| Antibody-immobilized column for quick cell separation based on cell rolling.
November 18, 2009 at 10:27 am
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| | Antibody-immobilized column for quick cell separation based on cell rolling. Biotechnol Prog. 2009 Nov 13; Authors: Mahara A, Yamaoka T Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010. PMID: 19918913 [PubMed - as supplied by publisher] |
| Fusion of uniluminal vascular spheroids: A model for assembly of blood vessels.
November 18, 2009 at 10:27 am
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| | Fusion of uniluminal vascular spheroids: A model for assembly of blood vessels. Dev Dyn. 2009 Nov 13; Authors: Fleming PA, Argraves WS, Gentile C, Neagu A, Forgacs G, Drake CJ We evaluated the self-assembly properties of uniluminal vascular spheroids having outer layers of vascular smooth muscle cells and a contiguous inner layer of endothelial cells lining a central lumen. We showed that while pairs of uniluminal vascular spheroids suspended in culture medium fused to form a larger diameter spheroidal structure, spheroids in collagen hydrogels formed elongated structures. These findings highlight the potential use of uniluminal vascular spheroids as modules to engineer blood vessels. We also demonstrate that uniluminal vascular spheroid fusion conforms to models describing the coalescence of liquid drops. Furthermore, the fusion of uniluminal vascular spheroids in vitro closely resembled the in vivo process by which the descending aorta forms from the fusion of the paired dorsal aortae during embryonic development. Together, the findings indicate that tissue liquidity underlies uniluminal vascular spheroid fusion and that in vivo anastomosis of blood vessels may involve a similar mechanism. Developmental Dynamics, 2009. (c) 2009 Wiley-Liss, Inc. PMID: 19918756 [PubMed - as supplied by publisher] |
| Do we really need cartilage tissue engineering?
November 18, 2009 at 10:27 am
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| | Do we really need cartilage tissue engineering? Swiss Med Wkly. 2009 Oct 17;139(41-42):602-9 Authors: Pelttari K, Wixmerten A, Martin I The in vitro engineering of functionally developed biological cartilage substitutes, based on cells and appropriate structural and soluble factors, is an attractive concept for the clinical treatment of cartilage injuries and degeneration. The field of cartilage tissue engineering has developed strongly in the last few years, bringing together the scientific, clinical and commercial interests of highly interdisciplinary communities. However, engineered grafts are still far from being the standard of care for cartilage repair. In this review we present some of the issues challenging the reproducible engineering of functional cartilage templates starting from human cells. We then discuss the need to identify the mode of action of cartilage tissue engineering approaches, which in turn is expected to define potency markers and quality controls for grafts capable of inducing durable cartilage regeneration. Finally, we propose the use of engineered cartilage tissues not only as implants to be implemented in the clinic, but also as models to understand mechanisms and processes related to cartilage development and repair. The knowledge generated using these models will be instrumental in moving to the next generation of cartilage repair approaches, namely those inducing regeneration in situ, based on the recruitment of resident cells. PMID: 19918699 [PubMed - in process] |
| Mesenchymal stem cells as anti-inflammatories: Implications for treatment of Duchenne muscular dystrophy.
November 18, 2009 at 10:27 am
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| | Mesenchymal stem cells as anti-inflammatories: Implications for treatment of Duchenne muscular dystrophy. Cell Immunol. 2009 Oct 19; Authors: Ichim TE, Alexandrescu DT, Solano F, Lara F, Campion RD, Paris E, Woods EJ, Murphy MP, Dasanu CA, Patel AN, Marleau AM, Leal A, Riordan NH Duchenne muscular dystrophy (DMD) is a lethal X-linked musculodegenerative condition consisting of an underlying genetic defect whose manifestation is augmented by inflammatory mechanisms. Previous treatment approaches using gene replacement, exon-skipping or allogeneic cell therapy have been relatively unsuccessful. The only intervention to mediate improvement in survival, albeit minor, is glucocorticoid treatment. Given this modality appears to function via suppression of underlying inflammation; we focus this review on the inflammatory response as a target for mesenchymal stem cell (MSC) therapy. In contrast to other cell based therapies attempted in DMD, MSC have the advantages of (a) ability to fuse with and genetically complement dystrophic muscle; (b) possess anti-inflammatory activities; and (c) produce trophic factors that may augment activity of endogenous repair cells. We conclude by describing one practical scenario of stem cell therapy for DMD. PMID: 19917503 [PubMed - as supplied by publisher] |
| Room temperature pulsatile perfusion of renal allografts with Lifor compared with hypothermic machine pump solution.
