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Objective To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. Methods The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers’ urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). Results An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers’ urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). Conclusions A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.
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Objective To investigate the mechanism of white matter damage (WMD) and the neuroprotective effect of Xenon on neonates with WMD.Methods Three-day-old SD rat pups (n =96) were randomly divided into the blank control group (n =24),the WMD control group (n =24),the Xenon intervention group A (n =24) and the Xenon intervention group B (n =24) by random number method according to their birth time.WMD rat models were successfully established by giving intraperitoneal injection of lipopolysaccharide(LPS) 0.05 mg/kg combined with carotid artery ligation and hypoxia for 1 hour in the WMD control group and the Xenon intervention groups.In the control group,only 9 g/L saline (0.05 mg/kg) was injected intraperitoneally,while carotid artery ligation and hypoxia were not administered.Rats in Xenon intervention group A and group B were given inhalation of 500 mL/L Xenon for 3 hours at 0 and 2 hours respectively after establishment of the models.Six rats in each group were randomly selected and decapitated at 0,24,48 and 72 hours after the intervention.The brain white matter on the right was analyzed by using HE staining and myelin basic protein(MBP) immunofluorescence staining,and real-time quantitative polymerase chain reaction was used to detect the expressions level of CLIC4 mRNA.Results (1) Brain tissue pathology:compared with the blank control group,the brain white matter on the right of the WMD control group and the Xenon intervention group A and group B had loose and disordered structure,nuclear pyknosis and cytoplasm loosening.However,the lesions in both Xenon intervention group A and group B were significantly less than those in the WMD control group,and there was no significant difference between the Xenon intervention group A and group B.(2) MBP measurement:the number of MBP-positive cells in the brain white matter on the right of WMD control group was significantly lower than that in the blank control group,while compared with WMD control group,they were significantly higher in Xenon intervention group A and group B.(3) CLIC4 mRNA expression level:compared with blank control group,the expressions levels of CLIC4 mRNA at most time point were higher both in the WMD control group and the Xenon intervention group A and group B (all P ＜ 0.05),except the time point 24 h in the Xenon intervention group A.The expressions of CLIC4 mRNA in group A and group B were significantly decreased compared with those in the WMD control group (all P ＜ 0.05).However,there were no significant differences between Xenon intervention group A and group B (P ＞ 0.05).Conclusions The expressions of CLIC4 mRNA in brain tissues on neonatal rats with WMD significantly increased,indicating that the mitochondrial pathway could be one of the pathological processes of WMD.Early Xenon intervention may reduce neonatal WMD by reducing the expression of CLIC4 mRNA,which plays a neuroprotective role.
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Premature infants are at high risk of vitamin D deficiency, thereby commonly treated with vitamin D supplementation after birth. However, the nutritional status of vitamin D in premature infants is not optimistic due to the fact of high incidence of vitamin D deficiency. Studies have found that vitamin D is associated with a variety of diseases in premature infants, including metabolic bone disease, respiratory distress syndrome, bronchopulmonary dysplasia, neonatal necrotizing enterocolitis and infection. Clinicians should be aware of the importance of vitamin D for premature infants and prescribe vitamin D supplementation as early as possible with the dose reference of serum 25-hydroxyvitamin D level.
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Objective To investigate the relationship between the mutation of ERG11 gene,a target of azole antifungal drugs (fluconazole,itraconazole,voriconazole),and azole-resistance in Candida albicans isolates from patients with AIDS.Methods Ninety-three Candida albicans strains were isolated from patients with AIDS.DNA was extracted from these isolates,and ERG11 gene was amplified by PCR followed by bidirectional sequencing.DNAman software was used to compare the resultant sequence with the reference sequence of ERG11 gene (GenBank accession no.X13296).Then,different base sequences were translated into amino acid sequences to determine whether missense mutations occured.Results A total of 40 mutation sites were identified in these isolates,including 27 silent mutations and 13 missense mutations.One or no missense mutation was detected in Candida albicans strains resistant to 1 antifungal agent,while those resistant to 2 or 3 antifungal agents simultaneously harbored 2 or 3 missense mutations.Conclusion The missense mutations in ERG11 gene are probably connected with azole resistance in Candida albicans.
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Objective To analyze the genotypic characteristics of T. violaceum, and offer evidence for molecular epidemiological study of causative agents of pediatric tinea capitis, in Xinjiang Uygur Autonomous Region. Methods The PCR-restriction fragment length polymorphisim (RFLP) of the nontranscribed spacer region of ribosomal DNA (rDNA-NTS) was assessed by using 5 restriction enzymes, including HaeⅢ, Bgl Ⅰ,MspⅠ, DdeⅠ and MboⅠ, for 30 clinical isolates of T. violaceum from children with tinea capitis in Xinjiang Uygur Autonomous Region. Nine strains of T. violaceum from Beijing and 2 strains from Taiwan served as the control.Results All the 41 strains of T. violaceum were classified into 12 genotypes with the restriction enzyme Ddel.Ten genotypes were revealed in the 30 strains from Xinjiang; among them, 17 strains showed 7 different genotypes (D, F, G, H, I, J and K) with a high intraspecies diversity in comparison with the Beijing and Taiwan isolates; the remaining 13 strains from Xinjiang showed 3 genotypes, which were shared by the Beijing and Taiwan isolates. Conclusions The T. violaceum isolates from children with tinea capitis in Xinjiang Uygur Autonomous Region have unique genotypes, but also show genetic homogeneity with the strains from Beijing and Taiwan, hinting the genetic diversity of T. violaceum.