Colorectal cancer cell-secreted exosomal miRNA N-72 promotes tumor angiogenesis by targeting CLDN18.

Angiogenesis is essential for the growth and metastasis of several malignant tumors including colorectal cancer (CRC). The molecular mechanism underlying CRC angiogenesis has not been fully elucidated. Emerging evidence indicates that secreted microRNAs (miRNAs) may mediate the intercellular communication between tumor cells and neighboring endothelial cells to regulate tumor angiogenesis. In addition, exosomes have been shown to carry and deliver miRNAs to regulate angiogenesis. miRNA N-72 is a novel miRNA that plays a regulatory role in the EGF-induced migration of human amnion mesenchymal stem cells. However, the relation between miRNA N-72 and cancer remains unclear. We here found that CRC cells could secrete miRNA N-72. A high miRNA N-72 level was detected in the serum of CRC patients and the cultured CRC cells. Moreover, the CRC cell-secreted miRNA N-72 could promote the migration, tubulogenesis, and permeability of endothelial cells. In addition, the mouse xenograft model was used to verify the facilitating effects of miRNA N-72 on CRC growth, angiogenesis, and metastasis in vivo. Further mechanism analysis revealed that CRC cell-secreted miRNA N-72 could be delivered into endothelial cells via exosomes, which then inhibited cell junctions of endothelial cells by targeting CLDN18 and consequently promoted angiogenesis. Our findings reveal a novel mechanism of CRC angiogenesis and highlight the potential of secreted miRNA N-72 as a therapeutic target and a biomarker for CRC.

Solubilization of luteolin in PVP40 solid dispersion improves inflammation-induced insulin resistance in mice.

Our previous studies have confirmed that luteolin (LU) has a good therapeutic effect on obesity and its complications. However, due to its poor water solubility, the bioavailability is low with limited clinical application. Therefore, the water-soluble solid dispersions (SD) of luteolin were prepared with polyvinylpyrrolidone (PVP) (K10, K40 & K90) by solvent evaporation. The polyvinylpyrrolidone K40 (PVP40) was selected as the ideal carrier to formulate polyvinylpyrrolidone K40-luteolin solid dispersion (PVP40-LU SD), thereby the solubility of luteolin increased about 250 times compared to the pure luteolin, without changing its physical stability and activity. The crystallinity of luteolin was reduced after the formation of solid dispersion, and no strong drug-polymer interactions were observed. This prepared water-soluble luteolin inhibits the polarization of inflammatory macrophages by decreasing the expression of pro-inflammatory cytokine genes interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in vitro. Moreover, it can improve glucose tolerance and insulin sensitivity quickly after intraperitoneal injection in mice.

Hsa_circ_0001696 modulates cell proliferation and migration in colorectal cancer.

Circular RNAs (circRNAs) are a novel class of endogenous non-coding RNA molecules that are extensively expressed in a variety of species. Recently, increasing evidence suggests that circRNAs have vital functions indifferent types of human cancer, such as gastric cancer, papillary thyroid cancer and lung cancer. However, the roles of circRNAs in the development of colorectal cancer (CRC) remain unclear. The present study aimed to determine the molecular mechanism underlying hsa_circ_0001696 on the proliferation and migration of CRC cells. Reverse transcription-quantitative PCR analysis was performed to detect hsa_circ_0001696 expression in 18 paired CRC tissues and matched adjacent normal tissues. RNA interference was also performed to decrease hsa_circ_0001696 expression, and its biological effects were further assessed via flow cytometry, wound healing, colony formation and western blot assays. The results demonstrated that hsa_circ_0001696 expression was significantly lower in CRC tissues compared with adjacent normal tissues. Furthermore, hsa_circ_0001696 knockdown promoted cell proliferation and migration, and the number of cell colonies significantly increased. In addition, western blot analysis demonstrated that the protein expression levels of cyclin-dependent kinase 4 (CDK4), cyclin D, cyclin E and matrix metalloproteinase 9 (MMP9) increased. Taken together, the results of the present study demonstrated that hsa_circ_0001696 expression was downregulated in CRC tissues, and inhibition of hsa_circ_0001696 promoted cell proliferation and migration by regulating the levels of CDK4, cyclin D, cyclin E and MMP9.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

BRAF activated non-coding RNA (BANCR) promoting gastric cancer cells proliferation via regulation of NF-κB1.

