by patients before and after treatment, and the scores were recorded. The scale consisted of 10 items and 18 questions (1–4 points per question) [Adverse reactions were evaluated every week during the treatment. Follow-ups were conducted 1 month after the treatment, every 3 months for the subsequent 2 years, and every 6 months in years 3–5. These evaluation include each patient’s symptoms, physical examinations, routine blood tests, biochemistry results, tumour markers and imaging results; gastroscopy and biopsy were performed if necessary.A statistical analysis was performed using SPSS™ Statistics v26.0 software, and the Kaplan–Meier method was used for the analysis of OS and PFS. The Wilcoxon rank–sum test was used to compare the difference in QLQ-OES18 scale scores for each patient before and after treatment.A total of 54 enrolled patients were treated with nimotuzumab combined with radiotherapy or chemoradiotherapy from December 2016 to November 2019. The median age of the patients was 75 (70–91) years, and the patient information is shown in Table Patients underwent a comprehensive evaluation after treatment. Six patients (11.1%) achieved a clinical complete response, 44 patients (81.5%) achieved a partial response, 2 patients (3.7%) had disease stability, and only 2 patients (3.7%) showed disease progression. The ORR was 92.6%. The response of each group is shown in Table The treatments were well tolerated. There were no Grade-5 adverse reactions, but 17/54 (31.5%) of patients had ≥ Grade-3 adverse reactions. The five most common adverse reactions were oesophagitis, hypoproteinaemia, fatigue, leukopenia and pneumonia.The incidence of ≥ Grade-3 haematologic reactions was 20.3% (11/54), including 2 patients with > Grade-3 leukocyte and platelet reduction. The incidence of hypoproteinaemia was 94.4% , which has not been reported in previous studies. The incidence of ≥ Grade-2 oesophagitis was 38.9% (21/54), including 3 patients (5.6%) with Grade-3 oesophagitis. The incidence of Grade-1–2 radiation pneumonitis was 46.2% (25/54), while it was 7.4% (4/54) for Grade 3. Other adverse reactions are shown in Table The last follow-up date was 2 November 2021. The median follow-up time was 35.1 months, and 13 patients (24.1%) survived at the last follow-up, 2 of whom were treated with re-irradiation because of lymph node recurrence. Of the 41 patients who died, 3 (7.3%) died from complications (including bleeding and pneumonia), 17 (41.5%) died from other underlying diseases , 6 (14.6%) had local and regional lymph node progression, and 15 (36.6%) had distant metastases. The median OS and PFS rates were 16.0 and 10.0 months, respectively. The 1- and 2-year OS rates were 61.1% and 35.2%, respectively, while the corresponding PFS rates were 42.6% and 16.7%, respectively , while other symptoms  worsened after therapy (p < 0.05). Some symptoms  did not change significantly (p > 0.05). The results are presented in Table The QLQ-OES18 is a scale specifically designed to evaluate the quality of life of patients with oesophageal carcinoma . In our Oesophageal carcinoma is usually at a late stage when it is diagnosed and is prone to metastasis, treatment resistance and frequent recurrence . The clip < 0.05). Some changes during treatment, such as dysphagia, were obvious, but sometimes, since patients may not always let medical staff know of their discomfort, some symptoms might have gone unnoticed. These symptom changes have a great influence on the overall quality of life, which is especially important for elderly patients. Therefore, the quality-of-life scale should be more widely used in patients with oesophageal carcinoma, especially in elderly patients.Elderly patients have a short life expectancy, and side effects during treatment directly influence their quality of life and survival to some extent. In this study, the QLQ-OES18 was scored before and after treatment in the enrolled patients, which was a great improvement compared with previous studies. The QLQ-OES18 is reliable, effective and acceptable for evaluating the quality of life in patients with oesophageal carcinoma . In thisIn our study, the ORR was 92.6%, the disease control rate was 96.3%, and the median OS and PFS durations were 16 and 10 months, respectively, which were similar to those reported in the NICE study in Brazil , 24. TheIn summary, nimotuzumab combined with chemoradiotherapy or radiotherapy was well tolerated in elderly patients with unresectable ESCC. This combination can achieve a good treatment response and enhance survival. The sample size of our study was relatively small because of the number of elderly patients admitted to the single centre. In addition, it was a retrospective study without a control group, so the results have limitations and need to be investigated further through large-sample prospective studies.


