Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Functions of the RIP kinase family members in the skin.

The receptor interacting protein kinases (RIPK) are a family of serine/threonine kinases that are involved in the integration of various stress signals. In response to several extracellular and/or intracellular stimuli, RIP kinases engage signaling cascades leading to the activation of NF-κB and mitogen-activated protein kinases, cell death, inflammation, differentiation and Wnt signaling and can have kinase-dependent and kinase-independent functions. Although it was previously suggested that seven RIPKs are part of the RIPK family, phylogenetic analysis indicates that there are only five genuine RIPKs. RIPK1 and RIPK3 are mainly involved in controlling and executing necroptosis in keratinocytes, while RIPK4 controls proliferation and differentiation of keratinocytes and thereby can act as a tumor suppressor in skin. Therefore, in this review we summarize and discuss the functions of RIPKs in skin homeostasis as well as the signaling pathways involved.

MariClus: Your One-Stop Platform for Information on Marine Natural Products, Their Gene Clusters and Producing Organisms.

BACKGROUND: The marine environment hosts the vast majority of living species and marine microbes that produce natural products with great potential in providing lead compounds for drug development. With over 70% of Earth's surface covered in water and the high interaction rate associated with liquid environments, this has resulted in many marine natural product discoveries. Our improved understanding of the biosynthesis of these molecules, encoded by gene clusters, along with increased genomic information will aid us in uncovering even more novel compounds. RESULTS: We introduce MariClus (https://www.mariclus.com), an online user-friendly platform for mining and visualizing marine gene clusters. The first version contains information on clusters and the predicted molecules for over 500 marine-related prokaryotes. The user-friendly interface allows scientists to easily search by species, cluster type or molecule and visualize the information in table format or graphical representation. CONCLUSIONS: This new online portal simplifies the exploration and comparison of gene clusters in marine species for scientists and assists in characterizing the bioactive molecules they produce. MariClus integrates data from public sources, like GenBank, MIBiG and PubChem, with genome mining results from antiSMASH. This allows users to access and analyze various aspects of marine natural product biosynthesis and diversity.

Modifying PCDH19 levels affects cortical interneuron migration.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.

Characteristic ERK1/2 signaling dynamics distinguishes necroptosis from apoptosis.

ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

RyR2 regulates Cx43 hemichannel intracellular Ca2+-dependent activation in cardiomyocytes.

AIMS: Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at -70 mV. METHODS AND RESULTS: Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of ∼220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity ligation studies indicated close Cx43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modelling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca2+ HC activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Interestingly, alterations in the concerned RyR sequence are known to be associated with primary familial hypertrophic cardiomyopathy. CONCLUSION: Our results demonstrate that Cx43 HCs are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation.

Novel Alkaloids from Marine Actinobacteria: Discovery and Characterization.

The marine environment is an excellent resource for natural products with therapeutic potential. Its microbial inhabitants, often associated with other marine organisms, are specialized in the synthesis of bioactive secondary metabolites. Similar to their terrestrial counterparts, marine Actinobacteria are a prevalent source of these natural products. Here, we discuss 77 newly discovered alkaloids produced by such marine Actinobacteria between 2017 and mid-2021, as well as the strategies employed in their elucidation. While 12 different classes of alkaloids were unraveled, indoles, diketopiperazines, glutarimides, indolizidines, and pyrroles were most dominant. Discoveries were mainly based on experimental approaches where microbial extracts were analyzed in relation to novel compounds. Although such experimental procedures have proven useful in the past, the methodologies need adaptations to limit the chance of compound rediscovery. On the other hand, genome mining provides a different angle for natural product discovery. While the technology is still relatively young compared to experimental screening, significant improvement has been made in recent years. Together with synthetic biology tools, both genome mining and extract screening provide excellent opportunities for continued drug discovery from marine Actinobacteria.

Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Functional exploration of the GH29 fucosidase family.

The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family's large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

Armc8 is an evolutionarily conserved armadillo protein involved in cell-cell adhesion complexes through multiple molecular interactions.

