Impact of nitrite exposure on oxidative stress and antioxidative-related genes responses in the gills of Procambarus clarkii.

Nitrite is the major environmental pollutant in the freshwater aquaculture environment, which has a negative impact on aquatic species growth. Currently, we know that the main way nitrite enters crustaceans is through their gills. In this study, a total of 96 h acute nitrite stress (60 mg/L) experiments were conducted, and the impact of the serum biochemical parameters, gill oxidase activity and oxidative-related gene expression of red swamp crayfish were evaluated. After exposure to nitrite for 0, 6, 12, 24, 48, and 96 h, hemolymph and gills samples were taken at each time point. In the serum, acute nitrite stress significantly increased glutamic-oxaloacetic transaminase (GOT) and alanine aminotransferase (ALT) activities after 6 h of exposure, decreased total protein (TP) and albumin (ALB) levels after 24 h and 48 h of exposure, respectively. In the gills, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were enhanced to the maximum level at 12 h, 24 h and 24 h, respectively. The contents of malondialdehyde (MDA) and lipid peroxide (LPO) were increased significantly after 12 h and 24 h exposure, respectively. In addition, the expression levels of antioxidative-related genes, including hsp70, fer and mt, were significantly upregulated in the gills after 6 h of exposure. The results indicated that acute nitrite stress changed the serum physiological status, induced oxidative stress and caused damage to gill cells in P. clarkii.

Dietary nucleotides can directly stimulate the immunity of zebrafish independent of the intestinal microbiota.

In this study, we firstly tested the effects of dietary nucleotides on the disease resistance and innate immunity of zebrafish. Further, we investigated the role of intestinal microbiota in the nucleotides-induced immunostimulatory effect by using a germ-free zebrafish model and microbiota transfer technique. Fish were fed control or nucleotides (NT)-supplemented diets (at 0.05%,0.1%, 0.15% or 0.2%, m/m) for 4 weeks, followed by immersion challenge with Aeromonas hydrophila NJ-1. The results showed that 0.1% NT group enhanced the resistance of zebrafish against A. hydrophila infection. We further observed that the relative expressions of mucin, claudin16, occlusin1, hepcidin, defensin beta-like, myeloperoxidase (Mpo), and serum amyloid A (Saa) increased in the 0.1% NT group compared with control (P < 0.05), indicating that dietary nucleotides enhanced the physical barrier and mucosal immunity in the intestine of zebrafish. Moreover, ROS level in the head kidney was significantly increased in NT fed zebrafish versus control (P < 0.05), indicating enhanced systematic immunity. Furthermore, dietary NT significantly elevated the relative expressions of mpo, saa and the ROS activity in germ-free zebrafish, while germ-free zebrafish colonized with NT-altered microbiota had no significant difference in the relative expressions of mpo, saa and the ROS activity compared with the control microbiota-colonized fish, suggesting that the immunostimulatory effect of dietary NT is mediated by direct action of NT and does not involve the microbiota. Consistently, dietary NT can protect germ-free zebrafish from pathogenic infection, whereas germ-free zebrafish colonized with NT microbiota showed no difference in disease resistance compared with control microbiota colonized counterparts. Together, these results indicated that the immunostimulatory and disease protection effect of dietary nucleotides in zebrafish was mediated by direct action of the nucleotides, and does not involve the intestinal microbiota.

Genetic correction of adipose tissue-derived mesenchymal stem cells mediated by TALEN targeting the GDF5 gene.

