This article describes the preparation of salmon calcitonin ultra-flexible liposomes and their hypocalcemia effect after intranasal administration in rats. Both the conventional liposomes and ultra-flexible liposomes were prepared by rotary evaporation-sonication and extrusion. The morphology of ultra-flexible liposomes was observed with transmission electronic microscope. The size and size distribution and their zeta potential were determined by dynamic light scattering. The mean size of ultra-flexible liposomes with DC-Chol was no more than 120 nm, while the mean size of the conventional liposomes was 256.5 nm. The results showed the content of sodium deoxycholate have significant effect on the mean particle size of liposomes. The ultra-flexible liposomes were intranasal administrated at the dose of 5.0 microg x kg(-1); the concentration of serum calcium was determined by OCPC method. The results showed that the salmon calcitonin solution only slightly lowered serum calcium levels and the conventional liposomes could improve the effect of decreased serum calcium level (D%), and the ultra-flexible liposomes had the best effect on the decreased serum calcium level, and the hypocalcemia effect was correlated with the content of sodium deoxycholate which was present in the liposomes. Moreover the ciliotoxicity of ultra-flexible nanoliposomes on nasal mucocilia was investigated with the electron microscope scanning. The results showed that the ultra-flexible liposomes markedly reduced the ciliotoxicity of sodium deoxycholate on nasal mucous. Thereby the ultra-flexible liposomes significantly enhanced the hypocalcemia effect of serum calcium after intranasal administration in rats. The ultra-flexible liposomes could be an effective carrier for intranasal delivery of the peptide and protein drugs.
Administration, Intranasal ; Animals ; Calcitonin ; administration & dosage ; pharmacology ; Calcium ; blood ; Liposomes ; Male ; Particle Size ; Rats ; Rats, Sprague-Dawley

The aim of this study was to obtain the capsid protein VP2 of human bocavirus (HBoV) identified in Beijing recently and construct virus-like particles (VLPs) in insect cells for further study of this virus. The full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector (pFastBac 1) to obtain the recombinant Bacmid, and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells. Then the recombinant VP2 protein was recognized by SDS-PAGE using Coomassie-blue staining and Western blot using hyper-immune serum against VP2 of HBoV from rabbit. The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titers to infect insect cells at a multiplicity of infection (MOI) of 0. 5. After 7-10 days or 4-5 days of the infection, the supernatants of culture or the cell lysates treated with lysing solution were harvested, and ultracentrifuged twice through 40% sucrose cushion to obtain purified VLPs, which were followed by Western blot and IFA for VLPs' composition and specificity analysis, by electron microscopy for VLPs' morphologic structure. The recombinant VP2 protein with molecular weight of approximately 61 kD expressed in recombinant baculoviruses was recognized by SDS-PAGE using Coomassie-blue staining and Western blot. The presence of VP2 on VLPs was demonstrated by Western blot and IFA from samples collected during the purification of VLPs from the supernatants of culture or the cell lysates, and the expression of VP2 in insect cells led to the formation of VLPs which formed the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. In conclusion, the recombinant baculoviruses were constructed, the HBoV VP2 protein was expressed in insect cells with high specific antigenicity and VLPs was formed successfully.
Animals ; Blotting, Western ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Fluorescent Antibody Technique, Indirect ; Human bocavirus ; genetics ; metabolism ; Microscopy, Electron, Transmission ; Polymerase Chain Reaction ; Spodoptera ; Virion ; genetics ; metabolism ; ultrastructure

OBJECTIVEThe present study was designed to explore the practical application of the rapid etiological diagnosis by detecting specific IgM antibody against common respiratory viruses in children with acute lower respiratory infections (ALRI).

METHODClinical specimens including nasopharyngeal aspirates and serum of acute phase from hospitalized children were collected from 207 infants and children with acute lower respiratory infections from March 2009 to September 2010. Seven common respiratory virus antigens were identified from the collected nasopharyngeal aspirates by direct immunofluorescence assay (DFA). ELISA was used to detect specific IgM antibody against RSV, ADV, IFVA, IFVB and PIV, while indirect immunofluorescence assay (IFA) was used to detect specific IgM antibody against RSV, ADV, IFVA, IFVB, PIV1, PIV2 and PIV3 in collected acute phase serum.

RESULTThe overall positive rates to detect viral antigen by using DFA, ELISA and IFA was 67.6%, 57.5% and 39.6%, respectively. The consistent rate of ELISA and IFA versus accepted DFA were 21.7% and 31.4%, respectively. The average days from onset of the symptoms to blood sample collection for those with the consistent results by ELISA and DFA were 12.0 d for ADV, 9.6 d for PIV2, 9.5 d for IFV, and 5.3 d for RSV, respectively, and by IFA and DFA were 15.0 d for PIV3, 9.2 d for ADV, and 7.4 d for RSV, respectively. Among all age groups, the consistent rate of serum viral IgM and antigen detections was highest in children younger than 3 years old.

CONCLUSIONAlthough there were differences between serum IgM antibody and viral antigen detections, specific IgM antibody detection was of value in early and rapid etiological diagnosis of pediatric ALRI, especially for young children. It could provide serologic evidence of respiratory virus infection. The diagnostic rate of pathogen could be improved if it was used in combination with viral antigen diagnostic methods.

Antibodies, Viral ; analysis ; blood ; Antibody Specificity ; Antigens, Viral ; analysis ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique ; Humans ; Immunoglobulin M ; analysis ; blood ; Infant ; Male ; Nasopharynx ; virology ; RNA Viruses ; genetics ; isolation & purification ; Respiratory Syncytial Virus Infections ; diagnosis ; virology ; Respiratory Syncytial Viruses ; genetics ; isolation & purification ; Respiratory Tract Infections ; diagnosis ; immunology ; virology ; Sensitivity and Specificity

To characterize the genomic sequence and arrangement of WU polyomavirus (WU virus) identified in clinical specimens collected from children with acute respiratory infections in Beijing, China, the sequences of capsid proteins VP1, VP2, and the large tumor antigen (LTAg), as well as the 5'-terminal sequence of WU virus, were amplified from the clinical specimen with ID number of BJF5276 which was determined as WU virus positive by PCR amplification. The PCR amplicons were sequenced, and genomic sequence analysis was performed by using the software DNAStar. In addition, VP2 coding-region sequences were amplified from other 21 clinical specimens identified as WU virus positive to investigate the gene diversity of WU virus. The genomic sequence of WU virus BJF5276 with accession number of HQ218321 in GenBank was 5,229 base pairs in length with 3 major coding domain sequences (CDS) sited on one strand coding for capsid proteins VP2, VP3 and VP1, and two CDS sited on the complementary strand coding for small tumor antigen (STAg) and LTAg; These 22 VP2 CDS sequences including 5 sequences submitted to GenBank were compared with 64 corresponding sequences downloaded from GenBank by MegAlign of DNAStar software, indicated that these sequences coming from children in Beijing shared high homology (over 98.8%) with those from GenBank. Phylogenetic analysis of these VP2 CDS by using Neighbor-joining (NJ) analyses with 2,000 bootstraps (Mega 4.0) showed that 20 sequences out of 22 belonged to clade Ia, and other 2 of them belonged to clade III, including 1 clustered in IIIa and 1 in a novel cluster proposed as IIIc. In conclusion, the genomic sequence of WU polyomavirus detected from clinical specimens from children in Beijing is closely related to other WU polyomaviruses in the feature of genomic coding region arrangement. Overall variation of VP2 CDS was very low, and there were different clades circulating in Beijing with a dominant clade Ia, which is different from dominated Ib circulating in other parts of the world reported previously, and a novel clade IIIc was proposed.
Acute Disease ; Child, Preschool ; China ; Female ; Genome, Viral ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Polyomavirus ; classification ; genetics ; isolation & purification ; Respiratory Tract Infections ; virology ; Viral Proteins ; genetics

