[Long-term outcomes of 328 patients with of autism spectrum disorder after fecal microbiota transplantation].

Objective: To evaluate the efficacy and safety of fecal microbiota transplantation (FMT) in the treatment of autism spectrum disorder (ASD). Methods: A longitudinal study was conducted. Clinical data from ASD patients with gastrointestinal symptoms and who underwent FMT in the Tenth People's Hospital affiliated to Tongji University or Jinling Hospital between May 2012 to May 2021 were retrospectively collected. Scores derived from the autism behavior checklist (ABC), the childhood autism rating scale (CARS), the Bristol stool form scale (BSFS), and the gastrointestinal symptom rating scale (GSRS) were analyzed at baseline and at the 1st, 3rd, 6th, 12th, 24th, 36th, 48th and 60th month after FMT. Records of any adverse reactions were collected. Generalized estimating equations were used for analysis of data on time points before and after FMT. Results: A total of 328 patients met the inclusion criteria for this study. Their mean age was 6.1±3.4 years old. The cohort included 271 boys and 57 girls. The percentage of patients remaining in the study for post-treatment follow-up at the 1st, 3rd, 12th, 24th, 36th, 48th and 60th month were as follows: 303 (92.4%), 284 (86.7%), 213 (64.9%), 190 (57.9%), 143 (43.6%), 79 (24.1%), 46 (14.0%), 31 (9.5%). After FMT, the average ABC score was significantly improved in the first 36 months and remained improved at the 48th month. However, the average score was not significantly different from baseline by the 60th month (1st-36th month, P<0.001; 48th month, P=0.008; 60th month, P=0.108). The average CARS score improved significantly during the first 48 months and remained improved at the 60th month (1st-48th month, P<0.001; 60th month, P=0.010). The average BSFS score was also significantly improved in the first 36 months (with an accompanying stool morphology that resembled type 4). This improvement was maintained at the 48th month. However, the average score was similar to baseline at the 60th month (1st-36th month, P<0.001; 48th month, P=0.008; 60th month, P=0.109). The average GSRS score was significantly improved during the first 24 months, but not afterwards (1st-24th month, P<0.001; 36th month, P=0.209; 48th month, P=0.996; 60th month, P=0.668). The adverse events recorded during treatment included abdominal distension in 21 cases (6.4%), nausea in 14 cases (4.3%), vomiting in 9 cases (2.7%), abdominal pain in 15 cases (4.6%), diarrhea in 18 cases (5.5%), fever in 13 cases (4.0%), and excitement in 24 cases (7.3%). All adverse reactions were mild to moderate and improved immediately after suspension of FMT or on treatment of symptoms. No serious adverse reactions occurred. Conclusion: FMT has satisfactory long-term efficacy and safety for the treatment of ASD with gastrointestinal symptoms.

Display of quintuple glucagon-like peptide 1 (28-36) nonapeptide on Bacillus subtilis spore for oral administration in the treatment of type 2 diabetes.

AIMS: To develop an oral delivery system of glucagon-like peptide 1 (GLP-1) (28-36) for treating type-2 diabetes, B.S-GLP-1(28-36), a recombinant Bacillus subtilis spores transformed with a plasmid vector encoding five consecutive GLP-1 (28-36) nonapeptides with an enterokinase site was constructed. METHODS AND RESULTS: GLP-1(28-36) nonapeptide was successfully expressed on the surface of B. subtilis spores and validated by Western blot and immunofluorescence. The therapeutic effect of oral administration of B.S-GLP-1(28-36) spores was evaluated in type 2 diabetic model mice. The efficacy of recombinant spores was examined for a period of 13 weeks after oral administration in diabetic mice. At the end of the sixth week, diabetic mice with oral administration of BS-GLP-1(28-36) spores showed decreased blood glucose levels from 2·4 × 10- 2  mol l-1 to 1·7 × 10- 2  mol l-1 . By the ninth week, the mean fasting blood glucose level in the experimental group was significantly lower than that in the control group 30 min after injection of pyruvate. At the end of the 10th week of oral administration, the blood glucose of the experimental group was significantly lower than that of the control group after intraperitoneal injection of glucose. By the 12th week, fasting blood glucose level and fasting insulin level were measured in all mice, the results showed that the recombinant spores increased the insulin sensitivity of mice. CONCLUSIONS: The results of pathological observation showed that the recombinant spores also had a certain protective effect on the liver and islets of mice, and the content of GLP-1(28-36) in the pancreas of the experimental group was increased. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study revealed that GLP-1(28-36) nonapeptides can reduce blood glucose and play an important role in the treatment of type 2 diabetes.

miR-382-3p Overexpression attenuates N-methyl-D-aspartate-induced HT22 cell apoptosis via the regulation of the RhoC/ROCK1 signaling pathway.

