Molecular cloning and characterization of Y-box gene (Rpybx) from Manila clam and its expression analysis in different strains under low-temperature stress.

Manila clam, Ruditapes philippinarum, is an economically important marine bivalve species. Y-box proteins are members of the cold shock proteins family and highly conserved from bacteria to humans. In this study, a novel Y-box gene (Rpybx) was cloned from the Manila clam and gene expression profiling was performed on three shell color strains (white, zebra and white zebra) and two wild populations (Southern and Northern) of R. philippinarum. The complete ORF length of Rpybx is 1367 bp, encoding 253 amino acids residues. Based on the amino acid sequence analysis and phylogenetic analysis, the Rpybx gene was identified as a member of the invertebrate Y-box proteins family. Rpybx has a similar tertiary structure to human Y-box protein YB-1. The Rpybx mRNA levels were analyzed by qPCR under acute and gradually varied cold stress. Under acute low-temperature stress, the expression of Rpybx mRNA in gills and hepatopancreas was significantly increased in all selected strains and populations (P < 0.05). The Northern population showed the lowest relative expression level of Rpybx. The expressions of Rpybx were greatly upregulated in gills and hepatopancreas of different stains and populations at 5 or -2°C under gradually varied temperature stress (P < 0.05). The results shed light on the biological function of the Rpybx gene in defending against low-temperature challenge and further exploring the molecular mechanism of cold tolerance and resistance in R. philippinarum.

[Association of Urinary Phenol Concentration and Blood Biochemical Indices in Coke Oven Workers].

Objective: To explore the association of urinary phenol concentration and blood biochemical indices in coke oven workers. Methods: From April to may 2019, we investigated 771 employing coke oven workers from a coke plant in Taiyuan city, and categorized into benzene-exposed group (n=402) and control group (n=369) based on their benzene exposures in workplace and urophenol concentrations. All subjects were interviewed face-to-face using a questionnaire including name, age (year) , gender, smoking and drinking habits, personal vocational history, working length (year) , and occupational protection, etc. Post-shift urine samples detected using a gas chromatography-hydrogen flame ionization detector. Fasting venous blood was drawn in the morning and centrifuged, the separated serum were detected the following items using an automatic blood biochemistry analyzer. Covariance and multiple linear regression were used to test the association of urinary phenol concentration and the levels of all the blood biochemical indices. Results: The subjects were predominantly males (n=719, 93.3%) , with an average age of (42.3±8.2) years and an average working length of (20.6±8.2) years. Compared with the control group, the benzene-exposed group were significantly different in age, working length of years, gender, smoking and drinking habits (P<0.05) . The median (interquartile interval) concentration of urinary phenol was 6.00 (0.00-33.00) µg/ml in the benzene-exposed group, which was significantly higher than that in the control group (P<0.05) . Covariance analysis indicated that the fasting blood glucose, total cholesterol and high density cholesterol in the benzene-exposed group were significantly reduced compared with the control group, yet the serum creatinine, serum uric acid and triglyceride were significantly increased (P<0.05) . Multiple linear regression showed that, an increase of each natural logarithm (Ln) transformed urinary phenol concentration was significantly associated with increases in serum uric acid level [9.82 (95%CI: 2.18-17.47) µmol/L] and cholesterol level[0.10 (95%CI:0.00-0.20) mmol/L]. An increase of each Ln-transformed accumulated benzene exposure levels was significantly associated with an increase in total cholesterol level[0.09 (95%CI: 0.01-0.17) mmol/L]. Conclusion: Occupational benzene exposure is possibly related to the variation of purine and total cholesterol metabolism in coke oven workers.

[Clinical application of enterostomy using running suture of dermis and seromuscular layer in laparoscopic-assisted radical resection for rectal carcinoma].

