Genomic, biochemical and expressional properties reveal strong conservation of the CLCA2 gene in birds and mammals

Recent studies have revealed the dynamic and complex evolution of CLCA1 gene homologues in and between mammals and birds with a particularly high diversity in mammals. In contrast, CLCA2 has only been found as a single copy gene in mammals, to date. Furthermore, CLCA2 has only been investigated in few mammalian species but not in birds. Here, we established core genomic, protein biochemical and expressional properties of CLCA2 in several bird species and compared them with mammalian CLCA2. Chicken, turkey, quail and ostrich CLCA2 were compared to their mammalian orthologues using in silico, biochemical and expressional analyses. CLCA2 was found highly conserved not only at the level of genomic and exon architecture but also in terms of the canonical CLCA2 protein domain organization. The putatively prototypical galline CLCA2 (gCLCA2) was cloned and immunoblotting as well as immunofluorescence analyses of heterologously expressed gCLCA2 revealed protein cleavage, glycosylation patterns and anchoring in the plasma membrane similar to those of most mammalian CLCA2 orthologues. Immunohistochemistry found highly conserved CLCA2 expression in epidermal keratinocytes in all birds and mammals investigated. Our results suggest a highly conserved and likely evolutionarily indispensable role of CLCA2 in keratinocyte function. Its high degree of conservation on the genomic, biochemical and expressional levels stands in contrast to the dynamic structural complexities and proposed functional diversifications between mammalian and avian CLCA1 homologues, insinuating a significant degree of negative selection of CLCA2 orthologues among birds and mammals. Finally, and again in contrast to CLCA1, the high conservation of CLCA2 makes it a strong candidate for studying basic properties of the functionally still widely unresolved CLCA gene family

Tailored Pig Models for Preclinical Efficacy and Safety Testing of Targeted Therapies

Despite enormous advances in translational biomedical research, there remains a growing demand for improved animal models of human disease. This is particularly true for diseases where rodent models do not reflect the human disease phenotype. Compared to rodents, pig anatomy and physiology are more similar to humans in cardiovascular, immune, respiratory, skeletal muscle, and metabolic systems. Importantly, efficient and precise techniques for genetic engineering of pigs are now available, facilitating the creation of tailored large animal models that mimic human disease mechanisms at the molecular level. In this article, the benefits of genetically engineered pigs for basic and translational research are exemplified by a novel pig model of Duchenne muscular dystrophy and by porcine models of cystic fibrosis. Particular emphasis is given to potential advantages of using these models for efficacy and safety testing of targeted therapies, such as exon skipping and gene editing, for example, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system. In general, genetically tailored pig models have the potential to bridge the gap between proof-of-concept studies in rodents and clinical trials in patients, thus supporting translational medicine

Interspecies diversity of chloride channel regulators, calcium-activated 3 genes

Members of the chloride channel regulators, calcium-activated (CLCA) family, have been implicated in diverse biomedical conditions, including chronic inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, the activation of macrophages, and the growth and metastatic spread of tumor cells. Several observations, however, could not be repeated across species boundaries and increasing evidence suggests that select CLCA genes are particularly prone to dynamic species-specific evolvements. Here, we systematically characterized structural and expressional differences of the CLCA3 gene across mammalian species, revealing a spectrum of gene duplications, e.g., in mice and cows, and of gene silencing via diverse chromosomal modifications in pigs and many primates, including humans. In contrast, expression of a canonical CLCA3 protein from a single functional gene seems to be evolutionarily retained in carnivores, rabbits, guinea pigs, and horses. As an accepted asthma model, we chose the cat to establish the tissue and cellular expression pattern of the CLCA3 protein which was primarily found in mucin-producing cells of the respiratory tract and in stratified epithelia of the esophagus. Our results suggest that, among developmental differences in other CLCA genes, the CLCA3 gene possesses a particularly high dynamic evolutionary diversity with pivotal consequences for humans and other primates that seem to lack a CLCA3 protein. Our data also help to explain previous contradictory results on CLCA3 obtained from different species and warrant caution in extrapolating data from animal models in conditions where CLCA3 may be involved

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype

Photorhabdus luminescens lectin A (PllA) : A new probe for detecting α-galactoside-terminating glycoconjugates

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa.We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wildtype pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues

Transgenic Expression of Human Thrombomodulin Inhibits HMGB1-Induced Porcine Aortic Endothelial Cell Activation.

BACKGROUND Transgenic expression of human thrombomodulin (hTBM), which has the potential to solve the problem of coagulation dysregulation in pig-to-primate xenotransplantation, may have additional benefits by neutralizing the proinflammatory cytokine high-mobility group box 1 (HMGB1). The aim of this study was to investigate HMGB1-mediated effects on porcine aortic endothelial cells (PAEC) from wild-type (WT) and hTBM transgenic pigs. METHODS Porcine aortic endothelial cells were treated with HMGB1, human (h)TNFα or lipopolysaccharide (LPS). Procoagulant and proinflammatory responses were assessed by measuring expression of cell surface markers (adhesion molecules, fibrinogen-like protein 2, plasminogen activator inhibitor (PAI)-1), secretion of porcine cytokines and chemokines (HMGB1, TNFα, IL-8, monocyte chemotactic protein-1), and formation of PAI-1/tissue plasminogen activator complexes. Thrombin-mediated degradation of HMGB1 in the presence of PAEC was examined by Western blot and functional assay. RESULTS High-mobility group box 1 potently activated WT PAEC, increasing the expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, fibrinogen-like protein 2, and PAI-1, the secretion of TNFα, IL-8, and monocyte chemotactic protein-1 and the formation of PAI-1/tissue plasminogen activator complexes. Human TNFα- or LPS-induced activation of WT PAEC was inhibited by treatment with rabbit anti-HMGB1 antibody. Transgenic expression of hTBM significantly reduced the activation of PAEC by HMGB1 or hTNFα, and significantly enhanced thrombin-induced HMGB1 cleavage. Chemically induced shedding of the lectin-like domain of TBM resulted in significantly increased HMGB1-induced PAEC activation. CONCLUSIONS High-mobility group box 1 exerts powerful proinflammatory and procoagulant effects on WT PAEC, and appears to be an important downstream mediator for the actions of hTNFα and LPS. Human thrombomodulin transgenic PAECs are less sensitive to activation by either HMGB1 or hTNFα, an effect that appears to be dependent on the lectin-like domain of TBM

Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood

Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno) transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO. hCD46. hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFa-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO. hCD46. hTBM PAEC. Coating of untreated or hTNFa-treated PAEC with CHC (100 mu g/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 +/- 16.1 min, p < 0.001) compared to WT PAEC (34.0 +/- 8.2 min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3 +/- 11.3 min, p < 0.001;GTKO. hCD46. hTBM: 146.2 +/- 20.4 min, p < 0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function

Remapping of the belted phenotype in cattle on BTA3 identifies a multiplication event as the candidate causal mutation

Background: It has been known for almost a century that the belted phenotype in cattle follows a pattern of dominant inheritance. In 2009, the approximate position of the belt locus in Brown Swiss cattle was mapped to a 922-kb interval on bovine chromosome 3 and, subsequently, assigned to a 336-kb haplotype block based on an animal set that included, Brown Swiss, Dutch Belted (Lakenvelder) and Belted Galloway individuals. A possible candidate gene in this region i.e. HES6 was investigated but the causal mutation remains unknown. Thus, to elucidate the causal mutation of this prominent coat color phenotype, we decided to remap the belted phenotype in an independent animal set of several European bovine breeds, i.e. Gurtenvieh (belted Brown Swiss), Dutch Belted and Belted Galloway and to systematically scan the candidate region. We also checked the presence of the detected causal mutation in the genome of belted individuals from a Siberian cattle breed. Results: A combined linkage disequilibrium and linkage analysis based on 110 belted and non-belted animals identified a candidate interval of 2.5 Mb. Manual inspection of the haplotypes in this region identified four candidate haplotypes that consisted of five to eight consecutive SNPs. One of these haplotypes overlapped with the initial 922-kb interval, whereas two were positioned proximal and one was positioned distal to this region. Next-generation sequencing of one heterozygous and two homozygous belted animals identified only one private belted candidate allele, i.e. a multiplication event that is located between 118,608,000 and 118,614,000 bp. Targeted locus amplification and quantitative real-time PCR confirmed an increase in copy number of this region in the genomes of both European (Belted Galloway, Dutch Belted and Gurtenvieh) and Siberian (Yakutian cattle) breeds. Finally, using nanopore sequencing, the exact breakpoints were determined at 118,608,362 and 118,614,132 bp. The closest gene to the candidate causal mutation (16 kb distal) is TWIST2. Conclusions: Based on our findings and those of a previously published study that identified the same multiplication event, a quadruplication on bovine chromosome 3 between positions 118,608,362 and 118,614,132 bp is the most likely candidate causal mutation for the belted phenotype in cattle

New Generation ENaC Inhibitors Detach Cystic Fibrosis Airway Mucus Bundles via Sodium/Hydrogen Exchanger Inhibition

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