Conus textile is a kind of highly toxic and abundantly existing conus in the South China Sea. The toxin from C.textile could act on sodium channels(δ-conotoxins)and calcium channels (ω-，ε-conotoxins), respectively. Their specific chemical structure and biological activity have attracted a lot of attention in recent years. This article briefly reviews their biochemical characteristics, isolation, gene cloning, biological and neuropharmacological activities, as well as their potential applications.

BACKGROUND: Many experiments indicate that the angiogenesis of tissue engineered bone graft plays a key role in the osteogenesis.OBJECTIVE: An experimental pattern was set up designed to prepare a kind of vascularized engineered-bone graft for repairing rhesus tibia defects and analyze the relation of angiogenesis and osteogenesis in vivo by rontgenographic and morphological approaches.DESIGN: Random controlled animal experiment.SETTING: Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University.MATERIALS: The composite graft was constructed by seeding the induced bone marrow stem cells (BMSCs) on to a beta-tricalcium phosphate(3-TCP) scaffold in vitro, a circular cylinder (20 mm × 8 mm diameter) with a slit (width 2 mm and length 3 mm ) open to both ends and slot. Porosity 60% and pore diameter 100-150 μm. Twenty-nine healthy rhesuses aged 4-5 years and weighted 3.5-5 kg were adopted without gender limitation.METHODS: The experiment was conducted in the Department of Orthopaedics and Traumatology, Nanfan Hospital, Southern Medical University from October 2003 to July 2005. ①Bone-periosteum defect of 20 mm was made in the middle part of right tibia of the 27 rhesuses, and randomly divided into 3 groups equally. ②The defect gaps in fascia-blood vessel group (A) were plugged with in vitro engineered composites constructed by bone marrow stem cells and 3-TCP scaffold, which were totally hugged by a sheet of pedicled deep fascia and additionally a corresponding portion of saphenous artery and veins. The gaps in fascia group (B) and control group(C), however, were inserted with fascia-coated tissue engineered bone and tissue engineered bone only, respectively. Furthermore, two rhesuses without filling materials on the defect were picked up as blanks fixed by steel pins. ③The angiogenesis and osteogenesis for each treatment was assessed by radioactive imaging, roentgenographic analyses, blocking density and vaso-area image analysis at time intervals of 4, 8 and 12 weeks postoperative.MAIN OUTCOME MEASURE: The score of radioactive imaging,roentgenographic, morphological and vaso-area image analyses RESULTS: Totally 29 rhesuses were involved in the result analysis.① General observation of samples: In group A, all the surfaces of the implanted material and the central part were wholly wrapped up or replaced by bonelike tissues which were hard and could not be broken. And 2/3 materials had been absorbed; In group B and C, partial materials of the medial surface and the front were not coated or replaced by bonelike tissues, which could be broken with force, and 1/3 material had been absorbed.②Histological observation of scaffolds: With time passing, the scaffold materials were absorbed to different degrees in group A, B and C, among which, group A was most significant; Under the microscope, the implanted materials at 12 weeks were completely coated with the bonelike tissues, while the blood vessels structures in the materials were mostly alveoli alike and multi-braches. In group B, most of the materials at 12 weeks were wrapped up by the new bone, and few blood vessels could be seen in the center of the materials. In group C, the implanted materials at 12 weeks were slightly absorbed. The new bone and the vascular structures were both increased a little, but still very few.③Analyses of vaso-area: The vaso-areas of both central and peripheral parts in group A were significantly bigger than those of group B and C (P ＜ 0.05). Furthermore, it tended to increase with the time.④X-rays observation: At 12 weeks, group A's images presented obviously decreased density which was lower than that of the normal bone in individual areas and the continual bony callus manifested. Whereas group B and C's images showed slightly decreased density and the continual bony callus appeared on the sections. ⑤The roentgenographic scores of bone defects: The results indicates that the scores of group A was better than those of group B and C at 4, 8 and 12 weeks, respectively (P ＜ 0.05).CONCLUSION: ①This study shows that a feasible and effective angiogenesis approach of tissue engineered bone can accelerate osteogenesis in vivo. ②The absorption level is positively related to local angiogenesis.

