Metastatic pattern and prognosis of gastrointestinal stromal tumor (GIST): a SEER-based analysis.

PURPOSE: This SEER-based study aimed to explore and analyze the relationship of metastasis of liver, lung and bone of GIST patients and their prognosis. METHODS: The data of GIST patients were from Surveillance, Epidemiology, and End Results (SEER) database from 2010 to 2015 and all the statistical analyses were conducted by statistical software package SPSS (Version 22.0). RESULTS: A total of 4224 GIST patients were identified, of which 388 (9.19%) patients with liver metastasis, 20 (0.47%) patients with bone metastasis and 32 (0.76%) patients with lung metastasis. There was no significant difference of risk of bone or lung metastasis between patients with and without liver metastasis (P = 0.935). The median overall survival of patients with liver, bone, or lung metastasis was, respectively, 49 months, 18 months, and 20 months, which were all shorter than that of patients without metastasis. The overall survival of patients with both liver and bone metastasis and those with metastasis of all three sites was not significantly different from that of patients with only liver metastasis. The multivariate analysis showed age of less than 65 years, female patients, married status and receiving surgery were all the beneficial factors for prognosis of GIST patients with liver metastasis. CONCLUSIONS: Patients with metastasis had a poorer prognosis than those without. Liver metastasis might have no relationship with bone or lung metastasis and liver might play a more dominant role than the other two sites in the prognosis of GIST patients with metastasis. So, more attention should be paid to liver status in diagnosis and treatment of GIST patients.

Isolation and characterization of microsatellite markers for Dendranthema morifolium (Asteraceae) using next-generation sequencing.

Dendranthema morifolium (Asteraceae) is a perennial herbaceous plant native to China. A long history of artificial crossings may have resulted in complex genetic background and decreased genetic diversity. To protect the genetic diversity of D. morifolium and enabling breeding of new D. morifolium cultivars, we developed a set of molecular markers. We used pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform, to isolate D. morifolium simple sequence repeats (SSRs). A total of 32,863 raw reads containing 2251 SSRs were obtained. To test the effectiveness of these SSR markers, we designed primers by randomly selecting 100 novel SSRs, and amplified them across 60 cultivars representing five different petal shape groups. Sixteen SSRs were polymorphic with the number of alleles ranging from 6 to 19, and their expected and observed heterozygosities ranging from 0.477 to 0.848, and 0.250 to 0.804, respectively. The polymorphism information content ranged from 0.459 to 0.854 and the inbreeding coefficient ranged from -0.119 to 0.759. An unweighted pair-group method arithmetic average analysis was performed to survey the phylogenetic relationships of these 60 cultivars and five clusters were identified. These markers can be used for investigating genetic relationships and identifying elite alleles through linkage and association analyses.

Relationship between the G75A polymorphism in the apolipoprotein A1 (ApoA1) gene and the lipid regulatory effects of pravastatin in patients with hyperlipidemia.

In this study, we investigated the relationship between the G75A polymorphism in the apolipoprotein A1 (ApoA1) gene and the lipid regulatory effect of pravastatin in patients with hyperlipidemia. A total of 179 patients were divided into two groups: the pravastatin (N = 97) and policosanol (N = 82) treatment groups. The total cholesterol (TC), triglyceride, low-density lipoprotein (LDL-c), high-density lipoprotein, ApoA, and ApoB concentrations in the serum were measured using an automatic biochemical analyzer before and after treatment for 12 weeks. The genotypes of the ApoA1 G75A SNP were detected by polymerase chain reaction-restriction fragment length polymorphism, and were subsequently statistically analyzed. Pravastatin treatment induced a significant decrease in the TC, LDL-c, and ApoB levels in patients expressing the ApoA1 AA+GA genotype (P < 0.05), and not in those expressing the GG genotype (P > 0.05). However, policosanol treatment induced a non-significant decrease in the serum TC levels (P > 0.05) and a significant decrease in the ApoB levels (P < 0.05), and did not induce a decrease in the LDL-c (P > 0.05) levels in patients with the AA+GA genotype. Policosanol also induced a significant decrease in the TC and LDL-c levels in patients with the GG genotype (P < 0.05). The various genotypes of the ApoA1 G75A SNP influence the efficacy of lipid regulation by pravastatin and policosanol in patients with hyperlipidemia.