Objective: To explore the clinical safety and feasibility of enterostomy using running suture of dermis and seromuscular layer in laparoscopic-assisted radical resection for rectal carcinoma. Methods: From May 1, 2017 to May 1, 2018, 46 patients who underwent laparoscopic-assisted radical resection for rectal carcinoma with enterostomy using running suture of dermis and seromuscular layer in Cancer Hospital, Chinese Academy of Medical Sciences were retrospectively enrolled in this study. Data regarding clinicopathologic characteristics, operation and postoperative outcomes, stoma-related complications and functions of stoma were collected and analyzed. Results: All of the 46 patients successfully underwent this operation. Among them, 30 patients underwent laparoscopic-assisted abdominoperineal resection for rectal cancer with sigmoidostomy and 16 patients underwent laparoscopic-assisted low anterior resection for rectal cancer with loop ileostomy. The mean operation time was 115.3 minutes and intraoperative blood loss was 86.1 ml. The mean time for enterostomy was 14.1 minutes. The average time to flatus, time to fluid diet intake and length of hospital stay were 1.8 days, 2.9 days and 6.5 days, respectively. During the follow-up period, three patients suffered from stomal edema, two patients suffered from parastomal hernia, and two patients suffered from skin inflammation surrounding stoma. None of re-operation related stoma and severe mobility such as stomal stenosis, stomal necrosis, stomal prolapse, stomal retraction and stomal mucocutaneous separation occurred. Thirty-five patients recovered with satisfactory stomal function, two with middle function and one with poor function. Conclusion: Enterostomy using running suture of dermis and seromuscular layer in laparoscopic-assisted radical resection for rectal carcinoma is a safe and feasible procedure with a satisfactory short-term effect.

Acute nutrient treatment causes alterations in intra-follicular antioxidation and AKT signaling.

This study aimed to determine if short-term nutrient alteration affects (1) ovarian morphology, (2) plasma and ovarian antioxidant capability and (3) cell apoptosis and AKT signaling within the ovary. After estrus synchronization, 24 Hu sheep were assigned to three groups based on the nutrient requirement recommended for maintenance (M): 1 × M (Control), 1.5 × M (S) and 0.5 × M (R) during days 7-14 of their estrous cycle. The results indicated that undernourishment significantly increased the counts and volume of follicles <2.5 mm and decreased the counts and volume of follicles ≥2.5 mm (P < 0.05). Feed restriction altered the plasma and follicular redox balance within follicles ≥2.5 mm by inhibiting total antioxidant capacity, increasing malondialdehyde concentration (P < 0.05) and reducing the mRNA expression levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GSH-PX), as well as the activities of total SOD and GSH-PX. Feed restriction also attenuated B-cell lymphoma-2 (BCL2) but enhanced Bcl-2-associated X protein (BAX) and BAX/BCL2 transcription and translation levels in granulosa cells (P < 0.05). Uniform staining intensities of AKT and P-AKT-Ser473 were observed in each follicle stage, whereas weaker P-AKT-Thr308 staining in the antral follicle than in the pre-antral follicle suggested possible involvement of P-AKT-Thr308 during the beginning of follicle development. P-AKT-Ser473 levels in follicles ≥2.5 mm was significantly reduced in the R group (P < 0.05). The results presented in this study demonstrate that suppressed folliculogenesis caused by feed restriction might be associated with attenuated AKT signaling, reduced follicular antioxidant capacity and enhanced granulosa cells apoptosis.

Preparation, characterization, and pharmacokinetics in swine of a florfenicol enteric formulation prepared using hot-melt extrusion technology.

The objective of this work was to manufacture an enteric formulation of florfenicol (FF) using hot-melt extrusion (HME) technology and to evaluate its in vitro dissolution and in vivo pharmacokinetics. For the HME process, hypromellose acetate succinate LG (HPMCAS-LG) was the enteric polymer mixed with FF, and the two components were extruded with a standard screw configuration at a speed of 50 rpm. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), powder X-ray diffraction (PXRD), and Fourier transform infrared spectroscopy (FT-IR) were performed to characterize the HME extrudate. The release percentage of the enteric formulation in the acidic stage was <10% of the loaded FF, whereas that in the phosphate buffer stage was >80%. Pharmacokinetic evaluations in swine revealed that the enteric formulation had a longer t1/2λ and MRT than commercially available FF powder (FULAIKA® ), indicating that the novel formulation exhibited enteric and sustained release properties. Compared with the commercial product, the relative bioavailability of the enteric formulation reached up to 117.2%. This study suggests that this formulation may have potential for future commercialization.