miR-382-3p can regulate apoptosis through multiple pathways, but the mechanism remains unknown. In this experiment, we explored whether miR-382-3p can modulate the N-methyL-D-aspartate (NMDA)- induced HT22 cell apoptosis by regulating the RhoC/ROCK1 signaling pathway. An excitatory neurotoxicity model of HT22 cells was induced in vitro with 2 mmol/L NMDA. The cells were divided into normal control, NMDA-induced, NMDA + miR-382-3p mimic, and NMDA + miR-382-3p inhibitor groups. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, Real-time PCR, Western blot, and flow cytometry were performed to investigate the mechanisms. The results found that NMDA can increase the oxidative stress of HT22 cells in a dose-dependent manner, downregulate the expression of miR-382-3p, upregulate the expression of mRNA and protein abundance of ROCK1 and RhoC, increase the expression levels of proapoptotic proteins Bax, Caspase-3, and Caspase-9, increase the apoptosis of HT22 cells, and reduce the activity and survival rate of HT22 cells. Compared with the NMDA-induced group, the miR-382-3p mimic-transfected HT22 cells increased the expression of miR- 382-3p, reduced the expression of the mRNA and protein abundance of ROCK1 and RhoC, inhibited the expression of proapoptotic proteins Bax, Caspase-3, and Caspase-9, reduced the apoptosis of HT22 cells, and increased the activity and survival rate of HT22 cells. The results suggest that increasing the expression of miR-382-3p can inhibit the activity of the RhoC/ROCK1 signaling pathway, reduce the expression of proapoptotic proteins, reduce the oxidative stress and apoptosis of HT22 cells, and increase the activity and survival rate of HT22 cells.

Expression of lncRNA AK058003 in esophageal carcinoma and analysis of its intervention effect.

OBJECTIVE: The aim of this study was to investigate the expression of long non-coding ribonucleic acid (lncRNA) AK058003 in esophageal carcinoma (EC) tissues, and to analyze its intervention effect. PATIENTS AND METHODS: The expression of lncRNA AK058003 in EC tissues and para-carcinoma tissues from 130 EC patients was detected via quantitative Polymerase Chain Reaction (qPCR). EC cell lines were selected for exogenous interference in lncRNA AK058003. Subsequently, the expression of lncRNA AK058003 in normal esophageal epithelial cell line (Het-1A) and EC cell lines (EC109, EC9706, KYSE-150, KYSE-30, and TE-1) was detected by qPCR. EC9706 cell lines with the highest expression of lncRNA AK058003 were selected and transfected with lncRNA AK058003 siRNA and lncRNA AK058003 control, respectively. After transfection, the expression of lncRNA AK058003 was determined using PCR. The changes in cell growth and proliferation were analyzed via cell growth curve and cell cycle assay. Meanwhile, the changes in cell migration and invasion were analyzed through wound healing assay. Protein expressions of matrix metalloproteinase-1 (MMP1) and MMP2 were determined by Western blot. Clinical data were collected from EC patients, and the association between lncRNA AK058003 expression and tumor-node-metastasis (TNM) stage was finally analyzed. RESULTS: LncRNA AK058003 was highly expressed EC tissues compared with para-carcinoma tissues (p<0.01). Compared with Het-1A cells, the expression of lncRNA AK058003 was significantly higher in EC109, EC9706, KYSE-150, KYSE-30, and TE-1 cells, with highest level in EC9706 cells (p<0.05). The expression of lncRNA AK058003 remarkably declined in lncRNA AK058003 siRNA group compared with lncRNA AK058003 control group (p<0.001). Compared with lncRNA AK058003 control group, the proliferation of EC cells was significantly weakened in lncRNA AK058003 siRNA group, with the greatest difference at 3 d. Flow cytometry results revealed that cell cycle was arrested in G0/G1 phase in lncRNA AK058003 siRNA group. Wound healing assay indicated that the intercellular distance became large, and cell migration ability was evidently enhanced in lncRNA AK058003 siRNA group with time (p<0.05). Besides, the protein expressions of MMP1 and MMP2 were remarkably lower in lncRNA AK058003 siRNA group than those in lncRNA AK058003 control group. This indicated remarkably declined invasion and metastasis ability. In addition, the postoperative prognosis was significantly worse in patients with higher expression of lncRNA AK058003 (p<0.05). All these findings suggested that lncRNA AK058003 could serve as a biomarker for EC prognosis. CONCLUSIONS: LncRNA AK058003 is highly expressed in EC patients, which promotes proliferation, migration, invasion, and metastasis of EC cells. In addition, the postoperative prognosis of EC patients with high expression of lncRNA AK058003 is relatively poor.

Phosphoproteome profiling revealed abnormally phosphorylated AMPK and ATF2 involved in glucose metabolism and tumorigenesis of GH-PAs.

