[Preparation technology comparison and performance evaluation of different protein A affinity chromatographic materials].

Protein A affinity chromatographic materials are widely used in clinical medicine and biomedicine because of their specific interactions with immunoglobulin G (IgG). Both the characteristics of the matrix, such as its structure and morphology, and the surface modification method contribute to the affinity properties of the packing materials. The specific, orderly, and oriented immobilization of protein A can reduce its steric hindrance with the matrix and preserve its bioactive sites. In this study, four types of affinity chromatographic materials were obtained using agarose and polyglycidyl methacrylate (PGMA) spheres as substrates, and multifunctional epoxy and maleimide groups were used to fix protein A. The effects of the ethylenediamine concentration, reaction pH, buffer concentration, and other conditions on the coupling efficiency of protein A and adsorption performance of IgG were evaluated. Multifunctional epoxy materials were prepared by converting part of the epoxy groups of the agarose and PGMA matrices into amino groups using 0.2 and 1.6 mol/L ethylenediamine, respectively. Protein A was coupled to the multifunctional epoxy materials using 5 mmol/L borate buffer (pH 8) as the reaction solution. When protein A was immobilized on the substrates by maleimide groups, the agarose and PGMA substrates were activated with 25% (v/v) ethylenediamine for 16 h to convert all epoxy groups into amino groups. The maleimide materials were then converted into amino-modified materials by adding 3 mg/mL 3-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in dimethyl sulfoxide (DMSO) and then suspended in 5 mmol/L borate buffer (pH 8). The maleimide groups reacted specifically with the C-terminal of the sulfhydryl group of recombinant protein A to achieve highly selective fixation on both the agarose and PGMA substrates. The adsorption performance of the affinity materials for IgG was improved by optimizing the bonding conditions of protein A, such as the matrix type, matrix particle size, and protein A content, and the adsorption properties of each affinity material for IgG were determined. The column pressure of the protein A affinity materials prepared using agarose or PGMA as the matrix via the maleimide method was subsequently evaluated at different flow rates. The affinity materials prepared with PGMA as the matrix exhibited superior mechanical strength compared with the materials prepared with agarose. Moreover, an excellent linear relationship between the flow rate and column pressure of 80 mL/min was observed for this affinity material. Subsequently, the effect of the particle size of the PGMA matrix on the binding capacity of IgG was investigated. Under the same protein A content, the dynamic binding capacity of the affinity materials on the PGMA matrix was higher when the particle size was 44-88 µm than when other particle sizes were used. The properties of the affinity materials prepared using the multifunctional epoxy and maleimide-modified materials were compared by synthesizing affinity materials with different protein A coupling amounts of 1, 2, 4, 6, 8, and 10 mg/mL. The dynamic and static binding capacities of each material for bovine IgG were then determined. The prepared affinity material was packed into a chromatographic column to purify IgG from bovine colostrum. Although all materials showed specific adsorption selectivity for IgG, the affinity material prepared by immobilizing protein A on the PGMA matrix with maleimide showed significantly better performance and achieved a higher dynamic binding capacity at a lower protein grafting amount. When the protein grafting amount was 15.71 mg/mL, the dynamic binding capacity of bovine IgG was 32.23 mg/mL, and the dynamic binding capacity of human IgG reached 54.41 mg/mL. After 160 cycles of alkali treatment, the dynamic binding capacity of the material reached 94.6% of the initial value, indicating its good stability. The developed method is appropriate for the production of protein A affinity chromatographic materials and shows great potential in the fields of protein immobilization and immunoadsorption material synthesis.

Antibodies against thyroid-stimulating hormone receptor cause maternal-neonatal transmission of Graves' Disease.

