Diet has a profound impact on the rumen microbiota, and the impact can vary among the different rumen ecological niches (REN). This study investigated the effects of dietary replacement of soybean meal (SBM) with dried distillers grains with solubles (DDGS) on the rumen microbiota occupying different REN of growing Hu lambs. After a 9-week feeding trial, 6 lambs from each dietary treatment (SBM vs. DDGS-based diets) were slaughtered for sample collection. The microbiota of the rumen solid, liquid, and epithelium fractions was examined using amplicon sequencing analysis of bacterial 16S rRNA gene, functional prediction, and qPCR.No interaction of dietary protein source (PS) and REN were detected for virtually all the measurements made in this study. The DDGS substitution resulted in very limited influence on bacterial community structure. However, the metabolic pathways predicted from 16S rRNA gene sequences varied greatly between SBM- and DDGS-based diets. The populations of rumen total bacteria, fungi, sulfate-reducing bacteria (SRB), and methanogens were not influenced by DDGS substitution, but the population of protozoa was reduced. The bacterial communities in rumen solid (RS) and liquid (RL) were similar in taxonomic composition but were different in relative abundance of some taxa. In contrast, the bacterial composition and relative abundance of rumen epithelium (RE) were greatly distinct from those of the RS and the RL. In alignment with the bacterial relative abundance, the metabolic pathways predicted from 16S rRNA genes also varied greatly among the different REN. The populations of total bacteria, protozoa, and methanogens attached to the RE were smaller than those in the RS and RL, and the fungal population on the rumen epithelium was smaller than that in the RS but similar to that in the RL. On the contrary, the SRB population on the RE was greater than that in the RS and RL.Substitution of SBM with DDGS had greater impact to the protozoa than to the other microbes, and the microbial community structure and functions at different REN are distinct and niche-adapted. Rumen microbiota determines animal health and production performance, and a better understanding of this complex and diverse microbiota, especially the relationship between the structure and function under different dietary conditions can lead to innovative interventions to improve animal productivity . DietaryWithin the rumen ecosystem, the environments create three different ruminal ecological niches (REN), namely the solid, liquid, and epithelium , 15. TheThe experimental design, diets, and management have been reported previously . Briefly2 area of the rumen epithelium were scraped using a sterilized glass slide and stored in liquid nitrogen immediately after collection. The RS, RL, and RE samples were stored at − 80 °C until further analysis.On days 6 and 7 of week 9, the six lambs each selected from the SBM-Control and DDGS-Control were slaughtered at 4–6 h after morning feeding. The whole rumen contents of each Hu lamb were first homogenized, mixed, and then strained through four layers of cheesecloth to separate the rumen liquid (RL) and the rumen solid (RS) fractions. Approximately 30 mL each of liquid and the solid fraction was collected into a sterilized tube and immediately stored in liquid nitrogen. To collect the rumen epithelial (RE) samples, the rumen walls were rinsed with cold sterile saline solution three times after the removal of the rumen contents. Epithelial tissue samples from an approximately 8 cmMetagenomic DNA of the rumen solid, liquid, and epithelium samples was extracted using the bead-beating and phenol-chloroform extraction method as previously described . The qua′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Unique barcodes were added to the 5′ end of both primers for multiplexing. PCR products were verified on agarose gel , and the expected bands were each extracted and purified using the AxyPrepDNA Gel Extraction Kit . The concentrations of the purified DNA amplicons were each quantified using a QuantiFluor® dsDNA kit . Amplicons from different samples were mixed in equal ratio and sequenced using the 2 × 300 paired-end kit on an Illumina MiSeq platform. The raw sequence reads were deposited into the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA565493.The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using primers 338F (5de novo clustered with a 97% sequence similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/), and possible chimeric sequences were identified and removed using UCHIME [Raw fastq files were de-multiplexed, quality-filtered, and analyzed using QIIME 1.9.1 . Operatig UCHIME . The mosg UCHIME . A PH Lag UCHIME  was usedg UCHIME . Principg UCHIME . The funmcrA, for methanogens), and dissimilatory sulfite reductase alpha subunit gene  in each sample was quantified in three technical replicate against respective standards, which were purified PCR products of known length and concentration. The absolute abundance of each microbial population was expressed as copies of the target gene/g of samples.The PCR primers used for real-time qPCR of total bacteria , fungi , protozoP ≤ 0.05.Analysis of similarity (ANOSIM) was used to assess the statistical significance of the PCoA analysis of overall microbiota across the treatments. The real-time PCR data were log transformed to improve normality. Residual analysis was used to determine if a transformation of variables was needed. If needed, cubic root transformations were performed. All data  were analyzed using the MIXED procedure of SAS version 9.4 . The model included PS and REN as fixed effects, with block as random effects. Degrees of freedom were calculated using the Kenward-Roger option. The mean separation test was performed using the Tukey multiple range test. Differences were considered statistically significant at P ≥ 0.06) between PS and REN for OTU numbers, Chao 1, or Shannon index. None of the alpha diversity measurements was affected (P ≥ 0.36) by dietary PS. The number of OTUs and Chao 1 richness estimate in the RS and the RL fractions were similar (P > 0.05), which were higher (P < 0.05) than in the RE fraction. In contrast, The Shannon index was higher (P > 0.05) in the RS than in the RL and the RE fraction.Across all 36 samples, a total of 1,275,639 quality-checked 16S rRNA gene sequences were obtained. On average, each sample had at least 35,434 sequences. Greater than 99.3% depth coverage was achieved for all the treatments .The PCoA analysis, based on either Weighted UniFrac or Bray-Curtis dissimilarity, indicated a clear separation between the epithelium and the liquid or solid fraction, but no dietary PS effects were observed Fig. . MoreoveP ≥ 0.59) of PS with REN was detected with respect to any of the bacterial phyla. None of the bacterial phyla was affected (P ≥ 0.11) by dietary PS, but all were greatly influenced by REN. The relative abundance of Bacteroidetes (52.15–55.01%) in the RS and the RL was higher (P < 0.01) than that in the RE fraction (27.41%). On the contrary, the relative abundance of Proteobacteria (0.64–0.93%) and Spirochaetes (0.76–2.45%) in the RS and the RL was lower (P < 0.01) than that in the RE fraction (18.06% and 6.23%). The relative abundance of Fibrobacteres was higher (1.16%) in the RS than in the RL and the RE fractions (0.12–0.30%). The relative abundance of Firmicutes (41.41–45.63%) and Actinobacteria (0.65–0.85%) were not influenced (P ≥ 0.55) by REN.In total 19 bacterial phyla were identified among all the treatments, with Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, Fibrobacteres, and Actinobacteria being the six most predominant phyla, each of which was represented by more than 0.5% of the total sequences in at least one treatment Table . No inteP ≥ 0.13) of PS with REN was detected on any of the bacterial abundance at the genus level except for the genera Pseudobutyrivibrio and Suttonella (P < 0.01). The relative abundance of Ruminococcaceae UCG-005 was higher (P < 0.01) for the lambs receiving SBM than those fed DDGS, while the relative abundance of other major bacterial genera was not influenced (P ≥ 0.08) by PS. In contrast, 39 out of the 49 major genera were significantly different (P < 0.05) among the three REN. Compared with the RS and the RL fraction, the relative abundance of 17 genera  was higher (P < 0.01), while that of five genera  was lower (P < 0.01) in the RE fraction than in the RS and the RL fractions. Seven genera, including Bacteroidales S24-7 group_norank, Succiniclasticum, Ruminococcus 1, Saccharofermentans, Eubacterium ruminantium group, Lachnospiraceae probable genus 10, and Fibrobacter, had a greater relative abundance (P < 0.01) in the RS than in the RL and RE fractions. In contrast, the RL fraction had a higher (P < 0.01) relative abundance of Bacteroidales BS11 gut group_norank, Bacteroidales RF16 group_norank, Ruminococcus 2, Eubacterium coprostanoligenes group, and Pseudobutyrivibrio, but a lower (P < 0.01) relative abundance of Ruminococcaceae UCG-014, than the RS and the RE fractions.A total of 238 bacterial genera were identified among all the treatments, but only 49 of them were each represented by more than 0.5% of the total sequences in at least one treatment (Table P ≥ 0.10) of PS with REN was detected for any of the pathways. However, the relative abundance of the predominant pathways was altered differently by dietary PS or REN. Among the pathways, 9 out of 20 were significantly influenced by PS. Compared with the SBM-based diets, the DDGS-based diets resulted in higher (P ≤ 0.02) relative abundance of the functions related to amino acid metabolism, lipid metabolism, and metabolism of terpenoids and polyketides, but lower (P ≤ 0.04) relative abundance of the functions related to nucleotide metabolism, replication and repair, translation, cell motility, folding, sorting and degradation, infectious disease , and cell growth and death. In contrast, 16 out of the predominant 20 pathways were significantly different (P < 0.05) among the three REN. Compared with the RS and RL, the RE fraction had greater (P < 0.01) predominance of the pathways related to amino acid metabolism, lipid metabolism, xenobiotics biodegradation and metabolism, and