November 18, 2009 at 10:27 am
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| | Room temperature pulsatile perfusion of renal allografts with Lifor compared with hypothermic machine pump solution. Transplant Proc. 2009 Nov;41(9):3571-4 Authors: Gage F, Leeser DB, Porterfield NK, Graybill JC, Gillern S, Hawksworth JS, Jindal RM, Thai N, Falta EM, Tadaki DK, Brown TS, Elster EA This pilot study compared the use of the Lifor Organ Preservation Medium (RTLF) at room temperature with hypothermic Belzer machine preservation solution (CMPS) and room in vitro temperature Belzer machine preservation solution (RTMPS) in a porcine model of uncontrolled donation after cardiac death (DCD). In this study, 5 porcine kidneys for each perfusate group were recovered under a DCD protocol. The kidneys were recovered, flushed, and placed onto a renal preservation system following standard perfusion procedures. The average flow rate for CMPS was 36.2 +/- 7.2549 mL/min, RTMPS was 90.2 +/- 9.7159 mL/min, and RTLF was 103.1 +/- 5.1108 mL/min. The average intrarenal resistance for CMPS was 1.33 +/- 0.1709 mm Hg/mL per minute, RTMPS was 0.84 +/- 0.3586 and RTLF was 0.39 +/- 0.04. All perfusion parameters were statistically significant (P < .05) at all time points for the CMPS when compared with both RTMPS and RTLF. All perfusion parameters for RTMPS and RTLF were equivalent for the first 12 hours; thereafter, RTLF became significantly better than RTMPS at 18 and 24 hours. It appears that both RTMPS and RTLF have equivalent perfusion characteristic for the initial 12 hours of perfusion, but LF continues to maintain a low resistance and high flow up to 24 hours. The results of this pilot study indicate that RTLF may represent a better alternative to pulsatile perfusion with CMPS and requires validation in an in vivo large animal transplant model. PMID: 19917346 [PubMed - in process] |
| Controlling the orientation and synaptic differentiation of myotubes with micropatterned substrates.
November 18, 2009 at 10:27 am
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| | Controlling the orientation and synaptic differentiation of myotubes with micropatterned substrates. Biophys J. 2009 Nov 18;97(10):2771-9 Authors: Gingras J, Rioux RM, Cuvelier D, Geisse NA, Lichtman JW, Whitesides GM, Mahadevan L, Sanes JR Micropatterned poly(dimethylsiloxane) substrates fabricated by soft lithography led to large-scale orientation of myoblasts in culture, thereby controlling the orientation of the myotubes they formed. Fusion occurred on many chemically identical surfaces in which varying structures were arranged in square or hexagonal lattices, but only a subset of patterned surfaces yielded aligned myotubes. Remarkably, on some substrates, large populations of myotubes oriented at a reproducible acute angle to the lattice of patterned features. A simple geometrical model predicts the angle and extent of orientation based on maximizing the contact area between the myoblasts and patterned topographic surfaces. Micropatterned substrates also provided short-range cues that influenced higher-order functions such as the localization of focal adhesions and accumulation of postsynaptic acetylcholine receptors. Our results represent what we believe is a new approach for musculoskeletal tissue engineering, and our model sheds light on mechanisms of myotube alignment in vivo. PMID: 19917231 [PubMed - in process] |
| ADIPOSE DERIVED STEM CELL DELIVERY INTO COLLAGEN GELS USING CHITOSAN MICROSPHERES.
November 18, 2009 at 10:27 am
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| | ADIPOSE DERIVED STEM CELL DELIVERY INTO COLLAGEN GELS USING CHITOSAN MICROSPHERES. Tissue Eng Part A. 2009 Nov 16; Authors: Natesan S, Baer DG, Walters TJ, Babu M, Christy R Integration of stem cells to injured tissues requires an appropriate delivery device and scaffolding system. In the present study we have developed an in vitro strategy to load and release adipose derived mesenchymal stem cells (ASC) from chitosan microspheres into a collagen gel scaffold. Porous chitosan microspheres of uniform size and composition were prepared and used as a stem cell carrier. Adipose derived stem cells were allowed to attach to the microspheres and infiltrate through the microsphere pores. The number of viable cells were measured in vitro, using MTT and Calcein AM assays, and showed a proportional increase with seeding density and reached a maximum cell number by 24 hours. The cells inside the microspheres remained metabolically active and viable, could be retrieved from the spheres and maintained expression of stem cell specific markers. Electron microscopic evaluation of the cell-microsphere complex showed that the chitosan microspheres were able to support cell attachment and that the cells had infiltrated into the pores of the microspheres. The ability of the cells to self-renew and differentiate into adipogenic and osteogenic-like precursors indicate that the cells have maintained their multipotency after migration out of the microspheres. To mimic cell delivery into a tissue, ASC-loaded chitosan microspheres were embedded in type-1 collagen scaffold by mixing them with type-1 collagen solution while inducing gelation. By 14 days the cells released into the collagen gel and were able to populate the entire scaffold. When observed through transmission electron microscopy, the cells align along the collagen fibrils with characteristic fibroblast-like morphology. This study provides a model to capture pluripotent stem cells, expand their cell number within a biomaterial scaffold in vitro and deliver within an appropriate matrix to repair damaged tissue. PMID: 19916819 [PubMed - as supplied by publisher] |
| Blocking of tumor necrosis factor activity promotes natural repair of osteochondral defects in rabbit knee.
November 18, 2009 at 10:27 am
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| | Blocking of tumor necrosis factor activity promotes natural repair of osteochondral defects in rabbit knee. Acta Orthop. 2009 Oct;80(5):606-11 Authors: Kawaguchi A, Nakaya H, Okabe T, Tensho K, Nawata M, Eguchi Y, Imai Y, Takaoka K, Wakitani S BACKGROUND AND PURPOSE: Osteochondral defects have a limited capacity for repair. We therefore investigated the effects of tumor necrosis factor (TNF) signal blockade by etanercept (human recombinant soluble TNF receptor) on the repair of osteochondral defects in rabbit knees. MATERIAL AND METHODS: Osteochondral defects (5 mm in diameter) were created in the femoral patellar groove in rabbits. Soon after the procedure, a first subcutaneous injection of etanercept was performed. This single injection or, alternatively, 4 injections in total (twice a week for 2 weeks) were given. Each of these 2 groups was divided further into 3 subgroups: a low-dose group (0.05 microg/kg), an intermediate-dose group (0.4 microg/kg), and a high-dose group (1.6 microg /kg) with 19 rabbits in each. As a control, 19 rabbits were injected with water alone. The rabbits in each subgroup were killed 4 weeks (6 rabbits), 8 weeks (6 rabbits), or 24 weeks (7 rabbits) after surgery and repair was assessed histologically. RESULTS: Histological examination revealed that the natural process of repair of the osteochondral defects was promoted by 4 subcutaneous injections of intermediate-dose etanercept and by 1 or 4 injections of high-dose etanercept at the various time points examined postoperatively (4, 8, and 24 weeks). Western blot showed that rabbit TNFalpha had a high affinity for etanercept. INTERPRETATION: Blocking of TNF by etanercept enabled repair of osteochondral defects in rabbit knee. Anti-TNF therapy could be a strategy for the use of tissue engineering for bone and cartilage repair. PMID: 19916697 [PubMed - in process] |
| Formulation and characterization of a covalently coated magnetic nanogel.