PURPOSE: Protein phosphorylation plays a key role in tumorigenesis and progression. However, little is known about the phosphoproteome profiles of growth hormone-secreting pituitary adenomas (GH-PAs). The aim of this study was to identify critical biomarkers and signaling pathways that might play important roles in GH-PAs and may, therefore, represent potential therapeutic targets. METHODS: The differential phosphoprotein expression patterns involved in GH-PAs were investigated by nano-LC-MS/MS in a group of samples. The phosphoprotein expression data were analyzed by bioinformatics. The expression levels of the candidate phosphorylated AMPK (ser496) and ATF2 (ser112) were validated by Western blot analysis in another group of samples. RESULTS: A total of 1213 phosphorylated protein sites corresponding to 667 proteins were significantly different between GH-PAs and healthy pituitary glands. Among these phosphorylated sites, 871 exhibited lower levels of phosphorylation in GH-PAs. Moreover, 140 novel phosphosites corresponding to 93 proteins were differentially phosphorylated between GH-PAs and healthy pituitary glands, 101 of which showed decreased phosphorylation in GH-PAs. The majority of differentially expressed phosphorylated proteins were significantly enriched in glycolysis and the AMPK signaling pathway in GH-PAs. The AMPK signaling pathway was demonstrated to be inhibited in GH-PAs by pathway activity analysis (z score = - 2.324). Notably, the phosphorylated levels of AMPK (ser496) and ATF2 (ser112) were significantly lower in GH-PAs than in healthy pituitary glands. CONCLUSION: These findings suggest that decreased phosphorylation of the AMPK/ATF2 pathway may be critical for glucose metabolism and tumorigenesis in GH-PAs.

Efficient production of coenzyme Q10 from acid hydrolysate of sweet sorghum juice by Rhodobacter sphaeroides.

In order to achieve efficient bioconversion of biomass-derived sugars, acid hydrolysate of sweet sorghum juice (SSJAH) containing abundant fermentable sugars was used for coenzyme Q10 (CoQ10) fermentation by Rhodobacter sphaeroides CQ-09-1. The synthesis of CoQ10 was facilitated when the initial concentration of total sugar was 80.00 g L-1. And the highest CoQ10 titer was obtained when the pH and temperature were maintained at 7.00 and 30.00 °C, respectively. Moreover, corn steep powder (CSP) was proved to be an efficient nitrogen & salt supplement to SSJAH. Under the optimized conditions, the titer of CoQ10 reached 141.95 mg L-1 in a fed-batch fermentation. The CoQ10 titer reported was about two times higher than that obtained in the previous study using wild strains. This process introduces a potential way to produce CoQ10 using the concept of biorefinery, while making full use of sweet sorghum juice (SSJ).

[Effects of low level laser irradiation on the osteogenic capacity of sodium alginate/gelatin/human adipose-derived stem cells 3D bio-printing construct].

OBJECTIVE: To explore the effects of low level laser irradiation (LLLI) on the osteogenic capacity of three-dimensional (3D) structure by 3D bio-printing construct used human adipose-derived stem cells (hASCs) as seed cells. METHODS: Using hASCs as seed cells, we prepared sodium alginate/gelatin/hASCs 3D bio-printing construct, and divided them into four groups: PM (proliferative medium), PM+LLLI, OM (osteogenic medium) and OM+LLLI, and the total doses of LLLI was 4 J/cm². Immunofluorescence microscopy was used to observe the viability of the cells, and analyze the expression of the osteogenesis-related protein Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). RESULTS: The 3D constructs obtained by printing were examined by microscope. The sizes of these 3D constructs were 10 mm×10 mm×1.5 mm. The wall thickness of the printed gelatin mold was approximately 1 mm, and the pores were round and had a diameter of about 700 µm. The cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct was high, and the difference among the four groups was not significant. On day 7, the expression of OCN from high to low was group OM+LLLI, PM+LLLI, OM and PM. There were significant differences among these groups (P<0.01), but there was no significant difference between group PM+LLLI and OM. On day 14, the expression of OCN in each group was higher than that on day 7, and there was no significant difference between group OM+LLLI and OM. The expression of Runx2 in group OM+LLLI was more than 90%, significantly higher than that in group OM (P<0.01). But the expression of Runx2 in group PM+LLLI and OM+LLLI were significantly lower than that in the non-irradiated groups. The expression of osteogenesis-related protein Runx2 and OCN were higher in OM groups than in PM groups. Furthermore, the irradiated groups were significantly higher than the non-irradiated groups. CONCLUSION: LLLI does not affect the cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct, and may promote the osteogenic differentiation of hASCs.

3D Black-Blood Luminal Angiography Derived from High-Resolution MR Vessel Wall Imaging in Detecting MCA Stenosis: A Preliminary Study.