Isolation and characterization of microsatellite markers for Eucommia ulmoides (Eucommiaceae), an endangered tree, using next-generation sequencing.

Eucommia ulmoides Oliver, a single extant species of Eucommiaceae, is an endemic dioecious tree in China. The natural resources of E. ulmoides have rapidly declined in recent years because of the over-collection of its cortex. To design a suitable protection strategy, it is necessary to develop a set of molecular markers to investigate genetic diversity and population structure of E. ulmoides. Pyrosequencing of an enriched microsatellite library by Roche 454 FLX+ platform was used to isolate simple sequence repeats (SSRs) for E. ulmoides. A total of 1568 SSRs that contained enough flanking sequences for primer pair design were identified from 45,236 raw sequence reads. One hundred SSRs were randomly selected to design primer pairs and polymerase chain reaction was performed. Among these 100 tested primer pairs, 16 were polymorphic across 18 individuals from three E. ulmoides populations. The number of alleles ranged from 3 to 8, with an average of 5.1. The expected heterozygosity ranged from 0.110 to 0.830, with an average of 0.648, and the observed heterozygosity ranged from 0.111 to 0.833, with an average of 0.524. The inbreeding coefficient ranged from -0.349 to 0.547. This set of microsatellite markers could be valuable for landscape genetic structure assessment and molecular marker-assisted breeding in E. ulmoides.

Analysis of the relationship between ribosomal DNA ITS sequences and active components in Rhodiola plants.

Rhodiola plants are a valuable resource in traditional Chinese medicine. The objective of this study was to evaluate the correlation between ribosomal DNA internal transcribed spacer (ITS) sequences and the three active components in Rhodiola plants. For this, we determined ITS sequence polymorphisms and the concentrations of active components salidroside, tyrosol, and gallic acid in different Rhodiola species from the Tibetan Plateau. In a total of 23 Rhodiola samples, 16 different haplotypes were defined based on their ITS sequences. Analysis of the active components in these same samples revealed that salidroside was not detected in species with haplotypes H4, H5, or H10, tyrosol was not detected with haplotypes H3, H5, H7, H10, H14, or H15, and gallic acid was detected in with all haplotypes except H14 and H15. In addition, the concentrations of salidroside, tyrosol and gallic acid varied between samples with different haplotypes as well as those with the same haplotype, implying that no significant correlation exists between haplotype and salidroside, tyrosol or gallic acid concentrations. However, a statistically significant positive correlation was observed for among these three active components.

Isolation and characterization of microsatellite markers for Osmanthus fragrans (Oleaceae) using 454 sequencing technology.

Osmanthus fragrans (Oleaceae) is an evergreen shrub or small tree that grows in south China. In this study, Roche 454 FLX+ sequencing combined with the magnetic bead enrichment method was used to isolate microsatellite markers from the genome of O. fragrans. A total of 1471 microsatellites that contained enough flanking sequences for primer pair design were identified from 89,633 raw sequencing reads. One hundred primer pairs were randomly chosen to test primer amplification efficiency. Among these tested primer pairs, 20 yielded polymorphic amplification products across 16 individuals from the Albus, Luteus, and Aurantiacus groups. The number of alleles ranged from 2 to 6, with an average of 3.7. The observed heterozygosity ranged from 0 to 0.813, with an average of 0.460. Shannon's information index ranged from 0.463 to 1.707, with an average of 0.975. Six loci (Of 05, Of 06, Of 08, Of 12, Of 15, and Of 19) deviated significantly from Hardy-Weinberg equilibrium (P < 0.05), which was due to an excess of homozygotes or heterozygotes. Nine pairs of loci (Of 01 and Of 05; Of 04 and Of 05; Of 01 and Of 06; Of 04 and Of 12; Of 02 and Of 13; Of 04 and Of 13; Of 12 and Of 13; Of 04 and Of 19; Of 05 and Of 19) showed significant linkage disequilibrium, which indicated significant allelic association between the loci. This set of microsatellite markers will be valuable for molecular marker-assisted breeding in O. fragrans.

Protective effects of bifidobacteria on intestines in newborn rats with necrotizing enterocolitis and its regulation on TLR2 and TLR4.