Armadillo-repeat-containing protein 8 (Armc8) belongs to the family of armadillo-repeat containing proteins, which have been found to be involved in diverse cellular functions including cell-cell contacts and intracellular signaling. By comparative analyses of armadillo repeat protein structures and genomes from various premetazoan and metazoan species, we identified orthologs of human Armc8 and analyzed in detail the evolutionary relationship of Armc8 genes and their encoded proteins. Armc8 is a highly ancestral armadillo protein although not present in yeast. Consequently, Armc8 is not the human ortholog of yeast Gid5/Vid28.Further, we performed a candidate approach to characterize new protein interactors of Armc8. Interactions between Armc8 and specific δ-catenins (plakophilins-1, -2, -3 and p0071) were observed by the yeast two-hybrid approach and confirmed by co-immunoprecipitation and co-localization. We also showed that Armc8 interacts specifically with αE-catenin but neither with αN-catenin nor with αT-catenin. Degradation of αE-catenin has been reported to be important in cancer and to be regulated by Armc8. A similar process may occur with respect to plakophilins in desmosomes. Deregulation of desmosomal proteins has been considered to contribute to tumorigenesis.

Neutralization of Junín virus by single domain antibodies targeted against the nucleoprotein.

The syndrome viral haemorrhagic fever (VHF) designates a broad range of diseases that are caused by different viruses including members of the family Arenaviridae. Prophylaxis for Argentine Haemorrhagic Fever (AHF), caused by the arenavirus Junín (JUNV), has been achieved by the use of a live attenuated vaccine, named Candid#1. The standard treatment of AHF is transfusion of convalescent human plasma. Our aim was to develop an alternative and safer treatment for AHF based on the use of virus-neutralizing single domain antibodies (VHHs). We describe the first reported VHHs directed against an arenavirus. These VHHs could neutralize Candid#1 by altering virion binding/fusion. Surprisingly, the neutralizing VHHs appeared to be specific for the viral nucleoprotein (N) that is not known to be involved in arenavirus entry. Candid#1 VHH-escape viruses had acquired a predicted N-glycosylation site in the surface glycoprotein GP1 that is present in highly pathogenic JUNV strains. Accordingly, the Candid#1-neutralizing VHHs could not neutralize pathogenic JUNV strains, but they could still bind to cells infected with a pathogenic strain or the escape mutant viruses. These results show that the attenuated strains of JUNV can be potently neutralized by nucleoprotein-specific VHHs.

Ancient Origin of the CARD-Coiled Coil/Bcl10/MALT1-Like Paracaspase Signaling Complex Indicates Unknown Critical Functions.

The CARD-coiled coil (CC)/Bcl10/MALT1-like paracaspase (CBM) signaling complexes composed of a CARD-CC family member (CARD-9, -10, -11, or -14), Bcl10, and the type 1 paracaspase MALT1 (PCASP1) play a pivotal role in immunity, inflammation, and cancer. Targeting MALT1 proteolytic activity is of potential therapeutic interest. However, little is known about the evolutionary origin and the original functions of the CBM complex. Type 1 paracaspases originated before the last common ancestor of planulozoa (bilaterians and cnidarians). Notably in bilaterians, Ecdysozoa (e.g., nematodes and insects) lacks Bcl10, whereas other lineages have a Bcl10 homolog. A survey of invertebrate CARD-CC homologs revealed such homologs only in species with Bcl10, indicating an ancient common origin of the entire CBM complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were only found in invertebrate organisms with CARD-CC/Bcl10, indicating that this pathway might be related to the original function of the CBM complex. Moreover, the type 1 paracaspase sequences from invertebrate organisms that have CARD-CC/Bcl10 are more similar to vertebrate paracaspases. Functional analysis of protein-protein interactions, NF-κB signaling, and CYLD cleavage for selected invertebrate type 1 paracaspase and Bcl10 homologs supports this scenario and indicates an ancient origin of the CARD-CC/Bcl10/paracaspase signaling complex. By contrast, many of the known MALT1-associated activities evolved fairly recently, indicating that unknown functions are at the basis of the protein conservation. As a proof-of-concept, we provide initial evidence for a CBM- and NF-κB-independent neuronal function of the Caenorhabditis elegans type 1 paracaspase malt-1. In conclusion, this study shows how evolutionary insights may point at alternative functions of MALT1.

GC Content of Early Metazoan Genes and Its Impact on Gene Expression Levels in Mammalian Cell Lines.

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure-function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

Nanos genes and their role in development and beyond.