BACKGROUND AND PURPOSE: 3D high-resolution vessel wall imaging is increasingly used for intracranial arterial diseases. This study compared the diagnostic performance of black-blood luminal angiography derived from 3D vessel wall imaging with source images of vessel wall imaging and TOF-MRA in detecting middle cerebral artery stenosis. MATERIALS AND METHODS: Sixty-two patients with suspected MCA atherosclerosis underwent TOF-MRA, vessel wall imaging, and CTA. Intracranial black-blood luminal angiography was created from source images of vessel wall imaging using minimum intensity projection. The degree and length of MCA stenosis were measured on source images of vessel wall imaging, TOF-MRA, and black-blood luminal angiography and compared using CTA as a reference standard. RESULTS: The image quality of black-blood luminal angiography was diagnostic in most patients. The intra- and interobserver agreement for both stenosis degree and length measurements was excellent for black-blood luminal angiography. It was comparable with that of source images of vessel wall imaging in grading stenosis. Compared with TOF-MRA, black-blood luminal angiography showed significantly higher sensitivity for the detection of severe stenosis (89.3% versus 64.3%, P = .039) and higher specificity for the detection of occlusion (95.4% versus 84.6%, P = .039). Lesion length estimated on source images of vessel wall imaging was significantly greater than that measured by CTA and black-blood luminal angiography (P < .001 and P = .010). CONCLUSIONS: Black-blood luminal angiography is better than TOF-MRA in detecting severe stenosis and occlusion of the MCA. Compared with source images of vessel wall imaging, it is more accurate in evaluating stenosis length. Black-blood luminal angiography can be produced as a derivative from vessel wall imaging and implemented as an adjunct to vessel wall imaging and TOF-MRA without extra acquisition time.

Effects of in ovo feeding of l-arginine on breast muscle growth and protein deposition in post-hatch broilers.

In ovo feeding (IOF) of l-arginine (Arg) can affect growth performance of broilers, but the response of IOF of Arg on breast muscle growth is unclear, and the mechanism involved in protein deposition remains unknown. Hense, this experiment was conducted to evaluate the effects of IOF of Arg on breast muscle growth and protein-deposited signalling in post-hatch broilers. A total of 720 fertile eggs were collected from 34-week-old Arbor Acres breeder hens and distributed to three treatments: (1) non-injected control group; (2) 7.5 g/l (w/v) NaCl diluent-injected control group; (3) 0.6 mg Arg/egg solution-injected group. At 17.5 days of incubation, fertile eggs were injected 0.6 ml solutions into the amnion of the injected groups. Upon hatching, 80 male chicks were randomly assigned to eight replicates of 10 birds each and fed ad libitum for 21 days. The results indicated that IOF of Arg increased relative breast muscle weight compared with those of control groups at hatch, 3-, 7- and 21-day post-hatch (P<0.05). In the Arg-injected group, the plasma total protein and albumen concentrations were higher at 7- and 21-day post-hatch than those of control groups (P<0.05). The alanine aminotransferase activity in Arg group was higher at hatch than that of control groups (P<0.05). The levels of triiodothyronine at four time points and thyroxine hormones at hatch, 7- and 21-day post-hatch in Arg group were higher than those of control groups (P<0.05). In addition, IOF of Arg increased the amino acid concentrations of breast muscle at hatch, 7- and 21-day post-hatch (P<0.05). In ovo feeding of Arg also enhanced mammalian target of rapamycin, ribosomal protein S6 kinase-1 and eIF4E-bindingprotein-1 messenger RNA expression levels at hatch compared with those of control groups (P<0.05). It was concluded that IOF of Arg treatment improved breast muscle growth, which might be associated with the enhancement of protein deposition.

[Role of vitamin K-dependent protein Gas6 in the expression of endothelial cell adhesion molecule-1 and chemokines induced by Porphyromonas gingivalis lipopolysaccharide].

OBJECTIVE: Growth-arrest-specific protein 6 (Gas6) is a vitamin K-dependent protein and involved in cell proliferation, survival, adhesion and migration . Also it has been shown to play an important role in the inflammatory response .The aim of present study was to investigate the role of Gas6 in the process of the expression of adhesion molecules and chemokines of human umbilical vein endothelial cells (HUVECs) induced by Porphyromonas gingivalis lipopolysaccharide(P.g-LPS). METHODS: After up-regulation and down-regulation of the expression of Gas6, the vascular endothelial cells were stimulated with 1 mg/L P.g-LPS for 3 h and 24 h. Real-time quantitative polymerase chain reaction(real-time PCR) was taken to detect the expression of the cell adhesion molecules:intercellular adhesion molecule-1 (ICAM-1) and E-selectin, as well as chemokines:interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1). Wound healing assay was taken to observe the migration ability of endothelium cells in different groups. RESULTS: After 3 h of P.g-LPS stimulation, the expression of adhesion molecules and chemokine in the down-regulation group was not significantly different from that in the control group,while in the up-regulation group the decrease of E-selectin, ICAM-1, IL-8 and MCP-1 was 81%±0%, 47%±3%, 76% ± 3%, 26% ± 6% respectively. After 24 h of P.g-LPS stimulation, the expression of adhesion molecules and chemokine in down-regulation group was significantly higher than that in control group (2.06±0.07, 1.99±0.11, 3.14±0.15, 1.84±0.03 flod), while these molecules in the down-regulation group was significantly lower than in the control group (29%±1%, 62%±3%, 69%±1%, 41%±2%). Differences were statistically significant (P<0.01). Wounding healing assay showed that down-regulation of Gas6 enhanced migration ability of endothelial cells while up-regulation of Gas6 weakened this ability,which was consistent with the trend of real-time PCR result. CONCLUSION: Down-regulation of the Gas6 gene enhanced the expression of ICAM-1, E-selectin, IL-8 and MCP-1 in HUVECs after P.g- LPS stimulating, while up-regulaiton of the Gas6 gene weakened the expression of ICAM-1, E-selectin, IL-8 and MCP-1 in HUVECs after P.g-LPS stimulating,suggesting that Gas6 may play a role in the process of endothelial cell adhesion.