Objective To investigate the changes of tight junction (TJ) protein occludin expression between the mierovascular endothelial cells of the blood-brain barrier (BBB) of rats after intracerebral hemorrhage (ICH). Methods Ninety-three male SD rats were randomly divided into normal control group and experimental group; rats of the experimental group were divided into 6 subgroups (6, 24, 48 and 72 h, and 7 and 14 d after intraeerebral hemorrhage). Rat models of intracerebral hemorrhage in the experimental group were established by autologous blood injection.Morphology of the brain tissues was detected by hematoxylin-eosin (HE) staining.The tight junctions between the microvascular endothelial cells of the BBB were sampled for ultrastructural observation using electron microcopy.Immunofluorescence and quantitative real time-PCR were used to analyze the protein and mRNA expressions of occludin,respectively. Results The brain edema was found in brain tissues around the hematoma. Necrosis and inflammatory infiltration could be found in perihematomal brain tissues and it was most obvious at 48 h after intracerebral hemorrhage. Obvious paracellular clefts were found between the adjacent endothelial cells in experimental groups. Strong expression of occludin was noted in the normal brain tissue; expression of occludin was positive 6 h after the injection and weak positive at 24, 48 and 72 h after the injection in the experimental groups.Quantitative real time-PCR showed that the mRNA expression of occludin in the experimental group was significantly reduced as compared with the normal brain tissue at 6,24,48 and 72 h after the injection with statistically significant difference (P＜0.05). Conclusion After intra-cerebral hemorrhage,as a major component of TJ of the BBB,occludin expression is down-regulated,which may be one of the most impotant molecular basis of disruption of BBB.

Metabolic syndrome (MetS) describes a set of risk factors that can eventually lead to the occurrence of cardiovascular and cerebrovascular disease. A detailed understanding of the MetS mechanism will be helpful in developing effective prevention strategies and appropriate intervention tools. In this article, we discuss the relationship between the clinical symptoms of MetS and differences in the gut microbial community compared with healthy individuals, characterized by the proliferation of potentially harmful bacteria and the inhibition of beneficial ones. Interactions between gut microbiota and host metabolism have been shown to be mediated by a number of factors, including inflammation caused by gut barrier defects, short-chain fatty acids metabolism, and bile acid metabolism. However, although we can clearly establish a causal relationship between gut microbial profiles and MetS in animal experiments, the relationship between them is still controversial in humans. Therefore, we need more clinical studies to augment our understanding of how we can manipulate the gut microbiota and address the role of the gut microbiota in the prevention and treatment of MetS.

OBJECTIVETo evaluate the influence of reconstructive surgery for male urethral stricture on erectile function and sexual life quality.

METHODSWe analyzed retrospectively the clinical data of 326 male patients who underwent urethroplasty for urethral stricture in our department and evaluated their erectile function and sexual life quality.

RESULTSA total of 172 groups of valid data were collected, with the mean follow-up of 28.5 months. The mean scores on IIEF-5 (P=0.002) and sexual life quality (P=0.026) were statistically significantly reduced after surgery. Erectile dysfunction was found in 88 (51.2%) of the patients after urethroplasty, as compared with 56 (32.6%) preoperatively.

CONCLUSIONThe location of urethral stricture, surgical method and urethral stricture recurrence may affect the erectile function and sexual life quality of the patient, but both can be gradually improved with the time after urethroplasty.

Adult ; Aged ; Coitus ; physiology ; psychology ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Penile Erection ; physiology ; psychology ; Quality of Life ; Reconstructive Surgical Procedures ; methods ; Retrospective Studies ; Surveys and Questionnaires ; Urethral Stricture ; physiopathology ; psychology ; surgery

OBJECTIVETo evaluate the selection of different procedures and the feasibility for the treatment of long segment urethral stricture.

METHODSSeventy-six patients with complex urethral stricture greater than 8 cm long underwent different procedures of urethroplasty. Of them various mucosa grafts urethral reconstruction were adopted in 42 cases (colonic mucosal graft, n = 26; buccal mucosal graft, n = 10; bladder mucosal graft, n = 6); One-stage pedicle flaps urethroplasty in 20; two-stage urethroplasty of Johanson procedure in 12; and penile urethra-prostatic urethra anastomosis, three-stage urethroplasty in 2.

RESULTSIn early followed up (within 6 months postoperatively), 67 patients (88%) voided well and complications developed in 10. Among the 70 patients who lasted more than 1 year after operation, 51 cases were followed up. Forty-four patients voided well, and complications developed in 8. Of the 8 cases urethral restructure developed in 2 (18%) for pedicle flaps urethroplasty, 2 for colonic mucosal urethroplasty (9%), 1 for buccal mucosal graft (1/7), 1 for bladder mucosal graft (1/3); penile chordee in 2 (2/5), and one of them was accompanied by hair bearing neourethra for two-stage urethroplasty of Johanson procedure.