Stem cells and other associated cell types may be a potential alternative to treat various genetic disorders that currently do not benefit from traditional approaches. Functional recovery of cells could be induced via directional differentiation or genetic manipulation. In this study, mesenchymal stem cells (MSCs) were obtained from a patient with osteoarthritis (OA) carrying a functional single­nucleotide polymorphism (SNP rs143383, C/T transition) within the 5'­UTR of growth and differentiation factor 5 (GDF5) gene. The SNP causes GDF5 expression to be reduced and thus increases OA susceptibility. Aiming to correct the dysfunctional gene, a pair of transcription activator­like effector nucleases (TALENs) were designed to cleave the DNA around the mutated locus, coupled with a short single stranded DNA complementary to the cleavage site. Following in vitro cell colony formation and selection, two genetically corrected MSC colonies were identified out of a total of 142. These MSCs were induced and differentiated into chondrocytes. As a result, genetically corrected chondrocytes exhibited normal morphology and lower levels of apoptosis compared with cells carrying the SNP. In cultured cells, the secretion of matrix metalloproteinases was suppressed and TIMP metallopeptidase inhibitor 1 was increased by correction of the mutation. Furthermore, the expression of GDF5 target genes, cell vitality­associated genes and extracellular matrix degrading genes were returned to normal levels in corrected cells compared with mutation­carrying cells, indicating the functional recovery of these corrected chondrocytes. The present study demonstrated that TALEN­mediated genetic correction can be used to edit genes in adipose­derived MSCs from patients with OA and may have clinical potential.

Hsa_circ_0045714 regulates chondrocyte proliferation, apoptosis and extracellular matrix synthesis by promoting the expression of miR-193b target gene IGF1R.

In recent years, some studies have been made on the effects of circular RNA (circRNA) in osteoarthritis (OA) and so on; however, its mechanisms remain to be further explored. Studies have shown that tumor necrosis factor-alpha can inhibit hsa_circ_0045714 expression in chondrocytes so as to upregulate miR-193b expression. Dual-luciferase reporter assay showed that insulin-like growth factor 1 receptor (IGF1R) is a key target gene of miR-193b. Hsa_circ_0045714 over-expression does not influence miR-193b expression, but can inhibit its transcriptional activity, thereby upregulating IGF1R expression. Hsa_circ_0045714 can promote the expression of type II collagen and aggrecan, and upregulate chondrocyte proliferation, while its linear sequences cannot. IGF1R has similar function, while miR-193b can inhibit the expression of type II collagen and aggrecan, and downregulate chondrocyte proliferation but enhance their apoptosis. IGF1R overexpression can reverse the effect of miR-193b, while miR-193b mimics or IGF1R siRNA can inhibit the function of hsa_circ_0045714. Therefore, hsa_circ_0045714 can regulate extracellular matrix synthesis as well as proliferation and apoptosis of chondrocytes by promoting the expression of miR-193b target gene IGF1R. The findings will provide new proofs for studies on the applications of circRNA in OA and other orthopedic diseases.

Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis.

Low dose antibiotics have been used as growth promoters in livestock and fish. The use of antibiotics has been associated with reduced pathogen infections in livestock. In contrast, antibiotic growth promoter has been suspected of leading to disease outbreaks in aquaculture. However, this phenomenon is circumstantial and has not been confirmed in experimental conditions. In this study, we showed that antibiotic olaquindox increased the susceptibility of zebrafish to A. hydrophila infection. Olaquindox led to profound alterations in the intestinal microbiota of zebrafish, with a drastic bloom of Enterobacter and diminishing of Cetobacterium. Moreover, the innate immune responses of zebrafish were compromised by olaquindox (P<0.05). Transfer of microbiota to GF zebrafish indicated that while the immuo-suppression effect of olaquindox is a combined effect mediated by both OLA-altered microbiota and direct action of the antibiotic (P<0.05), the increased pathogen susceptibility was driven by the OLA-altered microbiota and was not dependent on direct antibiotic effect. Taken together, these data indicate that low level of OLA induced gut microbiota dysbiosis in zebrafish, which led to increased pathogen susceptibility.

The Growth-Promoting Effect of Dietary Nucleotides in Fish Is Associated with an Intestinal Microbiota-Mediated Reduction in Energy Expenditure.

