Chondrogenic differentiation of amniotic fluid-derived stem cells

For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-β (TGF-β) supplementation, with TGF-β1 producing greater increases than TGF-β3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-β1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-β1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications.     

Oligonucleotide conjugate GRN163L targeting human telomerase as potential anticancer and antimetastatic agent

Telomerase is one of the key enzymes responsible for the proliferative immortality of the majority of cancer cells. We recently introduced a new telomerase inhibitor, a 13-mer oligonucleotide N3' --> P5'-thio-phosphoramidate lipid conjugate, designated as GRN163L. This compound inhibits telomerase activity in various tumor cell lines with IC(50) values of 3-300 nM without any cellular uptake enhancers. GRN163L demonstrated potent and sequence specific anti-cancer activity in vivo in multiple animal models. This compound was able to significantly affect not only the growth of primary tumors, but also the spread and proliferation of metastases. GRN163L is currently in Phase I and Phase I/II clinical studies in patients with solid tumors and CLL, respectively.     

Click-based synthesis of triazolobithiazole ΔF508-CFTR correctors for cystic fibrosis

Copper catalyzed azide-alkyne cycloaddition (CuAAC) chemistry is reported for the construction of previously unknown 5-(1H-1,2,3-triazol-1-yl)-4,5'-bithiazoles from 2-bromo-1-(thiazol-5-yl)ethanones. These novel triazolobithiazoles are shown to have cystic fibrosis (CF) corrector activity and, compared to the benchmark bithiazole CF corrector corr-4a, improved logP values (4.5 vs 5.96).     

A three-dimensional model of differentiation of immortalized human bronchial epithelial cells

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.     

Autosomal-Recessive Early-Onset Retinitis Pigmentosa Caused by a Mutation in PDE6G, the Gene Encoding the Gamma Subunit of Rod cGMP Phosphodiesterase

Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration, with a worldwide prevalence of 1 in 4000. Over 30 genes and loci have been implicated in nonsyndromic autosomal-recessive (ar) RP. Genome-wide homozygosity mapping was conducted in two sibships from an extended consanguineous Muslim Arab Israeli family segregating ar severe early-onset RP. A shared homozygous region on chromosome 17q25.3 was identified in both sibships, with an overlap of 4.7 Mb. One of the genes located in this interval is PDE6G, encoding for the inhibitory γ subunit of rod photoreceptor cyclic GMP-phosphodiesterase. Mutations in the genes encoding for the catalytic subunits of this holoenzyme, PDE6A and PDE6B, cause arRP. Sequencing of all coding exons, including exon-intron boundaries, revealed a homozygous single base change (c.187+1G>T) located in the conserved intron 3 donor splice site of PDE6G. This mutation cosegregated with the disease in the extended family. We used an in vitro splicing assay to demonstrate that this mutation leads to incorrect splicing. Affected individuals had markedly constricted visual fields. Both scotopic and photopic electroretinograms were severely reduced or completely extinct. Funduscopy showed typical bone spicule-type pigment deposits spread mainly at the midperiphery, as well as pallor of the optic disk. Macular involvement was indicated by the lack of foveal reflex and typical cystoid macular edema, proved by optical coherence tomography. These findings demonstrate the positive role of the γ subunit in maintaining phosphodiesterase activity and confirm the contribution of PDE6G to the etiology of RP in humans.     

Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol

The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay has been modified for real-time, quantitative PCR analysis. Here, we describe cost-effective procedures for detection of telomerase activity using a fluorescent-based assay as well as by using real-time PCR. These modified TRAP assays can be accomplished within 4 h (from lysis of samples to analysis of telomerase products).     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.     

Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.     

PREDICT modeling and in-silico screening for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets.