To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

To reveals the effects of tree species on polysaccharides content of epiphytic Dendrobium officinale. The polysaccharides content of D. officinale attached to living tress in wild or stumps in bionic-facility was determined by phenol-sulfuric acid method. There were extremely significant differences of polysaccharides content of D. officinale attached to different tree species, but the differences had no relationship with the form and nutrition of barks. The polysaccharides content of D. officinale mainly affected by the light intensity of environment, so reasonable illumination favored the accumulation of polysaccharides. Various polysaccharides content of D. officinal from different attached trees is due to the difference of light regulation, but not the form and nutrition of barks.
Dendrobium ; chemistry ; Light ; Plant Bark ; physiology ; Polysaccharides ; analysis ; Trees

AIMTo prepare amylopectin anchored dipyridamole (DIP) liposome and to study its tissue distribution in mice.

METHODSThe regular DIP liposomes were prepared by film-scatter method. The amphiphilic O-palmitoyl amylopectin was synthesized and added to modify the surface of liposome. The entrapping efficiency, zeta potential, mean diameter, span of modified and regular liposomes were assayed. The RP-HPLC was used for the determination of DIP concentration in mice tissue.

RESULTSAfter modification, the entrapping efficiency depressed, zeta potential was raised, mean diameter and span had no obvious change. The level of DIP in lung, liver and spleen for regular liposomes were higher than that of injections. Compared with regular liposomes, the modified liposomes increased the DIP level in lung, and decreased the DIP level in liver, spleen, moreover, lengthened the retention time of DIP in lung.

CONCLUSIONThe distribution of modified liposome in mice was markedly changed as compared with regular liposomes and injections. The modified liposomes had obvious lung targeting property.

Amylopectin ; analogs & derivatives ; chemistry ; Animals ; Area Under Curve ; Dipyridamole ; administration & dosage ; chemistry ; pharmacokinetics ; Drug Delivery Systems ; Liposomes ; Lung ; metabolism ; Male ; Mice ; Palmitates ; chemistry ; Particle Size ; Tissue Distribution

Animals ; Calcium Channel Blockers ; therapeutic use ; Dogs ; Hypothermia ; complications ; Male ; Nimodipine ; therapeutic use ; Ventricular Fibrillation ; etiology ; prevention & control

Objective·To investigate the correlation of single nucleotide polymorphism (SNP) of complement factor H (CFH) gene with schizophrenia in Chinese Han population.Methods·The genotype,allele,and haplotype frequencies of 5 SNP loci (rs800292,rs 1061170,rs 10801555,rs 10922096 and rs2019727) in CFH gene were compared between 418 patients with schizophrenia (case group) and 655 normal people (control group) by SNaPshot technique.Results·All SNP loci were well genotyped in the subjects.Correlation analysis showed that rs1061170 locus allele frequency distribution difference between case group and control group was statistically significant (corrected P=0.045),while genotype and allele frequencies of other SNP loci were not significantly different (all corrected P＞0.05).The frequency of haplotype C-A-T-A-A (rs800292-rs1061170-rs10801555-rs10922096-rs2019727) in case group was different from that in control group (corrected P=0.013).Conclusion·The allele polymorphisms of rsl061170 and the haplotype C-A-T-A-A of rs800292-rs 1061170-rs 10801555-rs 10922096-rs2019727 may be associated with schizophrenia in Chinese Han population.

Objective To screen and identify B cell epitopes in SAG1, SAG2, SAG3, GRA1, GRA6 and P35 antigens of Toxoplasma gondii. Methods The indexes such as hydrophilicity, accessibility, flexibility, secondary structure and polarity of the 6 antigen moleculars above mentioned were analyzed by BioSun system. Two B cell epitopes with high antigenicity from each antigen molecular were selected, and the total twelve pairs of oligonucleotide chains were designed according to the 12 B cell epitopes’ sequence and synthesized, then cloned into plasmid pET-32c. The 12 fragment B cell epitopes were expressed and the expressed fusion proteins were identified with Western blot. Results Twelve B cell epitopes from 6 Toxoplasma antigens (two from each antigen) were predicted and selected. The epitope genes were successfully cloned into pET-32c and expressed. Western blot results showed that 3 of 12 expressed fusion proteins could be recognized by the immunized rabbit sera with soluble antigen of Toxoplasma gondii, but not by the unimmunized rabbit sera Conclusion Three B cell epitopes of Toxoplasma[with potential diagnostic value are obtained.

Coronary artery disease (CAD) is a multifactorial disease in which inflammation plays a central role.This study aimed to investigate the association of inflammatory markers such as the neutrophil to lymphocyte ratio (NLR),the Global Registry of Acute Coronary Events (GRACE) score with in-hospital mortality of elderly patients with acute myocardial infarction (AMI) in an attempt to explore the prognostic value of these indices for elderly AMI patients.One thousand consecutive CAD patients were divided into two groups based on age 60.The laboratory and clinical characteristics were assessed retrospectively by reviewing the medical records.The NLR and GRACE score were calculated.In the elderly (≥60 years),patients with non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI) had significantly higher NLR than did those with unstable angina (UA) and stable angina pectoris (SAP) (P＜0.01).The NLR was considerably elevated in older AMI patients compared with their younger counterparts (＜60 years) (P＜0.05).In elderly AMI patients,the NLR was considerably higher in the high-risk group than in both the low-risk and medium-risk groups based on the GRACE score (P＜0.05 and P＜0.01,respectively),and the NLR was positively correlated with the GRACE score (r=0.322,P＜0.001).Either the NLR level or the GRACE score was significantly higher in the death group than in the surviving group (P＜0.05).By curve receiver operator characteristic curve (ROC) analysis,the optimal cut-off levels of 9.41 for NLR and 174 for GRACE score predicted in-hospital death [ROC area under the curve (AUC) 0.771 and 0.787,respectively,P＜0.001].It was concluded that an elevated NLR is a potential predictor of in-hospital mortality in elderly patients with AMI.