BACKGROUND AND OBJECTIVE: Long non-coding RNA, BANCR, has been demonstrated to contribute to the proliferation and migration of tumors. However, its molecular mechanism underlying gastric cancer is still unknown. In present study, we investigated whether BANCR was involved in the development of gastric cancer cells via regulation of NF-κB1. METHODS: Human gastric cancer tissues were isolated as well as human gastric cell lines MGC803 and BGC823 were cultured to investigate the role of BANCR in gastric cancer. RESULTS: BANCR expression was significantly up-regulated in gastric tumor tissues and gastric cell lines. Down-regulation of BANCR inhibited gastric cancer cell growth and promoted cell apoptosis, and it also contributed to a significant decrease of NF-κB1 (P50/105) expression and 3'UTR of NF-κB1 activity. Overexpression of NF-κB1 reversed the effect of BANCR on cancer cell growth and apoptosis. MiroRNA-9 (miR-9) targeted NF-κB1, and miR-9 inhibitor also reversed the effects of BANCR on gastric cancer cell growth and apoptosis. CONCLUSION: BANCR was highly expressed both in gastric tumor tissues and in cancer cells. NF-κB1 and miR-9 were involved in the role of BANCR in gastric cancer cell growth and apoptosis.

Anatomical characteristics of deer and sheep lumbar spines: comparison to the human lumbar spine / Características anatómicas de la columna vertebral de ciervos y ovejas: comparación con la columna vertebral humana

Deer and sheep spines are often used as models of the human spine. A prerequisite for the use of animal models is information regarding the interspecies differences in the parameters of general interest. This would clarify the limitations of each animal model and substantiate the applicability of the obtained results to humans. Since sufficient data appear to be currently unavailable, we sought to investigate the feasibility of using deer and sheep as animal models for studies on the human spine. The objective of this study was a thorough comparison of the anatomical parameters of deer and sheep spines with those of the human spine. We employed three-dimensional reconstructions of computed tomography images, generated using figure analysis software, which facilitated quantitative analysis of the linear and curvature parameters and the geometric index of the vertebral bodies. Our findings represent a comprehensive database of the anatomical characteristics of the deer and sheep lumbar spines and their comparisons with those of the human lumbar spine. This study provides insight into the similarities and differences in the vertebral geometries between the human spine and the deer and sheep spines. We found that the differences are minimal and that they do not greatly compromise the utility of deer and sheep lumbar spines as models of the human lumbar spine.

La columna vertebral de ciervos y ovejas se utiliza frecuentemente como modelo de la columna vertebral humana. Un requisito previo para el uso de modelos animales es la información con respecto a las diferencias entre especies en los parámetros de interés general, lo que aclara las limitaciones de cada modelo animal y fundamenta la aplicabilidad de los resultados obtenidos para los seres humanos. Debido a que existen datos suficientes actualmente, hemos intentado investigar la viabilidad de utilizar ciervos y ovejas como modelos animales para los estudios sobre la columna vertebral humana. El objetivo fue realizar una comparación exhaustiva de los parámetros anatómicos de las columnas de ciervos y ovejas, con los de la columna vertebral humana. Empleamos reconstrucciones tridimensionales de imágenes de tomografía computadorizada, mediante un programa de análisis de la figura, lo que facilitó el análisis cuantitativo de los parámetros lineales y de la curvatura y el índice geométrico de las vértebras. Nuestros hallazgos representan una amplia base de datos de las características anatómicas de la columna lumbar de los ciervos y ovejas y sus comparaciones con las de la columna lumbar humana. Este estudio proporciona información sobre las similitudes y diferencias en las geometrías vertebrales entre la columna vertebral humana y las columnas de venado y oveja. Se encontró que las diferencias son mínimas y que no comprometen el uso de la columna de ciervos y ovejas como modelos de la columna lumbar humana.