It has been shown that copper oxide nanoparticles (CuO NPs) induce pulmonary toxicity after acute or sub-acute inhalation exposures. However, little is known about the biodistribution and elimination kinetics of inhaled CuO NPs from the respiratory tract. The purposes of this study were to observe the kinetics of pulmonary inflammation during and after CuO NP sub-acute inhalation exposure and to investigate copper (Cu) biodistribution and clearance rate from the exposure site and homeostasis of selected trace elements in secondary organs of BALB/c mice.Sub-acute inhalation exposure to CuO NPs led to pulmonary inflammation represented by increases in lactate dehydrogenase, total cell counts, neutrophils, macrophages, inflammatory cytokines, iron levels in bronchoalveolar lavage (BAL) fluid, and lung weight changes. Dosimetry analysis in lung tissues and BAL fluid showed Cu concentration increased steadily during exposure and gradually declined after exposure. Cu elimination from the lung showed first-order kinetics with a half-life of 6.5 days. Total Cu levels were significantly increased in whole blood and heart indicating that inhaled Cu could be translocated into the bloodstream and heart tissue, and potentially have adverse effects on the kidneys and spleen as there were significant changes in the weights of these organs; increase in the kidneys and decrease in the spleen. Furthermore, concentrations of selenium in kidneys and iron in spleen were decreased, pointing to disruption of trace element homeostasis.Sub-acute inhalation exposure of CuO NPs induced pulmonary inflammation, which was correlated to Cu concentrations in the lungs and started to resolve once exposure ended. Dosimetry analysis showed that Cu in the lungs was translocated into the bloodstream and heart tissue. Secondary organs affected by CuO NPs exposure were kidneys and spleen as they showed the disruption of trace element homeostasis and organ weight changes.The online version contains supplementary material available at 10.1186/s12989-022-00480-z. Nanomaterials have been widely used in diverse fields due to their unique physicochemical properties including high surface-to-volume ratios, quantum effects, and flexible functionalization of surface structures , 2. Copp2O5, WO3, Al2O3, Fe2O3, TiO2, MgO, and CeO2, and revealed CuO NPs to exert the greatest cytotoxicity in a dose-dependent manner [3. Immediately after exposure, mice had significantly higher levels of bronchoalveolar lavage (BAL) cytokines, inflammatory cells, lactate dehydrogenase (LDH), as well as perivasculitis and alveolitis. At 3-weeks post-exposure, all toxicity parameters and histopathology returned to typical values except the number of inflammatory cells. The histopathology of the lung at 0- and 3-weeks post-exposure demonstrated the absence of Cu NPs, suggesting the possibility of Cu NPs translocation from the pulmonary site to other organs or Cu dissolution in the airways or airway surface liquid. Moreover, in an ovalbumin (OVA)-induced asthma mouse model, CuO NPs aggravated the increased airway hyperresponsiveness, inflammatory cell count, proinflammatory cytokines, reactive oxidation species, and immunoglobulin E induced by OVA exposure [With increasing applications for the use of CuO NP and the resulting concern their potentially harmful effects, nanomaterial toxicity research both in vitro and in vivo has dramatically increased , 12. Pret manner . In vitrt manner , 15. Mort manner . Therefot manner –20. We pt manner  the pulmexposure . Similarexposure , 20. Stuexposure . Howeverexposure .This study aimed to investigate the kinetics of pulmonary inflammation and biodistribution of inhaled CuO NPs during and after subacute exposure. Tracking Cu concentration in the lungs over time would deepen our understanding of CuO NP toxicity at the exposed site. Moreover, fluctuations in Cu and other trace element concentrations in secondary organs during and after exposure may lead to disruptions in trace element homeostasis and subsequently to systemic adverse effects since trace elements have essential roles in biochemical processes in the body and are heavily dependent on one another.3 using a nose-only exposure system for 4 h/day, 5 days/week over 2 weeks, and were necropsied on days 0, 3, 7, 12, 17, 22, and 27 . BAL fluids were collected to measure inflammatory cytokine/chemokines levels, numbers of white blood cells, and LDH. Lung histopathology was performed to evaluate inflammation. Whole blood, lung, brain, heart, kidneys, liver, spleen, urine, and BAL fluid were collected to measure Cu and selected trace element concentrations. Disruption of the elemental homeostasis in primary and secondary organs might be an indication of negative adverse effects in these biological systems. To better understand how CuO NPs are processed and mobilized once they are inhaled, dissolution studies in three artificial biological fluids including artificial lysosomal fluid , simulated epithelial lung fluid , and simulated gastric fluid  were conducted.In this study, female BALB/c mice were exposed to CuO NPs at 3.75 mg/m2 volume displacement) was 6.15 ± 0.0027 g/cm3. During inhalation exposure, the particle size distribution of CuO NP aerosol measured by a scanning mobility particles sizer (SMPS) spectrometer showed a geometric mean (GM) mobility diameter of 36.4 nm with a geometric standard deviation (GSD) of 1.67  Table . CuO NP 2 = 0.6243), while Cu concentrations were negatively associated with macrophage numbers (p = 0.0013 with R2 = 0.6785) when statistically adjusted for time.LDH levels increased for all time points compared to the control group, however, significant increases were observed on day 3 p < 0.01), 7 (p < 0.001), and 12 p < 0.05) which were the days of the exposure period Fig. a, b. Neu, 7 (p <  which weTwelve out of 23 cytokines/chemokines measured in BAL fluid showed significant changes during and after exposure and included: keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, eotaxin, RANTES, IL-1α, IL-6, IL-10, and IL-12(p40). The concentration of the following cytokines/chemokines was below the lowest limit of detection (LLOD): IL-1β, IL-2, IL-3, IL-4, IL-5, IL-9, IL-12 (p70), IL-17a. No significant changes were found in interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-13 concentrations. Murine KC is a potent chemoattractant, inducing the infiltration of neutrophils to inflammatory sites . G-CSF cMicrographs of lung tissues stained with hematoxylin and eosin (H&E) were observed under a bright-field microscope. The controls showed no pathology with clear alveolar spaces and no overt recruitment of lymphocytes or neutrophils. Development of progressive perivascular and periairway inflammation represented mainly by lymphocytes (indicating a chronic inflammation) was observed in the lungs over time starting at day 3 of exposure with increased signs of inflammation present at Day 27  at the exposure site was 6.5 days  to observe the time course of pulmonary inflammation during and after CuO NP sub-acute inhalation exposure, (2) to determine the biodistribution of Cu and its clearance rate from the lungs, and (3) to investigate the effect of inhaled Cu on secondary organs by monitoring concentrations of trace elements and organ weights.1/2 of 6.5 days. Finally, disruption in trace element homeostasis was observed in three organs: spleen, kidneys, and brain. Significant organ weight changes were found in kidneys, spleen, and lung.There were three major findings from these studies. First, sub-acute inhalation of CuO NPs (an estimated lung burden of 53 µg/mouse not considering lung clearance) led to pulmonary cytotoxicity (elevated LDH concentrations in BAL fluid) and inflammation . Observed progressive inflammation corresponded to the Cu concentration in lung tissue, and soluble Cu in BAL fluid, which started increasing during exposure and subsequently decreased post-exposure. The airway inflammation was still present at least 15 days post-exposure (day 27). Second, as expected, the lung as the route given the study design was the main organ that showed significant increases in Cu concentrations compared to controls at every time point. Cu concentrations declined in lungs with first-order kinetics and an elimination T3 (current OSHA PEL and NIOSH REL for copper dust and mist (excluding fumes)), breathing frequency of 15 breaths/min, tidal volume of 600 mL/breath, and pulmonary deposition fraction for 36 nm particles of 0.5.Inflammatory responses in lung after inhalation of CuO NPs have been recognized by many studies of ours , 32, 33 3 for 4 h exhibited decreases in macrophages viability [Klebsiella pneumoniae and induced a dose-dependent decrease in bacterial clearance from the lung [Neutrophils in BAL fluid significantly increased at all time points but maximally increased during exposure and declined after exposure. Similarly, as in our previous studies, neutrophils were the major cells recruited to the lung after inhalation exposure to Cu or CuO NPs , 19, 32 iability , in thisiability . Increasiability  performeiability . We prevThe measured amount of Cu in the lung at day 12 was fivefold lower than the estimated nominal lung burden of 53 μg/mouse. This discrepancy may be attributed to this measure excluding lung clearance, or translocation to extrapulmonary organs, in the estimation of deposition fraction of Cu from MPPD modeling. The measured soluble Cu in BAL fluid was less than 2.6% of total Cu in lung, indicating CuO NPs were slightly dissolved in BAL fluid , which led us to investigate hemolysis induced by Cu. Several studies reported that Cu could induce hemolysis –44. Coppand Co2+ . Seleniuand Co2+ , 50. Laiand Co2+ . Our indand Co2+  supporteand Co2+  and thusand Co2+ .Ceruloplasmin, an α-2-glycoprotein containing 95% of the Cu in plasma, functions as a major Cu transporter protein and multi-Cu oxidase protein, it mobilizes Fe from Fe storage cells into plasma . It is aRelative organ weight has been accepted as a valuable biomarker in toxicological studies to identify the potentially harmful effect of exposed substances , 65. AbsDissolution studies of CuO NPs conducted to help understand and predict toxicity showed that CuO NPs were dissolved in ALF and SGF, but dissolution in SELF was very low. Similar to Pettibone et al. and Lee et al., Cu NPs used in this study dissolved in ALF, SELF, and SGF at 100%, 2%, and 84% within 24 h, respectively , 31. FasSub-acute inhalation exposure to CuO NPs in BALB/c mice led to pulmonary toxicity represented by elevations of LDH, total cell number, neutrophils, macrophages, and inflammatory cytokines. The degree of pulmonary inflammation corresponded to the total amount of Cu in lung tissue, soluble Cu in BAL fluid, and Fe in BAL fluid, indicating that lung injury further increased during exposure and gradually declined after exposure. Decline in Cu concentrations in the lung post exposure exhibited as first-order kinetics. Whole blood and heart tissue showed marginal but significant increases in total Cu ion concentrations during exposure, exhibiting that dissolved Cu ions were absorbed into the bloodstream and heart tissue. The acute pulmonary toxicity was also observed by significant increases in the lung dry weight of exposed mice. Secondary organs affected by CuO NPs subacute inhalation were kidneys and spleen as they showed changes in organ weight and trace elements.This study confirmed our previous findings of a robust but reversible pulmonary inflammatory potential of CuO NPs after inhalation, which was correlated to Cu concentrations in the lungs. The findings on disruption of trace element homeostasis and organ weight changes in the kidneys and spleen suggest further investigation into additional toxicity outcomes on these organs caused by CuO NP inhalation exposure is warranted.Female BALB/c mice (4-week-old) from Jackson Laboratories  were acclimatized in the vivarium with a 12-h light/dark cycle with ad libitum access to food and water for 7 days before inhalation exposure. Animal protocols performed were approved by the University of Iowa Institutional Animal Care and Use Committee. This mouse strain was selected to complement other studies from our laboratory focused on a deeper investigation of an immunomodulatory effects of CuO NPs in asthmatic and allergen immunotherapy mouse models in females. In this work we sought to determine phases of pulmonary inflammation initiation, progression, and resolution.85Kr source, TSI Inc., Shoreview, MN) prior to splitting and entering two nose-only inExpose towers each holding 12 mice.Exposures to CuO NP aerosol were performed using a nose-only inExpose system . To minimize the stress induced by the exposure system, mice were acclimated to the nose-only holders for 3 days before the start of the exposure by placing them into nose-only holders connected to a tower with filtered air flow for an increasing time on each day as follows: 30 min, 1 h, and 3 h per day, respectively. Subsequently mice were exposed to CuO NP aerosols for 4 h/day from day 1 to day 12 with 2 days interruption on days 6 and 7. CuO NPs were provided and characterized by Nanomaterials Health Implications Research (NHIR) at the Engineered Nanomaterials Resource and Coordination Core (ERCC), HSPH-NIEHS Nanosafety Center, Harvard T.H. Chan School of Public Health, member of the Nanomaterials Health Implications Research (NHIR) Consortium. Nanoaerosols were generated as described previously . Dry pow3. The selection of exposure concentration was based on our previous studies [The CuO NP aerosol concentration was monitored during the exposure using a direct-reading aerosol photometer  to aid in controlling the concentration near the target concentration of 3.5 mg/m studies , 33, 67,To study sub-acute inhalation effects, mice were exposed for 4 h/day, 5 days/week for 2 weeks. Sentinel mice were used as controls for all comparisons. Mice were euthanized with an overdose of isoflurane, followed by cervical dislocation, thoracotomy, and exsanguination through the heart on day 0, 3, 7, 12, 17, 22, and 27 (n = 5 per time point). Controls were necropsied 3 days after the last time point investigated. Day 3, 7, and 12 were the days of CuO NP exposure period. BAL fluid was collected from the right lobes of the lung to assess the occurrence of pulmonary inflammation induced by CuO NP aerosols. The right lobes of the lung were perfused with 10% buffered formalin  through the cannulated trachea and then stored in 10% buffered formalin until further processing for histopathology. Dosimetry analysis for trace elements was performed on whole blood, left lobes of lung, brain, heart, spleen, liver, kidneys, BAL fluid, and urine. Whole blood was kept in blood collection tubes containing ethylenediaminetetraacetic acid (EDTA) as a chelating agent and stored at -80 °C. All organs were frozen in liquid nitrogen immediately after collection and stored at -80 °C. Urine samples were collected directly from the bladder with a syringe at necropsy. Since not all mice had urine in the bladder and due to limited volume of urine from each mouse, urine samples from mice in the same group were pooled for dosimetry analysis using ICP-MS.m, Dose per mouse (× 10–3 µg/mouse), C: Concentration (mg/m3), RMV, Respiratory minute volume (mL/min), t, Exposure time (min), The estimated deposited dose in the pulmonary region was calculated using the equation below:3. Considering breathing frequency of 165 breaths/min and tidal volume of 0.15 mL, the respiratory minute volume considered for this calculation was 24.8 mL/min. The pulmonary deposition fraction not considering clearance was calculated using the computational multiple path particle dosimetry (MPPD) model (software version 3.04 (Applied research associates (ARA), Albuquerque, NM) which was 0.24 [The average concentration of CuO NP aerosol over all exposure days was 3.75 mg/mwas 0.24 . Thus, eg for 5 min at 4 °C to separate the cellular and soluble components. The supernatant was stored at -80 °C to analyze LDH enzyme levels and cytokine/chemokine concentrations. Cell pellets from BAL fluid were resuspended in 200 µL Hank’s balanced salt solution  and stained with propidium iodide (PI) and the number of viable and dead cells counted using an automated cell counter . Briefly, 135 µL of 2.25 µg/mL PI in phosphate buffered saline, pH 7.4 containing 0.045% sodium azide  was added to 15 µL cell suspension, and incubated for 5 min. Then, 75 µL of sample was loaded into the MFS cassette (MXC020). The total viable cell count was measured using a fluorescence laser at 488 nm with a 561/LP filter. For differential cell counts, cells were fixed on microscope slides with fetal calf serum using a Cytospin™ 4 Cytocentrifuge , and stained using the Protocol® HEMA 3 stain set , which is similar to the traditional Wright and Wright-Giemsa stains. Cells were differentiated by type  in 400 cell counts per sample using a bright-field mode microscope .BAL fluid was collected from the right lobes of the lung by lavaging with 1 mL sterile sodium chloride solution  3 times. The collected BAL fluid was centrifuged at 800 × The levels of soluble substances in BAL fluid such as LDH and inflammatory cytokines/ chemokines were determined. LDH, a cytosolic enzyme released from cells upon cell membrane damage was measured as an indicator of cytotoxicity within the pulmonary region. LDH was spectrophotometrically measured using a cytotoxicity detection kit LDH . The following cytokines in the supernatant of BAL fluid; eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17A, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α were simultaneously determined using Bio-Plex Pro™ mouse cytokine 23-plex assay  according to the manufacturer’s protocol and measured using the Bio-Plex 200 system (Bio-Rad).After cardiac exsanguination and BAL fluid collection, the right lobes of the lung were perfused with 10% buffered formalin  through the cannulated trachea and then stored in 10% buffered formalin until further processing. The methods for lung histopathology have been previously described elsewhere , 69.63Cu), potassium (39K), magnesium (24Mg), calcium (44Ca), manganese (55Mn), Fe (56Fe), zinc (64Zn), and selenium (78Se) using ICP-MS  with helium gas in collision cell mode. Yttrium (70Y), rhodium (103Rh), tellurium (130Te), iridium (193Ir), and bismuth (209Bi) were utilized as internal standards. All reagents used for ICP-MS measurement are listed in Additional file The following elements were quantified in whole blood, lung, brain, heart, spleen, liver, kidneys, BAL fluid, and urine: Cu 3Cu, potaThe Centers for Disease Control and Prevention (CDC) method developed by Jones et al., 2017 was applied to determine trace element concentration in whole blood after CuO NP inhalation exposure . The dil3) and 1 mL 30% hydrogen peroxide (H2O2). The samples were acid digested using a microwave digestion system  set with a ramp to 210 °C for 20 min, and hold at 210 °C for 15 min. After microwave digestion, the samples were diluted with 40 mL deionized water. The digested samples were weighed with the digestion vessel and recorded as post-weighed samples to calculate the final solution mass. Acid-digested samples of the lung, kidney, and liver samples were diluted with 2% HNO3 10 times, while that of the brain samples were diluted 5 times. Heart and spleen acid-digested samples were measured without further dilutions. Measured elemental concentrations were expressed as µg/g dry tissue weight.All frozen organs were lyophilized using a freeze dryer  for 24 h, and weighed using an analytical balance . All tissue samples were digested using modified EPA method 3051 , 72 using  for 10 min to eliminate CuO NPs. The supernatants were diluted 5X with 2% HNO3 before ICP-MS measurement. The reported concentration of Cu represented soluble Cu in BAL fluid.BAL fluid collected from the right lobes was centrifuged at 194,400 × 3 and 1.5% v/v ethanol. Urine samples were diluted 20X with DI water and the diluent.Cu and other trace elements in urine samples were measured using a method from the CDC’s Division of Laboratory Sciences (Method No. 3031.1–01). The diluent reagent consisted of 10 µg/L Rh as an internal standard in 2% v/v HNOOur laboratory is enrolled in the Lead and Multielement Proficiency Program (LAMP), a laboratory standardization program run by the CDC . Each qug for 10 min to separate serum. Ceruloplasmin, an α-2-glycoprotein containing 95% of the Cu in plasma, functions as a major Cu transporter protein and multi-Cu oxidase protein [Whole blood collected by cardiac puncture was incubated at room temperature in a non-heparinized tube for at least 30 min and centrifuged at 2500 ×  protein . Since cPercentage body weight changes were calculated from initial body weight (before exposure) subtracted with the weight after exposure. All dry organs were weighed using an analytical balance . Relative organ weights (organ weight to body weight ratio) were presented for liver, kidney, heart, and spleen as the weight of these organs were associated with body weight change. In contrast, brain and lung were presented as the absolute organ weight as their weights were generally not affected by body weight , 75.Relevant dissolution studies of CuO NPs were conducted in three different simulated biological fluids including ALF (pH 4.5), SELF (pH 7.4), and SGF pH 1.5). The ALF fluid simulated the composition and pH of lysosomes in alveolar and interstitial macrophages while the SELF mimicked interstitial fluid in the lungs. Dissolution in SGF was performed to mimic bioavailability of Cu ions in the gastrointestinal tract since some portion of the NPs might be cleared out through mucociliary escalator activity and swallowed. The composition of SGF, ALF, and SELF can be found in the literature –78. The .5. The A g, then the supernatant was transferred to Beckman ultracentrifuge tubes and centrifuged for 15 min at 194,400 x g  to separate undissolved NPs out of the solution. The supernatant was collected and diluted with 2% HNO3 at 1:100 dilution for SELF samples, and 1:10,000 dilution for SGF and ALF samples. Samples were analyzed for Cu ions using ICP-MS .CuO NPs were dispersed at 2.0 mg/mL in 16 mL of ultrapure water. The CuO NP suspension was sonicated for the total of 1 min with the cycle of 30 s vortex and then 30 s sonication with the power 220 ~ 230 W. One mL CuO NPs suspension was added to 1 mL 2 × stock simulated fluids  to a final CuO NP concentration of 1 mg/mL. The samples were incubated on an orbital shaker at 37 °C, 250 rpm continuously for 4 h, 24 h, and 336 h (14 days) (SGF conditions were performed for only 4 and 24 h). The samples were centrifuged for 15 min at 18,800 xThe data were expressed as the mean ± standard deviation (SD). GraphPad Prism  was used for statistical analysis. Unpaired two-tailed Student’s t-tests were performed when two groups were compared. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used when more than two groups were compared. If data were not normally distributed, Kruskal–Wallis test was used. Significant differences were considered at p < 0.05. Statistical probability  in plots is expressed as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.Additional file 1. Figure S1. Percentage of Inflammatory cell numbers in BAL fluid of mice exposed to CuO NPs in aerosols: (a) Neutrophils, (b) Macrophages, (c) Lymphocytes, and (d) Eosinophils at different time points throughout or after exposure (Red-highlighted area indicates time during CuO exposure). Statistical analysis for % Neutrophil, % Lymphocyte, and % Macrophage were performed using one-way ANOVA with Dunnett’s post hoc test, while % Eosinophils was performed by Kruskal-Wallis test. Data are expressed as mean ± SD (n = 5). **** P < 0.0001, **P < 0.01,*P < 0.05.Additional file 2. Table S1. Total Cu in the lung (μg/mouse lung), total soluble Cu in the lung (μg/mouse lung), and % soluble Cu/ total Cu in the lung.Additional file 3. Table S2. Mean dried organ weights and body weights.Additional file 4. Table S3. Cu concentration and specific gravity in urine samples.Additional file 5. Figure S2. Dissolution of CuO NPs in simulated biological fluids: Simulated gastric fluid , artificial lysosomal fluid , and simulated epithelial lung fluid , n = 2. Percent dissolved Cu was calculated from dissolved Cu over total amount of CuO NPs.Additional file 6. Table S4. Reagents, sample composition, and calibrator concentrations for dosimetry analysis using ICP-MS.Additional file 7. Table S5. Performance data on NIST, LAMP, and QMEQAS analyses.Additional file 8. Table S6. Composition of simulated fluid including ALF (pH = 4.5), SELF (pH = 7.4), and SGF (pH = 1.5) for 500 mL solution preparation for each fluid.