Parasite classification and identification are central to controlling parasitosis. Traditional methods for identifying parasite species are based on morphological features, but these are time-consuming and inaccurate, especially for cryptic species. The purpose of the present study was to select molecular markers to promote the development of molecular systematic for parasites. The internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) falls in between 18S, 5.8S, and 28S rDNA sequences, including ITS-1 and ITS-2 sequences. Previous studies have demonstrated that rDNA ITS sequences provide useful genetic markers for identifying parasitic nematodes. With the ultimate goal of controlling parasite transmission, we identified Kalicephalus belonging to three species using ITS rDNA genes. The ITS genes (750–797 bp) of 21 Kalicephalus belonging to 3 species were cloned and sequenced. Intra- and interspecific identities were 98.4% and 80%–89%, respectively. The phylogenetic tree reconstructed with the neighbour-joining (NJ) method revealed that congener Kalicephalus form the same branch, which is far apart from other branches of other nematodes. This is consistent with morphological classifications, demonstrating the accuracy of our molecular method. This is the first report stating that ITS genes can be used to classify Kalicephalus, and it lays the foundation for identification, molecular epidemiology, and phylogenetics of Kalicephalus and related parasitic nematodes.

Trichuris suis infection in pigs is ubiquitous in intensive and extensive farms, which causes potential threat to human health. The objective of this research was to investigate the prevalence of T. suis in pigs in Hunan province. Total 2,267 fresh fecal samples distributed in 28 pig farms from 7 different administrative regions (Hunan province) were evaluated for the existence of T. suis eggs using saturated NaCl floating method. The average infection rate of T. suis in pigs was 8.91% in Hunan province. To determine genetic variation of the gained T. suis isolates in the present study, the internal transcribed spacer (ITS) regions from nuclear ribosomal DNA (rDNA) of 7 T. suis isolates were cloned and analyzed. Nucleotide diversities were 1.0–3.5% and 0–3.8% for ITS-1 and ITS-2, respectively. Phylogenetic analyses indicated that all isolates collected in the present study and T. suis available in Genbank generated a monophyletic clade. The present investigation revealed high infection rates of T. suis in pigs in Hunan province, which shed light on making effective measures to prevent and control T. suis infection in pigs in Hunan province.
Agriculture ; China ; Clone Cells ; Databases, Nucleic Acid ; DNA, Ribosomal ; Eggs ; Genetic Variation ; Humans ; Methods ; Ovum ; Prevalence ; Swine ; Trichuris

A preliminary survey of larvae and pupae of black flies (Diptera: Simuliidae) was conducted in three recreation parks [Templer Recreation Park (TRP), Congkak River Forest Reserve (CRFR) and Ampang Forest Reserve (AFR)] located in Selangor State, which is located 18 to 35 km from Kuala Lumpur city center, Malaysia. This study was initiated to determine the distribution and abundance of filarial vectors, Simulium spp. A total of 12 species of black flies belonging to three subgenera (Gomphostilbia, Simulium and Nevermannia) were collected. Simulium (Simulium) nobile was incriminated as the most dominant species in all recreation parks. This study is a first report on the distribution and abundance of black flies obtained from recreation parks in Malaysia.

OBJECTIVE To explore the genetic mechanism for a family affected with cardiac conduction block. METHODS Affected family members were screened for potential mutations of known candidate genes. As no pathogenic mutation was found, two patients and one healthy member from the family were further analyzed by exomic sequencing followed by Sanger sequencing. The pathogenicity of suspected mutation was analyzed using bioinformatics software. RESULTS Sequencing of the full exome has identified a c.G1725T mutation in the CLCA2 gene. Sanger sequencing has detected the same mutation in all five patients, but not in the normal member from the family. Bioinformatics analysis indicated that the mutation has resulted in substitution of the 575th amino acid cysteine (C) by tryptophan (W). The site is highly conserved and becomes pathogenic with the mutation. CONCLUSION The heterozygous c.G1725T mutation in exon 11 of the CLCA2 gene probably underlies the disease and fit the autosomal dominant pattern of inheritance.
Amino Acid Sequence ; Chloride Channels ; genetics ; Computational Biology ; Female ; Heart Block ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation

OBJECTIVETo explore the impact of Line-1 methylation on clinical features of non-small cell lung cancer and its connection with smoking and other living habits.

METHODSPyrosequencing was used to determine the extent of Line-1 methylation in cancer and adjacent tissues derived from 197 patients with primary non-small cell lung cancer. Non-conditional logistic regression analysis was performed to correlate the level of Line-1 methylation with clinical features and living habits of the patients.