In ovo feeding of L-arginine alters energy metabolism in post-hatch broilers.

This study aimed to investigate the effects of in ovo feeding (IOF) of L-arginine (Arg) on energy metabolism in post-hatch broilers. A total of 720 eggs was randomly assigned to 3 treatments: 1) non-injected control group, 2) 0.75% NaCl diluent-injected control group, and 3) 1.0% Arg solution-injected group. At 17.5 d of incubation, 0.6 mL of each solution was injected into the amniotic fluid of each egg of injected groups. After hatching, 80 male chicks were randomly assigned to each treatment group with 8 replicates per group. The results showed that IOF of Arg increased glycogen and glucose concentrations in the liver and pectoral muscle of broilers at hatch (P < 0.05). The plasma glucose and insulin levels were higher in the Arg group than in the non-injected and diluent-injected control groups (P < 0.05). Meanwhile, IOF of Arg enhanced the hepatic glucose-6-phosphatase (G6P) activity at hatch (P < 0.05). There was no difference in hexokinase (HK) or phosphofructokinase (PFK) enzyme activities in the pectoral muscle in all groups. Further, IOF of Arg increased the phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBP) mRNA expressions at hatch (P < 0.05). In addition, broilers in the Arg group had a higher mRNA expression of glycogen synthase and a lower expression of glycogen phosphorylase in the liver and pectoral muscles than in the non-injected controls at hatch (P < 0.05). In conclusion, IOF of Arg solution enhanced liver and pectoral muscle energy reserves at hatch, which might be considered as an effective strategy for regulating early energy metabolism in broilers.

In ovo feeding of creatine pyruvate modulates growth performance, energy reserves and mRNA expression levels of gluconeogenesis and glycogenesis enzymes in liver of embryos and neonatal broilers.

The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the growth performance, energy reserves and mRNA expression levels of gluconeogenesis and glycogenesis enzymes in liver of late-term embryos and neonatal broilers were investigated. After candling on 16 day of incubation, a total of 960 eggs were randomly assigned to three treatments: (i) non-injected control, (ii) saline group injected with 0.6 ml of 0.75% physiological saline and (iii) Creatine pyruvate group injected with 0.6 ml of physiological saline containing 12 mg CrPyr/egg. After hatching, 120 male chicks with average body weight (BW) were randomly allocated into each treatment group for a 7-day feeding trial. The results showed that broilers subjected to CrPyr treatment had higher BW than those of the control and saline groups on 1, 3 and 7 day post-hatch, as well as the yolk sac weight on 19 day of incubation (19 E), the day of hatch and 3 day post-hatch (p < .05). Compared with the control and saline groups, IOF of CrPyr increased the plasma creatine concentration on the day of hatch, and the plasma pyruvate concentration on the day of hatch and 3 day post-hatch (p < .05). Moreover, IOF of CrPyr increased the liver pyruvate and glucose concentrations on 19 E and the day of hatch, and the liver glycogen concentration during the experiment (p < .05). Broilers in the CrPyr group showed increased mRNA expression levels of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glycogen synthase 2 (GYS2) on 19 E and the day of hatch (p < .05). These results indicated that IOF of CrPyr increased energy reserves in liver of embryos and neonatal broilers possibly through upregulating the mRNA expression levels of PC, PEPCK and GYS2, which could benefit the increase of BW in broilers on 7 day post-hatch.

In ovo feeding of creatine pyruvate alters energy reserves, satellite cell mitotic activity and myogenic gene expression of breast muscle in embryos and neonatal broilers.

