Development of 107 SSR markers from whole genome shotgun sequences of Chinese bayberry (Myrica rubra) and their application in seedling identification.

Chinese bayberry (Myrica rubra Sieb. et Zucc.) is one of the important subtropical fruit crops native to the South of China and Asian countries. In this study, 107 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies, cultivar identification, and linkage map construction, were developed and characterized from whole genome shotgun sequences. M13 tailing for forward primers was applied as a simple method in different situations. In total, 828 alleles across 45 accessions were detected, with an average of 8 alleles per locus. The number of effective alleles ranged from 1.22 to 10.41 with an average of 4.08. The polymorphic information content (PIC) varied from 0.13 to 0.89, with an average of 0.63. Moreover, these markers could also be amplified in their related species Myrica cerifera (syn. Morella cerifera) and Myrica adenophora. Seventy-eight SSR markers can be used to produce a genetic map of a cross between 'Biqi' and 'Dongkui'. A neighbor-joining (NJ) tree was constructed to assess the genetic relationships among accessions, and the elite accessions 'Y2010-70', 'Y2012-140', and 'Y2012-145', were characterized as potential new genotypes for cultivation.

Development of simple sequence repeat (SSR) markers from a genome survey of Chinese bayberry (Myrica rubra).

BACKGROUND: Chinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species. RESULTS: The whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species. CONCLUSION: Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.

[Expression and significance of Notch-1 and Jagged-2 in patients with Hirschsprung disease].

OBJECTIVE: To investigate the expression of Notch-1 and Jagged-2 in the normal and spastic segments of colon in patients with Hirschsprung disease(HD), and to explore the correlation of Notch-1 and Jagged-2 with pathogenesis of HD. METHODS: From 2005 to 2010, resected colon specimens of 30 cases with HD were selected for this study. Normal colonic segments were served as control group, while the transitional and spastic segments as experimental group. Immunohistochemical staining, Western blotting, and RT-PCR were applied to detect the expression of Notch-1 and Jagged-2. RESULTS: A large number of Notch-1 and Jagged-2 positive gangliocytes were observed in the control group, while none was observed in spastic segments. Significantly less Notch-1 and Jagged-2 positive gangliocytes were found in the transitional segments. Western blotting revealed that Notch-1 and Jagged-2 protein levels in spastic segments (0.19±0.02 and 0.13±0.04) were less than that in transitional segments and normal segments (0.58±0.05 and 0.52±0.04, 0.72±0.04 and 0.69±0.04, respectively)(P<0.05). RT-PCR revealed that Notch-1 and Jagged-2 mRNA levels were consistent with protein expression. CONCLUSION: Notch-1 and Jagged-2 are not expressed in spastic colon segments, which may be associated with the pathogenesis of HD.

[Expression of Wnt5a in the terminal rectum of children with anorectal malformation].

OBJECTIVE: To study the expression of Wnt5a protein in the terminal rectum of children with anorectal malformation (ARM) and the possible association between Wnt5a and ARM. METHODS: Specimens were obtained from 20 children with ARM, 7 children with acquired rectovestibular fistula and 6 children with non-gastrointestinal tract disease (control group). The expression of Wnt5a protein in the terminal rectum was determined by immunohistochemistry and Western blot. RESULTS: Wnt5a was mainly expressed in the rectum of the myenteric nerve plexus, mucosal layer and submucosa in the control group. Compared with the control group, Wnt5a expression in the terminal rectum decreased significantly in the ARM group, and decreased more significantly in children with high ARM. The results of Western blot showed the expression of Wnt5a protein in the high, intermediate and low ARM groups were significantly lower than that in the acquired rectovestibular fistula and the control groups (P<0.01). The expression of Wnt5a protein in the high and the intermediate ARM groups were also lower than that in the low ARM group (P<0.01). There was no significant difference in the Wnt5a protein expression between the acquired rectovestibular fistula and the control groups. CONCLUSIONS: The expression of Wnt5a in the termina1 rectum decreases in children with ARM, suggesting Wnt5a may play an important role in the development of ARM.

The development of satellite cells and their niche in striated muscle complex of anorectal malformations rat embryos.