RESULTSLine-1 methylation for cancer tissue and adjacent tissue has measured 68.20±11.63 and 78.90±2.09, respectively (P < 0.01), and has been associated with TNM staging, smoking history and histopathological types.

CONCLUSIONLung cancer tissue Line-1 methylation level is closely related with clinical features and smoking. There is also a correlation between histopathological types of lung cancer and relative hypomethylation of Line-1.

Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; DNA Methylation ; Female ; Humans ; Long Interspersed Nucleotide Elements ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged

OBJECTIVESpinal muscular atrophy (SMA) is a common and fatal autosomal recessive disorder. Approximately 94% of SMA patients are caused by homozygous deletion of SMN1 gene. SMA carrier screening is recommended considering the high carrier frequency (1 in 35-50) as well as severity of the disease.

METHODSA prospective population-based cohort study was carried out on 4719 pregnant women from Shanghai region. Copy numbers of SMN1 and SMN2 genes were effectively determined with denaturing high performance liquid chromatography (DHPLC) technique. The method has detected 94% of SMA cases with deletion or conversion of the SMN1 genes.

RESULTSNinety SMA carriers with only one copy of the SMN1 gene were identified among the 4719 pregnant woman. The carrier rate was 1.9%. Respectively, 1.2% and 0.6% of the carriers were caused by SMN1 gene deletion and SMN1 gene conversion.

CONCLUSIONThrough this study, we have determined the frequency of SMA mutation carriers in a population of pregnant women. The result may provide a basis for genetic counseling in order to reduce the rate of SMA affected births.

Adult ; China ; Female ; Gene Deletion ; Genetic Testing ; methods ; Humans ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Pregnancy ; Prenatal Diagnosis ; methods ; Prospective Studies ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics ; Young Adult

OBJECTIVETo determine the karyotype of a patient with Prader-Willi-like syndrome features.

METHODSChromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region.

RESULTSNo abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient.

CONCLUSIONMolecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.

Child ; Chromosome Deletion ; Chromosomes, Human, Pair 1 ; genetics ; Female ; Humans ; Karyotyping ; Prader-Willi Syndrome ; genetics

OBJECTIVETo observe the revascularization process and histological changes in the dermal substitutes after grafting.

METHODSTwenty-one SD rats were enrolled in the study and were randomly divided into swine acellular dermal matrix (sADM), human acellular dermal matrix (hADM), and artificial dermal equivalent (Integra) groups according to different dermal substitutes grafted underneath the skin of SD rats. The gross appearance of the grafts was observed, and the tissue biopsies were harvested at 2, 3, 4, 7, 10, 14, 21, 30, 60, 90, 120, 150, 180 post grafting day (PGD) for the observation of the revascularization process and their histological changes.

RESULTSGross observation: The incision in each group healed well without local swelling and inflammatory response after grafting. The grafts had a compact contact with the wounds. The texture of the grafted dermis in each group was soft and could not be felt from the surface of the body on 90 PGD. The presence of the grafts could be discerned on 180 PGD in all the groups, but some of them became smaller in size and thinner, even hard to identify in a few. Fibroblasts, neutrophils and lymphocytes migrated into the grafts from 2PGD on. New capillary sprouts from the receiving beds could be observed after 3PGD. Affluent capillary nets formed in the grafts during 30 to 60 PGD. The structure of the grafts became nearly unidentifiable from the native dermis after 150 PGDs. Absorption and degradation to various degrees occurred in some of the grafts after 180 PGD.

CONCLUSIONThe revascularization of the three dermal substitutes could begin shortly after grafting. The sponge-like structure of the substitutes was advantageous for the migration of the host fibroblast into the substitute and for the secretion of the new extra-cellular matrix. The dermal substitutes could last in the wound for a long time with partial absorption and degeneration.

Animals ; Dermis ; transplantation ; Female ; Humans ; Neovascularization, Physiologic ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reconstructive Surgical Procedures ; Skin ; blood supply ; Skin Transplantation ; methods ; Skin, Artificial ; Swine ; Transplants ; Wound Healing

OBJECTIVETo observe the effects of the grafting of a platelet-derived growth factor gene-modified artificial composite skin on rat wounds with full thickness defect.

METHODSPlatelet derived growth factor-B (PDGF-B) eukaryotic expression plasmid was constructed, and the fibroblasts were transfected with it by liposome mediation. Artificial composite skins 1 and 2 were constructed respectively. The skin1 was composed of keratinocyte, porcine acellular dermal matrix and PDGF-B gene-transfected fibroblasts while the skin 2 contained keratinocyte, porcine acellular dermal matrix and fibroblasts. The two kinds of composite skin were grafted onto wounds on the rat back to form composite skin group 1 (C1) and 2 (C2), respectively, with 18 rats in each group. Eight rats with wounds without treatment served as control (C) group. The survival rate of the composite skin was observed at 2 post-operative weeks (POWs). The rat wounds were examined grossly on 2, 4 and 6 POWs for the calculation of wound contraction rate. Wound tissue samples were harvested for histological examination.