We investigated the effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on energy reserves, satellite cell mitotic activity (SCMA) and myogenic gene expression in breast muscle of embryos and neonatal broilers. A total of 960 eggs were randomly allocated into three treatments: 1) non-injected control group, 2) saline group injected with 0.6 mL of physiological saline (0.75%), and 3) CrPyr group injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr/egg at 17.5 d of incubation. After hatching, a total of 120 male chicks were randomly assigned to each treatment group, with eight replicate sets per group. Selected chicks had body BW close to the average of their pooled group. Our results showed that the total and relative breast muscle weights of broilers subjected to CrPyr treatment were higher than those in the control and saline groups on 19 d of incubation (19 E), the day of hatch, 3 and 7 d post-hatch (P < 0.05). The myofiber diameter and cross-sectional area of individuals in the CrPyr group were higher than those in other treatments on 3 and 7 d post-hatch (P < 0.05). Moreover, IOF of CrPyr increased (P < 0.05) creatine concentrations on 19 E, the day of hatch and 3 d post-hatch, the same treatment increased phosphocreatine concentrations on 19 E. Broilers in the CrPyr group showed higher expression of myogenic differentiation 1 (MyoD) (P < 0.05), myogenin and paired box 7 (Pax7), as well as higher index of SCMA on 3 d post-hatch. However, myostatin mRNA expression in CrPyr-treated broilers was down-regulated on 3 d post-hatch (P < 0.05). These results indicated that IOF of CrPyr increased energy reserves of embryos and SCMA of broilers on 3 d post-hatch, which led to enhanced muscle growth in the late embryos and neonatal broilers. Additionally, IOF of CrPyr increased the activity of satellite cells possibly through up-regulating MyoD, myogenin, and Pax7 mRNA expression and down-regulating myostatin mRNA expression.

Effect of in ovo feeding of l-arginine on the hatchability, growth performance, gastrointestinal hormones, and jejunal digestive and absorptive capacity of posthatch broilers.

This study was conducted to investigate the effects of in ovo feeding (IOF) of Arg solution on the hatchability, growth performance, gastrointestinal hormones, serum AA, activities of digestive enzymes, and mRNA expressions of sensing receptors and nutrient transporters in the jejunum of posthatch broilers. One thousand two hundred embryonated eggs with similar weight were randomly allocated to 5 groups consisting of 8 replicates of 40 eggs each. The 5 treatments were arranged as a noninjected control, a diluent-injected (0.75% NaCl solution) group, and Arg solution-injected groups with 0.5%, 1.0%, and 2.0% Arg, all dissolved in diluent. At 17.5 d of incubation, 0.6 mL of IOF solution was injected into the amniotic fluid of each egg of the injected groups. Results showed the hatchability of the 2% Arg group was lower (linear, = 0.025) than that of the other groups, and the BW of 21-d-old broilers increased (linear, = 0.008; quadratic, = 0.003) with increasing IOF concentration of Arg. The ADFI (linear, = 0.005; quadratic, = 0.001) and ADG (linear, = 0.010; quadratic, = 0.004) increased during d 1 to 21 with increasing IOF concentration of Arg. For 7- and 21-d-old broilers, the weights of digestive organs increased (linear, < 0.05) with increasing IOF concentrations of Arg; the greatest values were observed in the 1% Arg group. For 21-d-old broilers, IOF of the 1% Arg solution increased ( < 0.05) the concentrations of ghrelin and glucagon-like peptide 2; the activities of digestive enzymes, alkaline phosphatase, maltase, and sucrase in the jejunum; and the concentrations of serum AA of Val, Met, Ile, Leu, Arg, and Pro compared with those of the noninjected control and diluent-injected group. In ovo feeding of the 1% Arg solution also increased ( < 0.05) the mRNA expressions of jejunal sensing receptors of taste receptor type 1 members 1 and 3; the G protein-coupled receptor, class C, group 6, subtype A; nutrient transporters of solute carrier family 7, members 4, 6, and 7; sodium-glucose transporter 1; and fatty acid-binding protein 1. In conclusion, the 1% Arg solution was the appropriate injection level. In ovo feeding of the 1% Arg solution did not affect the hatchability but facilitated the release of gastrointestinal hormones, increasing the digestive and absorptive capacity and finally improving the growth performance of 21-d-old broilers. Therefore, IOF of the appropriate Arg solution could be an effective technology for regulating early nutrition supply and subsequent growth development in the poultry industry.

[Effect of low-level laser irradiation on proliferation and osteogenic differentiation of human adipose-derived stromal cells].

OBJECTIVE: To examine the in vitro effects of low-level laser irradiation (LLLI) on proliferation and differentiation of human adipose-derived stromal cells (hASCs). METHODS: Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (980 nm; 100 mW-12 W power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated for four consecutive days with laser doses of 2, 4, 6 or 8 J/cm2, the cells without irradiation were used as controls. Half of the cells were changed to osteogenic medium (OM) when they had grown to 70% confluence. The hASCs both with and without osteogenic supplements were divided into three groups, and each group was irradiated at doses of 0, 2 and 4 J/cm2. In order to examine the in vitro effects of LLLI on osteogenic differentiation of hASCs, the alkaline phosphatase activity was assessed on day 7, and alizarin red staining (AR-S) and quantitative detection were assessed on days 14 and 21. The expression of osteoblast master genes (ALP and Runx2) were tested on days 7 and 14. RESULTS: The proliferation medium(PM)+LLLI4 J/cm2 group had the highest multiplication rate. In the groups with osteogenic supplements, LLLI increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of ALP and Runx2. Furthermore, the effect became more obvious at high dose. CONCLUSION: Our data demonstrated that hASCs proliferation and osteogenic differentiation were enhanced by LLLI. With the increase of laser dose, the effect of LLLI would be enhanced at first, and then be decreased after reaching a peak.