[Effect of maternal high-fat diet before and during pregnancy on bone growth of neonatal offspring rats].

OBJECTIVE: To explore the mechanism and effect of maternal high-fat diet before and during pregnancy on bone growth of neonatal offspring rats. METHODS: Forty female Sprague-Dawley rats were divided into high-fat diet and control groups (n=20) that were fed with 35% high-fat diet and standard chow, respectively. After 8 weeks, 8 female rats from each group were sacrificed for liver pathological examinations and the other female rats were mated with male rats and fed continuously with 35% high-fat diet and standard chow throughout gestation, respectively. The body lengths (from apex nasi to end of tail) of the offspring rats from both groups were measured within 24 hours after birth. Enzyme-linked immunosorbent assay was used to detect serum insulin-like growth factor (IFG-I) levels. Liver pathological changes were observed under a light microscope. The expression of insulin receptor substrate 1 (IRS-1) and phosphorylation IRS-1 (Phospho-IRS-1) in tibia and femur samples were detected by immunohistochemistry. The expression of mitogen-activated protein kinase (MAPK) and phosphorylation MAPK (Phospho-MAPK), phosphatidylinositol 3-kinase (PI3K) and phosphorylation PI3K (Phospho-PI3K), protein kinase B (AKT1) and phosphorylation AKT1 (Phospho-AKT1) in tibia and femur samples were detected by Western blot. RESULTS: The offspring rats from the high-fat diet group showed a significant shorter body length compared with those from the control group (P<0.05). The level of serum IGF-I in offspring rats from the high-fat diet group decreased by 20.1% in comparison to those from the control group, but there was no significant difference between the two groups (P>0.05). Fatty degeneration was found in livers of both high-fat diet-fed maternal rats and their offspring rats under a light microscope. There were no significant differences in IRS-1 and Phospho-IRS-1 expression in chondrocytes of tibia and femur samples between the offspring rats of the two groups (P>0.05). The protein expression of MAPK in chondrocytes of tibia and femur samples of offspring rats from the high-fat diet group was higher than that from the control group (P<0.05). There were no significant differences of PI3K and AKT1/Phospho-AKT1 between the offspring rats of the two groups (P>0.05). CONCLUSIONS: A maternal high-fat diet before and during pregnancy may affect the bone growth of offspring rats in utero, which is possibly associated with the decreased IGF-I level. However, further study on the exact mechanism of IGF-I on the bone growth is needed.

[Production of mature red blood cell by using peripheral blood mononuclear cells].

Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.

Identification of genes required for nonhost resistance to Xanthomonas oryzae pv. oryzae reveals novel signaling components.

BACKGROUND: Nonhost resistance is a generalized, durable, broad-spectrum resistance exhibited by plant species to a wide variety of microbial pathogens. Although nonhost resistance is an attractive breeding strategy, the molecular basis of this form of resistance remains unclear for many plant-microbe pathosystems, including interactions with the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzae (Xoo). METHODS AND FINDINGS: Virus-induced gene silencing (VIGS) and an assay to detect the hypersensitive response (HR) were used to screen for genes required for nonhost resistance to Xoo in N. benthamiana. When infiltrated with Xoo strain YN-1, N. benthamiana plants exhibited a strong necrosis within 24 h and produced a large amount of H(2)O(2) in the infiltrated area. Expression of HR- and defense-related genes was induced, whereas bacterial numbers dramatically decreased during necrosis. VIGS of 45 ACE (Avr/Cf-elicited) genes revealed identified seven genes required for nonhost resistance to Xoo in N. benthamiana. The seven genes encoded a calreticulin protein (ACE35), an ERF transcriptional factor (ACE43), a novel Solanaceous protein (ACE80), a hydrolase (ACE117), a peroxidase (ACE175) and two proteins with unknown function (ACE95 and ACE112). The results indicate that oxidative burst and calcium-dependent signaling pathways play an important role in nonhost resistance to Xoo. VIGS analysis further revealed that ACE35, ACE80, ACE95 and ACE175, but not the other three ACE genes, interfered with the Cf-4/Avr4-dependent HR. CONCLUSIONS/SIGNIFICANCE: N. benthamiana plants inoculated with Xoo respond by rapidly eliciting an HR and nonhost resistance. The oxidative burst and other signaling pathways are pivotal in Xoo-N. benthamiana nonhost resistance, and genes involved in this response partially overlap with those involved in Cf/Avr4-dependent HR. The seven genes required for N. benthamiana-mediated resistance to Xoo provide a basis for further dissecting the molecular mechanism of nonhost resistance.

Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses.

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.

[Influence of cytokine combinations on proliferation and differentiation of umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro].

In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.

[To identify a novel HLA-DRB1 allele in Chinese by sequencing].

OBJECTIVE: To identify a novel HLA-DRB1 allele in Chinese. METHODS: A novel HLA-DR allele was detected by PCR-SSP and SBT in a patient with leukemia. RESULTS: The sequence of the novel allele was different from all other known alleles. The novel allele differed from the closet matching allele HLA-DRB1*1404 by one nucleotide substitution in exon 2, at position 33 T>C, this resulted in an amino acid change from Tyr to His at codon 17. CONCLUSION: The novel allele is confirmed as a new HLA allele and it was officially named HLA-DRB1*1461 by WHO Nomenclature Committee in May, 2006.

[Lateral thoracic flaps pedicled with subscapular vessels for defects in the upper extremities].

OBJECTIVE: To investigate the feasibility and therapeutic effect of lateral thoracic flaps pedicled with subscapular vessels for defects in the upper extremities. METHODS: From June 2003 to September 2009, 5 cases with large soft tissue defects in the upper extremities were treated with lateral thoracic flaps pedicled with subscapular vessels. The flap size ranged from 23 cm x 8 cm to 40 cm x 20 cm. The subscapular vessels, the thoracodorsal vessels, the lateral branch and the cutaneous perforators of thoracodorsal vessels were all included in the flap. A muscular sleeve of 2-3 cm in width was preserved to protect the musculocutaneous perforator. The defects in the donor area were closed directly or covered by skin graft. RESULTS: The lateral thoracic flaps were used in four cases. A combination of lateral thoracic flap and paraumbilical flap was used in one case. Partial necrosis happened at the distal portion of the flap in one case. All the other flaps survived completely. 4 cases were followed up for two to fourteen months with satisfactory cosmetic and functional results. The color, texture and thickness of the flaps were also satisfactory. CONCLUSIONS: The lateral thoracic flap pedicled with the subscapular vessel is flexible for repairing defects in the upper extremities with a reliable blood supply, leaving less morbidity to donor site.

[Investigation on induced expansion of erythroid cells from cord blood CD34(+) cells in vitro].

This study was aimed to investigate a beneficial approach for resolving the deficiency of blood source, preventing the infection resulting from blood transfusion and overcoming the knotty match of patients with rare blood group by using massive expansion of erythroid cells from cord blood CD34(+) cells in vitro. The CD34(+) cells from human cord blood were cultured in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) for 1 week, then expansion and differentiation of CD34(+) cells into erythroid cells were supported by co-culture with human mesenchymal stem cells (MSCs) derived from bone marrow for 2 weeks. The results indicated that after culture for 23 days, the expansion multiple of total cell number reached 2.52 x 10(5), and over 95% of these cells were erythroid cells as compared with less than 1% of myelomonocytic (CD14(+) or CD15(+)) cells and megakaryocytic (CD41(+)) cells. However, the culture system without MSC support was significantly disadvantaged both in expansion ability and ratio of erythroid cells when compared with MSC supporting system. It is concluded that the erythroid cells can be produced from CD34(+) cells in large scale by culturing in the system comprised of cytokine sets and MSC feeders, in which MSCs can support the proliferation and differentiation of erythroid cells.

[A recombination event occurring between HLA-B and DRB1 loci within a Chinese Han family].