metabolism of terpenoids and polyketides, but lower (P < 0.01) predominance of the function related to membrane transport, signal transduction, and cell motility. The pathways related to replication and repair, biosynthesis of other secondary metabolites, and endocrine system were higher (P ≤ 0.02) in the RS than in the RL and RE fractions. In contrast, a higher (P < 0.01) relative abundance of the pathways related to the metabolism of cofactors and vitamins, and a lower (P < 0.01) relative abundance of the pathway related to the metabolism of other amino acids were observed in the RL than in the RS or the RE fractions.The top 20 predominant level 2 metabolic pathways were shown in Table P ≥ 0.16) between PS and REN was detected with respect to the absolute abundance of total bacteria, SRB, methanogens, fungi, or protozoa (Table P = 0.04) population of protozoa than those fed SBM, but the population of total bacteria, SRB, fungi, and methanogens were similar (P ≥ 0.41). The populations of total bacteria and methanogens were lower (P < 0.05) in the RL fraction than in the RS fraction, but higher (P < 0.05) than in the RE fraction. The RS had a larger (P < 0.05) fungal population than the RL and the RE fractions, but no difference (P > 0.05) in the fungal population was noted between the RL and the RE fractions. Compared with the RS and the RL fractions, the RE fraction had a larger (P < 0.05) SRB population but a smaller (P < 0.05) protozoal population. However, no difference (P > 0.05) was observed in the populations SRB or protozoa between the RS and the RL fractions.No interaction , and again, they are virtually undetected or only detected at much lower relative abundance in the RS and or the RL. These results support the notion that the distinctive epithelial bacteria assigned to different phyla may have additional functions other than feed digestion [In line with previous studies , 18, 26,igestion .Desulfobulbus, one genus of SRB [Butyrivibrio 2 (15.51%) on RE was about 3 times higher than that of Desulfobulbus. Members of Butyrivibrio  are important butyrate producing bacteria ubiquitous in the rumen [In vitro cell culture experiments have shown that butyrate could regulate colonic proliferation and treat ulcerative colitis caused by sulfide [Butyrivibrio spp., may help repair the damage to the RE caused by the sulfide produced by SRB . Based on this, we speculate that there is a self-regulating mechanism between the epithelial microbes and the host that helps the homeostasis of RE function.Corroborating the finding of previous studies , 48, 49,s of SRB , was thes of SRB , 52. Howhe rumen . In vitr sulfide . TherefoRumen epithelial microbiota play an important role in digesting and recycling the keratinized distal cells of the epithelium . In the This study, combing high-throughput sequencing, functional prediction, and qPCR, for the first time, revealed the difference of structure and function of rumen microbiota occupying different REN of growing Hu lambs in response to alterations in dietary PS. The results of the present study indicate that substitution of SBM with DDGS had greater impact to protozoa than to other microbes, and the microbiota structure and function in different REN are specific and niche-adapted. In order to have a better understanding of the complex rumen ecosystem, in addition to bacteria, the structure and function of other microbes such as protozoa, fungi, and methanogens should also be studied.Additional file 1 : Table S1. Primers used for real-time PCR quantification of rumen target organisms. Figure S1. Box plots showing within-group similarity and between-group dissimilarity of rumen microbiota based on Bray-Curtis dissimilarity in different ruminal ecological niches of growing Hu lambs in response to alterations in dietary protein sources. The different letters denote significant differences . DDGS: dried distillers grains with solubles, SBM: soybean meal, RE: rumen epithelium, RS: rumen solid, RL: rumen liquid.


The successfully established breast precancerous lesion rat model and normal healthy rats were randomly assigned into the blank (BLA), model (MOD), XTJY-low (LD), XTJY-medium (MD), XTJY-high (HD), and tamoxifen (TAM) groups. Different concentrations of XTJY and saline were supplied by intragastric administration for 4 consecutive weeks to assess the protective effect of XTJY on the progress of the breast precancerous lesion in rats involving the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway.  In this study, it determined that 10 mg/each rat DMBA-combined estrogen and progesterone induction for 10 weeks was the optimal condition for the establishment of the breast precancerous lesion rat model. In vivo administration of XTJY or TAM was found to inhibit the development of the breast precancerous lesion, and the occurrence rate of breast invasive carcinomas was decreased by about 50%. Furthermore, XTJY or TAM markedly reduced protein expressions of PI3K and p-Akt and increased protein expressions of PTEN.  These data indicated that XTJY can significantly alleviate the development of breast precancerous lesions by inhibiting the activation of the PI3K/Akt signaling pathway. XTJY may be a promising drug for the treatment of precancerous lesions in breast cancer. Breast cancer (BC) is the most common cancer in women, accounting for approximately 29% of female malignancy, and is the leading cause of cancer-related death in women ,2. A prePrecancerous lesions are diseases that have greater than 20% risk of developing into cancer; notably, breast precancerous lesions mainly include atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), ductal carcinoma, and papillary tumor, which resulted in approximately 50% probability of breast cancer occurrence . TherefoBupleurum, fried Atractylodes, Hangbaishao, tangerine peel, Pinellia ternate, Poria cocos, Angelica sinensis, and Indian Iphigenia Bulb. Above with the use of herbs could invigorate the circulation of Qi and dissipate phlegm and resolve mass. At present, modern pharmacological research had shown that activating blood, removing phlegm, and dredging the liver to use qi medicine had their unique antitumor mechanism [Traditional Chinese medicine (TCM) had been extensively used in China and proved effective in the prevention and treatment of various diseases. “Xiaotan Jieyu (XTJY) prescription” is a TCM prescription established by professor Pin-Kang Wei after summarizing years of clinical experience. Clinical observation shows that XTJY can effectively treat breast precancerous lesions, reduce or even reverse the pathological state, and extend the survival time of patients. XTJY consisted of 12 traditional Chinese herbs, including rhizoma arisaematis, echanism , 8. Our echanism . HoweverIn the present study, breast precancerous lesion model rats induced by DMBA-combined estrogen and progesterone were treated with different concentrations of XTJY to investigate the biological effects of XTJY on antibreast precancerous lesions. The data demonstrated that the incidence of premalignant breast disease was 91.7% induced by DMBA-combined estrogen and progesterone. Besides, it has been proved that XTJY significantly alleviated breast precancerous lesions of rats at least partially through the PI3K/Akt signaling pathway.7, 12-Dimethylbenz anthracene  was obtained from Tokyo Chemical Industry Co., Ltd . Estradiol benzoate injection (lot number: B130902) was purchased from Ningbo Sansheng Pharmaceutical Co., Ltd. Progesterone injection (lot number: H31021401) was purchased from Shanghai General Pharmaceutical Co., Ltd. Tamoxifen  was purchased from Yangtze River Pharmaceutical Group . Additional reagents employed in the present study were commercially available and of analytical purity.The DMBA was precisely weighed, dissolved in sesame oil according to the proportion of 10 mg/ml, placed in an ultrasonic constant temperature water bath box , and completely dissolved by ultrasonic oscillation.∗4) of TAM were dissolved in 50 ml of normal saline and prepared into tamoxifen solution with a concentration of 0.8 mg/ml.Four tablets . XTJY was prepared according to the following procedures: these herbs were boiled for 2 h 8 times (v/w) with distilled water, then these herbs were boiled 2 times with distilled water again (each 1 h), and then the filtrate was collected and mixed with the previously collected filtrate and concentrated. The concentrate was precipitated 2 times (v: v) with ethyl alcohol for 48 h. The supernatant was collected and then further concentrated to 3.35 g/ml.A total of 123 female Sprague Dawley rats  were purchased from Shanghai SIPPR/BK Laboratory Animals Ltd., Shanghai, China (Production license: SCXK (Shanghai) 2013–0016). All animals were housed in polycarbonate cages in an animal laboratory with 12 h light/12 h dark cycles under conditions of controlled temperature 23 ± 2°C and in a humidified atmosphere (55 ± 15%). All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals . The experimental protocol was subject to approval by the Animal Ethics Committee of Second Military Medical University.n = 15), DMBA-low , DMBA-medium , and DMBA-high  groups. The breast precancerous lesion SD rat model was induced using DMBA in combination with estrogen and progesterone as described previously [n = 5), DLD (n = 12), DMD (n = 12), and DHD (n = 12) groups were sacrificed by cervical dislocation after being anesthetized by an intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg).Following a week of adaptation, rats were randomly divided into four groups as follows: the blank , model , XTJY-low , XTJY-medium , XTJY-high , and TAM (n = 20) groups. Treatment intervention was launched 11 weeks after the establishment of the rat model of the breast precancerous lesion. The rats in the LD, MD, and HD groups received 2 g/kg, 4 g/kg, and 8 g/kg XTJY prescription intragastrically. The rats in the TAM group received 8 mg/kg TAM by intragastric administration. The rats in the BLA and MOD groups received 10 ml/kg saline by intragastric administration. The treatment in each group was given once a day for 4 consecutive weeks. The rats were evaluated once a week, and their life characteristics were observed every day after treatment. Once treatment was complete, rats were sacrificed by cervical dislocation after being anesthetized by an intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg) and tumors measured. The thymus and spleen tissues were collected and weighed for measuring the thymus index and spleen index. The final animal numbers for this research were n = 20 (BLA), n = 20 (MOD), n = 19 (LD), n = 19 (MD), n = 20 (HD), and n = 20 (TAM).Rats were randomly divided into six groups as follows: the blank  staining. For IHC staining, the paraffin sections were deparaffinized with xylene and 3% hydrogen peroxide for antigen retrieval at room temperature for 10 min. Then, the sections were incubated with primary antibodies against PTEN , PI3K p85 , and p-Akt  at 4°C overnight followed by incubation with the appropriate amount of biotin-conjugated goat anti-rabbit IgG (BA1003) or biotin-conjugated goat anti-mouse IgG  for 30 min at 37°C. Next, the reaction was visualized using AEC  and counterstained with hematoxylin. Positive immune staining was presented as brown or yellow granules in the cytoplasm or the nucleus.Breast tissues from the rats were fixed in 4% paraformaldehyde at room temperature for 48–72 h. The breast tissue was embedded in paraffin, and 5 μg) were separated by 10% SDS-PAGE and then transferred to a PVDF  membrane. Following blocking with 5% BSA, the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies included rabbit anti-PTEN , mouse anti-PI3K p85 , rabbit anti-p-Akt , and mouse anti-β-actin . Then, the membranes were rinsed with TBST and incubated with HRP-conjugated goat anti-rabbit IgG  or HRP-conjugated goat anti-mouse IgG  at room temperature for 2 h. β-Actin was used as an inner loading control. The protein bands were identified by an ECL chemiluminescence kit (Millipore) and analyzed by Image-Pro Plus software.The protein samples were drawn from breast tissues of rats by using radioimmunoprecipitation assay lysis buffer . Protein was quantified with a BCA protein quantitative kit (Boster). The protein samples . The normal distribution data among multiple groups were compared by one-way analysis of variance (ANOVA), and differences between two groups were compared by the least significant difference. In order to identify the optimal dose and time for inducing the breast precancerous lesion in the rat model, different doses of DMBA-combined estrogen and progesterone were used to treat the rats for 8, 10, and 12 weeks. Results of body weight change are summarized in After DMBA administration, some rats developed mild diarrhea, which gradually disappeared after 2–4 weeks. The rats in the BLA group had good hair color, agile activities, and no obvious enlargement of the mammary gland. The rats in each model group showed dark hair color, slow reaction, and irritability after DMBA induced more than 4 weeks (data not shown). From the 8th week, tumors were visible in each model group, and the number of tumors in the DMD and DHD groups was significantly higher than that in the DLD group . MorpholRats in the LD, MD, and HD groups were treated with XTJY of different concentrations to assess the therapeutic effect of XTJY on breast precancerous lesion rats. Results of body weight change after XTJY or TAM treatment are summarized in The effects of XTJY on the expression of PI3K/Akt signaling pathway key proteins were detected using a western blot assay. As shown in Breast cancer is the most common invasive cancer in women worldwide, and it imposes a heavy burden on patients . ClinicaIn order to make an animal model of breast precancerous lesion which corresponds with the demand of the research, the models must simulate a continuous process containing normal, hyperplasia, atypical hyperplasia, and carcinoma . CurrentPinellia ternata, and Indian Iphigenia Bulb were the main drugs to eliminate phlegm; Bupleurum, fried Atractylodes, Angelica sinensis, and hangbaishao are complementary drugs for relieving depression; and peppermint as adjuvant can regulate Qi function. Above with the use of herbs is mainly to dissolve phlegm and detoxify, as well as to regulate Qi depression [For breast precancerous lesions, professor Pin-Kang Wei believes that the poison of phlegm-blood stasis and the depression of Qi are the basic pathogenesis of breast precancerous lesions. “XTJY prescription” is created by professor Pin-Kang Wei after summarizing years of clinical experience. In the prescription, rhizoma arisaematis, pression . Clinicapression . In addipression . HoweverAs a critical regulator of apoptosis, the PI3K/Akt signaling pathway regulates important biological processes including tumorigenesis , 23. TheIn summary, the present study suggests that XTJY can effectively alleviate the development of breast precancerous lesions through inhibiting the PI3K/Akt signaling pathway, indicating that XTJY may be a promising TCM prescription for the prevention or treatment of breast precancerous lesions. This study provides an experimental basis for the further application of XTJY prescription in clinical treatment of breast precancerous lesions.


Hyperlipidemia is one of the strong risk factors for ischemic heart disease. Using the Taiwan Biobank (TWB) database, we evaluated the risk of hyperlipidemia and its interaction with sex and rs688 polymorphism on the low-density lipoprotein receptor (LDLR) gene. Data collection in the biobank started in 2008 and is ongoing. Data analysis was performed on the participants’ data collected between 2008 and 2015. In general, 27.92% of the 9237 female participants and 32.65% of the 8690 male participants were identified with hyperlipidemia. Compared to the C/C genotype, C/T and T/T genotypes were not significant risk factors for hyperlipidemia  in the general model. However, there was a significant interaction between sex and rs6888 on hyperlipidemia risk (p-interaction = 0.0321). With the male sex/CC genotype being the reference group, only the female sex/CT and T/T genotypes were closely associated with hyperlipidemia, with respective ORs of 1.153 (CI: 1.014–1.311) and 1.423 (CI: 1.056–1.917). Our data indicate that rs688 C/T and T/T genotypes may be associated with increased risk of hyperlipidemia in Taiwanese women. These findings may be relevant in lipid-modification therapy. Hyperlipidemia, defined as elevated total cholesterol TC ≥ 240 mg/dL), elevated triglyceride (TG ≥ 200 mg/dL), or both, is a well-established risk factor for atherosclerosis, which is closely associated with cardiovascular diseases (CVD) ,2. The r40 mg/dL,Several factors ranging from genetic, environmental, and lifestyle factors are associated with hyperlipidemia. Some of its risk factors, such as excessive alcohol intake, smoking, high blood pressures, and others, can be controlled . SecondaGenetic mutations in several genes play a significant role in the development of hyperlipidemia . EffectsThe sample consisted of participants who were recruited from 2008 to 2015 into the Taiwan Biobank and whose baseline information was linked to the National Health Insurance Research databases (NHIRD) using encrypted personal identification numbers. Ethical approval was obtained from the Institutional Review Board of Chung Shan Medical University (CS2-16114).Of the 17,985 biobank participants aged 30–70 years, we excluded those with incomplete information (n = 54) or missing genetic information (n = 4). The final analytic sample included 5416 cases with hyperlipidemia and 12,511 controls individuals.2). The TC/HDL was obtained by dividing total cholesterol by HDL cholesterol.The diagnosis coding in the NHIRD was based on ICD-9 codes. We identified patients with hyperlipidemia (ICD-9-CM: 272), diabetes mellitus (ICD-9-CM: 250), and hypertension (ICD-9-CM: 401–405) who must have had either two outpatient visits or one admission from 1998 to 2015. Alcohol drinkers were defined as persons who reported drinking more than 150 mL of alcohol per week for more than six months. Former drinkers included adults who drank in their lifetime but who had abstained from drinking for over 6 months. Smokers were defined as people who smoked continuously for more than six months. Never smokers included adults who never smoked or did not continuously smoke for 6 months or more while former smokers included those who smoked in their lifetime but who had not smoked in the last 6 months. The body-mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/mp < 1.0 × 10−3). We further excluded those with minor allele frequency < 0.05.LDLR rs688 was selected based on a literature search. We restricted to this variant because of its significant associations with plasma cholesterol in different populations as cited above. Genotyping in the Taiwan Biobank was performed by Affymetrix using the Axiom™ Genome-Wide Array Plate System . Following the Taiwan Biobank (TWB) quality control, we excluded SNPs with low call rates (< 95%) and those that deviated from the Hardy–Weinberg equilibrium  gene variants on hyperlipidemia. The odds ratios (ORs) with their 95% confidence intervals were estimated. Statistical analyses were performed using Statistical Analysis System (SAS) software (version 9.4) and PLINK 1.09 beta.p < 0.005). Compared to the C/C genotype, C/T and T/T genotypes were not significant risk factors for hyperlipidemia  in the general model, as shown in p-interaction = 0.0321). Across the three rs688 genotypes, we found significant associations only among rs688 T/T male carriers; that is, the OR for hyperlipidemia was significant only in T/T  but not C/T and CC carriers. It is not certain why the rs688 allele with the higher risk associated with hyperlipidemia was significantly lower in our male population. Nevertheless, this may be attributed to rs688 actions on splicing efficiency, which has been shown to significantly decrease in women compared to men [In this study, we evaluated the relationship between rs688 and hyperlipidemia in Taiwanese men and women aged 30–70 years. Our preliminary results showed that of the overall samples, rs688 C/T and T/T genotypes did not show significant associations with hyperlipidemia when C/C genotype was used as the reference genotype. However, we detected an interaction between the rs688 polymorphism and sex on hyperlipidemia  these individuals.


Orientia tsutsugamushi and Rickettsia typhi isolations from this laboratory over a period of 6 years between 2008 and 2014.In the Lao People’s Democratic Republic (Laos), rickettsial infections, including scrub and murine typhus, account for a significant burden of fevers. The Mahosot Hospital Microbiology Laboratory in Vientiane, Laos, routinely performs rickettsial isolation from hospitalized patients with suspected rickettsioses using mammalian cell culture systems. We review the clinical and laboratory factors associated with successful  Orientia tsutsugamushi and Rickettsia typhi isolations from this laboratory over a period of 6 years between 2008 and 2014. The overall isolation success was 7.9% for all samples submitted and 17.3% for samples for which the patient had a positive O. tsutsugamushi or R. typhi rapid diagnostic test (RDT), serology, or PCR. The frequency of successful isolation was highest for samples submitted in November, at the end of the wet season (28.3%). A longer median duration of reported illness, a positive result for a concurrent Orientia or Rickettsia spp. quantitative PCR, and the use of antibiotics by the patient in the week before admission were significantly associated with isolation success (P < 0.05). Buffy coat inoculation and a shorter interval between sample collection and inoculation in the laboratory were associated with a higher frequency of isolation (both P < 0.05). This frequency was highest if cell culture inoculation occurred on the same day as blood sample collection. Factors related to the initial rickettsial bacterial concentration are likely the main contributors to isolation success. However, modifiable factors do contribute to the rickettsial isolation success, especially delays in inoculating patient samples into culture.In the Lao People’s Democratic Republic (Laos), rickettsial infections, including scrub and murine typhus, account for a significant burden of fevers. The Mahosot Hospital Microbiology Laboratory in Vientiane, Laos, routinely performs rickettsial isolation from hospitalized patients with suspected rickettsioses using mammalian cell culture systems. We review the clinical and laboratory factors associated with successful  Orientia tsutsugamushi and Rickettsia typhi are responsible for a significant burden of treatable febrile illnesses. Scrub typhus, caused by O. tsutsugamushi, is the main contributor to this burden and an underrecognized disease affecting predominantly rural areas with poor health infrastructure  rickettsia are also present within the region. Spread through flea and tick species, these diseases tend to be milder in comparison with scrub typhus, though nonetheless severe disease occurs . However, known limitations in IFA performance, the requirements for convalescent-phase sera, and a lack of standardization can make interpretation of serology difficult . Other tic tests , 14. Howic tests .O. tsutsugamushi and R. typhi. We therefore undertook a retrospective review of laboratory data at Mahosot Hospital for samples collected for rickettsial culture over 6 years between January 2008 and December 2014.The Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), based within the Mahosot Hospital Microbiology Laboratory in Vientiane, Lao People’s Democratic Republic (Laos), has been routinely performing rickettsial isolation since 2008. It is currently the only laboratory within Laos equipped for this purpose. Blood samples from febrile patients with suspected rickettsiosis at Mahosot Hospital and regional hospitals in Laos undergo incubation with mammalian cell lines for up to 8 weeks, with the aim of isolating rickettsial organisms. Although there are studies which describe culture conditions used in rickettsial isolation , there ag for 10 min. Inoculation was performed directly in 12.5-cm2 flasks containing L929 (ATCC number CCL-1) or Vero (ATCC number CCL-81) cell monolayers at 80% confluence, and subsequently centrifuged for 30 min at 50 × g. The flask was incubated for 2 h at 35°C in a 5% CO2 atmosphere and then the inoculum was washed and replaced with RPMI 1640-10% fetal calf serum medium. Incubation duration was at least eight weeks at 35°C in a 5% CO2 atmosphere. Media were changed twice weekly, and subculture was performed at week four by resuspending cells and making a 1:2 or 1:10 dilution into 12.5-cm2 or 75-cm2 flasks containing L929 or Vero cells at 80% confluence. A scraping of the inoculated cell cultures underwent indirect IFA using pooled antibodies against scrub typhus, spotted fever, and typhus group Rickettsia every week after four weeks of incubation or earlier   or EDTA buffy coat fraction (200 μl) mixed with 5 ml of RPMI 1640 medium  and supplemented with 10% fetal calf serum  was used as an inoculum. The buffy coat layer was prepared by centrifugation of EDTA-whole blood at 1,600 × ctively) . StatistEthical approvals for collection of samples for determination of the etiology of fever were granted by the Lao National Ethics Committee for Health Research and the Oxford Tropical Research Ethics Committee.n = 1,064; 33.0%), Luang Namtha (northern Laos) , and Salavan (southern Laos)  between January 2008 and December 2014 .A total of 3,227 EDTA-anticoagulated whole blood samples were received for rickettsial isolation from 3,200 patients recruited into fever studies conducted in Vientiane  (O. tsutsugamushi was isolated from 231/256 (90.2%) samples and R. typhi from 24/256 (9.4%) samples, and isolations were confirmed through IFA and qPCR. One blood sample yielded both O. tsutsugamushi and R. typhi, while the species could not be determined for two IFA-positive isolates (0.8%). No spotted fever group rickettsia were cultured. The median duration between incubation start and a positive result was 28 days (interquartile range [IQR]: 23 to 34 days), with no significant difference between organisms cultured  or the cell line used  (Table S2). Cell culture contamination frequency was 373/6,452 (5.8%). The median age of patients was 26 years  and was significant between successful and unsuccessful isolations . The majority of patients were male, with 45.2% female  samples. n = 1,126; 34.9%) with other laboratory evidence for rickettsial infection were selected for further analysis. These samples came from patients who had a confirmed positive result for scrub typhus group (STG) or typhus group (TG) rickettsiae by one or more of the following diagnostic tests: (i) Dip-S-Ticks murine typhus RDT  (43.8%); (ii) scrub typhus IgM RDT  (50.0%); (iii) O. tsutsugamushi 47 kDa qPCR ;kDa qPCR , 20 , with the fewest in February . However, the frequency of successful isolations was highest from samples submitted in November  and lowest during February  (P < 0.001) (O. tsutsugamushi from STG-positive patients was significantly associated with month (P < 0.001), with the highest isolation rate seen in December (41.5%) (R. typhi from TG-positive patients (11.6%) but with no significant relationship (P = 0.804) (P < 0.001), a reduced incidence of CNS involvement , a history of taking an antibiotic during the previous week (P = 0.022), and sample type being buffy coat (P = 0.020)  (R. typhi was only associated with a history or presence of a cough (P = 0.011) or a greater use of antibiotics in the preceding week (P < 0.001) (P = 0.760) (Table S4). In the 1,006 patients with data, the antibiotic classes prescribed or purchased in the week prior to sampling included beta-lactams (47.3%), tetracyclines (23.8%), quinolones (7.6%), and macrolides (4.4%), with 19.2% of patients taking combination therapy.The frequency of successful isolation from the STG/TG-positive subset was 17.3% , of which 171 were < 0.001) . Isolati (41.5%) , while N= 0.804) . From al= 0.020) . In addiO. tsutsugamushi 47 kDa qPCR or Rickettsia spp. 17 kDa qPCR) on a patient’s EDTA buffy coat sample as a way to select samples for culturing was significantly associated with a higher rate of isolation success for the respective pathogens , although no significance was seen in relation to actual threshold cycle (TC) values and isolation success . Similarly, samples from patients with positive IgM IFAs were more likely to have successful isolation for both pathogens , while a positive IgG IFA was only significant for R. typhi isolation   for all STG/TG-positive patients, and 2 days (IQR: 1 to 4) versus 4 days (IQR: 2 to 8)  for all STG-positive patients. For all samples from STG/TG-positive patients, the frequency of successful isolation was highest if the blood sample was inoculated on the same day as collection  and decreased with samples inoculated on subsequent days  (P = 0.809).The majority of blood samples (72.6%) were received by the laboratory for inoculation into cell lines within 5 days. Successful isolation was associated with shorter median interval between blood collection and cell inoculation of 2 days (IQR: 1 to 4) versus 3 days (IQR: 1 to 7) (= 0.032) . AlthougP < 0.001). Isolation of O. tsutsugamushi from STG-positive patients was significantly associated with a positive O. tsutsugamushi PCR , while the presence of a cough was significant for R. typhi isolation from TG-positive patients  (see Tables S7 to S10).A multivariate logistic regression model for isolation of either organism from STG/TG-positive patients identified that a positive PCR was significantly associated with isolation success, although the confidence interval was large , indicating low bacterial loads. Although comparisons between TC values of successful and unsuccessful isolations do not show any statistical significance, there is a significant relationship between overall qPCR positivity and isolation success in the regression model, as well as a higher isolation frequency through the use of the buffy coat, suggesting an association with bacterial load. The dynamics of rickettsial infection are complex. An early, limited bacteremic phase for O. tsutsugamushi before intracellular entry has been described from animal models (O. tsutsugamushi and R. typhi infections (R. typhi) or a rash (for O. tsutsugamushi) is significantly associated with successful isolation. We also found a significant association between isolation and antibiotic use in the preceding week before admission, although, unexpectedly, this was related to an increase in successful isolations. One possible explanation for this is that patients with more severe disease had a higher chance of seeking antibiotics, but the majority (74%) of those antibiotics taken would not be efficacious against either rickettsial pathogen , the majority of attempted isolations were carried out based on positive RDT results; therefore, it is difficult to interpret their usefulness without discounting the risk of bias in the data. This information highlights the benefit of using diagnostic tests to guide the selection of samples for isolation.O. tsutsugamushi reflect the observed seasonality of scrub typhus, which appears after onset of the annual monsoon season (R. typhi isolation. Although not statistically significant, seasonal changes are most likely due to the same factors because seasonality in regional flea abundance has been previously identified (Temporal changes in isolation success for n season , 8. Isoln season . A similentified .ex vivo blood with time. Delays occur because samples collected from study sites have to be transported to the laboratory in Vientiane, and rickettsial isolation was not performed during weekends. The impact of delayed inoculation has been demonstrated for R. conorii, for which the frequency of isolation through shell-vial culture was reduced when clinical samples were not inoculated on the same day as blood draw (A shorter time between blood collection and inoculation was associated with higher rates of isolation and may relate to decline in viability of the bacteria in ood draw . Systemsood draw  may imprO. tsutsugamushi and R. typhi isolation success in Laos. Similar to the culture of conventional bacteria from blood, we found that host factors such as illness duration and factors indicative of initial blood bacterial concentration are strongly associated with isolation success. It will be important to further define the role of culture technique to aid staff training and reduce delayed sample inoculation to improve the isolation yield and support research in this group of diseases.In summary, we describe the clinical and laboratory factors associated with


Forage plays critical roles in milk performance of dairy. However, domestic high-quality forage such as alfalfa hay is far from being sufficient in China. Thus, more than 1 million tons of alfalfa hay were imported in China annually in recent years. At the same time, more than 10 million tons of corn stover are generated annually in China. Thus, taking full advantage of corn stover to meet the demand of forage and reduce dependence on imported alfalfa hay has been a strategic policy for the Chinese dairy industry. Changes in liver metabolism under different forage resources are not well known. Thus, the objective of the present study was to investigate the effect of different forage resources on liver metabolism using RNAseq and bioinformatics analyses.P <  0.01). KEGG analysis for differently expressed genes (DEG) in liver  showed that pathways associated with glycan biosynthesis and metabolism and amino acid metabolism was inhibited by the CS diet. In addition, results from DAVID and ClueGO indicated that biological processes related to cell-cell adhesion, multicellular organism growth, and amino acid and protein metabolism also were downregulated by feeding CS. Co-expression network analysis indicated that FAM210A, SLC26A6, FBXW5, EIF6, ZSCAN10, FPGS, and ARMCX2 played critical roles in the network. Bioinformatics analysis showed that genes within the co-expression network were enriched to “pyruvate metabolic process”, “complement activation, classical pathway”, and “retrograde transport, endosome to Golgi”.The results of this study showed that the cows fed a diet with corn stover (CS) as the main forage had lower milk yield, DMI, milk protein content and yield, milk fat yield, and lactose yield than cows fed a mixed forage (MF) diet (Results of the present study indicated that feeding a low-quality forage diet inhibits important biological functions of the liver at least in part due to a reduction in DMI. In addition, the results of the present study provide an insight into the metabolic response in the liver to different-quality forage resources. As such, the data can help develop favorable strategies to improve the utilization of corn stover in China.The online version contains supplementary material available at 10.1186/s12864-020-07332-0. Forage is the largest component of the diet of lactating cows and could affect dry matter intake (DMI)  and consWhile high-quality forage such as alfalfa hay is still a bottleneck for the development of the dairy industry in China, it has become one of the largest dairy producers in the world . In 2019L. chinensis and cornstalk) on the rumen microbiota of dairy cows [In the last 10 years, a large number of studies to evaluate the nutritive value of corn stover or Chinese wild rye grass have been conducted , 4, 7, 8In ruminants, liver contributes to more than 80% of the glucose produced via gluconeogenesis , 10. In P <  0.01) and efficiency  was lower with CS than MF. In addition, DMI , milk protein content and yield , milk fat yield , and lactose yield  were all decreased by CS compared with MF.As shown in Table A total of 8582 unigenes were detected in the liver of dairy cows and 504 DEG  were identified between cows consumed CS and MF  were enriched by downregulated DEG and upregulated DEG respectively. For the KEGG analysis, there were 8 terms enriched among DEG in total, with 5 terms enriched with downregulated DEG (P ≤ 0.05).Results of DAVID analysis are shown in Fig. P ≤ 0.05).The GO_BP analysis was also performed using ClueGO Fig. . The resFAM210A, SLC26A6, FBXW5, EIF6, ZSCAN10, FPGS, ARMCX2) with higher degree and betweenness centrality  of mid-to-late lactating cows is able to meet glucose required for milk lactose synthesis and maintenance , 16. In P = 0.07) decreased [P = 0.01) [Consistent with a previous study where milk yield of cows fed more alfalfa than those fed corn stover  , suggestIn this study, all categories and subcategories of the KEGG pathways in liver were overall inhibited to different extents in CS vs. MF cows Fig. . FurtherAs shown in Fig. Among the top 20 impacted pathways, “Glycosphingolipid biosynthesis – globo series” and “Glycosphingolipid biosynthesis – lacto and neolacto series” were highly inhibited in CS vs. MF cows  with higher degree and betweenness centrality  which had the highest degree has a crucial role in regulating bone structure and function [SLC26A6 belongs to the solute carrier 26 family, and encodes a protein involved in transporting chloride, oxalate, sulfate and bicarbonate [SLC26A6 indicated a decreased transporting ability of chloride, oxalate, sulfate and bicarbonate in liver of CS cows  in Mz-ChA-2 and TFK-1 cell lines inhibit cell proliferation and induced apoptosis [EIF6, the results of the present study suggested an inhibited protein synthesis in liver of cows fed low-quality forage.Among the 7 genes, function . SLC26A6arbonate –39. Thusows Fig. . FBXW5 ipoptosis , while Epoptosis . HoweverFPGS is a gene encoding the folylpolyglutamate synthetase enzyme. This enzyme has a central role in establishing and maintaining both cytosolic and mitochondrial folylpolyglutamate concentrations and, thus, is essential for folate homeostasis and the survival of proliferating cells [FPGS . The two diets were formulated to meet their nutrient requirement (net energy lactation) according to NRC (2001) [The field experiment of this study was performed in Beijing ZhongDi Dairy Farm  and the cows used in this study were all from this farm. Thirty-two healthy lactating Holstein cows  were selected and divided into two groups based on average daily milk yield, body weight and days in milk. The two groups were randomly assigned to two diets: (i) mixed forage diet (MF), and (ii) corn stover diet (CS). Each treatment included 16 cows . The offered total mixed rations were sampled twice per week, and the samples were pooled for each week and analyzed for DM, CP, NDF, ADF, EE, Starch, Ca, and P content as previously described . Five coTotal RNA was extracted with TRIzol reagent  according to the manufacturer’s protocol. Integrity and concentration of total RNA were then assessed using a 2100 Bioanalyzer  with the RNA 6000 Nano Kit . Complementary DNA (cDNA) was synthesised and used to construct a library with the NEBNext Ultra RNA Library Prep Kit . The libraries were sequenced on the Illumina HiSeq2000 platform via 2 × 50-bp paired-end sequencing at BGI Tech Solutions Co., Ltd. . The RNA-Seq, libraries were sequenced at BGI Tech Solutions Co., Ltd. .ftp://ftp.ensembl.org/pub/release-89/fasta/bos_taurus/dna/) and (ftp://ftp.ensembl.org/pub/release-89/gtf/bos_taurus) respectively. Index of the reference genome was built using Bowtie2 v2.2.8. HISAT2 (v2.0.4) was used to align paired-end clean reads to the reference genome [The reads containing adapter, poly-N and low-quality reads in the raw data were removed to obtain the clean reads. All the downstream analyses were based on the clean data with high quality. Reference bovine genome and gene model annotation files were downloaded from , with treatment used as fixed effect, cow used as random effect, and day used as repeated effect.The mapped reads count table of each gene and each sample was used for the analysis of DEG with DESeq2 package 1.26.0) in R (3.6.1) as the standard workflow instructed . The com.26.0 in DIA) was used to perform the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [P-value, Padj), expression ratio, and P-value was uploaded and Entrez Gene ID were used as background. Adjusted P-value < 0.05 were used as cut-off. The output of DIA was exported in Additional file Dynamic Impact Approach  by CS vs MF; 2) down-regulated DEG  by CS vs MF; and 3) both up- and down-regulated DEG (Padj < 0.05) of CS vs MF. Results were downloaded using both Functional Annotation Chart and Functional Annotation Clustering , Cellular components (GO_CC) and Molecular function (GO_MF) was run by Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 . For thiRead counts of each gene and each sample were normalized by median normalization using the EBSeq (3.10) R package. The correlation and correlation significance of every pair DEG (Padj < 0.01) was calculated using logarithmic matrix of read counts with the R package “psych” (1.8.12). Pearson was chosen as the correlation method. Then, the table containing the statistically significant correlations across the whole data set for every pair of DEGs was generated  of CS vs. MF.Additional file 2. Output of Dynamic Impact Approach (DIA) analysis of the DEG.Additional file 3. Output of Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of the DEG.Additional file 4. The table containing the statistically significant correlations across the whole data set for every pair of DEGs.Additional file 5. Co-expression network statistics including correlation of the DEGs, the degree and betweenness centrality of the nodes.


Oxt was the only gene with metabolic, inflammation, and neurological relevance upregulated by both soybean oil diets compared to both control diets. Oxytocin immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus was reduced, whereas plasma oxytocin and hypothalamic Oxt were increased. These central and peripheral effects of soybean oil diets were correlated with glucose intolerance but not body weight. Alterations in hypothalamic Oxt and plasma oxytocin were not observed in the coconut oil diet enriched in stigmasterol, a phytosterol found in soybean oil. We postulate that neither stigmasterol nor LA is responsible for effects of soybean oil diets on oxytocin and that Oxt messenger RNA levels could be associated with the diabetic state. Given the ubiquitous presence of soybean oil in the American diet, its observed effects on hypothalamic gene expression could have important public health ramifications.Soybean oil consumption has increased greatly in the past half-century and is linked to obesity and diabetes. To test the hypothesis that soybean oil diet alters hypothalamic gene expression in conjunction with metabolic phenotype, we performed RNA sequencing analysis using male mice fed isocaloric, high-fat diets based on conventional soybean oil , a genetically modified, low-LA soybean oil (Plenish), and coconut oil . The 2 soybean oil diets had similar but nonidentical effects on the hypothalamic transcriptome, whereas the coconut oil diet had a negligible effect compared to a low-fat control diet. Dysregulated genes were associated with inflammation, neuroendocrine, neurochemical, and insulin signaling.  Oxt and the related neuropeptide, vasopressin (Avp), is increased both in induced and spontaneous models of diabetes  disrupt energy balance and cause changes in body weight regulation and an upregulation of proinflammatory cytokines and insulin resistance in the hypothalamus and other tissues . AlthougIndividual risk factors such as the consumption of fat-rich foods and a sedentary lifestyle are thought to contribute significantly to the rapid rise in the prevalence of obesity and comorbid diseases such as diabetes , 11. WheAgrp) and anorexigenic  neuronal populations in the hypothalamus  (Nr1h4) (Non–fatty acid components in SO include phytosterols such as stigmasterol (ST), campesterol, and β-sitosterol. Of these, ST has been shown to have metabolic effects such as increasing insulin synthesis and secretion and causing cholesterol efflux in macrophage foam cells , 27. TheNr1h3) , 29, whi (Nr1h4) . Because (Nr1h4) , 32, ST Oxt, and causes an elevated level of circulating oxytocin. Although these effects correlated significantly with diabetes as measured by a glucose tolerance test (GTT), they did not correlate with body weight nor seem to involve either LA or ST.In this study, we hypothesized that an SO-rich diet, such as that currently consumed in the United States, affects hypothalamic gene expression and oxytocin peptide levels differentially from other HFDs, that those changes correlate with obesity and/or diabetes, and that they involve one or both of its major components, LA and ST. To test these hypotheses, we subjected male C57BL/6N mice to 4 isocaloric HFDs composed of CO (largely medium-chain saturated fats), conventional SO (high in LA), a genetically modified SO low in LA  or CO supplemented with ST. We performed RNA-sequencing (RNA-seq) analysis on the hypothalamus of the mice fed the first 3 diets (and a low-fat control diet) and analyzed hypothalamic and peripheral levels of oxytocin in mice fed all 4 diets. The results show that compared to the CO and low-fat control diets, the 2 SO-based diets resulted in a significant dysregulation of more than 100 hypothalamic genes including those involved in neurochemical and neuroendocrine pathways and metabolic and neurological disorders. With only 2 exceptions, the changes in gene expression were not significantly different between the high-LA and low-LA SO diets. Importantly, oxytocin was significantly upregulated on both the RNA level in the hypothalamus and the peptide level in the plasma in both SO diets, but not the CO diet. In contrast, immunohistochemical (IHC) staining of the SON and PVN in the hypothalamus showed reduced OXT protein levels in both sets of SO-fed mice, consistent with increased peripheral release of OXT hormone. The ST diet did not affect hypothalamic oxytocin expression or plasma levels nor promote obesity or diabetes compared to the CO diet. Taken together, our results support the hypothesis that an SO-enriched diet affects gene expression in the hypothalamus, including Three isocaloric diets with 40 kcal% fat   were forDiets were provided to the animals in pellet form, twice weekly for up to 24 weeks; the amount of food consumed was calculated weekly on a per-cage basis. Food intake  was not significantly different between any of the HFD groups. The Viv chow group consumed a greater quantity of food by mass because it has a much lower caloric density.2 inhalation and their brains snap-frozen. Others were subjected to tail blood draw or cardiac puncture and humanely killed by cervical dislocation, or transcardial perfusion under isoflurane anesthesia (see as follows), in accordance with stated National Institutes of Health guidelines.Care and treatment of animals was in accordance with guidelines from and approved by the University of California, Riverside Institutional Animal Care and Use Committee . All mice had ad libitum access to food and water (other than the indicated fasting times). At the end of the study, some mice were humanely killed by COMale C57BL/6N mice (Charles River Laboratories) were bred in-house and maintained on a 12:12–hour light-dark cycle in a specific pathogen-free vivarium for the 40 kcal% diet experiment (cohort 1) . Pups we2 inhalation) at 24 weeks after weaning and their brains harvested and stored at –80°C for subsequent transcriptomic analysis. To isolate the hypothalamus, brains were slightly thawed in a dry-ice filled container and blocked manually at the anterior commissure rostrally, at mammillary bodies caudally , and at the top of third ventricle dorsally, and then the entire hypothalamus was removed. Glucose tolerance, food consumption, and body weight data from these mice have been published previously  experiments . Weekly GTT was performed as described previously . Briefly+ RNA (4 μg) having an RNA Integrity Number (RIN) of greater than 7.5 was used to construct sequencing libraries with the TruSeq Long RNA Sample Prep Kit (Illumina). RNA libraries were validated for RNA integrity by Bioanalyzer, pooled in equimolar amounts, and sequenced on an Illumina HiSeq 2000 at the UCR Genomics Core to generate 50 base, paired-end reads. Three biological replicates each for Viv Chow, CO, SO + CO, and PL + CO were sequenced yielding approximately 15 million reads per sample.Total RNA was isolated from hypothalamic homogenates using an miRNeasy kit  and evaluated for purity and concentration by NanoDrop (NanoDrop Technologies) and for quality using Agilent 2100 Bioanalyzer. Poly(A)Padj less than or equal to .5 were included in the heat map; data were row-normalized before plotting.Reads were aligned to the mouse reference genome (mm10) with TopHat v2.1.1 using the default parameters with the exception that only one unique alignment for a given read was allowed. Raw read counts were calculated at the gene level for each sample using HTSeq v0.6.1. Library normalization was performed with EDASeq ; within-The gene expression patterns of the hypothalamic samples from 2 biological conditions can be characterized by a high-dimensional vector that consists of the read counts of all differentially expressed genes (DEGs). To visualize the similarity in the gene expression patterns, we conducted a principal component analysis (PCA) to transform the high-dimensional vectors into ones in a 3-dimensional space. In the PCA, the read counts in a vector were normalized by the size of the corresponding RNA-seq library. The 3 dimensions of the transformed space were chosen by the 3 principal components with the highest Eigenvalues. The Euclidean distance in the 3-dimensional space was used to measure the similarity of the gene expression patterns between the corresponding treatment group samples.P and Padj or q ≤ .05, at least 1.5 fold-change) dysregulated between any 2 dietary comparisons were uploaded to Panther (http://pantherdb.org)   for funcrdb.org) . Lists ordb.org) . Venn dirdb.org) . The prordb.org) .2 inhalation and decapitation. Whole brains were collected, snap-frozen in 2-methylbutane over dry ice, and stored at –80°C until later use. From each brain, the hypothalamus was dissected in half and homogenized in TRIzol Reagent (Thermo Fisher Scientific). Total hypothalamic RNA was prepared using a modified phenol-chloroform extraction protocol with the Qiagen RNeasy Plus Mini Kit. RNA concentration and purity were evaluated using a spectrophotometer . Reverse transcriptase (RT)-qPCR was performed with the SensiFAST SYBR No Rox One-Step Kit (Bioline) on the CFX Connect Real-Time PCR Detection System (Bio-Rad) using the following 2-step cycling protocol: 45°C for 10 minutes, 95°C for 2 minutes, followed by 40 cycles at 95°C for 5 seconds, 20 seconds at primer’s ideal annealing temperature, and melt curve analysis at 65°C and 95°C for 5 seconds. Each reaction was run in triplicate using 10 ng of RNA per reaction well.Following 18 to 26 weeks of chronic diet and GTT, mice were sacrificed by COOligonucleotide primer sequences  were cusOxt relative to Actb (β-actin) was determined using the Pfaffl method and biological replicates from CO as the control group  plus 0.9 g% NaCl  followed by 4% paraformaldehyde (PFA) in phosphate buffer (pH 7.4). Brains were dissected, sucrose-infiltrated (30% sucrose), until brains were submerged (about 24 hours). Brains were cryosectioned into 30-μm sections and mounted onto gelatin-subbed microscope slides. Sections were washed free of PFA and subjected to a permeabilization/blocking step using 4% normal donkey serum, 1% bovine serum albumin, and 0.3% Triton-X in PBS. Sections were then incubated with 1 of 2 primary antibodies: mouse monoclonal anti-oxytocin neurophysin   for 48 hg for 20 minutes using a refrigerated centrifuge at 4°C. Samples underwent solid-phase extraction using 200 mg C18 Sep-Pak columns (Waters) using a modification of a published protocol  as indicated by manufacturer (Enzo Life Sciences) (Blood was collected by cardiac puncture and the plasma was separated at 2000 × protocol . Columnsciences) .Avp. Cross-reactivity of the kit is reported as mesotocin (7%), OXT (100%), Arg8-Vasotocin (7.5%), and less than 0.02% for other related molecules. Specifications of this kit are 15 pg/ml (or 1.5 pg/well) for limits of detection and 9% and 7% for intra-assay and interassay variability, respectively. The lyophilized sample was reconstituted in 0.12 mL of assay buffer provided in the kit and diluted 1:4 before processing. Briefly, standards and samples (100 μl) were loaded in duplicate onto a 96-well ELISA plate and processed as directed by kit specifications. Optical density (OD) was read at 405 nm; absorbance values were manually corrected using average OD values obtained from the control “blank” wells. OXT concentration values were determined according to a 4-parameter logistic fit standard curve. Values were reported as pictogram (pg) per mL. Extraction efficiency (recovery > 80%) was determined by spiking plasma with the 200 pg/ml OXT standard and comparing to standard plasma. Values obtained for control mouse subjects were similar to those reported previously .Data are presented as mean ± standard error of the mean. Hypothesis testing was conducted using statistical analysis (GraphPad Prism 6.0) using the following tests: repeated-measures two-way for effects of diet and time on body weight gain and food consumption, and one-way ANOVA for effects of diet on GTT, ELISA, and qPCR were followed by Tukey, Fisher least significant difference, and Bonferroni post hoc tests for multiple comparisons. For simple planned comparisons we used 1-tailed, unpaired Student t test. Statistical significance was accepted at an α level .05. Linear regression analysis was performed between body weight, glucose tolerance, and messenger RNA (mRNA) and plasma levels of OXT. The following cutoffs were used to determine significance: Pearson coefficient r greater than 0.5 with Transcriptomic analysis (RNA-seq) was performed on hypothalami obtained from adult male C57BL6/N mice used in a previous study (cohort 1) in which we reported that an HFD made with conventional SO (SO + CO) induces more obesity than isocaloric diets made with either CO or a genetically modified low-LA soybean oil, PL (PL + CO)  Fig. 1AFig. 1A. Padj ≤ .5) dysregulated in at least 1 of the 3 HFDs vs Viv chow revealed a nearly identical transcriptome in the CO diet and Viv chow: only 5 genes were significantly different  (see [Kcng1 (1700018M17Rik (not shown). Kcng1 is a potassium voltage-gated channel modifier (subfamily G member 1) that belongs to a family of proteins that regulate neurotransmitter release, among other physiological functions: its expression was downregulated approximately 2-fold in the conventional SO compared to the Viv chow but not changed by either CO or PL.PCA showed that both SO diets significantly alter gene expression in the hypothalamus compared to the CO diet, which was very similar to the low-fat Viv chow control diet . In fact3a) , a voltage-gated potassium channel (Kcnab2), and a mitochondria reductase (Msra) , glucokinase (Gck), and a peroxidase (Gpx3) , whereas the bottom half contains the genes that have lower levels of expression in Viv chow–fed hypothalami (“Down in Viv Chow”). Gene ontology (GO) analysis of the molecular functions associated with the dysregulated genes revealed that the majority of the genes in “Up in Viv Chow” (down in SO + CO and PL + CO) are categorized as “binding” and “catalytic activity” genes  top. Thee (Msra)  bottom. e (Gpx3) . . Aspn, which is involved in regulating inflammatory responses (Ankk1 were the most downregulated genes in SO + CO (~3-fold) and PL + CO (~21-fold) vs CO, respectively . Interestingly, Ankk1 alters dopamine D2 receptor signaling; genetic variants with it have been associated with obesity . Th. Th37). assembly , was incfunction ,48, was function  top. Foresponses , and Ankectively , bottom. obesity , neurops obesity ,52.Hdc), thyrotropin-releasing hormone receptor (Trh), gonadotropin-releasing hormone receptor (Gck), and sex steroid hormone biosynthesis (Hsd17b7)  (Irs4 (insulin receptor substrate 4) and Prkcg (protein kinase C γ) , as well as the genes uniquely dysregulated by SO + CO (69 genes) and PL + CO (35 genes)  37). Of. Of37). Hsd17b7) . Dysregu (TGF-β) . Dysreguase C γ) . In addihe brain . The expain Hdc , 57, Trh, TrhHdc,Hdc  (both increased ~3-fold compared to CO)   are asso the SON  . The transcriptional regulators that were dysregulated in SO + CO vs CO include Neurod6 (down), Etv1 (down), and Six6 (up). Neurod6 is known to play a role in neurogenesis in the hypothalamus  , both of which are linked to inflammation. This suggests that the medium-chain fatty acids in CO not only have less of an impact on hypothalamic gene expression in general, but also in terms of gene expression related to metabolic and inflammatory diseases, than the 2 SO diets , and inflammation to the hypothalamic DEGs. Eighteen disease-associated genes were identified across the 4 categories for SO + CO vs CO, of which 11 were uniquely dysregulated by SO + CO: -change) . In cont PL + CO . The gennd Abhd8 . Interesnd Abhd8 , whereasSO diets .Alzheimer disease, anxiety, autism, depression, pain, Parkinson disease, and schizophrenia as search terms in PubMed. As with inflammation and metabolic diseases, more genes in the SO + CO vs CO comparison were identified in one or more categories than the PL + CO vs CO comparison. No genes associated with neurological diseases were identified among the CO vs Viv chow DEGs , Gal , Nr4a2 , Mt1 , Dio2 , Ghsr  (Hdc (metabolic disease and anxiety), Avp , Oxt    and Hdc nd pain) ,C. Imporisorders .Oxt (and many other genes), we designed a second cohort of mice to determine whether another component of the oils—ST—might play a role in the altered Oxt expression and obesity and diabetes. Mice in cohort 2  and diet . Post hoc group comparisons indicated that the SO + CO group gained the most weight (36.6 vs 24.3 g for Viv chow)  and genetically modified, low-LA (PL) SOs revealed similar effects on the expression of cohort 2  were fedcohort 2 . Consistcohort 2 , 14, we iv chow) . Howeveriv chow) , as we siv chow) . This diiv chow) . Anotheriv chow) , also coiv chow) .4,28 = 7.130, P < .001). The reduced weight gain was not due to lower food consumption by the ST + CO mice because the mice on all the HFDs displayed similar food intake . Taken together, we conclude that the obesogenic and diabetogenic component of conventional SO is a compound other than ST. As noted previously, LA could be contributing to obesity but not to glucose intolerance  as those on the CO and PL + CO diets. The ST + CO-fed mice also showed no signs of glucose intolerance in the GTT and were trending toward even less glucose tolerance than the CO and Viv chow diets  and 6B. d intake . Only thOxt expression, we performed RT-qPCR on RNA isolated from the hypothalamus of male mice fed the various diets . Post hoc analysis revealed lower Oxt expression in the ST-enriched diet (ST + CO) compared to either of the 2 SO diets (P < .05). In contrast, Pfaffl ratios for Oxt transcript levels were significantly upregulated in PL + CO (P < .05) but were just below the level of significance in SO + CO (P = .06) relative to CO, consistent with the RNA-seq results.To determine whether ST affected us diets . A one-w4,24 = 12.84, P < .001, N = 3-10/diet). Post hoc group comparisons revealed that plasma OXT in SO + CO (P < .05) and PL + CO diets (P < .001) was significantly elevated compared to CO, consistent with the notion that these changes are likely due to a component of SO. That component, however, was not ST because the ST + CO-fed mice had similar levels to those in the CO diet group but significantly lower plasma OXT compared to the PL + CO group (P < .001).Blood drawn at the time of death was used to measure plasma levels of oxytocin using an ELISA . One-wayOxt mRNA vs GTT . The plasma OXT vs GTT was trending toward significance . In contrast, there were no significant correlations with body weight. Similarly, a heat map of individual body weights of mice used for RNA-seq analysis did not show the same pattern as the gene expression profiles in An analysis was performed to correlate gene expression and plasma levels of OXT with body weight and GTT in each of the dietary groups . The onlTo determine the abundance of OXT protein in the brain, hypothalamic tissue sections were incubated with an antibody that recognizes OXT-neurophysin, a carrier protein for OXT: Cell bodies, dendrites, and axonal projections of OXT-positive neurons were observed in the SON and PVN of the hypothalamus . Within Oxt. We also observed elevated levels of circulating OXT peptide in the plasma of SO-fed mice. An isocaloric CO diet did not affect the expression of Oxt either in the brain or the circulation, and had a minimal effect on hypothalamic gene expression compared to the low-fat control diet. Furthermore, the level of neither LA nor ST in the diets had a significant effect on hypothalamic Oxt mRNA or plasma OXT. All told, our results demonstrate that different dietary oils can have differential effects on hypothalamic gene expression and raise the possibility that the SO-rich American diet may be not only contributing to increased rates of metabolic disease but also affecting neurological function.The consumption of SO, one of the most ubiquitous components of the American diet, has increased dramatically over the last several decades and parallels the increase in prevalence both of obesity and diabetes in humans , 18. We Avp synthesis were common to both because of 2 genes: Oxt and Avp.Chronic SO + CO and PL + CO diets produced similar hypothalamic transcriptomic profiles and dysregulated more genes than CO when compared to the low-fat control Viv Chow diet . Althougcentral OXT within the PVN, an area critical for energy homeostasis and susceptible to the effects of HFD, can influence body weight regulation, as has been shown for OXT and other hypothalamic neuropeptide systems previously  and low-LA Plenish (PL + CO) had effects both on the peripheral and central components of the oxytocinergic system. Plasma OXT peptide levels were increased in the blood: ~250% (PL + CO) and ~200% (SO + CO) relative to both the high-fat CO and low-fat control (Viv chow) diets. Although culation , 4. Othe ex vivo , suggestOxt. They did report, however, an increase in expression of other hypothalamic neuropeptide genes such as POMC, GALP, MCH, preORX, Agrp, and Cart  as well as changes in proinflammatory genes Akt, Ikk, IL6, Icam1, and Cx3cl1. We did not observe a change of expression in any of these genes with either SO diet, with the exception of Cartpt, Pmch  and Akt1s1, a substrate of AKT, reinforcing the notion that the oxytocin-related changes observed in the SO diets are likely due to some component other than LA.Regardless of which aspect of OXT function—central or peripheral—is more relevant to the metabolic effects of SO, what is clear is that LA does not appear to play a role. Mice fed conventional SO, high in LA (~55%), or the genetically modified PL, low in LA (7.4%), displayed essentially identical effects on every aspect of oxytocin examined—elevated RNA levels in the hypothalamus, low protein immunoreactivity in SON and PVN, and high peptide levels in plasma. Although others have reported that PUFAs from various dietary sources  induce obesity in rodents , 23, 24,Oxt RNA in the hypothalamus nor OXT peptide in the plasma. However, the mice fed the ST + CO diet did show light hypothalamic OXT immunoreactivity, as did the 2 SO diets, suggesting that ST could be affecting the synthesis and/or central release from the