Effects of in ovo feeding of creatine pyruvate on the hatchability, growth performance and energy status in embryos and broiler chickens.

The effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on the hatchability, growth performance and energy status of embryos and broilers (Arbor Acres) were investigated. Five treatments were arranged as non-injected treatment (Control), 0.6 ml physiological saline (0.75%) injected treatment (Saline), and IOF treatments injected with 0.6 ml physiological saline (0.75%) containing 3, 6 or 12 mg CrPyr (CrPyr3, CrPyr6 or CrPyr12) into the amnion per fertile egg on day 17.5 of incubation. After hatching, 80 male chicks from each treatment with similar weight close to the average BW of their pooled group were selected and randomly assigned into eight replicates of 10 chicks each. The results showed that the hatchability was not affected among groups, whereas the hatching weight of broilers in CrPyr12 was significantly higher than the control and saline groups (P0.05). Irrespective of dosage, the concentrations of creatine and phosphocreatine, and activities of creatine kinase in embryos were enhanced in CrPyr treatments at 19 E when compared with the control and saline groups (P<0.05). The activities of glucose-6-phosphatase in liver in CrPyr6 and CrPyr12 treatments were higher than the control and saline groups at 19 E (P<0.05). In conclusion, these results indicated that IOF of CrPyr, especially at the level of 12 mg/egg, could improve energy status of embryos and hatchlings, which was useful for enhancing hatching weight, BW and pectoral muscle weight until the end of the experiments at 21 days post-hatch in broilers.

Combination of IL-6, IL-10, and MCP-1 with traditional serum tumor markers in lung cancer diagnosis and prognosis.

Early detection and treatment is critically important for lung cancer patients. Inflammatory mediators such as IL-6, IL-10, and MCP-1 participate in lung cancer regulation. CEA, CA125, and ProGRP are commonly used serum tumor markers for lung cancer. In this study, we assessed the sensitivity and specificity of CEA, CA125, and ProGRP when used in combination with IL-6, IL-10, and MCP in lung cancer diagnosis. Serum from three different groups (healthy controls, individuals with high risk for lung cancer, and lung cancer patients) was collected. Electrochemiluminescence was used to detect expressions of CEA, CA125, and ProGRP; ELISA was used to examine serum levels of IL-6, IL-10, and MCP-1. Specificity and sensitivity of single as well as combination markers in lung cancer diagnosis were determined. Results indicated that CEA, CA125, ProGRP, and MCP-1 were significantly up-regulated in lung cancer patients as compared to those in controls and high risk individuals. Higher IL-6 and IL-10 levels were observed in both lung cancer patients and high-risk individuals as compared to those in controls. Highest sensitivity (95.2%) in cancer diagnosis was achieved when all six markers were used. This was followed by a combination of IL-6, IL-10, CEA, CA125, and ProGRP (92.6%). The most sensitive (88.6%). Four-marker combination was composed of IL-6, CEA, CA125, and ProGRP. As the combined usage of CEA, CA125, ProGRP, IL-6, IL-10, and MCP-1 significantly improved sensitivity of lung cancer detection; this biomarker arrangement may be beneficial for early diagnosis, treatment, and prognosis of lung cancer.

[Quantitative evaluation of fabricating complete denture by computer numerical control in manufacturing dentition and baseplate separately plus adhesive molding].