Carcass traits, meat quality, antioxidant status and antioxidant gene expression in muscle and liver of Hu lambs fed perilla seed.

The effects of perilla (Perilla frutescens L.) seed on carcass traits, meat quality, antioxidant status and antioxidant gene expression in the liver and muscle of Hu lambs were investigated in this study. Sixty Hu lambs (23.02 ± 1.36 kg) were randomly divided into four experimental groups receiving diets containing 0%, 5%, 10% or 15% perilla seed (CD, 5%PFSD, 10%PFSD and 15%PFSD, respectively). The addition of perilla seed had no significant impacts on carcass traits (p > .05). There were no differences in pH, meat colour, drip loss, cooking loss or shear force among the four treatments (p > .05). Addition of perilla seed increased (p < .05) deposition of intramuscular lipids but had no effect on other chemical components in the longissimus dorsi (LD) (p > .05). The 15%PFSD diet decreased the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content in the liver (p < .05 for both) but increased the activity of these antioxidant enzymes in LD (p < .05 for both). Compared to CD, addition of perilla seed increased superoxide dismutase (SOD) and glutathione peroxidase (GPX) expression in the liver and LD (p < .05 for all). These results indicate that perilla seed supplementation in lambs' diets can increase deposition of intramuscular lipids and improve muscular oxidative status and meat quality.

Metabolomic profiling in umbilical venous plasma reveals effects of dietary rumen-protected arginine or N-carbamylglutamate supplementation in nutrient-restricted Hu sheep during pregnancy.

Maternal nutrient restriction during pregnancy is a major problem worldwide for human and animal production. Arginine (Arg) is critical to health, growth and reproduction. N-carbamylglutamate (NCG), a key enzyme in arginine synthesis, is not extensively degraded in rumen. The aim of this study was to investigate ameliorating effects of rumen-protected arginine (RP-Arg) and NCG supplementation on dietary in undernourished Hu sheep during gestation. From day 35 to 110 of gestation, 32 Hu ewes carrying twin foetuses were randomly divided into four groups: a control (CG) group (n = 8; fed 100% National Research Council (NRC) requirements for pregnant sheep), a nutrient-restricted (RG) group (n = 8; fed 50% NRC requirements, which included 50% mineral-vitamin mixture) and two treatment (Arg and NCG) groups (n = 8; fed 50% NRC requirements supplemented with 20 g/day RP-Arg or 5 g/day NCG, which included 50% mineral-vitamin mixture). The umbilical venous plasma samples of foetus were tested by 1 H-nuclear magnetic resonance. Thirty-two differential metabolites were identified, indicating altered metabolic pathways of amino acid, carbohydrate and energy, lipids and oxidative stress metabolism among the four groups. Our results demonstrate that the beneficial effect of dietary RP-Arg and NCG supplementation on mammalian reproduction is associated with complex metabolic networks.

[Effects of modified three-step procedure for anatrophic nephrolithotomy in the treatment of complex staghorn renal calculi].