RESULTS(1) Up to 2 POWs, 14 grafts in C1 group survived completely, 3 with partial survival and 1 failure. In C2 group, 10 skin grafts survived completely, 4 with partial survival and 4 failures. (2) A scab was formed in the wound at 2 POW in C group. The surface of the grafted skin in C1 group was smooth, elastic, and showed good anti-friction properly, and it was better in quality compared with that in other two groups at 6 POW. (3) The wound contraction rate of the grafts in C group of rats was higher than that in C1 and C2 groups at 2, 4 and 6 POWs, while that in C1 was lower than that in C2 group. (4) Capillary formation was more intense in the grafted skins in C1 group at 2 POWs, and the epithelia differentiated well into 7 to 10 layers of epithelial cells with compact and orderly arrangement and evenly distributed fibrous tissue at 6 POWs.

CONCLUSIONRepair of the wound with artificial composite skin containing PDGF-B gene could improve the quality of wound healing.

Animals ; Cells, Cultured ; Female ; Humans ; Proto-Oncogene Proteins c-sis ; genetics ; Rats ; Rats, Sprague-Dawley ; Skin Transplantation ; methods ; Skin, Artificial ; Swine ; Tissue Engineering ; Transfection ; Wound Healing

OBJECTIVETo analyze several methods of wound repair for deep partial thickness burn wounds retrospectively, so as to evaluate the significance of improvement of wound microcirculation on wound healing.

METHODS(1) 2,976 burn patients admitted to our department were enrolled in the study, among them 614 undertook tangential excision, 32, eschar abrasion, 86 allo-skin coverage after debridement, 1836 tropical application of silver sulfadiazine and 408 with traditional Chinese medicine (Jing Wan Hong ointment) with gauze bandage. The results of the management with different methods were compared. (2) Rat model with deep partial thickness burn was reproduced and topical application of silver sulfadiazine was given. The rats were randomly divided into control (n = 10, with normal saline injected via caudal vein within 5 minutes postburn), and treatment (n = 10, with batroxobin injected via caudal vein within 5 minutes postburn) groups. The blood flow perfusion unit in the wound skin was measured before burn and at 0.5 to 72 postburn hours by Laser Doppler. The wound healing rate, contraction rate and wound healing time in each group were calculated on 14 and 18 postburn days (PBDs). The number of hair follicles after wound healing was observed by histological method.

RESULTS(1) The burn wound treated by tangential excision healed within 2 to 3 post operation weeks (POWs), with the healing rate of 94.8% in patients with burn covering 50% - 70% TBSA and 93.4% in those with burn of 80% approximately 98% TBSA. The healing time of patients with allo-grafts coverage after eschar abrasion was 13.8 +/- 2.1 days without scar formation. The wound healing time was 18.0 +/- 2.3 day in 82 patients with allo-graft coverage after debridement, and it was 26.0 +/- 3.2 days with subeschar healing in 1658 patients with topical application of silver sulfadiazine. Infection in burn wound was encountered in most patients undergoing traditional Chinese medicine bandage treatment with wound healing time of 26.0 +/- 2.8 days in the lower extremities. (2) The blood flow perfusion unit of the rats in the treatment group was significantly higher than that in the control group (P < 0.01). The wound healing rate in treatment group on 14 and 18 PBD was obviously higher than that in the control group (P < 0.01). But the wound contraction rate in the two groups was similar (P > 0.05). The wound healing time in treatment group was much shorter than that in control group (P < 0.01). A few hair follicles remained in the dermis of the rats in the control group on 30 PBD, and the number was evidently smaller than that in the treatment group (P < 0.01).

CONCLUSIONEarly tangential excision and eschar abrasion remained better methods in the management of deep partial thickness burn wounds, as they could ameliorate burn wound infection, shorten treatment period, raise wound healing rate and quality. Application of batroxobin could accelerate wound healing rate by improving wound microcirculation in deep partial thickness burn wound.

Adult ; Animals ; Batroxobin ; therapeutic use ; Burns ; pathology ; surgery ; therapy ; Female ; Humans ; Male ; Microcirculation ; Rats ; Rats, Wistar ; Retrospective Studies ; Skin ; blood supply ; Skin Transplantation ; methods ; Wound Healing