Background: Nucleotides have been used as functional nutrients to improve the growth and health of animals, including fish. The mechanism involved in the growth-promotion effect of nucleotides is still unclear.Objective: We investigated the bioenergetic mechanism underlying the growth-promotion effect of nucleotides in zebrafish and the associated roles played by the intestinal microbiota.Methods: Larval zebrafish were fed a control or a 0.1% mixed nucleotides-supplemented diet for 2 wk. Standard metabolic rate, the minimal rate of energy expenditure by animals at rest, was evaluated by oxygen consumption with the use of a respirometer. The expressions of fasting-induced adipose factor (Fiaf), inflammatory cytokines, and genes involved in fatty acid (FA) oxidation were tested by quantitative reverse transcriptase-polymerase chain reaction. The intestinal microbiota from the nucleotide-fed fish (NT fish) or control fish was transferred to 3-d postfertilization germ-free zebrafish in which oxygen consumption and expression of cytokines and fiaf were evaluated.Results: Compared with controls, nucleotide supplementation at 0.1% increased the weight and energy gains of zebrafish by 10% and 25%, respectively (P < 0.01). Standard metabolic rate was 28% lower in NT fish than in controls (P < 0.001). Nucleotide supplementation downregulated the inflammatory tone in the head kidney of the fish. Moreover, NT fish had a 51% lower intestinal expression of fiaf than did controls (P < 0.05), which was consistent with decreased expression of key genes involved in FA oxidation [carnitine:palmitoyl transferase 1a (cpt1a) and medium-chain acyl coenzyme A dehydrogenase (mcad)] in liver and muscle. Germ-free zebrafish colonized with microbiota from NT fish had a 25% lower standard metabolic rate than did those colonized by control microbiota (P < 0.01), whereas direct nucleotide feeding of germ-free zebrafish did not affect standard metabolic rate relative to germ-free controls that were not fed nucleotides. Furthermore, germ-free zebrafish colonized with nucleotide microbiota exhibited downregulated inflammatory tone and 33% lower fiaf expression compared with their control microbiota-colonized counterparts.Conclusions: The growth-promoting effect of dietary nucleotides in zebrafish involves 2 intestinal microbiota-mediated mechanisms that result in reduced standard metabolic rate: 1) lower inflammatory tone and 2) reduced FA oxidation associated with increased microbial suppression of intestinal fiaf.

Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway.

The quenching enzyme AIO6 (AiiO-AIO6) has been reported as a feed additive preparation for application in aquaculture and biological control of pathogenic Aeromonas hydrophila. We developed an economical strategy to express AIO6BS (AiiO-AIO6BS, codon optimized AIO6 in Bacillus subtilis) in Bacillus subtilis for facilitating its widespread application. Promoter p43 without the signal peptide was used for secretory expression of AIO6BS in B. subtilis. Western blotting analysis demonstrated that AIO6BS was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tatAC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. The expression analysis of the various N- or C-terminal truncated gene products indicated that AIO6BS probably acts as an export signal to direct its self-secretion across the cell membrane.

[Application of 3D digital orthopedic techniques in treatment of acetabular fracture].

OBJECTIVE: To evaluate the therapeutic effect of three-dimensional digital orthopedic techniques in treatment of acetabular fractures. METHODS: We retrospectively analyzed 50 cases of acetabular fracture treated between March, 2007 and December, 2013. The lamellar CT scanning data were imported into Mimics software, and 3D anatomical models of the pelvic and proximal femur were reconstructed. Computer-assisted analysis was carried out to understand the condition of fractures and simulate fracture reduction. The pelvic models were manufactured by rapid prototyping technique for definite diagnosis and typing of acetabular fractures and subsequent surgical treatment. RESULTS: Three-dimensional reconstruction images and rapid prototyping pelvic models faithfully represented the findings in operations. Preoperative simulation of the operation shortened the time of operation and reduced the volume of bleeding in the operation. All the patients were followed up for 6 to 24 months. According to Matta imaging score, anatomical reduction was achieved in 41 cases and satisfactory reduction in 9 cases. According to the Harris functional criteria, 32 patients had excellent, 12 had good and 6 had acceptable outcomes with a rate of excellent and good outcomes of 88%. CONCLUSION: Three-dimensional digital orthopedic techniques allow accurate display of the acetabulum and the spatial relation of the anatomic structures to assist in fracture diagnosis, typing and treatment.