Objective: To investigate the effects of modified three-step procedure for anatrophic nephrolithotomy in the treatment of complex staghorn renal calculi. Methods: A total of 22 patients with complex staghorn renal calculi between June 2013 and June 2016 at Department of Urology in Guangzhou General Hospital of Guangzhou Military Command were retrospective analyzed. There were 13 males and 9 females, ranging from 35 to 62 years old with mean age of 47 years. There were 17 patients with dull pain, and 5 patients who were found through physical examinations. Kidney calculi located in left kidney in 15 patients, right kidney in 7 patients. All patients were treated with modified three-step procedure for anatrophic nephrolithotomy. The operation time, blood loss, time of intraoperative renal ischemia, and postoperative complications were recorded. Serum creatinine (Scr), blood urea nitrogen(BUN), ß(2)-microglobulin(ß(2)-MG), diseased side glomerular filtration rate(GFR) , and renal cortical thickness of the diseased kidney in preoperative and postoperative were compared. The clinical data were compared by paired sample t test between pre-operation and post-operation. Results: The calculi were completely removed in 22 patients, the mean operation time was 84 minutes (50 to 126 minutes), the mean time of intraoperative renal ischemia was 31 minutes (20 to 56 minutes), the mean blood loss was 246 ml (150 to 360 ml). There were no secondary bleeding or urinary fistula happened, the perinephric drainage tub was removed in 3 to 7 days postoperative, the mean hospitalization time was 7 days.Compared with the preoperative, the Scr ((172.7±21.3)µmol/L vs. (146.4±22.8)µmol/L, t=7.197, P=0.000), BUN ((9.2±1.8)mmol/L vs. (8.0±0.5)mmol/L, t=3.798, P=0.001) and ß(2)-MG ((203.0±32.0)µg/L vs. (175.6±23.8)µg/L, t=5.009, P=0.000) in postoperative decreased, the diseased side GFR increased ((28.6±4.0) ml/min(31.8±3.3) ml/min, t=-3.521, P=0.002). There were no significant difference of diseased renal cortical thickness between preoperative and postoperative(t=-1.323, P=0.200). There were 12 patients with postoperative pain, 2 patients with vomiting, 3 patients with fever, and 2 patients with wound infection. The follow-up time was 6 months, no residual stones in 22 patients. Conclusion: The modified three-step procedure for anatrophic nephrolithotomy has high stone free rates with less effects on renal function and fewer complications, the method could be widely applied.

A novel label-free fluorescence strategy for methyltransferase activity assay based on dsDNA-templated copper nanoparticles coupled with an endonuclease-assisted signal transduction system.

Evaluating DNA methyltransferase (MTase) activity has received considerable attention due to its significance in the fields of early cancer clinical diagnostics and drug discovery. Herein, we proposed a novel label-free fluorescence method for MTase activity assay by coupling double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) with an endonuclease-assisted signal transduction system. In this strategy, dsDNA molecules were first methylated by DNA adenine methylation (Dam) MTase and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved DNA fragments could not act as efficient templates for the formation of fluorescent CuNPs and thus no fluorescence signal was produced. Under optimized experimental conditions, the developed strategy exhibited a sensitive fluorescence response to Dam MTase activity. This strategy was also demonstrated to provide an excellent platform to the inhibitor screening for Dam MTase. These results demonstrated the great potential for the practical applications of the proposed strategy for Dam MTase activity assay.

Impact of Food Restriction on the Expression of the Adiponectin System and Genes in the Hypothalamic-Pituitary-Ovarian Axis of Pre-Pubertal Ewes.

Adiponectin, a cytokine secreted typically by adipocytes, has been implicated as a molecular switch between female reproduction and energy balance. The present study was undertaken to investigate the expression of adiponectin system and patterns of genes in the hypothalamic-pituitary-ovary (HPO) axis of food-restricted pre-pubertal ewes. Eighteen 2-month-old female ewes were assigned to 3 groups after a pre-feeding ad libitum for 10 days (six in each group): the control group (C), the low-food-restricted group (LR) and the high-food-restricted group (HR), which were fed with 100%, 70% and 50% of ad libitum food intake, respectively. The hypothalamus, pituitary, ovary and serum were collected after food restriction for 2 months. Results by ELISA showed that food restriction increased serum adiponectin concentrations. Quantitative real-time PCR showed that the gene transcriptions for adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) were enhanced in the hypothalamic-pituitary-ovarian (HPO) axis, while KISS-1/GPR-54 and gonadotropin-releasing hormone (GnRH) in the hypothalamus and luteinizing hormone ß-subunit (LHß) and follicle-stimulating hormone ß-subunit (FSHß) in the pituitary were reduced after food restriction. Immunohistochemistry results demonstrated that AdipoR1 localized in the oocytes of follicles in the ovary. These results suggest that the alterations in the expression of adiponectin and its receptors in response to food restriction might negatively influence the HPO axis.