The present study aimed to investigate whether the thyroid-stimulating hormone receptor (TSHR) autoantibodies (Ab) from mothers with Graves' disease (GD) could cause neonatal thyroid disease and the underlying mechanisms of this. An adenovirus expressing the TSHR A-subunit and a control adenovirus expressing ß-galactosidase was constructed by Beijing Sino Geno Max Co., Ltd. The sequences were subsequently verified and amplified via PCR. A GD model was established in female BALB/c mice (n=90) by three intramuscular injections of a TSHR-expressing adenovirus (Ad-TSHR). Mice injected with Ad-ß-galactosidase served as a sham immunization group. The immunized females were paired with unimmunized males to generate offspring. The serum levels of TSHR-Ab and thyroxine (T4) of mothers and neonates were measured after delivery. Breast milk was collected from the stomachs of neonatal mice to determine the TSHR-Ab levels. The positive rate of serum TSHR-Ab (>0.3 IU/l) in the TSHR group was 99% (89/90) and 0% in the sham group. The mother mice in the TSHR group had elevated serum T4 levels and the thyroid pathological features of Graves' hyperthyroidism.GD mice gave birth to smaller newborns with thyroid pathological changes and higher serum levels of TSHR-Ab and T4, compared to the offspring in the sham group. The TSHR-Ab levels in breast milk from the GD mice declined with time. Mice immunized with Ad-TSHR exhibited the clinicopathological features of human GD and give birth to neonates with thyroid disease at birth.

AP39 ameliorates high fat diet-induced liver injury in young rats via alleviation of oxidative stress and mitochondrial impairment.

Non-alcoholic fatty liver disease (NAFLD) is a complication of childhood obesity and an oxidative stress-related multisystem disease. A mitochondria-targeting hydrogen sulfide (H2S) donor AP39 has antioxidant property, while the mechanism underlying the function of AP39 on pediatric NAFLD remains undefined. Here, 3-week-old SD rats were received a high-fat diet (HFD) feeding and injected with AP39 (0.05 or 0.1 mg/kg/day) via the tail vein for up to 7 weeks. AP39 reduced weight gain of HFD rats and improved HFD-caused liver injury, as evidenced by reduced liver index, improved liver pathological damage, decreased NAFLD activity score, as well as low alanine transaminase (ALT) and aspartate transaminase (AST) activities. AP39 also reduced serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) concentrations but increased high-density lipoprotein-cholesterol (HDL-C). Moreover, AP39 prevented reactive oxygen species (ROS) generation, reduced MDA content and increased glutathione (GSH) level and superoxide dismutase (SOD) activity. Furthermore, AP39 increased H2S level, protected mitochondrial DNA (mtDNA), reduced mitochondrial swelling, and restored mitochondrial membrane potential (MMP) alteration. Notably, AP39 diminished HIF-1α mRNA and protein level, possibly indicating the alleviation in mitochondrial damage. In short, AP39 protects against HFD-induced liver injury in young rats probably through attenuating lipid accumulation, oxidative stress and mitochondrial dysfunction.

The epidemic characteristics of short stature in school students.

BACKGROUND: There was no large-scale population survey on the prevalence of short stature in Anhui province yet. To further acquainting the epidemiological character of teenage children short stature in Anhui province, and to provide basis for exploring reasonable intervention measure about children's height, we took this survey on short stature in primary and middle school students of Anhui province. METHODS: Twelve thousand nine primary and secondary school students in urban and rural aged from 7 to 18 years were recruited. The comparison of short stature in different genders, regions and age groups was done according to genetic metabolic endocrine group of pediatrics branch in Chinese Medical Association (CMA). RESULTS: The average detection rate of short stature in primary and middle school students was 3.16% in Anhui province. The detection rate was higher in rural area than in urban area, higher in economic backward area than in economic developed areas. CONCLUSION: There was no significant difference in detection rate in gender and age.

Isolation and characterization of exopolysaccharide with immunomodulatory activity from fermentation broth of Morchella conica.