oxytocinergic neurons. Finally, it is worth noting that although we have examined only male mice in this study, the effects of SO diets on the female hypothalamus still needs to be addressed in future studies. We do not a priori expect sex differences produced by dietary SO on solely this basis because SO is free of phytoestrogens, such as isoflavanoids  did not increase the levels of avanoids . HoweverOxt expression rather than the constituents of the diets, or their metabolites, per se. Correlation analysis between body weight gain and glucose intolerance vs hypothalamic Oxt mRNA and circulating OXT peptide shows that although the obese state per se is not significantly correlated with Oxt levels, the diabetic state is (as measured by glucose intolerance). Indeed, others have reported that both spontaneous and induced diabetes results in an increase in hypothalamic Oxt mRNA and an increase in hypothalamic OXT protein immunointensity  of the changes in expression of non-metabolic-associated genes observed in the SO + CO and PL + CO diets  (Cartpt (cocaine- and amphetamine-regulated transcript) gives rise to bioactive neuropeptides that are associated with food intake, body weight maintenance, and energy balance  (Abhd8) (Other genes commonly dysregulated in both SO diets linked to metabolic disorders include gulated) . Cartpt  balance . Hcrt eneostasis . The rem (Mmp11) , 85 and  (Abhd8) .Atp2a1 and Rgs16 . These genes are different from the ones reported by others: IKKbeta, NF-kB, and c-Jun N-terminal kinase (Chronic HFD feeding can lead to diabetes and obesity via activation of proinflammatory pathways in the hypothalamus , 8, 87. nd Rgs16 . Rgs16 hrophages  but in trophages . Five ofIn addition to being linked to metabolic disorders, OXT signaling is associated with neurological disorders and behavioral and psychiatric functions , 90. ForThe results of 2 recent studies support the notion that HFD affects some aspect of central OXT signaling and reveal the behavioral implications of such an effect. In the first study, reduced OXT levels in the prefrontal cortex of rats fed an HFD were associated with reduced functionality of OXT signaling, namely impaired social preference, memory, and synaptic potentiation, effects that were normalized by prefrontal injections of an OXT agonist . In the Avp, associated with schizophrenia (Hcrt, related to anxiety, depression, and pain (Pcsk1n, which is predictive of Alzheimer’s disease via association with the amyloid antiaggregant protein, proSAAS (Ankk1, which has a role in dopamine receptor signaling, was downregulated but only in the PL diet (Kcng1, the potassium voltage-gated channel modifier that is the only gene with a significantly different level of expression between SO + CO and PL + CO (In addition to OXT, several other genes associated with neurological diseases were also dysregulated in both the SO diets but not the CO diet . The uprophrenia  and deprophrenia , 100 (Fiophrenia ; Hcrt, rand pain , 102 (Fiand pain ; and Pcs proSAAS  (Fig. 2C PL diet . NotablyThe results presented here demonstrating the impact of dietary SO on gene expression in the hypothalamus lead to the provocative suggestion that dietary fat, in general, and SO, in particular, may have an impact on mental as well as metabolic health. The results also clearly indicate that additional studies are needed to determine the effects of both high-LA and low-LA SO on hypothalamic and potentially other brain function and underscore the need for a careful evaluation of the extensive use of SO-based food products, including infant formula, animal feed, and other processed foods.


Clinical assessment of EAE was performed daily using a 15-point scoring system following immunization. Training groups performed the swimming program for 30 min/day, 5 times/week, for 4 weeks. Mice had access to a treadmill for one hour per day, 5times/week, for 4 weeks in individual cage. The mice were scarified, and the plantaris and soleus muscles were then isolated for investigation of proteins expression using IHC. An analysis of the preventive exercise (before) and recovery exercise (after) of the EAE was performed. Images of the stained sections were taken using a fluorescent microscope. Quantitative image analysis was performed using ImageJ software package. The obtained data from the mean percentage expression of PSA and NCAM in pre- and post-soleus and plantaris muscles showed that the highest and lowest expression levels of PSA and NCAM belonged to control and swim EAE (SE) groups, respectively. The low expression levels of PSA and NCAM were detected in rat with MS without intervention. In conclusion, the relationship between increasing levels of NCAM and PSA protein expression and voluntary and compulsory activity were detectable both in pre and post-soleus and plantaris. However, voluntary activity resulted in more expression levels of NCAM and PSA than that of compulsory. In conclusion, since it has been suggested that depletion of NCAM is one of the factors associated with or possibly responsible for MS progression, these findings show exercise MS progression may be reduced by increasing expression of exercise-related adhesion molecule such as NCAM and PSA (a glycan modification of the NCAM).It has been suggested that depletion of adhesion molecules is one of the factors associated with or possibly responsible for multiple sclerosis (MS) progression. The aim of this study was to investigate the effect of forced and voluntary training before and after induction of experimental autoimmune encephalomyelitis (EAE) on accumulation of neural cell adhesion molecule (NCAM) and polysialic acid (PSA) in neuromuscular junction denervation in plantaris and soleus muscles in C57BL/6 female mice. A total of 40 female C57BL/6 mice, 10-week-old, were randomly divided into four groups, including induced control groups without EAE induction, induced EAE without training, and forced and voluntary training groups. Myelin oligodendrocyte glycoprotein peptide 35–55 (300  Multiple sclerosis (MS) is a chronic complex T cell-mediated autoimmune disease of the central nervous system (CNS). It is characterized by demyelination, axonal degeneration, and neuronal inflammation with a prominent neurodegenerative component . In MS, It has also been suggested that not only regular exercise may benefit cognitive function by rescuing some hippocampal cellular and molecular impairments but also it has a direct neuroprotective effect in EAE, an animal model of MS. Several molecules such as NCAM and PAX7 are commonly used for detecting myogenic precursor cells and identifying their activation status. Caldow et al.  have demμg in saline; MOG 35–55; KJ Ross-Petersen ApS, Denmark) was emulsified in an equal volume of complete Freund's adjuvant  containing 500 μg of heatkilled Mycobacterium tuberculosis. This emulsion was then injected subcutaneously at the base of the tail of each mouse. The mice also received intraperitoneal injection of pertussis toxin  at the time of immunization and the same was repeated 48 hours later. To confirm EAE induction, animal clinical assessment of EAE was performed daily using a 15-point scoring system following immunization as reported previously by Giuliani et al. [A total of 40 female C57BL/6 mice, 10-week-old, were randomly divided into four groups, including induced control groups without EAE induction, induced EAE without training, and forced and voluntary training groups. All animals have started training 4 weeks before experimental autoimmune encephalitis- (EAE-) induction. The mice were intraperitoneally anesthetized with an injection of xylazine (5 mg/kg) and ketamine (50 mg/kg). Myelin oligodendrocyte glycoprotein peptide 35–55   to identify the neuromuscular junction. The slides were mounted with 4′, 6-diamidino-2-phenylindole (DAPI) .Localization of NCAM and PSA in plantaris and soleus muscles was studied using IHC. Tissue samples were fixed in 10% natural buffered formalin embedded in paraffin. Paraffin sample sections with a thickness of 10 i et al. . In brieImages of the stained sections were taken using a fluorescent microscope. Quantitative image analysis was performed using ImageJ software package.p <  0.05. Data are presented as the mean ± SD.The data were analyzed by SPSS version 22. Differences between the mean expressions of NCAM and PSA in various groups were analyzed by one-way ANOVA. Effects were considered significant at P < 0.0001; 25.50 ± 3.34%, P < 0.0001, respectively), ER , and ES  groups  and ES (P < 0.0001) groups  showed significantly higher mean percentage expression of PSA compared to SE  groups . Unlike P < 0.0001; 25.87 ± 3.69%, P < 0.0001, respectively), ER , and ES .The mean percentage expression of NCAM in pre- and post-soleus muscle showed that the control group  expressed significantly higher percentage expression of NCAM than that in SE  and ES (P < 0.0001) groups (The mean percentage expression of NCAM in pre- and post-soleus was significantly downregulated in the SE group compared with that in ER  groups . However) groups  and 4.P < 0.0001; 27.66 ± 4.22%, P < 0.0001%, respectively), ER , and ES  groups (P < 0.0001) and ES (P < 0.0001) groups. There was no significant difference in the mean percentage expression of NCAM of pre- and post-EDL in the SE group compared with that in ER and ES groups  compared with that in SE  groups . The meaP < 0.0001; 24.41 ± 1.75%, P < 0.0001%, respectively), ER , and ES  groups (P < 0.0001) and ES (P < 0.0001) groups. There was no significant difference in the mean percentage expression of NCAM of pre- and post-EDL in the SE group compared with that in ER and ES groups  compared with that in SE  groups . The meaThe aim of this study was to investigate the effect of forced and voluntary training pre- and postinduction of EAE on accumulation of NCAM and PSA in neuromuscular junction denervation in plantaris and soleus muscles in C57BL/6 female mice. A growing body of evidence has implicated NCAM and PSA as risk factors for major neuropsychiatric and neurodegenerative disorders . The obtIt has been suggested that PSA-NCAM may modulate the functional interaction between neurotrophin brain-derived neurotrophic factor (BDNF) and its high and low affinity receptors. So, the distribution of the BDNF and PSA-NCAM modulates BDNF signaling . It has The endoplasmic reticulum (ER) modulates various physiological processes including protein folding and calcium homeostasis to maintain cellular homeostasis . MultiplIn conclusion, the relationship between increasing levels of NCAM and PSA protein expression and voluntary and compulsory activities was detectable both in pre- and post-soleus and plantaris. However, voluntary activity resulted in more expression levels of NCAM and PSA than those of the compulsory one. In conclusion, since it has been suggested that depletion of NCAM is one of the factors associated with or possibly responsible for MS progression, these findings show exercise MS progression may be reduced by increasing expression of exercise-related adhesion molecule such as NCAM and PSA (a glycan modification of the NCAM).