[Relationship between autophagy of melanocytes in patients with vitiligo and clinical types].

OBJECTIVE: To evaluate autophagy of melanocytes and its mechanism in patients with vitiligo and to analyze its correlation with clinical types of vitiligo. METHODS: Nine cases of segmental vitiligo (SV) and 11 cases of generalized vitiligo (GV) were recruited in Hangzhou Third Hospital between May 2014 and June 2015. Six people with healthy skin were recruited as controls. Epidermal melanocytes were obtained from the normal colour skin around the white spot area in SV and GV patients and from foreskin in controls for culture in vitro. Cultures for each group contained negative control and rapamycin (30 nmol/L) sub-groups. The autophagy was observed using transmission electron microscopy (TEM) and immunofluorescence laser scanning confocal microscope (LSCFM). Protein expressions of microtube-associated protein light chain 3 (LC3Ⅱ) and microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase related protein (TYRP)1, and TYRP2 were detected by Western blot. RESULTS: (1)Autophagy of melanocytes was observed under TEM and LSCFM in both SV and control groups before rapamycin treatment, but not in GV patients. The expressions of autophagosome and LC3Ⅱ protein were increased in melanocytes in SV, GV and control groups after autophagy induction(SV group 0.58±0.10 vs 0.37±0.06; GV group 0.57±0.16 vs 0.22±0.08; control group 0.67±0.09 vs 0.46±0.12), and the autophagy intensity was higher in the GV group compared to the SV group and the control group. (2)Before autophagy induction, the expressions of MITF, TYR, TYRP1, and TYRP2 protein were lower in the GV and SV patients compared with the control group; after autophagy induction, the expressions of MITF, TYR, and TYRP1 statistically significantly increased in melanocytes in all the three groups(all P<0.05), while TYRP2 protein expression was not significantly changed (all P>0.05). CONCLUSIONS: Autophagy of melanocytes may be present in vitiligo and affect the expression of functional molecules, and is related with clinical type of vitiligo.

Sevoflurane preconditioning-induced neuroprotection is associated with Akt activation via carboxy-terminal modulator protein inhibition.

BACKGROUND: Sevoflurane preconditioning has a neuroprotective effect, but the underlying mechanism is not fully understood. The aim of the present investigation was to evaluate whether sevoflurane-induced cerebral preconditioning involves inhibition of carboxy-terminal modulator protein (CTMP), an endogenous inhibitor of Akt, in a rat model of focal cerebral ischaemia. METHODS: Male Sprague-Dawley rats were exposed to 2.7% sevoflurane for 45 min. One hour later, rats were subjected to 60 min of focal cerebral ischaemia. The phosphoinositide 3-kinase inhibitors wortmannin and LY294002 were administered 10 min before preconditioning. Rats in the lentiviral transduction group received an intracerebroventricular injection of lentiviral vector Ubi-MCS-CTMP 3 days before ischaemia. Neurological deficits and infarct volumes were evaluated 24 h and 7 days after reperfusion. Phosphorylation of Akt, glycogen synthase kinase-3ß (GSK3ß), and expression of CTMP were determined at 1, 3, 12, and 24 h after reperfusion. Akt activity was measured at 3 h after reperfusion. RESULTS: Sevoflurane preconditioning improved neurological score and reduced infarct size at 24 h of reperfusion. Pretreatment with wortmannin or LY294002 attenuated these neuroprotective effects. Expression of CTMP correlated with reduced Akt activity after ischaemia, while sevoflurane preconditioning preserved Akt activity and increased phosphorylation of GSK3ß. CTMP over-expression diminished the beneficial effects of sevoflurane preconditioning. CONCLUSIONS: Activation of Akt signalling via inhibition of CTMP is involved in the mechanism of neuroprotection provided by sevoflurane preconditioning.

Analysis of gene expression patterns and levels in maize hybrids and their parents.