UNLABELLED: BACKGROUND AND THE PURPOSE OF THIS STUDY: Mushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. In vitro and in vivo studies suggest that certain polysaccharides affect immune system function. Morchella conica (M. conica) is a species of rare edible mushroom whose multiple medicinal functions have been proven. Thus, the objective of this study is to isolate and characterize of exopolysaccharide from submerged mycelial culture of M. conica, and to evaluate its immunomodulatory activity. METHODS: A water-soluble Morchella conica Polysaccharides (MCP) were extracted and isolated from the fermentation broth of M. conica through a combination of DEAE-cellulose and Sephacryl S-300 HR chromatograph. NMR and IR spectroscopy has played a developing role in identification of polysaccharide with different structure and composition from fungal and plant sources, as well as complex glycosaminoglycans of animal origin. Thus, NMR and IR spectroscopy were used to analyze the chemical structure and composition of the isolated polysaccharide. Moreover, the polysaccharide was tested for its immunomodulatory activity at different concentrations using in vitro model. RESULTS: The results showed that MCP may significantly modulate nitric oxide production in macrophages, and promote splenocytes proliferation. Analysis from HPLC, infrared spectra and nuclear magnetic resonance spectroscopy showed that MCP was a homogeneous mannan with an average molecular weight of approximately 81.2 kDa. The glycosidic bond links is →6)-α-D-Man p-(1→. CONCLUSION: The results suggested that the extracted MCP may modulate nitric oxide production in macrophages and promote splenocytes proliferation, and it may act as a potent immunomodulatory agent.

Effects of growth environments and two environmental endocrine disruptors on children with idiopathic precocious puberty.

OBJECTIVE: The incidence of idiopathic precocious puberty (IPP) might have an increasing trend. But the causes and risk factors of IPP are unknown. The objective of our study is to evaluate the effects of growth environments and two environmental endocrine disruptors (EDCs), zearalenone (ZEA), and 1,1-dichloro-2,2,bisethylene (p,p'-DDE), on patients with IPP. DESIGN: Case-control study. METHODS: The study consisted of 78 IPP patients at diagnosis and 100 control children matched for age and sex. A questionnaire was designed to collect data on growth environments, and serum ZEA and p,p'-DDE were tested in all subjects. We analyzed data on growth environments, two EDCs, and biological interaction between growth environments and EDCs. RESULTS: In growth environments, small for gestational age, maternal physical disease during pregnancy, early maternal menarche, early puberty of same-degree relatives, and father's absence in 4- to 6-year olds were risk factors for children with IPP (P<0.05). Serum ZEA concentration, ZEA, and p,p'-DDE-positive rates in the IPP group were significantly higher than those in the control group (P<0.05). There was a biological interaction between growth environments and ZEA (relative excess risk due to interaction =34.562, attributable proportion due to interaction =0.745, synergy index =4.193). CONCLUSIONS: Results suggest possible effects of growth environments and two EDCs on the development of IPP. In addition, growth environments and ZEA have biological interaction that might increase the risk of developing IPP.

[Comparison of proximal femoral nail and dynamic hip screw for treating intertrochanteric fractures].

OBJECTIVE: To discuss characters of proximal femoral nail and dynamic hip screw for treating type A1, A2, A3 of intertrochanteric fractures. METHODS: We review 104 patients with intertrochanteic fractures, 33 patients were treated with proximal femoral nail (PFN), including 13 males and 20 females with an average age of 76 years (ranging from 63 to 87 years). 12 cases of type A1; 18 cases of type A2 and 3 cases of type A3; and 71 patients were treated with dynamic hip screw (DHS), including 29 males and 42 females with an average age of 74.5 years (ranging from 61 to 92 years), 32 cases of type A1, 34 cases of type A2 and 5 cases of type A3. Comparision in an average time of operations, the length of incision, blood loss, weight loading time and complications between two groups. RESULTS: An average time of operation was (51.5 +/- 4.4) min in PFN; (68.8 +/- 5.9) min in DHS. The length of incision was (9.6 +/- 0.9) cm in PFN; (15.5 +/- 1.5) cm in DHS. The blood loss was (179.0 +/- 12.9) ml in PFN; (269.3 +/- 40.0) ml in DHS. Varus collapse was none in PFN, 1 case in DHS. The collodiaphyseal angle of 7 cases decreased in DHS. Lateral hip pain caused by proximal screw removal was 6 cases in PEN. CONCLUSION: The therapeutic effect of DHS and PEN was primitively same in treating type A1 of intertrochanteric fracture. Operative injuries of PFN were less than that of DHS and anti-tonia was more stronger which is more suitable for type A2 and A3 of intertrochateric fractures.