BACKGROUND: It has been demonstrated that different degrees of pelvic floor muscle (PFM) maldevelop in anorectal malformations (ARMs); yet the development of satellite cells, the myogenic stem cells responsible for muscle growth, repair, and maintenance remains elusive during the embryogenesis of PFM. Striated muscle complex (SMC) is one of the most important components of PFM. The objective of this study was to observe the development pattern of satellite cells and their niche of SMC and investigate its possible role in PFM dysplasia in ARMs. METHODS: Immunohistochemistry, cell culture, transmission electron microscopy (TEM), real-time quantitative PCR, and Western blot were performed to trace the dynamic development pattern of satellite cells during the morphogenesis of PFM in ethylenethiourea (ETU)-induced ARMs rat embryos. RESULTS: In ARMs rat embryos, earlier presentation and higher number of Pax7-expressing cell were observed in SMC. The expression of Pax7 and vimentin were up-regulated, while the expression of myogenin, vWF, and neurofilament were down-regulated. Ultrastructure analysis of SMC was characterized by increased amount of nuclear heterochromatin of satellite cell nuclei, thickened basal lamina, widened gap between satellite cell and myofiber, and disarrangement of muscle fibers. The satellite cells demonstrated abnormal differentiation after they were isolated and cultured in vitro. CONCLUSIONS: Our results suggest that premature origination of satellite cell from myogenic progenitor or precursor may result in the depletion of myogenic precursor and cessation of muscle growth; intrinsic defect in satellite cell structure, and extrinsic impairment of microenvironment compromised the myogenic competence of satellite cell, which might contribute substantially to the hypoplastic SMC in ARMs.

Ileal duplication with extensive gastric heterotopia in a girl.

BACKGROUND: Gastrointestinal duplications are rare congenital abnormalities known to occur at any level of the alimentary tract from the mouth to the anus. The cause of intestinal duplication has not been established. Several theories have been put forward to explain different types of duplications. Some of these duplications are large sized and giant, and only 4 cases have been reported. METHODS: A 4-year-old girl was referred to our hospital with a history of abdominal pain, abdominal distension, and diarrhea mixed with black blood for 20 days. Technetium-99m scintigraphy identified heterotopic gastric mucosa at the middle and lower abdominal region. Enteric duplication was suspected. RESULTS: Operatively, duplication was found to be located at the ileum with abnormal hypertrophy in shape, 50 cm of the ileum was resected, and an ileoileal end-to-end anastomosis was made. Stomach-like mucosa and some ring structures were identified instead of the normal intestinal mucosa when opening this ileal duplication. Microscopically, most of mucosa showed gastric corpusfundic glands. CONCLUSIONS: This is an unusual case of enteric duplication. Ultrasonography, computed tomography and technetium-99m scintigraphy are helpful in the diagnosis of duplication.

Apoptosis during the development of pelvic floor muscle in anorectal malformation rats.

PURPOSE: Fecal incontinence and constipation still remain as major postoperative complications after procedures for anorectal malformations (ARM). The striated muscle complex (SMC) is one of the most important factors that influence defecation. Previous studies have demonstrated different degrees of the muscle complex dysplasia dependent on the complexity of ARM. To explore the mechanisms of maldevelopment of SMC in ARM, apoptosis was investigated during pelvic floor muscle development in rat embryos with ARM. METHODS: Anorectal malformations in rat embryos were induced by treating pregnant rats with ethylenethiourea on the 10th embryonic day (E10). Normal and ARM rat embryos from E16 to E21 were serial-sectioned transversely or sagittally, and SMCs were dissected and snap frozen. TdT mediated dUTP Nick Ending Labeling (TUNEL) staining and DNA ladder analysis were performed to identify apoptosis and expression of Bax/Bcl-2 were confirmed with immunohistochemical staining and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis. RESULTS: Hypoplastic and disordered SMC sling shifted cephalad, ventrally, and converged inferior to the rectourethral fistula and infiltrated connective tissue in ARM embryos. In the normal group, TUNEL-positive cells became evident on E17; sporadic positive staining was mainly localized in 2 areas as follows: the junction area between SMC and bulbocarvernosus muscle and posterior to the rectum where bilateral SMC converged. In the ARM group, massive positive staining of nuclei was observed from E16 to E21 and was mainly distributed in the dorsal part of the SMC. Electrophoresis of DNA samples yielded a "ladder" pattern of migration both in normal and the ARM group from E17 to E21, the ladders were stronger in the ARM group. In both groups, the expression of Bax/Bcl-2 was detectable on E17, the immunoreactivity increased on E19 and E21. Compared with the normal group, the expression of Bax was increased, whereas Bcl-2 was declined in the ARM group. Significant upregulation of Bax messenger RNA (mRNA) levels and downregulation of Bcl-2 mRNA levels were observed in ARM embryos. CONCLUSIONS: In the current study, abnormal apoptosis and disturbed expression of Bax/Bcl-2 were identified during SMC development in ARM embryos. It is suggested that precocious, excessive, and dislocated apoptosis might be a fundamental pathogenesis for the maldeveloped SMC in ARM rats. The temporospatial expressions of Bax/Bcl-2 indicate they may have an important role in the regulation of apoptosis of SMC.