Heterosis has greatly contributed to conventional plant breeding and is widely used to increase crop plant productivity. However, although some studies have explored the mechanisms of heterosis at the genomic and transcriptome level, these mechanisms still remain unclear. The growth and development of maize seedlings and immature embryos have an important impact on subsequent production. This study investigated differentially expressed genes (DEGs) between parents and reciprocal hybrids in the seedling leaves, roots, and immature embryo 15 days after pollination using amplified fragment length polymorphism (AFLP)-based transcript profiling (cDNA-AFLP). We isolated 180, 170, and 108 genes from the leaves, roots, and immature embryos, respectively, that were differentially expressed between hybrids and parents. Sequencing and functional analysis revealed that 107 transcript-derived fragments in the roots and leaves and 90 in the immature embryos were involved in known functions, whereas many DEGs had roles in plant growth and development, photosynthesis, signal transduction, and seed germination. Quantitative reverse-transcription polymerase chain reaction analysis of relative expression levels between reciprocal hybrids and both parental genotypes of selected genes produced results that were consistent with cDNA-AFLP. We validated the expression patterns of 15 selected genes related to heterosis formation and revealed that most showed non-additive expression in one or both hybrids, including dominant, underdominant, and overdominant expression. This indicates that gene-regulatory interactions among parental alleles play an important role in heterosis during the early developmental stages of maize.

Effect of Age on Energy Requirement for Maintenance and Growth of Dorper and Hu Crossbred F1 Ewes Weighing 20 to 50 kg.

This research aimed to define the energy requirement of Dorper and Hu Hybrid F1 ewes 20 to 50 kg of body weight, furthermore to study energy requirement changes with age and evaluate the effect of age on energy requirement parameters. In comparative slaughter trial, thirty animals were divided into three dry matter intake treatments (ad libitum, n = 18; low restricted, n = 6; high restricted, n = 6), and were all slaughtered as baseline, intermediate, and final slaughter groups, to calculate body chemical components and energy retained. In digestibility trial, twelve ewes were housed in individual metabolic cages and randomly assigned to three feeding treatments in accordance with the design of a comparative slaughter trial, to evaluate dietary energetic values at different feed intake levels. The combined data indicated that, with increasing age, the net energy requirement for maintenance (NEm) decreased from 260.62±13.21 to 250.61±11.79 kJ/kg(0.75) of shrunk body weight (SBW)/d, and metabolizable energy requirement for maintenance (MEm) decreased from 401.99±20.31 to 371.23±17.47 kJ/kg(0.75) of SBW/d. Partial efficiency of ME utilization for maintenance (km, 0.65 vs 0.68) and growth (kg, 0.42 vs 0.41) did not differ (p>0.05) due to age; At the similar condition of average daily gain, net energy requirements for growth (NEg) and metabolizable energy requirements for growth (MEg) for ewes during late fattening period were 23% and 25% greater than corresponding values of ewes during early fattening period. In conclusion, the effect of age upon energy requirement parameters in the present study were similar in tendency with previous recommendations, values of energy requirement for growth (NEg and MEg) for Dorper and Hu crossbred female lambs ranged between the NRC (2007) recommendation for early and later maturating growing sheep.

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai).

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker-centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72-h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second-division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y-values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y-values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.

Impact of food restriction on ovarian development, RFamide-related peptide-3 and the hypothalamic-pituitary-ovarian axis in pre-pubertal ewes.

RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibiting hormone, has been implicated as a mediator between reproduction and energy balance. This study aimed to investigate the physiological effects of RFRP-3 on the process of ovarian development in food-restricted pre-pubertal ewes. The results showed that food restriction significantly inhibited the ovarian development and follicular growth. The data of qPCR in the hypothalamic-pituitary-ovarian (HPO) axis showed that food restriction not only upregulated RFRP-3 mRNA expression but also downregulated the mRNA expression of gonadotropin-releasing-hormone receptor, follicle-stimulating hormone receptor and luteinizing hormone receptor (LHR). Immunohistochemistry of RFRP-3 in the ovaries suggested that RFRP-3 may regulate the follicular development. These results suggested that the changes of RFRP-3 in response to food restriction might influence the HPO axis and inhibit ovarian development.