We established a necrotizing enterocolitis (NEC) rat model and explored the role of bifidobacteria in the intestines of the rats and its regulation on intestinal Toll-like receptors (TLRs). Seventy-five newborn Sprague-Dawley rats were randomly divided into 5 groups (15 rats/group): group A, artificial feeding group (formula-fed); group B, NEC model (LPS + formula-fed); group C, bifidobacterium (LPS + formula-fed + bifidobacterium microcapsules, intragastric administration); group D, artificial feeding + bifidobacterium (formula-fed + bifidobacterium microcapsules gavage); group E, rat breast-feeding group (rat breast-feeding). After 3 days of feeding, rats were placed in incubators, fasted for 12 h, and killed by decapitation. The ileocecal proximal segment ileum was fixed and sliced; pathological examination was conducted, and TLR2, TLR4, and nuclear factor-kB p65 protein expression in the intestinal tissue was detected by immunohistochemistry. There was a statistically significant difference in pathological scores between groups C and B (H = 21.789, P = 0.000), and the former was lower than the latter. TLR2, TLR4, and nuclear factor-kB p65 expression in intestinal tissue was determined in groups A-E. There were statistically significant differences between groups C and B (P = 0.001; P = 0.000; P = 0.000). Bifidobacteria had a protective effect on the intestines of newborn rats with NEC, which showed reduced NEC and intestinal damage severity. This observation may be related to the reduced levels of TLR2, TLR4, and nuclear factor-kB P65 observed during the inflammatory response.

Effect of Bifidobacterium on the mRNA expression levels of TRAF6, GSK-3ß, and microRNA-146a in LPS-stimulated rat intestinal epithelial cells.

We investigated the effect of inactivated Bifidobacterium on the mRNA expression of TRAF6, GSK-3ß, and microRNA-146a in lipopolysaccharide (LPS)-stimulated rat small intestinal epithelial cells (IEC-6s). IEC-6s were randomly divided into an LPS group, a culture supernatant group, and an inactivated bacteria group. After stimulation with LPS for 5 h, the three groups were treated as follows: the LPS group was cultured for 24 h with sterile saline; the culture supernatant group was cultured with Bifidobacterium (infantis strain) culture supernatant for 24 h; and the inactivated bacteria group was cultured with inactivated infantis Bifidobacterium for 24 h. Reverse transcription polymerase chain reaction was used to determine mRNA expression levels. The mRNA expression levels of TRAF-6 and GSK-3ß in the culture supernatant group were lower, and microRNA-146a expression was higher, compared with the LPS group (t = 5.278, P = 0.000; t = 6.316, P = 0.000; t = 13.218, P = 0.000, respectively). GSK-3ß mRNA expression in the inactivated bacteria group was lower than in the LPS group (t = 4.837, P = 0.000). There was no difference in the mRNA expression levels of TRAF-6 and microRNA-146a between the two groups (t = 0.732, P = 0.472 and t = 1.463, P = 0.164). Both the culture supernatant and the inactivated Bifidobacterium had a protective effect on LPS-stimulated IEC-6s. The protective effect of Bifidobacterium may be achieved through increased microRNA-146a by reducing levels of TRAF6 and GSK-3ß; the protective effect of inactivated Bifidobacterium may be achieved by reducing levels of GSK-3ß.

CMTM3 is reduced in prostate cancer and inhibits migration, invasion and growth of LNCaP cells.

PURPOSE: A novel tumor suppressor gene CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) is reduced or undetectable in many kinds of cancers and relates tumor malignant features. We detected its role in prostate cancer for possibility of target therapy as accumulating evidence has shown that CMTM3 is a promising tumor suppressor gene (TSG) for gene therapy. METHODS: The expression of CMTM3 detected in prostate tissue microarray, specimens and cell lines were evaluated by immunohistochemistry and semi-quantitative PCR and Western blot, respectively. After being transfected with CMTM3 adenovirus or vector (mock), the proliferation and migration and invasion of LNCaP cells were detected by transwell assay and matrigel assay, respectively. Furthermore, the effects of CMTM3 on tumor growth were performed in nude mice xenograft in vivo. RESULTS: We found CMTM3 was reduced in PCa tissues and cells compared with BPH tissues, and its expression in PCa tissues was related to the Gleason score. Moreover, after being transfected with adenovirus, ectopic expression of CMTM3 in LNCaP cells led to significant inhibition of cell proliferation and migration and invasion compared with the control (P < 0.05), which may be attributed to decreased Erk1/2 activity as p-Erk1/2 was remarkably reduced when CMTM3 was overexpressed. Finally, restoration of CMTM3 significantly suppressed xenograft tumor growth in vivo (P < 0.01).

