[Effects of Low-density Polyethylene Microplastics on the Growth and Physiology Characteristics of Ipomoea aquatica Forsk].

Microplastic pollution in soil and its toxicological effects have attracted increasing attention from researchers, but the mechanisms of microplastics affecting crop growth and physiology remain unclear. A pot experiment was conducted to evaluate the impacts of various mass concentrations (0%, 0.2%, 5%, and 10%) of low-density polyethylene microplastics (LDPE MPs) on the germination rate, photosynthetic pigment content, biomass, antioxidant enzyme activity, soluble protein, and soluble sugar content of water spinach (Ipomoea aquatica Forsk). The results showed that LDPE MPs significantly inhibited (P<0.05) the seed vigor of water spinach, and the inhibitory effect increased with increasing concentration of LDPE MPs. However, the 5% LDPE MPs significantly promoted the aboveground biomass of water spinach. The 0.2% and 10% LDPE MPs significantly improved the superoxide dismutase (SOD) activity and catalase (CAT) and peroxidase (POD) activities, respectively. Further, malondialdehyde (MDA) content decreased with increasing concentration of LDPE MPs, and the reductions reached 15.53%-27.39% in comparison to that in the control. The LDPE MPs also significantly increased the soluble sugar content of water spinach leaves. In summary, LDPE MPs could inhibit the seed vigor and promote biomass accumulation in water spinach. Water spinach could relieve the oxidative stress caused by LDPE MPs by regulating antioxidant enzyme activity and soluble protein content. Therefore, this study may provide basic information for assessing the influences of microplastics on vegetables.

Parental involvement in postoperative pain management among children in a urology ward: A best practice implementation project.

AIM: Postoperative pain has adverse effects on children with urological problems, including sleep disturbances, incision dehiscence, bleeding and delayed recovery. Accurate parental assessment of children's behaviours and responses could help to manage postoperative pain. We aimed to implement evidence-based practice for parental involvement in a urology ward, to increase parents' participation in children's postoperative pain management. DESIGN: The project was conducted in a paediatric urology ward using the framework and methods of the Fudan University Evidence-Based Nursing Center's Evidence-based Continuous Quality Improvement Model. METHODS: Fifteen audit criteria were used to represent best practice recommendations for parental involvement in postoperative pain management. A pre-implementation audit was conducted with 211 randomly sampled children and parents. Obstacles, promoting factors and key strategies were analysed, and evidence-based interventions implemented to improve compliance. A follow-up audit using the same audit criteria was conducted with 202 children and parents to assess the effect of targeted strategies on compliance with best practice. The SQUIRE guidelines were followed. RESULTS: At the baseline audit, compliance with the evidence-based criteria was 0%-71.5%; only five audit criteria achieved a compliance rate > 60%. After best practice implementation, the follow-up audit showed compliance improvements for all criteria; compliance for three criteria improved to 100%. PATIENT OR PUBLIC CONTRIBUTION: This best practice implementation project improved parents' participation in children's postoperative pain management. The findings demonstrate how audits can promote best practice in postoperative pain management for children. Additional studies will be conducted to address children's postoperative life quality based on best practice.

Development and validation of an UHPLC-Orbitrap-HRMS method for rapid determination of endogenous L-carnitine in patients on hemodialysis/peritoneal dialysis and its application to promote rational drug use.

Background: L-carnitine is an endogenous vitamin-like amino acid derivate which plays an essential role in energy metabolism and can be easily lost via dialysis. Deficiency of L-carnitine has great effects on many aspects of bodily functions. To determine the deficiency degree and adjust the supplementation dose, a rapid, sensitive, and specific method for the detection of endogenous L-carnitine in the plasma of dialysis patients using ultra-high performance liquid chromatography-Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap-HRMS) was developed and validated. Methods: The plasma samples were processed by protein precipitation and centrifugation before analysis using UHPLC-Orbitrap-HRMS. Sample separation was achieved with a hydrophilic interaction liquid chromatography (HILIC) column, using an isocratic elution with a runtime of 5 min. The separated analytes were detected by positive ionization mode in full scan mode and targeted-single ion monitoring (t-SIM) mode. Mildronate was used as the internal standard (IS). Results: All the plasma could be detected in the range of 6.169 to 197.394 µM, with adequate accuracy, precision, and recovery. The method was validated in fortified validation with relative standard deviations (RSD) 5.15-8.74%. This method was applied to the analysis of 105 dialysis patients and 39 healthy participants, the results revealed that peritoneal dialysis patients without L-carnitine supplementation should pay more attention to L-carnitine monitoring, meanwhile, all the hemodialysis patients were advised to be routinely given a full dose of L-carnitine, no matter whether they had taken L-carnitine or not. Conclusions: This study developed a simple and rapid UHPLC-Orbitrap-HRMS method for detection of endogenous L-carnitine in dialysis patients, which could be useful to promote rational drug use.