Effects of different dietary intake on mRNA levels of MSTN, IGF-I, and IGF-II in the skeletal muscle of Dorper and Hu sheep hybrid F1 rams.

MSTN, IGF-І(insulin-like growth factor-І) and IGF-II (insulin-like growth factor-II) regulate skeletal muscle growth. This study investigated the effects of different dietary intake levels on skeletal muscles. Sheep was randomly assigned to 3 feeding groups: 1) the maintenance diet (M), 2) 1.4 x the maintenance diet (1.4M), and 3) 2.15 x the maintenance diet (2.15M). Before slaughtering the animals, blood samples were collected to measure plasma urea, growth hormone, and insulin concentrations. After slaughtering, the longissimus dorsi, semitendinosus, semimembranosus, gastrocnemius, soleus, and chest muscle were removed to record various parameters, including the mRNA expression levels of MSTN and IGFs, in addition to skeletal muscle fiber diameter and cross-sectional area. The result showed that as dietary intake improved, the mRNA expression levels of MSTN and IGF-II decreased, whereas IGF-Іexpression increased. The mRNA expression levels of MSTN and IGFs were significantly different in the same skeletal muscle under different dietary intake. The skeletal muscle fiber diameter and cross-sectional area increased with greater dietary intake, as observed for the mRNA expression of IGF-І; however, it contrasted to that observed for the mRNA expression of MSTN and IGF-II. In conclusion, dietary intake levels have a certain influence on MSTN and IGFs mRNA expression levels, in addition to skeletal muscle fiber diameter and cross-sectional area. This study contributes valuable information for enhancing the molecular-based breeding of sheep.

A study of microemboli monitoring of atherosclerotic thrombotic cerebral infarction and artery stenosis.

This study aimed to assess the relationship between the recurrence and prognosis of patients with acute middle cerebral artery infarction, atherosclerotic brain infarction, and the existence of microemboli. We continuously enrolled patients with acute atherosclerotic thrombotic cerebral infarction artery stenosis. We performed transcranial Doppler color ultrasound micro emboli monitoring, color Doppler ultrasound carotid artery tests, intracranial and carotid artery magnetic resonance angiography, impairment evaluation of nerve function, and registration of stroke recurrence and stroke mortality. Of the 49 patients enrolled in the study, 123 main arteries presented atherosclerotic stenosis or formed plaques, and 33 patients had symptomatic stenosis. Patients with symptomatic stenosis have a higher incidence of microemboli than patients with asymptomatic stenosis (P = 0.009). The microembolus-positive rate increased in patients with unstable plaques (P = 0.001). Patients who were microembolus-negative were more likely to show a neural function deficient NIHSS (National Institutes of Stroke Scale) score improvement than patients who were microembolus-positive at one week (P = 0.026). However, we found no significant difference between mRS (modified rankin scale) score (P = 0.319), relapse, and death (P = 0.179). The rate of microembolus-positivity increased in patients with atherosclerotic thrombotic cerebral infarction and unstable plaques. Patients who were microembolus-negative were more likely to show an improvement of neural function deficiency than patients with microembolus-positivity at one week (P = 0.026).

[Effects of periodontitis on bone and tryptophan metabolism of gut microbiota in estrogen-deficient mice].