Selection of a novel DNA thioaptamer against HER2 structure.

PURPOSE: Human epithelial growth factor receptor 2 (HER2) is over-expressed in several malignancies and represents an important therapeutic target. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand with excellent affinity and specificity for targeted cancer therapy. However, aptamers need to have nuclease resistance in order to function in vivo. The aim of this study was to generate a novel HER2 thioaptamer with enhanced nuclease resistance. METHODS: The HER2 thioaptamer is selected in an evolutionary process called systematic evolution of ligands by exponential enrichment. RESULTS: The thioaptamer could bind to the extracellular domain of HER2 with a K d of 172 nM and had minimal cross reactivity to trypsin or IgG. Moreover, the thioaptamer was found capable of binding with the HER2-positive breast cancer cells SK-BR-3 and MDA-MB-453, but not the HER2-negative cells MDA-MB-231. Notably, the thioaptamer HY6 largely maintained its structural integrity facing the nucleases in serum, while regular DNA aptamers were mostly digested. Additionally, the thioaptamer retained the capability of binding with the HER2-positive cells in the presence of serum, whereas non-thionated HER2 aptamer lost the binding function. CONCLUSION: The results indicated that the selected thioaptamer was more resistant to nuclease than regular DNA aptamers and might potentially function as a HER2-targeting ligand in complicated environment.

Assessment of the genetic diversity of tomato yellow leaf curl virus.

The objective of the present study was to analyze the genetic diversity of tomato yellow leaf curl virus (TYLCV). Representative TYLCV sequences were searched in the National Center for Biotechnology Information database. Comprehensive analysis of TYLCV was performed using bioinformatics by examining gene structure, sequence alignments, phylogeny, GC content, and homology. Forty-eight representative TYLCV sequences were selected from 48 regions in 29 countries. The results showed that all TYLCV sequences were 2752-2794 nucleotides in length, which encoded 6 open reading frames (AV1, AV2, AC1, AC2, AC3, and AC4). GC content ranged from 0.41-0.42. Sequence alignment showed a number of insertions and deletions within these TYLCV sequences. Phylogenetic tree results revealed that the sequences were divided into 10 classes; homology of the sequences ranged from 72.8 to 98.6%. All 48 sequences contained the typical structure of TYLCV, including open reading frames and intergenic regions. These results provide a theoretical basis for the identification and evolution of the virus in the future.

Toll-like receptor 4 promotes fibrosis in bleomycin-induced lung injury in mice.

The specific role of Toll-like receptor 4 (TLR4) in bleomycin-induced lung fibrosis of mice, a model of human idiopathic pulmonary fibrosis, has not been characterized. We injected bleomycin intratracheally into TLR4 knockout (TLR4(-/-)) and wild-type (WT) mice. Twenty-one days after injection, mice were sacrificed and their lungs were harvested for pathological, hydroxyproline, mRNA expression, and collagen I analyses. Body weight changes and mortality were observed. Light microscopy showed that lung fibrosis was minimal in TLR4(-/-) compared to that in WT mice on day 21 after bleomycin instillation. The Ashcroft score was significantly lower in TLR4(-/-) than in WT mice (3.667 ± 0.730 vs 4.945 ± 0.880, P < 0.05). Hydroxyproline content was significantly lower in TLR4(-/-) than in WT mice on day 21 after bleomycin injection (0.281 ± 0.022 vs 0.371 ± 0.047, P < 0.05). Compared to WT mice, bleomycin-treated TLR4(-/-) mice expressed significantly lower type I collagen mRNA levels (mesenchymal marker; 11.069 ± 2.627 vs 4.589 ± 1.440, P < 0.05). Collagen I was significantly lower in TLR4(-/-) than in WT mice (0.838 ± 0.352 vs 2.427 ± 0.551, P < 0.05). Bleomycin-treated TLR4(-/-) mice had a significantly lower mortality rate on day 21 than WT mice (33 vs 75%, P < 0.05). Body weight reduction was lower in TLR4(-/-) mice than in WT mice; this difference was not statistically significant (-3.735 ± 5.276 vs -6.698 ± 3.218, P > 0.05). Thus, bleomycin-induced pulmonary fibrosis is TLR4-dependent and TLR4 promoted fibrosis in bleomycin-challenged mice.