Ureteropelvic junction obstruction (UPJO) is characterized by decreased flow of urine down the ureter and increased fluid pressure inside the kidney. Open pyeloplasty had been regarded as the standard management of UPJO for a long time. Laparoscopic pyeloplasty reports high success rates, for both retroperitoneal and transperitoneal approaches, which are comparable to those of open pyeloplasty. However, open and laparoscopic pyeloplasty have yielded disappointing failure rates of 2.5%-10%. The main causes for recurrent UPJO are severe peripelvic and periureteric fibrosis due to urinary extravasation, ureteral ischemia, and inadequate hemostasis. In addition, failing to diagnose lower pole crossing vessels before or during the primary procedure is also responsible for recurrent UPJO. In addition, poor preoperative split renal function, hydronephrosis, presence of renal stones, patient age, diabetes, prior endopyelotomy history, and retrograde pyelography history were considered as predictors of pyeloplasty failure. The failure is usually defined by persistent pain, persistent radiographic obstruction (infection or stones), continued decline in split renal function, or a combination of the above. And the failure of pye-loplasty often occurs in the first 2 years after the surgery. The available options for managing recurrent UPJO with a salvageable renal unit include endopyelotomy, re-do pyeloplasty, stent implantation, percutaneous nephrostomy, ureterocalicostomy, and nephrectomy. Re-do pyeloplasty has such merits as high successful rates and rare complications, compared with endopyelotomy or ureterocalicostomy. And some investigators think that re-do pyeloplasty should be regarded as the gold standard for secondary therapy if feasible. Open pyeloplasty can enlarge the operating field, facilitate the exposure of the ureteropelvic junction, reduce the difficulty of operation, and thus reduce the occurrence of complications. There are no significant differences among the success rates of re-do pyeloplasty under open approach, traditional laparoscopy and robot-assisted laparoscopy, according to previous reports. However, traditional laparoscopic and robot-assisted pyeloplasty give advantages of cosmetology, small trauma, less postoperative pain, speedy recovery and shorter hospitalization, fewer complications and lower recurrent rates. If the primary pyeloplasty is an open operation in retroperitoneal approach, the traditional laparoscopic and robotic operation with retroperitoneal approach should be considered for secondary repair. The cause of recurrent UPJO should be evaluated before surgery and identified intraoperatively to minimize the possibility of recurrence.
Humans ; Hydronephrosis ; Kidney Pelvis ; Laparoscopy ; Ureter ; Ureteral Obstruction/surgery* ; Urologic Surgical Procedures

The chitosan/PHEA-blended hydrogels were prepared from PHEA and chitosan in various blend ratios. The water contents of the hydrogels were in the range of 50%-80% (wt). The attachment and growth of fibroblast cells(L929) on the hydrogels were studied. The results indicated the PHEA content in hydrogels has great effect on cell attachment but has little effect on the growth of L929 cells.
Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; physiology ; Cell Division ; physiology ; Cells, Cultured ; Chitin ; analogs & derivatives ; chemistry ; Chitosan ; Fibroblasts ; cytology ; physiology ; Hydrogel, Polyethylene Glycol Dimethacrylate ; chemistry ; Mice ; Peptides ; chemistry ; Water ; chemistry

The clinical manifestations of five children with paroxysmal kinesigenic dyskinesia (PKD) were retrospectively analyzed and their gene mutations were analyzed by high-throughput sequencing and chromosome microarray. The 5 patients consisted of 4 males and 1 female and the age of onset was 6-9 years. Dyskinesia was induced by sudden turn movement, scare, mental stress, or other factors. These patients were conscious and had abnormal posture of unilateral or bilateral extremities, athetosis, facial muscle twitching, and abnormal body posture. The frequency of onset ranged from 3-5 times a month to 2-7 times a day, with a duration of <30 seconds every time. Electroencephalography showed no abnormality in these patients. Three patients had a family history of similar disease. The high-throughput sequencing results showed that a heterozygous mutation in the PRRT2 gene, c.649_650insC (p.R217PfsX8), was found in two patients; the mutation c.436C>T (p.P146S) was found in one patient; a splice site mutation, IVS2-1G>A, was found in one patient. The two mutations c.436C>T and IVS2-1G>A had not been reported previously. The chromosome microarray analysis was performed in one patient with negative results of gene detection, and the chromosome 16p11.2 deletion (0.55 Mb) was observed. Low-dose carbamazepine was effective for treatment of the 5 patients. PKD is a rare neurological disease. The detection of the PRRT2 gene by multiple genetic analysis can help the early diagnosis of PKD.
Carbamazepine ; therapeutic use ; Child ; Chromosome Deletion ; Chromosomes, Human, Pair 16 ; Dystonia ; complications ; diagnosis ; drug therapy ; genetics ; Electroencephalography ; Female ; Humans ; Male ; Membrane Proteins ; genetics ; Mutation ; Nerve Tissue Proteins ; genetics