OBJECTIVE: To quantitatively evaluate the assembly precision of fabricating complete denture by computer numerical control (CNC) in manufacturing dentition and baseplate separately plus adhesive molding. METHODS: The 3D surface data of a standard edentulous maxilla plaster cast model and the temporary base-plate were obtained using an Activity 880 3D scanner. The data (data1) of a complete denture were designed using a set of computer aided design (CAD) software developed by the research group of this study. The pins without undercut were designed as 3D shape of the joining area of the dentition and the baseplate by using the software of Imageware 13.2 and Geomagic Studio 2013. Zero in the top and 0.05 mm in the rest surfaces of the retention pins were set for adhesive clearance. Zenotec T1 (5-axis milling machine) was employed to manufacture polymethyl methacrylate (PMMA) dentition and baseplate. Double sides posterior and one anterior "union teeth" were got. The teeth were inserted into the retention pins in the baseplate and cemented with self-curing resin (Huge Dental Material Co., Ltd). The denture was scanned with the 3D scanner to obtain dataset Data4. Data2 and Data3 registration was set in Data4, Data2 and Data3 were united to gain Data 5. The adhesive clearance on the top of the retentional pins was measured, which was originally designed into 0 mm, and the assembly precision of dentition and baseplate obtained. RESULTS: The average clearance measurements between the dentition and the baseplate: left molar teeth (0.44±0.04) mm, max 0.52 mm, min 0.29 mm; right molar teeth (0.52±0.07) mm, max 0.64 mm, min 0.28 mm; anterior teeth (0.60±0.10) mm, max 0.81 mm, min 0.40 mm; total average clearance (0.52±0.10) mm. CONCLUSION: The adhesive clearance can be controlled to the level of 0.5 mm when the joining part of the artificial teeth and the base was designed into the shape of retentional pins and the artificial dentition divided into 3 parts. We succeeded in using the CAD/ computer aided manufacturing (CAM) technology to fabricate the complete denture. Although the assembly precision of the dentition and the baseplate is not perfect, the results have proved that the technical routes are workable.

[A preliminary study for the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells mixture 3D bio-printing].

OBJECTIVE: To study the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells(hASCs) mixture 3D bio-printing for cells' proliferation and osteogenesis. METHODS: P5 hASCs were used as seed cells, 10 g/L nano hydroxyapatite was added into the cell-sodium alginate-gelatin mixture (concentration: 20 g/L sodium alginate, 80 g/L gelatin; cell density: 1×106/mL), then the mixture was printed by 3D bio-printer as the experimental group. And the cell-sodium alginate-gelatin mixture without nano hydroxyapatite was printed as the control group. Respectively, both the experimental and control groups were detected by microscope, CCK-8, Western blot and PCR at certain time pointsafter being printed, whose cells' proliferation and osteogenic differentiation were analyzed. RESULTS: The microscopic observation and CCK-8 results showed that the cells of the experimental group and the control group both had a good proliferation 24 h and 7 d after being printed. The Western blot results showed that 14 d after printing, the expression of Runt-related transcription factor 2 (RUNX2) had no statistical difference between the experimental group and control group. The PCR results showed that 14 d after printing, the expression of osteogenesis-related genes (RUNX2, osterix, and osteocalcin) was significantly higher in the experimental group than in the control group. CONCLUSION: Nano hydroxyapatite can increase osteogenic differentiation of the hASCs mixture after bio-printing, in which the cells still have a good proliferation.

Impact of Axis of GHRH and GHRH Receptor on Cell Viability and Apoptosis of the Placental Choriocarcinoma Cell Line.

Although GHRH and GHRH-R are recognized as key factors in placental development, little is known about the mechanism(s) of the regulation in trophoblastic cells during placental development. The objective of this study is to determine the potential relationship between the expression levels of GHRH-R and the placental and JEG-3 cell function. Furthermore, we aim to investigate the downstream pathways of GHRH/GHRH-R axis in the control of the JEG-3 cell viability and apoptosis. In this study, we detected the expression pattern of GHRH-R in human chorionic villous tissues and JEG-3 cell. Then, we evaluated the effects of GHRH/GHRH-R and the downstream pathways by using GHRH antagonist (JMR-132) on JEG-3 cell. Our present study found the expressions of GHRH-R in placental villous tissues and JEG-3 cell, and the expression levels of GHRH-R was significantly lower in villous tissues of early pregnancy loss when compared to normal controls. JMR-132 inhibited cellular viability and induced apoptosis in JEG-3 cell in a time and dosedependent manners through activation of caspase-3, p38, and p53, as well as inhibition of phosphorylation of Akt. Interestingly, ER stress markers such as GRP78, ubiquitinated proteins and phospho-eIF2α were significantly increased in JEG-3 cell after being treated with JMR-132. Conversely, pretreated with salubrinal (a selective inhibition of protein phosphatase 1-mediated eIF2α dephosphorylation), JEG-3 cells were rescued from JMR-132-mediated cell growth inhibition, and abolished JMR-132-induced cleaved caspase-3, CHOP, phospho-p53, and ubiquitinated proteins accumulation. Knockdown of endogenous GHRH-R significantly abolished the JMR-132-induced cleaved caspase-3 and activation of p38. In conclusion, our results, for the first time, demonstrated the expression levels of GHRH-R were closely related to the placental function. Inhibition of GHRH-R by using GHRH antagonist in JEG-3 cell may reduce cell viability and induce apoptosis through inactivation of Akt and ER stress via phosphorylation of eIF2α. These observations have enriched our understanding on the function of GHRH/GHRH-R axis and the downstream pathways in the control of the placental development. The Most Important Aspect of the Paper: Our present study for the first time provided evidences that GHRH and GHRH-R loops involve in JEG-3 cell viability and apoptosis through Akt and eIF2α pathways.