CONCLUSIONSColonic mucosal and buccal mucosal grafts urethroplasty are feasible procedures for the treatment of long segment urethral stricture, and Colonic mucosal graft urethroplasty may be considered when more conventional procedures fail or complicated urethral strictures greater than 10 cm long.

Adolescent ; Adult ; Aged ; Follow-Up Studies ; Humans ; Intestinal Mucosa ; surgery ; Male ; Middle Aged ; Mouth Mucosa ; surgery ; Surgically-Created Structures ; Treatment Outcome ; Urethral Stricture ; pathology ; surgery ; Urologic Surgical Procedures, Male ; methods

OBJECTIVETo find out the nasendoscopic changes of velopharyngeal configuration and movement after palatoplasty with or without velopharyngeal muscle reconstruction.

METHODSThe nasendoscopy was taken in forty-one patients with palatoplasty, 22 repaired by velopharyngeal muscle reconstruction and 19 with modified von Langenbeck's procedure (non-reconstructive group).

RESULTSIn patients with velopharyngeal muscle reconstruction, the velopharyngeal ports are smooth and full with a definite reduction in size than patients without velopharyngeal muscle reconstruction. During phonation, the complete and marginal velopharyngeal competence rate in reconstructive group (90.91%) is higher than the group of non-reconstruction (37.31%) The major velopharyngeal closure is circular movement in reconstructive group, otherwise coronal closure in nonconstructive group.

CONCLUSIONSBased the observation of nasendoscopy, the velopharyngeal muscle reconstruction in palatoplasty has more definite improvement to velopharyngeal closure than non-reconstructive procedure. Palatoplasty with velopharyngeal muscle reconstruction could reduce the size of velopharyngeal port and make the complete velopharyngeal closure easier.

Adolescent ; Child ; Child, Preschool ; Cleft Palate ; surgery ; Endoscopy ; methods ; Humans ; Infant ; Nose ; surgery ; Pharyngeal Muscles ; abnormalities ; surgery ; Reconstructive Surgical Procedures ; methods

BACKGROUNDPelizaeus-Merzbacher disease (PMD) is a rare X-linked recessive disorder with symptoms including nystagmus, impaired motor development, ataxia, and progressive spasticity. The proteolipid protein 1 (PLP1) gene is the only pathogenic gene of PMD. Duplication of the PLP1 gene is the most frequent gene defect, accounting for 50%-70% of PMD cases, whereas point mutations in the coding sequence or the splice sites account for 10%-25% of PMD cases. This study aimed to identify PLP1 mutations in nine unrelated Chinese patients (P1-9) with PMD, and 14 subjects from the family of patient 2 were also described.

METHODSGenomic DNA was extracted from peripheral blood samples. Gene dosage was determined using the multiplex ligation-dependent probe amplification (MLPA). All 7 exons and exon-intron boundaries of the PLP1 gene were amplified and analyzed using direct DNA sequencing.

RESULTSOf these nine patients, there were four transitional, four classical, and one connatal PMD according to their clinical and radiological presentations. PLP1 duplications were identified in patients 1-7 with PMD. Their mothers were PLP1 duplications carriers as well. Both duplication carriers and normal genotypes of PLP1 were identified in the family members of patient 2. A c.517C > T (p. P173S) hemizygous missense mutation in exon 4 was found in patient 8 with PMD, and his mother was shown to be a heterozygote of this mutation.

CONCLUSIONSWe identified seven genomic duplications and one missense mutation (p. P173S) of the PLP1 gene in eight Chinese patients with PMD. This is the report about PLP1 mutations in PMD patients from the mainland of China.

Child, Preschool ; Female ; Gene Duplication ; Humans ; Infant ; Male ; Mutation ; Myelin Proteolipid Protein ; genetics ; Nucleic Acid Amplification Techniques ; Pelizaeus-Merzbacher Disease ; genetics ; Sequence Analysis, DNA