Objective To identify a novel pathogenicity mutation of acid alpha‐glucosidase(GAA) gene in a Chinese family with two siblings affected with juvenile onset form glycogen storage disease Ⅱ(GSD Ⅱ) .Methods The clinical and family data of two siblings presenting recurrent respiratory tract infections ,respiratory failure associated with systemic muscle weakness ,were an‐alyzed and diagnosed with GSD Ⅱ by detecting alpha‐1 ,4‐glucosidase activity .DNA was extracted from peripheral blood of the proband ,younger brother and his parents .All 20 exons and the intron‐exon splice sites of GAA gene were amplified by polymerase chain reaction (PCR) .Mutations were detected by direct sequencing the PCR products .Results The younger brother was found to be compound heterozygous for two mutations in the GAA gene :c .1216G>A (p .Asp406Asn) missense mutation in the exon 8 from his father and c .1935C>A (p .Asp645Glu) missense mutation in the exon 14 from his mother .Conclusion The compound hetero‐zygous c .1216G>A and c .1935C>A mutations caused the juvenile onset form GSD Ⅱ characterized by dyspnea and cardiac hyper‐trophy .The novel c .1216G>A mutation may be related to the juvenile onset form GSD Ⅱ .

The single-chain antibody gene (ox26-scFv) to transferrin receptor (TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20 degrees C, after 0.02mM IPTG induction using the strain E. coli BL21 (DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography (Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.
Animals ; Antibodies ; administration & dosage ; genetics ; metabolism ; Antibody Formation ; Base Sequence ; Blood-Brain Barrier ; drug effects ; Drug Delivery Systems ; Genetic Vectors ; Molecular Sequence Data ; Rats ; Rats, Sprague-Dawley ; Receptors, Transferrin ; immunology

Objective:To explore the efficacy and safety of Rasburicase therapy in critically ill children su-ffering from advanced Burkitt′s lymphoma.Methods:A retrospective analysis of children with advanced Burkitt′s lymphoma was admitted to Pediatric Intensive Care Unit, the First Affiliated Hospital of Sun Yat-Sen University, from January 2015 to May 2020 and accepted treatment.According to the uric acid-lowering therapies, patients were divided into 2 groups, namely Rasburicase group (Group R) and traditional treatment group (Group T), to compare the effects of hypouricemic treatment and the prognosis between the 2 groups.Results:Twenty-nine children with advanced Burkitt′s lymphoma were included in this study, with 13 cases (44.83%) of stage Ⅲ and 16 cases (55.17%) of stage Ⅳ.Abdominal mass/ abdominal distension (13 cases, 44.83%) and abdominal pain (7 cases, 24.14%) were the main reasons of initial medical visit attendance.The most common primary tumor site was abdominal/ pelvic cavity (21 cases, 72.41%), followed by head or neck (6 cases, 20.69%). There were 15 cases in Group R and 14 cases in group T. No significant differences in serum creatinine, lactate dehydrogenase and uric acid were detected between the 2 groups (all P>0.05). The proportion of serum uric acid recovery rate of 24 hours and 72 hours after initial treatment in Group R were significantly higher than those in T group (85.71% vs.25.00%, 100.00% vs.25.00%, all P<0.01). Although there were no obvious differences in the incidence of tumor lysis syndrome between the 2 groups (33.33% vs.64.29%, P=0.096), the incidence of acute renal injury, renal replacement therapy requirement, serious complications and the 28 day mortality in Group R were remarkably lower than those in Group T (33.33% vs.85.71%, 13.33% vs.64.29%, 20.00% vs.78.57%, 0 vs.35.71%, all P< 0.05). Conclusions:Rasburicase can effectively reduce the serum uric acid level and decrease the incidence of acute kidney injury and other severe complications, thus improving the prognosis of children experiencing advanced Burkitt′s lymphoma.

ObjectiveTo verify the reliability of the mouse model of cerebral cortical microinfarct induced by two-photon microscopy and to explore its pathological changes.MethodsSeventeen male C57BL/6J mice were randomly divided into a microinfarct group (n=11) or a sham operation group (n=6).A thinned cranial window of 3 mm diameter was performed over the cerebral cortex with a high-speed micro-drill until the small blood vessels were clearly observed under a dissecting microscope.Then, a permanent single cortical penetrating arteriole occlusion was induced with a gradually enhanced ultrashort laser irradiation through the thinned cranial window with two-photon microscopy.At 7 days after modeling, the cerebral microinfarct volume was measured with HE staining, and the neuron loss, activation of glial cells and deposition of 3-nitrotyrosine were assessed using immunohistochemistry.ResultsThe target vessels of cerebral cortex in 8 (72.7%) mice were occluded and the microinfarcts formed in the microinfarct group, and the average microinfarct volume was 317.23±20.29 μm3.There were remarkable neuron loss and microglia infiltration in the infarcted core, a large number of reactive astrocytes surrounding the infarcted lesion, and massive deposition of 3-nitrotyrosine in the peri-infarct area.No infarcts were observed in the sham operation group.The deposition of 3-nitrotyrosine in the sham operation group was significantly less than that in the microinfarct group (8.00±1.48 vs.98.38±9.10;t=23.962, P<0.001).Conclusions The mouse model of cerebral cortical microinfarct induced by two-photon microscopy is reliable, and its histopathologic changes are consistent with the pathologic features of cerebral microinfarct.

Anatomic structure of acetabular fractures are complex and operative exposure and fixation are extremely difficult. For those obviously displaced acetabular fractures, close reduction is doomed to cause deformed healing. Open reduction with internal fixation (ORIF) not only results in anatomic reduction, but also brings complications. No matter which method will be adopted, traumatic arthritis or avascular necrosis of femoral head might occur. In order to treat acetabular fractures more effectively, orthopedic surgeons should be required to fully master the acetabular anatomy, biomechanics, classification and the necessary knowledge for complication prevention.
Acetabulum ; diagnostic imaging ; injuries ; surgery ; Fracture Fixation, Internal ; methods ; Fractures, Bone ; classification ; diagnostic imaging ; surgery ; Humans ; Orthopedic Procedures ; Radiography

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.
Enzyme-Linked Immunosorbent Assay ; Genes, Synthetic ; genetics ; physiology ; Models, Theoretical ; Peptide Library ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transcription Factors ; chemical synthesis ; chemistry ; metabolism ; Zinc Fingers ; genetics ; physiology

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

Objective:To validate five-year risk prediction models for atherosclerotic cardiovascular di-sease (ASCVD) in a contemporary rural Northern Chinese population.Methods: Totally 6 489 rural adults aged 40 to 79 years without clinical ASCVD were enrolled at baseline between June and August 2010, and followed up through January 2017.Expected prediction risk using the China-PAR (prediction for ASCVD risk in China) model was compared with the pooled cohort equations (PCE) reported in the American College of Cardiology / American Heart Association guideline.Kaplan-Meier analysis was used to obtain the observed ASCVD event (including nonfatal myocardial infarction, coronary heart disease death, nonfatal or fatal stroke) rate at 5 years, and the expected-observed ratios were calculated to eva-luate overestimation or underestimation in the cohort.The participants in the cohort were divided into 4 categories (<5.0%, 5.0%-7.4%, 7.5%-9.9%, and ≥10.0%) for comparisons based on ASCVD prediction risk.The models were assessed by discrimination C statistic, calibration χ2, and calibration charts and plots for illustration as well.Results: Over an average 5.82 years of follow-up in this validation cohort with 6 489 rural Chinese participants, 955 subjects developed a first ASCVD event.Recalibrated China-PAR model overestimated ASCVD events by 22.2% in men and 33.1% in women, while the overestimations were much higher for recalibrated PCE as 67.3% in men and 53.1% in women.Gender-specific China-PAR model had C statistics of 0.696 (95%CI, 0.669-0.723) for men and 0.709 (95%CI, 0.690-0.728) for women, which were similar to those of 0.702 (95%CI, 0.675-0.730) for men and 0.714 (95%CI, 0.695-0.733) for women in the PCE.Calibration χ2 values in China-PAR were 17.2 and 54.2 for men and women, respectively;however, the PCE showed poorer ca-libration (χ2=192.0 for men and χ2=181.2 for women).In addition, the calibration charts and plots illustrated good agreement between the observations and the predictions only in the China-PAR model, especially for men.Conclusion: In this validation cohort of rural Northern Chinese adults, the China-PAR model had better performance of five-year ASCVD risk prediction than the PCE, indicating that recalibrated China-PAR model might be an appropriate tool for risk assessment and primary prevention of ASCVD in China.