OBJECTIVE: To investigate a recombination event occurring between the HLA-B and DRB1 loci in a Chinese family with a leukemia patient. METHODS: HLA class I (-A and -B) low resolution typing was carried out by polymerase chain reaction-sequence specific oligonucleotide, PCR-SSO). HLA class II low resolution typing was performed by PCR-sequence specific primer (PCR-SSP). And HLA class I and II high resolution typing was done by sequencing-based typing (SBT). Then the recombination event was analyzed by family study. RESULTS: The 2 haplotypes of the patient were A*3101-B*1301-DRB1*0701 and A*3303-B*4403-DRB1*1302. His father's 2 haplotypes were A*3001-B*1302-DRB1*0701 and A*3101-B*1301-DRB1*1501. Family study demonstrated that the HLA-A*3101-B*1301 was from one of his father's chromosome and the DRB1*0701 was from the other chromosome of his father. So the result indicated that the recombination event occurred between the HLA-B and -DRB1 loci during meiosis of his father and resulted in a new HLA haplotype that was transferred to the son. CONCLUSION: A HLA-B/DR recombination event occurring between the HLA-B and -DRB1 loci has been found in a Chinese family, which may help further study of the mechanism of HLA recombination.

Genome-wide association study suggested copy number variation may be associated with body mass index in the Chinese population.

Obesity is a major public health problem characterized with high body mass index (BMI). Copy number variations (CNVs) have been identified to be associated with complex human diseases. The effect of CNVs on obesity is unknown. In this study, we explored the association of CNVs with BMI in 597 Chinese Han subjects using Affymetrix GeneChip Human Mapping 500K Array Set. We found that one CNV at 10q11.22 (from 46.36 Mb to 46.56 Mb) was associated with BMI (the raw P=0.011). The CNV contributed 1.6% of BMI variation, and it covered one important obesity gene-pancreatic polypeptide receptor 1(PPYR1). It was reported that PPYR1 was a key regulator of energy homeostasis. Our findings suggested that CNV might be potentially important for the BMI variation. In addition, our study suggested that CNV might be used as a genetic marker to locate genes associated with BMI in Chinese population.

Genome-wide copy-number-variation study identified a susceptibility gene, UGT2B17, for osteoporosis.

Osteoporosis, a highly heritable disease, is characterized mainly by low bone-mineral density (BMD), poor bone geometry, and/or osteoporotic fractures (OF). Copy-number variation (CNV) has been shown to be associated with complex human diseases. The contribution of CNV to osteoporosis has not been determined yet. We conducted case-control genome-wide CNV analyses, using the Affymetrix 500K Array Set, in 700 elderly Chinese individuals comprising 350 cases with homogeneous hip OF and 350 matched controls. We constructed a genomic map containing 727 CNV regions in Chinese individuals. We found that CNV 4q13.2 was strongly associated with OF (p = 2.0 x 10(-4), Bonferroni-corrected p = 0.02, odds ratio = 1.73). Validation experiments using PCR and electrophoresis, as well as real-time PCR, further identified a deletion variant of UGT2B17 in CNV 4q13.2. Importantly, the association between CNV of UGT2B17 and OF was successfully replicated in an independent Chinese sample containing 399 cases with hip OF and 400 controls. We further examined this CNV's relevance to major risk factors for OF (i.e., hip BMD and femoral-neck bone geometry) in both Chinese (689 subjects) and white (1000 subjects) samples and found consistently significant results (p = 5.0 x 10(-4) -0.021). Because UGT2B17 encodes an enzyme catabolizing steroid hormones, we measured the concentrations of serum testosterone and estradiol for 236 young Chinese males and assessed their UGT2B17 copy number. Subjects without UGT2B17 had significantly higher concentrations of testosterone and estradiol. Our findings suggest the important contribution of CNV of UGT2B17 to the pathogenesis of osteoporosis.

Reimplantation combined with transplantation of transgenic neural stem cells for treatment of brachial plexus root avulsion.

OBJECTIVE: To explore a new method to treat brachial plexus root avulsion experimentally by reimplantation combined with transplantation of neural stem cells (NSCs) modified by neurotrophin-3 gene (NT-3). METHODS: The total RNA was extracted from neonatal rat striatum and the NT-3 cDNA was obtained by reverse transcription and amplified by polymerase chain reaction. The NT-3 gene was transferred into NSCs via the pLEGFP-C1, an expression plasmid vectors. The untransfected NSCs, the pLEGFP-C1 treated NSCs, and the pLEGFP-C1-NT-3 treated NSCs were transplanted into corresponding spinal cord segment with brachial plexus root avulsion. The survival, differentiation, and migration of the transplanted cells were determined under confocal laser scanning microscope or by immunohistochemistry method. The nerve regeneration was evaluated by gross observation, electrophysiological examination and reverse horseradish peroxidase tracing. RESULTS: The NT-3 gene was successfully amplified and transferred into neural stem cells via the plasmid vectors. The transplanted cells survived, differentiated, and migrated and NT-3 was expressed within the spinal cord. The animals regained some muscle strength which was less than 3-degree muscular strength according to the British Medical Research Council (BMRC) evaluating system. The results of electrophysiological examination and reverse horseradish peroxidase tracing were superior in the pLEGFP-C1-NT-3 group to the NSCs untransfected group or the pLEGFP-C1 group. CONCLUSION: Transplantation of NSCs modified by NT-3 gene combined with reimplantation is a relatively effective way to treat brachial plexus root avulsion experimentally. It still need further study to improve the results.

[Tetrahydrobiopterin loading test in differential diagnosis among hyperphenylalaninemia patients].

OBJECTIVE: To perform tetrahydrobiopterin (BH(4)) loading test and to further understand its usefulness in differential diagnosis among hyperphenylalaninemia(HPA) patients. METHODS: BH(4) loading test was carried out in 73 HPA patients, including the positive cases unveiled by neonatal screening and the clinically suspected cases. These patients, 47 males and 26 females, were at a mean age of 1.93 months. BH(4) (20 mg/kg) loading test was performed in all patients, and a combined phenylalanine (Phe)(100 mg/kg) and BH(4) loading test was performed among the patient who had a basic blood Phe concentration less than 600 micro mol/L. The urine pterine profile analysis and the dihydropteridine reductase activity in dry blood filter spot were tested simultaneously. RESULTS: During BH(4) loading test or combined Phe and BH(4) loading test, the patients with classic phenylketonuria showed no response to BH(4), the patients with moderate HPA caused by Phe hydroxylase deficiency decreased 32.8% of blood Phe level and the patients with BH(4) deficiency showed a prompt reduction in blood Phe level and it decreased to normal level at 4 h and lasted until 24 h. Twenty-two cases were diagnosed as classic phenylketonuria, 39 were moderate phenylketonuria and 12 were BH(4) deficiency. CONCLUSION: Hyperphenylalaninemia may be caused by deficiency of Phe hydroxylase or by deficiency of co-factor BH(4). Early diagnosis is important. BH(4) loading test is a safe and fast test in vivo. It is sensitive, easy-to-do, and is highly useful in differential diagnosis for suspected cases of HPA.

Biological effect of velvet antler polypeptides on neural stem cells from embryonic rat brain.

BACKGROUND: Velvet antler polypeptides (VAPs), which are derived from the antler velvets, have been reported to maintain survival and promote growth and differentiation of neural cells and, especially the development of neural tissues. This study was designed to explore the influence of VAPs on neural stem cells in vitro derived from embryonic rat brain. METHODS: Neural stem cells derived from E12-14 rat brain were isolated, cultured, and expanded for 7 days until neural stem cell aggregations and neurospheres were generated. The neurospheres were cultured under the condition of different concentration of VAPs followed by immunocytochemistry to detect the differentiation of neural stem cells. RESULTS: VAPs could remarkably promote differentiation of neural stem cells and most neural stem cells were induced to differentiate towards the direction of neurons under certain concentration of VAPs. CONCLUSION: Neural stem cells can be successfully induced into neurons by VAPs in vitro, which could provide a basis for regeneration of the nervous system.