The effectiveness of a parent participation in postoperative pain management programs for children in a urology ward: A randomized controlled trial.

BACKGROUND: Postoperative pain has adverse effects on children after urology treatment, including sleep disturbance, incision dehiscence, bleeding, and delayed recovery. Parents, as the most direct caregivers of children, can make accurate assessments of children´s personal behaviors and responses, which is very important for the management of postoperative pain in children. PURPOSE: The purpose of the current study was to develop a Parent Participation in Postoperative Pain Management Program for children in a urology ward and to evaluate its effects on children's postoperative pain scores and other outcome indicators. DESIGN: This research comprised two phases. The first phase was the development of a Parent Participation in Postoperative Pain Management Program. The second phase was a randomized controlled trial between two groups, and was carried out in a 45-bed inpatient urology ward of a tertiary children's hospital in China. In the trial, 211 children and their parents were randomly selected as a control group between July 1 and August 15, 2019, and 202 children and their parents were randomly selected as an intervention group between August 16 and September 15, 2019. METHODS: Following the framework and methods of the Evidence-based Continuous Quality Improvement Model developed at Fudan University Evidence-Based Nursing Center, we systematically gathered evidence regarding parental involvement in postoperative pain management in children to construct the program. To evaluate the program's effectiveness, the control group performed routine postoperative pain management, while the intervention group underwent the Parent Participation in Postoperative Pain Management Program. The management period was during hospitalization, and generally ranged 3-7 days. The Statistical Table of Pain Assessment for Children after Urology was employed by researchers. FINDINGS: The results revealed no significant differences in demographic characteristics between the two groups of children and their parents. Children's pain scores during dressing removal (Z = -3.108, p = 0.002), at discharge (Z = -2.185, p = 0.029) and during catheter removal (Z = -6.553, p = 0.000) were significantly lower in the intervention group compared with the control group. CONCLUSIONS AND CLINICAL RELEVANCE: The Parent Participation in Postoperative Pain Management Program was found to be effective for alleviating postoperative pain scores among children, and provided useful information regarding postoperative pain management in children involving four aspects of parental involvement: cognition, guidance, documentation and support.

[Soil denitrifying enzyme activity and its influencing factors in a bamboo forest riparian zone in the upper reaches of the Taihu Lake Basin, China]. / å¤ªæ¹–æµåŸŸä¸Šæ¸¸ç«¹æž—æ²³å²¸å¸¦åœŸå£¤åç¡åŒ–é ¶æ´»æ€§åŠå ¶å½±å“å› ç´ .

Soil denitrifying enzyme activity (DEA) was measured by acetylene inhibition technique, along with exploration of factors influencing DEA in a bamboo forest riparian zone in the upper reaches of the Taihu Lake Basin during summer. Our aim was to provide important insights into the assessment of ecological functions of bamboo forest riparian zone on reducing nitrogen pollution in rivers. The results showed that the riparian soil DEA ranged from 6.32 to 23.22 µg N·kg-1·h-1, with a mean value of 14.65 µg N·kg-1·h-1. The vertical distribution (0-40 cm soil profile) of DEA was affected by several factors, such as soil organic carbon (SOC), total nitrogen (TN), nitrate nitrogen (NO3--N), soil water content, and activity of carbon and nitrogen hydrolase, which resulted in decreased DEA with increasing soil depth. The horizontal changes in DEA (at the same soil depth but at different distances from river) was mainly governed by the variation in SOC concentration. In this area, the concentration of soil dissolved organic carbon was relatively low, which might inhibit the soil DEA during summer.

Combination of high-frequency ultrasound and virtual touch tissue imaging and quantification improve the diagnostic efficiency for mild carpal tunnel syndrome.

BACKGROUND: Carpal tunnel syndrome (CTS) is the most common entrapment symptom in the peripheral nerves. High-frequency ultrasound (HFUS) is widely used in the diagnosis of CTS. Virtual Touch Tissue Imaging and Quantification (VTIQ), which provides more information about the hardness of organization, is used to diagnose CTS. However, the data of diagnostic value of them in various degrees of CTS are limited. Whether the combination of HFUS and VTIQ can improve the diagnostic efficiency also remains unknown. The study aimed to explore the diagnostic value of HFUS and VTIQ in various degrees of CTS and whether combination of HFUS and VTIQ could improve the diagnostic efficiency of CTS. METHODS: A collection and analysis of 133 CTS patients and 35 volunteers from January 2016 to January 2019 were performed. We compared the clinical characteristics, cross-sectional area (CSA) value and shear wave velocity SWVmean value of CTS group with volunteer group. RESULTS: The CSA value and SWVmean value of CTS cohort were significantly higher than volunteer group (10.79 ± 2.88 vs. 8.06 ± 1.39, p < 0.001, 4.36 ± 0.95 vs. 3.38 ± 1.09, p < 0.001, respectively). The area under the curve (AUC) of receiver operating characteristic (ROC) curve of CSA value and SWVmean value were 0.794 and 0.757, respectively. Hierarchical analysis of CSA value and SWVmean value showed that the AUC in the moderate and severe CTS group were higher than in mild CTS group. Furthermore, the CSA value combined with SWVmean value used to diagnose mild CTS was 0.758, which was higher than that of single CSA value or single SWVmean value. CONCLUSIONS: Both HFUS and VTIQ technology were feasible to evaluate CTS. HFUS was suitable for use in diagnosis of moderate and severe CTS. For mild CTS, combination of HFUS and VTIQ was relevant to improve the diagnostic efficiency of CTS.

Identification of novel SMN1 subtle mutations using an allelic-specific RT-PCR.

Spinal muscular atrophy (SMA) is caused by homozygous deletions of the SMN1 gene in approximately 95% of patients. The remaining 5% of patients with SMA retain at least one copy of the SMN1 gene carrying insertions, deletions, or point mutations. Although molecular genetic testing for most SMA patients is quite easy, diagnosing "nondeletion" SMA patients is still compromised by the presence of a highly homologous SMN2 gene. In this study, we analyzed the SMN1/SMN2 copy number by quantitative PCR and multiplex ligation-dependent probe amplification (MLPA). Further, common primers for both SMN1 and SMN2 sequences were used to screen DNA intragenic mutations. To confirm whether the identified mutations occurred in SMN1 or SMN2, we improved the traditional RT-PCR method by only amplifying SMN1 transcripts using an allelic-specific PCR (AS-RT-PCR) strategy. We identified six SMN1 point mutations and small indels in 8 families, which included c.683T>A, c.22dupA, c.815A>G, c.19delG, c.551_552insA and c.401_402delAG. To the best of our knowledge, the latter three have never been previously reported. The most common mutation in Chinese patients is c.22dupA, which was identified in three families. In this work, we demonstrated AS-RT-PCR to be reliable for identifying SMN1 subtle mutations, especially the prevalent mutation c.22dupA in Chinese SMA patients. By reviewing published papers and summarizing reported SMN1 mutations, a distinct ethnic specificity was found in SMA patients from China. Our research extends the SMN1 mutation spectrum.

Novel motif is capable of determining CCR and CCR-like proteins based on the divergence of CCRs in plants.

Cinnamoyl-coenzyme A reductases (CCRs) have been reported as key enzymes involved in monolignol biosynthesis. In this study, a motif-aware workflow based on a new signature motif effectively distinguished CCRs from CCR-like proteins. The divergence of CCRs and CCR-like sequences in Populus tomentosa Carr, Panicum virgatum L, Oryza sativa L and Selaginella moellendorffii Hieron suggests that NWYCY is not efficient for CCR recognition. The novel motif H202(X)2K205 (CCR-SBM or CCR substrate binding motif) was introduced to distinguish between CCRs and CCR-like proteins. The site-directed mutant R205K in Os(I)CCR-like and H202 in PtoCCR7 resulted in the rescue and loss of activity, respectively, further validating the fact that CCR-SBM is critical for maintaining CCR activity. The molecular docking using feruloyl-cinnamoyl-coenzyme A (CoA) as the ligand and binary PhCCR-NADP structures as receptors indicated an interaction between H202 and K205 with CoA moiety. The genuine CCRs and CCR-like proteins from several angiosperms and gymnosperms were screened using a motif-aware workflow and were validated using a biochemical assay. Our results suggest that the motif-aware workflow is efficient and effective for the identification of CCRs and CCR-like proteins in land plants and can be used as a more accurate way of identifying genuine CCRs among land plants.

Association between SCN1A polymorphism rs3812718 and valproic acid resistance in epilepsy children: a case-control study and meta-analysis.

Resistance to valproic acid (VPA), a first-line antiepileptic drug (AED), is occurring at an alarming rate, particularly in children. Signal nucleotide polymorphisms are considered crucial in this process. Therefore, we investigated whether the SCN1A polymorphism rs3812718 could be associated with VPA resistance. A total of 231 children with epilepsy who were solely administered VPA were enrolled. DNA was extracted from the peripheral blood samples and was genotyped by the Mass Array method. Furthermore, a meta-analysis was conducted between the drug responsive and resistant patients who were exposed to voltage-gated sodium channels. Results revealed that the TT genotype was associated with a higher risk of developing drug resistance (OR = 2.636, 95% CI 1.08-6.433, P = 0.033). After adjusting for the risk factors, a significant difference was still observed between the responsive and resistant groups (OR = 2.861, 95% CI 1.141-7.174, P = 0.025). Moreover, the recessive model was associated with a decreased drug resistance (OR = 0.402, 95% CI 0.167-0.968, P = 0.042) after correcting the risk factors. Meta-analysis of nine studies revealed similar results. In conclusion, our results proved that the rs3812718 TT genotype was associated with a high risk of developing drug resistance, and the recessive model could decrease the risk of VPA resistance.

Two distinct cinnamoyl-CoA reductases in Selaginella moellendorffii offer insight into the divergence of CCRs in plants.

MAIN CONCLUSION: Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Selaginella moellendorffii were evaluated, and of these, SmCCR2-1, which has both distinct sequence motifs and catalytic properties, was clustered into a new CCR subgroup. Cinnamoyl-coenzyme A reductases (CCRs) have been reported in many land plants to have critical functions in monolignol biosynthesis. In this study, we performed a genome-wide screen and obtained two distinct SmCCRs from S. moellendorffii. Phylogenetic analysis indicated that SmCCR2 (both SmCCR2-1 and 2-2) and SmCCR3 together with PpaCCR belong to a distinct subgroup of genuine CCRs with variations in the NAD(P)H-binding motif. Enzymatic assays showed detectable activity by both SmCCR1 and SmCCR2-1 toward four hydroxycinnamoyl-CoA esters. SmCCR1, which clustered with reported CCRs from angiosperms and gymnosperms, exhibited specificity toward feruloyl-CoA, while SmCCR2-1 showed a preference for sinapoyl-CoA. Interestingly, the reaction temperature profiles for SmCCR1 and SmCCR2-1 are complementary. Homology models and molecular simulations suggest that the variations in NADPH-binding motifs, especially R(X)6K instead of R(X)5K, affect the NADP+ conformation. Notably, the signature motif NWYCY was replaced with NGYCL in SmCCR1 and with EWYCL in SmCCR2-1, while the signature residues H202 and R253, reported in a previous study, were conserved in SmCCR1 and SmCCR2-1 but varied in SmCCR-like genes. It is likely that NWYCY is not a reliable signature for CCRs in plants. The detectable activity of site-direct mutant S123T of SmCCR1 suggested that S123 which consists of catalytic triad is changeable. Possible evolution process for the emergence of two subgroups of genuine CCRs was also revealed. Altogether, these findings revise our understanding of CCRs with regard to divergence and active sites.

Expression of Nitric Oxide Synthase Isoenzyme in Lung Tissue of Smokers with and without Chronic Obstructive Pulmonary Disease.

BACKGROUND: It has been demonstrated that only 10%-20% cigarette smokers finally suffer chronic obstructive pulmonary disease (COPD). The underlying mechanism of development remains uncertain so far. Nitric oxide (NO) has been found to be closely associated with the pathogenesis of COPD, the alteration of NO synthase (NOS) expression need to be revealed. The study aimed to investigate the alterations of NOS isoforms expressions between smokers with and without COPD, which might be helpful for identifying the susceptibility of smokers developing into COPD. METHODS: Peripheral lung tissues were obtained from 10 nonsmoker control subjects, 15 non-COPD smokers, and 15 smokers with COPD. Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) mRNA and protein levels were measured in each sample by using real-time polymerase chain reaction and Western blotting. RESULTS: INOS mRNA was significantly increased in patients with COPD compared with nonsmokers and smokers with normal lung function (P < 0.001, P = 0.001, respectively). iNOS protein was also higher in COPD patients than nonsmokers and smokers with normal lung function (P < 0.01 and P = 0.01, respectively). However, expressions of nNOS and eNOS did not differ among nonsmokers, smokers with and without COPD. Furthermore, there was a negative correlation between iNOS protein level and lung function parameters forced expiratory volume in 1 s (FEV1) (% predicted) (r = -0.549, P = 0.001) and FEV1/forced vital capacity (%, r = -0.535, P = 0.001). CONCLUSIONS: The expression of iNOS significantly increased in smokers with COPD compared with that in nonsmokers or smokers without COPD. The results suggest that iNOS might be involved in the pathogenesis of COPD, and may be a potential marker to identify the smokers who have more liability to suffer COPD.

Haplotype-based approach for noninvasive prenatal tests of Duchenne muscular dystrophy using cell-free fetal DNA in maternal plasma.

PURPOSE: This study demonstrates noninvasive prenatal testing (NIPT) for Duchenne muscular dystrophy (DMD) using a newly developed haplotype-based approach. METHODS: Eight families at risk for DMD were recruited for this study. Parental haplotypes were constructed using target-region sequencing data from the parents and the probands. Fetal haplotypes were constructed using a hidden Markov model through maternal plasma DNA sequencing. The presence of haplotypes linked to the maternal mutant alleles in males indicated affected fetuses. This method was further validated by comparing the inferred single-nucleotide polymorphism (SNP) genotypes to the direct sequencing results of fetal genomic DNA. Prenatal diagnosis was confirmed with amniocentesis, and those results were interpreted in a blinded fashion. RESULTS: The results showed an average accuracy of 99.98% for the total inferred maternal SNPs. With a mean depth of 30× achieved in the 10-Mb target region of each sample, the noninvasive results were consistent with those of the invasive procedure. CONCLUSION: This is the first report of NIPT for DMD and the first application of a haplotype-based approach in NIPT for X-linked diseases. With further improvements in accuracy, this haplotype-based strategy could be feasible for NIPT for DMD and even other X-linked single-gene disorders.

A novel mutation identified in PKHD1 by targeted exome sequencing: guiding prenatal diagnosis for an ARPKD family.

