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Mutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named hereditary persistence of fetal hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3′ end of the β-globin locus, including a CTCF binding site termed 3′HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3′HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies. [ABSTRACT FROM AUTHOR]

There are many ways in which cells may not adequately behave or respond to their environment, and the molecular mechanisms leading to these defects are as diverse as they are many. In this review, we report on how spatial chromatin organization contributes to the proper expression of genes, relating how CTCF — one of its main architects — contributes to gene regulation. We also touch on the emerging role of long noncoding RNAs in shaping chromatin organization and activity. The HOX gene clusters have been used as paradigm in the study of various biological pathways, and the overview we provide gives emphasis to what research on these loci has revealed about chromatin architecture and its regulation in the control of gene expression. [ABSTRACT FROM AUTHOR]

Summary: It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl− conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl− conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3−/− mice. Systematically decreasing [Cl−]i to mimic postnatal [Cl−]i regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl−-dependent change in synaptic strength is absent in clc-3 −/− mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl− dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons. [Copyright &y& Elsevier]

Background: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. Results: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. Conclusion: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames. [ABSTRACT FROM AUTHOR]

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription–polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and 1 d old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes. [ABSTRACT FROM AUTHOR]

Background: Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. Results: Here, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2, a long non-coding RNA that enhances gene expression during cellular differentiation. Conclusions: 2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis. [ABSTRACT FROM AUTHOR]

Focuses on the identification of GLI1 oncogene in the etiology of basal cell carcinoma. Increase in expression of GlI1 in basal cell carcinoma; Frequency of the nucleotide changes in the general population; Variation between wild-type GLI1 and the original human GlI1 clone.

Phycocyanin is a blue fluorescent protein with multi-bioactive functions. However, the multi-bioactivities and spectral stability of phycocyanin are susceptible to external environmental conditions, which limit its wide application. Here, the structure, properties, and biological activity of phycocyanin were discussed. This review highlights the significance of the microcapsules' wall materials which commonly protect phycocyanin from environmental interference and summarizes the current preparation principles and characteristics of microcapsules in food and pharma industries, including spray drying, electrospinning, electrospraying, liposome delivery, sharp-hole coagulation baths, and ion gelation. Moreover, the major technical challenge and corresponding countermeasures of phycocyanin microencapsulation are also appraised, providing insights for the broader application of phycocyanin. [ABSTRACT FROM AUTHOR]

Abstract: The interaction of nanocarriers with cells including their transcellular behavior is vital not only for a drug delivery system, but also for the safety of nanomaterials. In an attempt to clarify how the structures of polymers impact the transport mechanisms of their nanocarriers in epithelial cells, three amphiphilic polymers (PEEP–PCL, PEG–PCL and PEG–DSPE) with different hydrophilic or hydrophobic blocks were synthesized or chosen to form different micelle systems here. The endocytosis, exocytosis, intracellular colocalization, paracellular permeability and transcytosis of these micelle systems were compared using Förster resonance energy transfer analysis, real-time confocal images, colocalization assay, transepithelial electrical resistance study, and so on. All micelle systems were found intact during the studies with cells. The endocytosis and exocytosis studies with undifferentiated MDCK cells and the transcytosis study with differentiated MDCK monolayers all indicated the fact that PEG–DSPE micelles achieved the most and fastest transport, followed by PEG–PCL and PEEP–PCL in order. These might be because DSPE has higher hydrophobicity than PCL while PEG has lower hydrophilicity than PEEP. Different in hydrophilic or hydrophobic structures, all kinds of micelles demonstrated similar pathways during endocytosis and exocytosis, both caveolae- and clathrin-mediated but with difference in degree. The colocalization studies revealed different behaviors in intracellular trafficking among the three polymer micelles, suggesting the decisive role of hydrophilic shells on this process. Finally, all micelle systems did not impact the paracellular permeability of test cell monolayer. In conclusion, the hydrophilic and hydrophobic structures of test micelles could influence their transport ability, intracellular trafficking and the transport level under each pathway in MDCK cells. [Copyright &y& Elsevier]

Summary: Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea. [Copyright &y& Elsevier]