Solution structure of the carboxy-terminal Tudor domain from human Coilin

AbstractThe Cajal body is a dynamic eukaryotic nuclear organelle that is known primarily as an organizational center for the assembly of snRNAs involved in transcript splicing. One of the most critical components of the Cajal body is the scaffolding protein, Coilin. Here, we demonstrate by NMR methods that the carboxy-terminal region contains a Tudor domain. The Tudor domain is atypical due to the presence of several unstructured loops, one greater than thirty amino acids in length. Tudor domains have been noted previously to bind DNA, RNA and modified amino acids. The absence of these sequence and structural signatures in the Coilin Tudor domain supporting these established functions suggests an alternative role

Single-Cell Transcriptomes Reveal a Complex Cellular Landscape in the Middle Ear and Differential Capacities for Acute Response to Infection.

Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens

Active Transport of Peptides Across the Intact Human Tympanic Membrane.

We previously identified peptides that are actively transported across the intact tympanic membrane (TM) of rats with infected middle ears. To assess the possibility that this transport would also occur across the human TM, we first developed and validated an assay to evaluate transport in vitro using fragments of the TM. Using this assay, we demonstrated the ability of phage bearing a TM-transiting peptide to cross freshly dissected TM fragments from infected rats or from uninfected rats, guinea pigs and rabbits. We then evaluated transport across fragments of the human TM that were discarded during otologic surgery. Human trans-TM transport was similar to that seen in the animal species. Finally, we found that free peptide, unconnected to phage, was transported across the TM at a rate comparable to that seen for peptide-bearing phage. These studies provide evidence supporting the concept of peptide-mediated drug delivery across the intact TM and into the middle ears of patients

Esophageal cancer-related gene 4 at the interface of injury, inflammation, infection, and malignancy

In humans, esophageal cancer-related gene 4 (ECRG4) is encoded by four exons in the c2orf40 locus of chromosome 2. Translation of ECRG4 messenger ribonucleic acid produces a 148 amino acid-secreted 17 KDa protein that is then processed to 14, ten, eight, six, four, and two KDa peptides, depending on the cell in which the gene is expressed. As hypermethylation at the c2orf40 locus inhibits ECRG4 gene expression in many epithelial cancers, several investigators have speculated that ECRG4 is a candidate tumor suppressor. Indeed, overexpression of ECRG4 inhibits cell proliferation in vitro, but it also has a wide range of effects in vivo beyond its antitumor activity. ECRG4 overexpression affects apoptosis, senescence, cell migration, inflammation, injury, and infection responsiveness. ECRG4 activities also depend on its cellular localization, secretion, and post-translational processing. These cytokine/chemokine-like characteristics argue that ECRG4 is not a traditional candidate tumor suppressor gene, as originally predicted by its downregulation in cancer. We review how insights into the regulation of ECRG4 gene expression, knowledge of its primary structure, and the study of its emerging physiological functions come together to support a much more complex role for ECRG4 at the interface of inflammation, infection, and malignancy

Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation

The inhibitory role of lignin in the enzymatic hydrolysis of softwoods

Ethanol from biomass is one of the most promising technologies for the production of a renewable and environment friendly liquid biofuel. Currently, industrial production of ethanol is mostly based on sugar or starch-based substrates. However, ethanol produced through the bioconversion of lignocellulosic substrates such as woody and agricultural residues by enzymatic hydrolysis and fermentation, is a promising way of producing a green fuel. Softwoods are characterized by a high lignin content, which makes their enzymatic hydrolysis and eventually conversion to ethanol extremely difficult. Pretreatment of softwoods makes the substrate more accessible to cellulose-degrading enzymes (cellulases) and facilitates the cellulose degradation by modifying the physicochemical properties of the substrate. Different studies have shown that the ability of cellulases to hydrolyze pretreated softwoods is limited by various substrate factors such as, porosity, cellulose crystallinity, available surface area and the physicochemical characteristics of the residual lignin. Although the influence of cellulose properties on the enzymatic hydrolysis of lignocellulose has been studied extensively, the role of lignin as a substrate limiting factor has been more difficult to elucidate. This dissertation has focused on aspects relevant to the improvement of the enzymatic hydrolysis of softwood substrates. The first series of experiments were designed to address the possible inhibitory role of lignin in the enzymatic hydrolysis of lignocelluloses. A quantitative approach to compare the inhibition ability of lignin with other classical cellulase inhibitors was developed and assessed. In a related series of experiments, novel cellulase complexes, characterized by their higher hydrolytic ability on lignocellulosic substrates were also evaluated. A quantitative evaluation of the impact of lignin on the hydrolytic ability of various carbohydrases was performed. The study demonstrated that the magnitude of the lignin inhibition, on a concentration basis, was comparable to that of classical cellulase inhibitors. The inhibition by lignin followed a mixed-type pattern (competitive or uncompetitive, depending on the enzyme and substrate assayed). The second part of the research showed that a novel Penicillium sp. cellulase complex was more effective in the enzymatic hydrolysis of the pretreated softwood than commercially available cellulases. It was apparent that the Penicillium sp. cellulases yielded up to 2.5-fold more glucose from softwood substrates than was obtained when hydrolysis was carried out using Trichoderma sp. enzymes. Thus, a novel enzyme complex with a particularly high hydrolytic ability was identified and its application to the hydrolysis of pretreated softwood was demonstrated. Naturally occurring high levels of xylanase and β-glucosidase activities and the presence of weaker lignin-binding cellulases were postulated to be the reasons for the better performance of the Penicillium sp. enzymes. It was suggested that the increase in β-glucosidase activity reduces the cellobiose end-product inhibition, while the increase in xylanase activity increases the enzyme accessibility to cellulose by removing the shielding xylan. Weaker lignin-cellulase interactions lead to more free enzymes in solution. If this hypothesis is confirmed in the future, it could be used as a basis for further improvement of the commercially available cellulase complexes for lignocellulose bioconversion.Forestry, Faculty ofGraduat