Ultrasonic imaging characteristics of transplanted kidneys with delayed graft function.

We investigated the ultrasonic imaging characteristics of transplanted kidneys with delayed graft function (DGF). Ultrasonography was performed in 44 patients after kidney transplantation, and a time-intensity analysis was performed to compare the differences between patients with normal graft function (NGF) and those with DGF. Compared with the NGF group, the DGF group had earlier arrival time, shorter time to peak, and higher arrival intensity and peak intensity (P < 0.05). The variation-of-intensity parameters in different renal cortices increased, whereas the variation-of-time parameter decreased, in those with DGF (P < 0.05). In conclusion, compared with the NGF group, the microcirculation perfusion of transplanted kidneys in the DGF group showed higher perfusion with earlier arrival time, shorter time to peak, and higher arrival intensity and peak intensity. In addition, the intensity variations of contrast agent in different renal cortices from patients with DGF were greater, whereas the variations in perfusion time were smaller than those in patients with NGF.

Vitamin D receptor genetic variants are associated with susceptibility of gallbladder adenocarcinoma in a Chinese cohort.

The aim of this study was to test for the possible association between vitamin D receptor (VDR) genetic variants and susceptibility to gallbladder cancer (GBC). A total of 291 GBC cases were recruited and 396 gender- and age-matched healthy volunteers were enrolled as controls. The VDR gene polymorphisms were determined in all subjects. The genotype and the allele frequencies of ApaI, BsmI, and TaqI polymorphisms were not significantly different between GBC subjects and controls. However, the genotype and allele frequencies of the FokI C>T polymorphism were significantly different between GBC subjects and controls. The FokI TT genotype was in markedly higher frequency in GBC subjects compared to controls (38.14 vs 22.73%, P < 0.001). Using TT as the reference genotype, multivariate logistic regression analysis showed that CC genotype carriers had a higher risk of GBC (adjusted odds ratio (OR) = 3.423, adjusted P = 0.001) with adjustment for age, gender, smoking status, alcohol use, and gallstone presence, as well as the serum 1,25(OH)2D level. Carriers of the CT genotype also had a higher risk of GBC (adjusted OR = 1.992, adjusted P = 0.003). Multivariate logistic regression analysis did not reveal any association between the ApaI, BsmI, and TaqI polymorphisms and GBC risk (all P > 0.05).

Differentiation of neonatal dorsal root ganglion-derived neural stem cells into oligodendrocytes after intrathecal transplantation into a cauda equina lesion model.

Cauda equina syndrome (CES) is characterized by varying patterns of low back pain, sciatica, lower extremity sensorimotor loss, and bowel and bladder dysfunction. The prognosis for complete recovery of CES is dependent on not only the time before surgical intervention with decompression but also the severity of the nerve damage. Delayed or severe nerve compression impairs the capability of nerve regeneration. Transplantation of neural stem cells (NSCs) may facilitate axon regeneration and functional recovery in a spectrum of neurological disorders. Our study shows that the NSCs derived from early postnatal dorsal root ganglion (DRG) are able to proliferate to form neurospheres and differentiate into O4(+) oligodendrocytes but not glial fibrillary acidic protein (GFAP(+)) astrocytes or ßIII-tubulin(+) neurons in vitro. After intrathecal transplantation into the lumbar spinal canal stenosis animal model, most of the GFP-expressing NSCs were induced to differentiate into oligodendrocytes in vivo. Although the recovery of sensorimotor function was not significantly improved in rats with transplantation therapy, our results implied that subarachnoid microinjection of NSCs may promote axon regeneration of DRG neurons in the cauda equina model after nerve injury.