[DESIGN AND CLINICAL APPLICATION OF LESSER TROCHANTERIC REDUCTION FIXATION SYSTEM].

OBJECTIVE: To design and produce a lesser trochanteric reduction fixation system and verify its value and effectiveness. METHODS: A lesser trochanteric reduction fixation system was designed and produced according to the anatomical features of the lesser trochanteric fractures. Sixty-six patients with intertrochanteric fractures of Evans type III were included between January 2010 and July 2012. Of 66 patients, 32 were treated with dynamic hip screw (DHS) assisted with the lesser trochanteric reduction fixation system (study group), and 34 cases were treated with DHS only (control group). The 2 groups were comparable with no significant difference in gender, age, the reasons, and the types of the fractures (P > 0.05). The operation time, intraoperative blood loss, neck-shaft angle, bone healing time, ratio of successful fixations, and the functional evaluation of the hip joint after operation were compared between 2 groups. RESULTS: The study group had shorter operation time [(58.4 ± 5.3) minutes] and less intraoperative blood loss [(186.3 ± 6.6) mL than the control group [(78.5 ± 6.2)minutes and (246.2 ± 8.7) mL], showing significant differences (t = -14.040, P = 0.000; t = -31.145, P = 0.000). There was no significant difference in neck-shaft angle between study group [(138.6 ± 3.0)] and control group [(139.4 ± 2.9) degrees] (t = -1.044, P = 0.301). The wounds healed by first intention in both groups. The 30 and 31 patients were followed up 12 to 24 months (mean, 15 months) in the study group, and 13 to 25 months (mean, 16 months) in the control group, respectively. All fractures healed well in 2 groups. The study group had significantly shorter healing time [(8.8 ± 2.0) weeks] than the control group [(10.7 ± 3.4) weeks] (t = -2.871, P = 0.006). At 12 months after operation, coxa vara happened in 2 cases of the study group with a successful fixation ratio of 93.3% and in 10 cases of the control group with a successful fixation ratio of 67.7%, showing significant difference (Χ2 = 6.319, P = 0.022). According to Harris hip score, the excellent and good rate was 83.3% in the study group (25/30) and was 58.1% in the control group (18/31), showing significant difference (Χ2 = 4.680, P = 0.049). CONCLUSION: The application of the lesser trochanteric reduction fixation system can reduce stripping of the soft tissue around the fracture fragments, shorten the operation time and the healing time, and preserve the function of the hip joint maximumly.

Effects of lipid-lowering pharmaceutical clofibrate on lipid and lipoprotein metabolism of grass carp (Ctenopharyngodon idellal Val.) fed with the high non-protein energy diets.

This study investigated the effects of clofibrate treatment on blood lipids, hepatic enzyme activities and relative expression of genes involved in lipid metabolism of grass carp fed with high non-protein energy diets. For that purpose, five diets were formulated: a commercial-like diet (Control), a high-carbohydrate diet (HC), a high-fat diet (HF) and two diets identical to the HC and HF diets, but supplemented with 1.25 g kg(-1) clofibrate (HC + Clo and HF + Clo diets). Grass carp fed the HC and HF diet exhibited increases in blood lipids and body fat compared with the control group after 4 weeks. In the clofibrate treatment groups, there was a marked decrease in triacylglycerol and cholesterol concentrations of plasma, and total lipids of the whole body, mesentery adipose tissue and liver tissue. Fish treated with clofibrate exhibited increased hepatic acyl-CoA oxidase activity, but did not show any changes in carnitine palmitoyltransferase (CPT) I activity compared with HC and HF diets without clofibrate. Clofibrate treatment had no effect on peroxisome proliferator-activated receptor alpha and CPT I mRNA expression. However, there was an increase in lipoprotein lipase expression in the clofibrate-treated groups. In addition, the relative mRNA expression levels of hepatic de novo lipogenic enzymes (fatty acid synthetase and acetyl coenzyme-A carboxylase) were significantly higher in the fish fed the HC diet than those of other groups, and clofibrate inhibited this increase. These results suggest that clofibrate has the hypolipidaemic effects and affects lipid metabolism in grass carp.