Autosomal recessive polycystic kidney disease (ARPKD) is a rare hereditary renal cystic disease involving multiple organs, mainly the kidney and liver. Parents who had an affected child with ARPKD are in strong demand for an early and reliable prenatal diagnosis to guide the future pregnancies. Here we provide an example of prenatal diagnosis of an ARPKD family where traditional antenatal ultrasound examinations failed to produce conclusive results till 26th week of gestation. Compound heterozygous mutations c.274C>T (p.Arg92Trp) and c.9059T>C (p.Leu3020Pro) were identified using targeted exome sequencing in the patient and confirmed by Sanger sequencing. Further, the mother and father were revealed to be carriers of heterozygous c.274C>T and c.9059T>C mutations, respectively. Molecular prenatal diagnosis was performed for the current pregnancy by direct sequencing plus linkage analysis. Two mutations identified in the patient were both found in the fetus. In conclusion, compound heterozygous PKHD1 mutations were elucidated to be the molecular basis of the patient with ARPKD. The newly identified c.9059T>C mutation in the patient expands mutation spectrum in PKHD1 gene. For those ultrasound failed to provide clear diagnosis, we propose the new prenatal diagnosis procedure: first, screening underlying mutations in PKHD1 gene in the proband by targeted exome sequencing; then detecting causative mutations by direct sequencing in the fetal DNA and confirming results by linkage analysis.

[A pilot study on spinal muscular atrophy carrier screening in Shanghai region using real-time PCR].

OBJECTIVE: To develop a screening program for spinal muscular atrophy (SMA) carriers, and to assess the carrier frequency and detection rate in Shanghai region. METHODS: Quantitative analysis of the SMN1 gene by real-time PCR was developed using specimens from 15 SMA patients and 76 SMA parents from 38 affected nuclear families. A pilot screening was carried out for 1741 asymptomatic pregnant women. Frequencies of SMN1 alleles were determined with the Hardy-Weinberg equilibrium. RESULTS: Forty five out of the 1741 women were identified as SMA carriers by the presence of single copy of SMN1. The frequencies of no copy, 1 copy, 2 copy and 3 copy alleles were 1.37 U+00D7 10-2, 9.45 U+00D7 10-1, 2.80 U+00D7 10-2 and 1.27 U+00D7 10-2, respectively. The adjusted SMA carrier frequency was 1:35 with a detection rate of 94.49%. For those with a negative screening result, individuals with 3 copies carried a higher residual risk. CONCLUSION: The incidence of SMA carriers in Shanghai region is similar with that in Caucasian populations. Carrier screening has high detection efficiency. An effort should be made to further distinguish SMN1 gene copy numbers for those with more than 2 copies, since accurate determination of 2 and 3 copy allele frequencies is essential for post-screening genetic consulting.

[Molecular and cytogenetic characterization of six 46, XX males due to translocations between the short arms of X and Y chromosomes].

OBJECTIVE: To characterize molecular and cytogenetic abnormalities in six 46, XX males, and to investigate the clinical manifestations and underlying mechanisms in such patients. METHODS: Clinical data of six XX male patients were collected. Karyotyping, multiple polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) were utilized to detect and locate the sex determining region (SRY) gene. RESULTS: PCR and FISH showed that all patients were SRY-positive XX males. All patients have their SRY gene located at the tip of derivative X chromosomes, which have resulted from translocation between short arms of X and Y chromosomes. High resolution karyotyping at 550-750 band level has revealed that the translocation breakpoints were at Xp22.33 and Yp11.2 in three patients. In the remaining patients, the breakpoints were either at Xp22.32 and Yp11.31 or Xp22.31 and Yp11.2. The breakpoints at Xp22.32, Xp22.31 and Yp11.31 were rarely reported. Genotype-phenotype correlation analysis indicated that the clinical manifestations were age-specific. Four adult patients have come to clinical attention due to infertility, with typical features including azoospermia and testis dysgenesis, whereas poorly developed secondary sexual characteristics and short stature were main complaints of adolescence patients, and short stature was the sole symptom in a child patient. CONCLUSION: Combined karyotyping, PCR and FISH are important for the analysis of XX males. Particularly, high resolution karyotyping is valuable for the refinement of chromosome breakpoints and detailed analysis of genotype-phenotype correlation.

[A case with partial trisomy 7 (q34→qter) derived from a paternal reciprocal translocation t(7;14)(q34;q32)].

OBJECTIVE: To determine the origin of chromosomal aberrants in a mentally retarded children, and to correlate the karyotype with phenotype. METHODS: Routine G-banding were performed to analyze the karyotype of the patient and her parents, and array comparative genomic hybridization (array CGH) were used for finely mapping the aberrant regions. RESULTS: The mother had a normal karyotype. The father had an apparently balanced translocation involving chromosome 7q and 14q, the karyotype was 46, XX, t(7;14) (q34;q32), the karyotype of the child was then ascertained as 46, XX, der(14) t(7;14) (q34;q32.33) pat. Array CGH finely mapped the duplication to 7q34-qter, a 17.09 Mb region, and a very small associated deletion of distal chromosome 14 to 14q32.33-qter, a 2.27 Mb region. The patient presented some frequently seen features in partial trisomy 7q cases such as mental retardation, low birth weight, small nose, cleft palate, low-set ears and short neck. CONCLUSION: This result suggested that partial trisomy 7q exert mainly phenotypic effect on the patient. Parental karyotype analysis could help define the aberrant type.

[Clinical application of multiplex ligation-dependent probe amplification for the detection exonic copy number alterations in the Dystrophin gene].

OBJECTIVE: To clarify advantages and disadvantages of using multiplex ligation-dependent probe amplification (MLPA) for detecting exonic deletions and duplications of the Dystrophin gene, and to explore the appropriate management for single-exon abnormality detected by MLPA. METHODS: MLPA were performed to detect exonic copy number changes in 70 Duchenne/Becker muscular dystrophy (DMD/BMD) patients diagnosed by clinical and histological findings. PCR, DNA sequencing and real-time PCR were applied to the samples in which MLPA indicated single-exon deletion or duplication. RESULTS: Of all 70 patients, MLPA detected exonic deletions in 42 (60%), including 12 with single-exon deletion and one with ambiguous single-exon deletion. Exon duplications were found in 7 patients (10%), among which two were single-exon duplication. 21 patients showed normal results (30%). For the 12 patients with single-exon deletion, MLPA results were confirmed by PCR in 11. In one patient, a deletion of two nucleotides (c.4470-4471delAA) was found by sequencing. A novel two-nucleotide deletion (c.4746-4747delCT) was identified in the patient with the ambiguous single-exon deletion. For the two patients showed single-exon duplication, MLPA results were confirmed by real-time PCR. CONCLUSION: MLPA should be the first choice in detecting Dystrophin gene exon deletions and duplications. Single-exon deletion/duplication resulted from MLPA should be further evaluated by other methods.

Three new compounds from soil actinomycete Streptomyces albospinus 15-4-2.

Three new compounds, 2-methyl-2,5,6-bornantriol (1), 4,4'-(3-hydroxypropane-1,1-diyl)diphenol (2), and 7-(4-methoxybenzyl)-4,5,6,7-tetrahydro-1,3-oxazepine-5,6-diol (3), were isolated from the fermentation broth of the soil actinomycete Streptomyces albospinus 15-4-2. Their structures were completely elucidated using the combination of 1D, 2D NMR techniques (COSY, HMQC, HMBC, and ROESY), and HR-ESI-MS analysis. None of the compounds 1-3 showed any inhibitory effect on Fusarium oxysporum f.sp. cubense race 4.

[Optimization of polymerase chain reaction assay combined with capillary electrophoresis to detect the pre- and full mutation of FMR1 gene].

OBJECTIVE: To develop an efficient, high resolution PCR assay suitable for detection of the (CGG)n repeats of the fragile X mental retardation 1 (FMR1) gene by optimizing the PCR system in combination with capillary electrophoresis. METHODS: Three standard samples and twelve samples that were verified by Southern blot analysis including both male and female in the normal, pre- and full mutation range were used in this study to evaluate the enhanced PCR system. All amplicons were electrophoresed by agarose, polyacrylamide and capillary electrophoresis to compare the results. RESULTS: The enhanced PCR assay developed in this study was able to detect a full mutation with (CGG)n being larger than 260 repeats in a male. An expanded pre-mutation allele with (CGG)n as large as 183 repeats in a female was also amplified. The capillary electrophoresis method used in this study was able to distinguish two alleles with 1 CGG repeat difference and the results were reproducible. CONCLUSION: A high resolution PCR assay is developed, which is much more efficient than the general PCR systems. It is suitable for the clinical screening of FMR1 gene and will greatly reduce the number of Southern blot analysis needed in clinical application.