Temporal and spatial expression of caudal-type homeobox gene-1 in the development of anorectal malformations in rat embryos.

PURPOSE: The aim of this study was to determine caudal-type homeobox gene-1 (Cdx1) expressions during anorectal development in normal and anorectal malformation (ARMs) embryos and investigate the possible role of Cdx1 in the pathogenesis of ARM. MATERIALS AND METHODS: Anorectal malformation was induced by ethylenethiourea on the 10th gestational day (GD10) in rat embryos. Cesarean deliveries were performed to harvest embryos from GD13 to GD21. The temporal and spatial expression of Cdx1 was evaluated in normal rat embryos (n = 334) and ARM embryos (n = 328) from GD13 to GD20 using immunohistochemistry staining, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: Immunostaining revealed that in normal embryos, on GD13.5, Cdx1 expression was mainly located on the epithelium of the dorsal urorectal septum (URS), cloacal membrane, and the hindgut. On GD15, increased positive tissue staining was noted on the fused tissue of URS, especially in the very thin anal membrane. In the ARM embryos, however, the epithelium of the cloaca, URS, and anorectum was negative or faint for Cdx1. In Western blot and RT-PCR, in the normal group, Cdx1 protein and Cdx1 messenger RNA expression showed time-dependent changes in the developing hindgut, on GD14, GD14.5, and GD15. The expression level reached a peak when the anus was forming. Once the anus was open, Cdx1 expression gradually decreased. In addition, the expression level of Cdx1 in the ARM group from GD13 to GD16 was significant lower than that of the normal group (P < .05). CONCLUSIONS: In ARM embryos, an imbalance of spatiotemporal expression of Cdx1 was noted during anorectal morphogenesis from GD13 to GD16. This suggests that downregulation of Cdx1 at the time of cloacal separation into rectum and urethra might be related to the development of ARM.

Presacral extrarenal Wilms tumor in a child.

OBJECTIVES: Wilms tumor (WT) is one of the most common solid tumors in children. It also is the most frequent tumor found in the kidneys. In 5% of cases, it affects both kidneys simultaneously. Extrarenal WTs are extremely rare. The diagnosis of extrarenal WT is almost always made after surgical intervention. The tumor can be located in the retroperitoneum, uterus, cervix, testes, skin, and even the thorax. METHODS: We report on a 3-year-old girl with an extrarenal WT located in the presacral region. RESULTS: The exact mechanism whereby a WT occurs in extrarenal tissues is unknown. The presence of ectopic metanephric blastema cells or the WT gene causing transformation of extrarenal primitive mesonephric or pronephric remnants into WT have both been considered in the embryogenesis. Although imaging examinations are helpful in the definition of retroperitoneal tumors, no characteristic findings are available to diagnose an extrarenal WT before surgery. CONCLUSIONS: As a rule, the diagnosis of extrarenal WT is made after surgery. Surgical excision is the treatment of choice and the same general therapeutic rules should be followed as when the kidney has been affected. The use of Stage III guidelines for chemotherapy and radiotherapy are appropriate for these patients.

Clinical images. Primary non-Hodgkin's lymphoma of the sigmoid colon in a child.