Programming effects of high-carbohydrate feeding of larvae on adult glucose metabolism in zebrafish, Danio rerio.

The aim of the present study was to determine the potential long-term metabolic effects of early nutritional programming on carbohydrate utilisation in adult zebrafish (Danio rerio). High-carbohydrate diets were fed to fish during four ontogenetic stages: from the first-feeding stage to the end of the yolk-sac larval stage; from the first-feeding stage to 2 d after yolk-sac exhaustion; after yolk-sac exhaustion for 3 or 5 d. The carbohydrate stimuli significantly increased the body weight of the first-feeding groups in the short term. The expression of genes was differentially regulated by the early dietary intervention. The high-carbohydrate diets resulted in decreased plasma glucose levels in the adult fish. The mRNA levels and enzyme activities of glucokinase, pyruvate kinase, α-amylase and sodium-dependent glucose co-transporter 1 were up-regulated in the first-feeding groups. There was no significant change in the mRNA levels of glucose-6-phosphatase (G6Pase) in any experimental group, and the activity of G6Pase enzyme in the FF-5 (first feeding to 2 d after yolk-sac exhaustion) group was significantly different from that of the other groups. The expression of phosphoenolpyruvate carboxykinase gene in all the groups was significantly decreased. In the examined early programming range, growth performance was not affected. Taken together, data reported herein indicate that the period ranging from the polyculture to the external feeding stage is an important window for potential modification of the long-term physiological functions. In conclusion, the present study demonstrates that it is possible to permanently modify carbohydrate digestion, transport and metabolism of adult zebrafish through early nutritional programming.

[Double volume internal fixation for correction of adult post-traumatic cubitus varus deformity].

OBJECTIVE: Supracondylar closing wedge osteotomy is a standard operation for the management of post-traumatic cubitus varus deformity. There are many fixation methods for the broken ends of bone. However, most of these fixation methods are fraught with various complications. To evaluate the methods and functional results of double volume internal fixation for correction of adult post-traumatic cubitus varus deformity. METHODS: The clinical data were retrospectively reviewed, from 22 cases of adults post-traumatic cubitus varus deformity between June 2007 and December 2010. There were 16 males and 6 females, aged 18-29 years (mean, 21 years) and they all had a history of supracondylar fracture. The deformities of cubitus varus appeared at 6 months (range, 3 months to 1 year) after fracture, and the operations were carried out at 4-17 years (mean, 8 years) after deformity occurrence. The valgus angle were 16-25 degrees (mean, 20.6 degrees) and the Flynn functional scores were all poor before operation. Supracondylar closing wedge osteotomies were performed. Two reconstruction plates were moulded and placed to the media and lateral volumes of the humerus to fix the broken ends of the osteotomy surfaces. External fixation was not needed and early rehabilitation was performed postoperatively in all cases. RESULTS: Incisions healed by first intention. All cases were followed up 6 to 24 months (mean, 13 months). At last follow-up, the valgus angle was 0-10 degrees (mean, 7.5 degrees). All cases got bone union at 8-13 weeks (mean, 10 weeks) after operation. No related complications occurred, such as infection, nervous or vein injury, and loosening or breakage of internal fixator; and no cubitus varus recurred. The Flynn scores were excellent in 17 cases, good in 3 cases, and fair in 2 cases; the excellent and good rate was 91%. CONCLUSION: The operation of supracondylar osteotomy with double plates internal fixation for the correction of adult post-traumatic cubitus varus deformity can rigidly stabilize distal humerus, which is helpful to functional training just after operation and satisfactory restoration of the elbow function.