Objective: To study the effects of periodontitis on bone and tryptophan metabolism of gut microbiota in the context of estrogen deficiency. Methods: Thirty-two female C57BL6/J mice were randomly divided into four groups based on table of random numbers (n=8 in each group): Sham group, in which mice were given sham surgery; Sham_Lig group, in which mice were given sham surgery and were induced to periodontitis by ligating the bilateral maxillary second molars with 5-0 silk threads at the fourth week; Ovx group, in which mice were given bilateral ovariectomy; Ovx_Lig group, in which mice were given bilateral ovariectomy and were induced to periodontitis at the fourth week. After 8 weeks of ligation, the mice of 4 groups were euthanized for collecting the samples of femur, tibia, mandible and skull. Those samples were scanned by micro-CT to measure the bone mineral density (BMD), bone volume versus total volume ratio (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp). The cecum contents of 4 groups of mice were collected for gut microbiota 16S rRNA gene sequencing. The tryptophan and its metabolites in intestinal tracts were detected by liquid chromatography-mass spectrometry. Pearson correlation analysis was performed to analyze the correlation between the abundance of gut microbiota and the content of tryptophan and its metabolites. Results: Femur BMD [(82.23±3.97) mg/cm3], BV/TV [(9.25±1.37)%] and Tb.Th [(70.95±5.70) µm] in Ovx_Lig group were significantly lower than Ovx group [(96.30±3.76) mg/cm3 (P=0.004); (14.45±1.55)% (P=0.022) and (87.58±8.02) µm (P<0.001), respectively]. The ß-diversity analysis of gut microbiota based on Bray-Curtis distance showed that samples of Ovx_Lig group and Ovx group were obviously grouped. Linear discriminant analysis effect size (LEfSe) showed that Alistipes was the representative genus in Ovx_Lig group. The relative abundance of Alistipes in Ovx_Lig group [(0.42±0.14)%] were significantly higher than that in Ovx group [(0.17±0.05)%] (t=4.45, P<0.001). Tryptophan metabolism analysis showed that the content of kynurenic acid [(531.12±158.60) ng/g] in Ovx_Lig group were significantly higher than that in Ovx group [(400.42±57.96) ng/g] (t=2.19, P=0.046). And the content of indole-3-carbaldehyde [(383.37±144.06) ng/g] in Ovx_Lig group were significantly lower than Ovx group [(701.72±141.93) ng/g] (t=4.45, P<0.001). Correlation analysis showed that relative abundance of Alistipes was positively correlated with kynurenic acid (r=0.32, P=0.088), while negatively correlated with indole-3-carbaldehyde (r=-0.32, P=0.088). Conclusions: Periodontitis can induce bone destruction of femur in estrogen-deficient mice, the mechanism of which may be related to Alistipes in gut and the tryptophan metabolites kynurenic acid and indole-3-carbaldehyde.

Analysis of the mechanism of Sophorae Flavescentis Radix in the treatment of intractable itching based on network pharmacology and molecular docking.

OBJECTIVE: Sophorae Flavescentis Radix (Kuh-seng, SFR), a Traditional Chinese Medicine (TCM), is widely used alone or within a TCM formula to treat pruritus, especially histamine-independent intractable itching. In the previous study, potential antipruritic active components of the SFR were screened based on cell membrane immobilized chromatography (CMIC), revealing oxymatrine (OMT) as an antipruritic agent. However, the low oral bioavailability (OB) of OMT cannot explain the antipruritic effect of SFR when administered orally in clinic. In this study, we investigated the antipruritic effects and underlying mechanisms of orally administered SFR. MATERIALS AND METHODS: A network pharmacology and molecular docking were employed to screen the active components of SFR and predict their binding to disease-related target proteins, while the potential mechanisms were explored with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The binding energy between components and target proteins was calculated by molecular docking. RESULTS: The SFR-components-targets-intractable itching Protein-Protein Interactions (PPI) network was established, and 22 active components and 42 targets were screened. The GO enrichment analysis showed that the key target genes of SFR were related to nuclear receptors, transcription factors, and steroid hormone receptors. The results of the KEGG enrichment pathway analysis include Hepatitis B, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance, advanced glycation end product (AGE)-receptor for AGE (RAGE) signaling pathway in diabetic complications, etc. Molecular docking showed that three key target proteins in the network, the vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor (EGFR) and caspase-3 (CASP3), have higher binding activities with inermine, phaseolin and kushenol O, respectively; the binding energy of each pair is stronger than that of the target protein-corresponding inhibitors. CONCLUSIONS: The complexity of the SFR-components-targets-intractable itching network demonstrated the holistic treatment effect of SFR on intractable itching. The partial coherence between results screened by CMIC in the previous study and network pharmacology demonstrated the potential of network pharmacology in active component screening. Inermine screened from both CMIC and network pharmacology is a VEGFA inhibitor, which possibly accounts for the antipruritic effect of orally administered SFR.