November 18, 2009 at 10:27 am
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| | Formulation and characterization of a covalently coated magnetic nanogel. J Nanosci Nanotechnol. 2009 Jul;9(7):4128-34 Authors: Rahimi M, Yousef M, Cheng Y, Meletis EI, Eberhart RC, Nguyen K The aim of this study was to develop a novel method to encapsulate magnetic nanoparticles (MNPs) with polymer via covalent bonding, in order to increase the magnetic nanoparticle stability and ease the synthesis process. In this technique, silane coated MNPs act as a template for polymerization of the monomer N-isopropylacrylamide, (NIPA) via radical polymerization. Transmission and scanning electron microscopy indicated the size of the original MNP was approximately 10 nm, the silane-coated MNP was 40 nm and the NIPA silane-coated MNP was 100 +/- 10 nm. Chemical composition and chemical state analysis of NIPA MNPs by FTIR and XPS showed that the MNPs were actually encapsulated by silane and NIPA. Furthermore, the magnetic properties of different layers on the MNP, analyzed by SQUID, indicated a decrease in saturation magnetization for each layer. The results demonstrate the feasibility of encapsulation of the MNP with NIPA by means of silane covalent bonding. Future work will investigate the phase transition and biocompatibility properties of the NIPA-coated MNP for drug delivery and tissue engineering applications. PMID: 19916419 [PubMed - in process] |
| De novo follicular regeneration of the skin by wingless int 3 and bone morphogenetic protein 2 genes introduced into dermal fibroblasts and fibroblast growth factor-2 protein.
November 18, 2009 at 10:27 am
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| | De novo follicular regeneration of the skin by wingless int 3 and bone morphogenetic protein 2 genes introduced into dermal fibroblasts and fibroblast growth factor-2 protein. Wound Repair Regen. 2009 May-Jun;17(3):436-46 Authors: Ono I, Akasaka Y, Kamiya T, Sato M, Kobune M, Hamada H, Yamashita T In this study, we regenerated skin and its appendages by transplanting cultured normal dermal fibroblasts, into which morphogen genes had been introduced. We cultured normal dermal fibroblasts obtained from Fisher 344 rats on the surface of hydroxyapatite beads, and then adsorbed them onto the surface of a collagen sponge, which was transplanted into a full-thickness skin defect prepared on the backs of rats. Before transplantation, genes were introduced into the dermal fibroblasts via adenovirus vector (ad)-bone morphogenetic protein 2 and ad-wingless int 3 genes in addition to fibroblast growth factor-2 protein. By Week 4, the appearance of follicle germs or primitive hair germs was observed only in the ad-bone morphogenetic protein 2+ad-wingless int 3 combined with the fibroblast growth factor-2 protein group. By Week 16, in that same group, hair follicles having mature pilosebaceous systems with equally spaced localization had formed in the ulcer wound. PMID: 19660053 [PubMed - indexed for MEDLINE] |
| Staphylococcal biofilms impair wound healing by delaying reepithelialization in a murine cutaneous wound model.
November 18, 2009 at 10:27 am
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| | Staphylococcal biofilms impair wound healing by delaying reepithelialization in a murine cutaneous wound model. Wound Repair Regen. 2009 May-Jun;17(3):354-9 Authors: Schierle CF, De la Garza M, Mustoe TA, Galiano RD Bacterial biofilms have gained increasing visibility in recent years as a ubiquitous form of survival for microorganisms in myriad environments. A number of in vivo models exist for the study of biofilms in the setting of medically relevant implanted foreign bodies. Growing evidence has demonstrated the presence of bacterial biofilms in the setting of a number of chronic wound states including pressure sores, diabetic foot ulcers, and venous stasis ulcers. Here we present a novel murine cutaneous wound system that directly demonstrates delayed reepithelialization caused by the presence of a bacterial biofilm. We established biofilms using either Staphylococcus aureus or Staphylococcus epidermidis in splinted cutaneous punch wounds created on the backs of normal C57Bl6/J mice. Wound reepithelialization was significantly delayed by bacterial biofilms. This effect was specifically dependent on the ability of the bacteria to form biofilms as demonstrated by exogenous administration of biofilm inhibiting peptides and the use of mutant Staphylococcus spp. deficient in biofilm formation. This represents the first direct evidence for the effect of bacterial biofilms on cutaneous wound healing. PMID: 19660043 [PubMed - indexed for MEDLINE] |
| Regeneration of epidermal appendages.