Primary non-Hodgkin's lymphomas of the gastrointestinal tract are rare in children, and few of these lymphomas are located in the sigmoid colon. The preoperative diagnosis rate is low. Complete resection is indicated if it can be done safely. Combination chemotherapy after resection is indicated.

Mesenteric Castleman disease.

Castleman disease is a rare benign lymph node hyperplasia usually presenting as an asymptomatic mediastinal mass in children. It can present at any extrathoracic site where there is lymphoid tissue. The sites include retroperitoneum, mesentery, axilla, and pelvis. Mesenteric Castleman disease is very rare in children.

Solid cystic papillary tumor of pancreas in eight children.

OBJECTIVE: To estimate the clinical and pathological features of pancreatic solid cystic papillary tumor (SCPT) in children. METHODS: From 2000 to 2005, 8 cases with SCPT of the pancreas were analyzed retrospectively. All cases but one were females. Average age was 12.8 years. By case review, we discussed the clinical and pathological features of SCPT in children. RESULTS: The chief complains were abdominal pain and palpable mass. There were 3 cases in the head, 1 case in the body, and 4 cases in the tail of pancreas. The procedures employed included local resection (1 case), distal pancreatectomy (5 cases), pancreaticoduodenectomy (1 case), and biopsy (1 case). Histological examination showed solid with cystic areas and papillary protrusions in the 8 cases; as for immunohistochemical examinations, the positive rate was 100% for alpha-antitrypsin (AACT), 87.5% for vinmentin, and 62.5% for neuron-specific enolase (NSE). The patients were followed up for 2 months to 4 years but one was lost by follow-up and all were alive postoperatively. SCPT in 2 cases relapsed. CONCLUSION: Occurring predominantly in young females, SCPT is usually curable by surgical resection with a favorable prognosis.

Diagnosis and treatment of mediastinal enterogenous cysts in children.

OBJECTIVE: To investigate the diagnosis and therapy of mediastinal enterogenous cysts in children. METHODS: Clinical data of 17 cases with mediastinal enterogenous cysts within 19 years in our hospital were retrospectively analyzed. RESULTS: One case was intramural esophageal cyst and 16 cases were enteric cysts, two among which were complicated with abdominal enteric duplications. Most cases presented with symptoms of respiratory distress. Twelve cases were complicated with vertebral anomalies. Ultrasound of 12 cases and magnetic resonance imaging of 4 cases were helpful in confirming the cystic nature of these lesions. Eight cases had technetium-99m pertechnetate scintigraphy of posterior mediastinum. CONCLUSIONS: Most patients present with symptoms of respiratory distress, complicated with vertebral anomalies. Ultrasonography and magnetic resonance imaging may be helpful in confirming the cystic nature of these lesions. Technetium-99m pertechnetate scintigraphy is the most effective method for differentiation of the disease from other mediastinal cysts.

[Solid cystic papillary tumor of pancreas in children---a report of eight cases with literature review].

BACKGROUND & OBJECTIVE: Solid cystic papillary tumor (SCPT) of the pancreas in children is rare with limited references of its clinical features and treatment. Therefore, to estimate the clinicopathologic features of SCPT of the pancreas in children is necessary. METHODS: Clinical data of 8 children with pathologically confirmed SCPT of the pancreas, treated from 2000 to 2005 with surgery, were analyzed retrospectively. The clinicopathologic features were analyzed with literature review. RESULTS: All patients were girls but one, with the average age of 12.8 years. The chief complaints were burbulence, abdominal pain, and palpable mass. The tumors were mainly located in the head or tail of the pancreas. Of the 8 patients, 1 received local resection, 5 received distal pancreatectomy, 1 received pancreatico-duodenectomy, and 1 received only biopsy. Seven patients were followed-up for 2 months to 4 years and were still alive; 2 patients suffered recurrence. All cases showed solid with cystic areas and papillary protrusions. Immunohistochemically, the positive rates were 100% for alpha-antitrypsin, 87.5% for vinmentin, and 62.5% for neuron-specific enolase. CONCLUSIONS: SCPT of the pancreas occurs predominantly in young girls, and its pathogenesis is still unknown. Surgical resection is usually curative, and the prognosis is good.