November 18, 2009 at 10:27 am
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| | Regeneration of epidermal appendages. Wound Repair Regen. 2009 May-Jun;17(3):293 Authors: Hebda PA PMID: 19660034 [PubMed - indexed for MEDLINE] |
| Microfabricated electrospun collagen membranes for 3-D cancer models and drug screening applications.
November 18, 2009 at 10:27 am
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| | Microfabricated electrospun collagen membranes for 3-D cancer models and drug screening applications. Biomacromolecules. 2009 Aug 10;10(8):2019-32 Authors: Hartman O, Zhang C, Adams EL, Farach-Carson MC, Petrelli NJ, Chase BD, Rabolt JF Invasive epithelial tumors form from cells that are released from their natural basement membrane and form 3-D structures that interact with each other and with the microenvironment of the stromal tissues around the tumor, which often contains collagen. Cancer cells, growing as monolayers on tissue culture plastic, do not reflect many of the properties of whole tumors. This shortcoming limits their ability to serve as models for testing of pharmacologically active compounds, including those that are being tested as antineoplastics. This work seeks to create new 3-D cellular materials possessing properties similar to those in native tissues surrounding cancers, specifically electrospun micro- and nanofibrous collagen scaffolds that support tumor growth in 3-D. We hypothesize that a 3-D culture system will provide a better replica of tumor growth in a native environment and, thus, better report the bioactivity of antineoplastic agents. In addition, we optimized conditions and identified physical characteristics that support growth of the highly invasive, prostate cancer bone metastatic cell line C4-2B on these matrices for use in anticancer drug studies. The effects of matrix porosity, fiber diameter, elasticity, and surface roughness on growth of cancer cells were evaluated. Data indicates that while cells attach and grow well on both nano- and microfibrous electrospun membranes, the microfibrous membrane represented a better approximation of the tumor microenvironment. It was also observed that C4-2B nonadherent cells migrated through the depth of two electrospun membranes and formed colonies resembling tumors on day 3. An apoptosis study revealed that cells on electrospun substrates were more resistant to both antineoplastic agents, docetaxel (DOC), and camptothecin (CAM) compared to the cells grown on standard collagen-coated tissue culture polystyrene (TCP). Growth, survival, and apoptosis were measured, as well as the differences in the apoptotic capabilities, of the two above-mentioned compounds compared to known clinical performance. We conclude that 3-D electrospun membranes are amenable to high throughput screening for cancer cell susceptibility and combination killing (Banerjee, S.; Hussain, M.; Wang, Z.; Saliganan, A.; Che, M.; Bonfil, D.; Cher, M.; Sarkar, F.H. Cancer Research, 2007, 67 (8), 3818-26). PMID: 19624098 [PubMed - indexed for MEDLINE] |
| Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol.
November 18, 2009 at 10:27 am
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| | Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol. Cell Biol Int. 2009 Oct;33(10):1079-86 Authors: Stefanova N, Staneva G, Petkova D, Lupanova T, Pankov R, Momchilova A Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells. PMID: 19589391 [PubMed - indexed for MEDLINE] |
| Novel and common BRCA1 mutations in familial breast/ovarian cancer patients from Lithuania.
November 18, 2009 at 10:27 am
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| | Novel and common BRCA1 mutations in familial breast/ovarian cancer patients from Lithuania. Breast Cancer Res Treat. 2009 Sep;117(2):467-9 Authors: Janavicius R, Pepalyte I, Kucinskas V PMID: 18763032 [PubMed - indexed for MEDLINE] |
| A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism.
November 18, 2009 at 4:27 am
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| | A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism. Hum Mutat. 2009 Sep;30(9):E855-65 Authors: Qiao J, Han B, Liu BL, Chen X, Ru Y, Cheng KX, Chen FG, Zhao SX, Liang J, Lu YL, Tang JF, Wu YX, Wu WL, Chen JL, Chen MD, Song HD Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537-3 C>A). Computational analysis of the missense mutation in the three-dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice-acceptor mutation (c. 537-3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene. PMID: 19551906 [PubMed - indexed for MEDLINE] | | | 
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