The hallmark of Nanos proteins is their typical (CCHC)2 zinc finger motif (zf-nanos). Animals have one to four nanos genes. For example, the fruit fly and demosponge have only one nanos gene, zebrafish and humans have three, and Fugu rubripes has four. Nanos genes are mainly known for their evolutionarily preserved role in germ cell survival and pluripotency. Nanos proteins have been reported to bind the C-terminal RNA-binding domain of Pumilio to form a post-transcriptional repressor complex. Several observations point to a link between the miRNA-mediated repression complex and the Nanos/Pumilio complex. Repression of the E2F3 oncogene product is, indeed, mediated by cooperation between the Nanos/Pumilio complex and miRNAs. Another important interaction partner of Nanos is the CCR4-NOT deadenylase complex. Besides the tissue-specific contribution of Nanos proteins to normal development, their ectopic expression has been observed in several cancer cell lines and various human cancers. An inverse correlation between the expression levels of human Nanos1 and Nanos3 and E-cadherin was observed in several cancer cell lines. Loss of E-cadherin, an important cell-cell adhesion protein, contributes to tumor invasion and metastasis. Overexpression of Nanos3 induces epithelial-mesenchymal transition in lung cancer cell lines partly by repressing E-cadherin. Other than some most interesting data from Nanos knockout mice, little is known about mammalian Nanos proteins, and further research is needed. In this review, we summarize the main roles of Nanos proteins and discuss the emerging concept of Nanos proteins as oncofetal antigens.

ConTra v3: a tool to identify transcription factor binding sites across species, update 2017.

Transcription factors are important gene regulators with distinctive roles in development, cell signaling and cell cycling, and they have been associated with many diseases. The ConTra v3 web server allows easy visualization and exploration of predicted transcription factor binding sites (TFBSs) in any genomic region surrounding coding or non-coding genes. In this updated version, with a completely re-implemented user interface using latest web technologies, users can choose from nine reference organisms ranging from human to yeast. ConTra v3 can analyze promoter regions, 5΄-UTRs, 3΄-UTRs and introns or any other genomic region of interest. Thousands of position weight matrices are available to choose from for detecting specific binding sites. Besides this visualization option, additional new exploration functionality is added to the tool that will automatically detect TFBSs having at the same time the highest regulatory potential, the highest conservation scores of the genomic regions covered by the predicted TFBSs and strongest co-localizations with genomic regions exhibiting regulatory activity. The ConTra v3 web server is freely available at http://bioit2.irc.ugent.be/contra/v3.

Evolution and diversity of cadherins and catenins.

Cadherin genes encode a superfamily of conserved transmembrane proteins that share an adhesive ectodomain composed of tandem cadherin repeats. More than 100 human cadherin superfamily members have been identified, which can be classified into three families: major cadherins, protocadherins and cadherin-related proteins. These superfamily members are involved in diverse fundamental cellular processes including cell-cell adhesion, morphogenesis, cell recognition and signaling. Epithelial cadherin (E-cadherin) is the founding cadherin family member. Its cytoplasmic tail interacts with the armadillo catenins, p120 and ß-catenin. Further, α-catenin links the cadherin/armadillo catenin complex to the actin filament network. Even genomes of ancestral metazoan species such as cnidarians and placozoans encode a limited number of distinct cadherins and catenins, emphasizing the conservation and functional importance of these gene families. Moreover, a large expansion of the cadherin and catenin families coincides with the emergence of vertebrates and reflects a major functional diversification in higher metazoans. Here, we revisit and review the functions, phylogenetic classifications and co-evolution of the cadherin and catenin protein families.

Metazoan evolution of the armadillo repeat superfamily.

The superfamily of armadillo repeat proteins is a fascinating archetype of modular-binding proteins involved in various fundamental cellular processes, including cell-cell adhesion, cytoskeletal organization, nuclear import, and molecular signaling. Despite their diverse functions, they all share tandem armadillo (ARM) repeats, which stack together to form a conserved three-dimensional structure. This superhelical armadillo structure enables them to interact with distinct partners by wrapping around them. Despite the important functional roles of this superfamily, a comprehensive analysis of the composition, classification, and phylogeny of this protein superfamily has not been reported. Furthermore, relatively little is known about a subset of ARM proteins, and some of the current annotations of armadillo repeats are incomplete or incorrect, often due to high similarity with HEAT repeats. We identified the entire armadillo repeat superfamily repertoire in the human genome, annotated each armadillo repeat, and performed an extensive evolutionary analysis of the armadillo repeat proteins in both metazoan and premetazoan species. Phylogenetic analyses of the superfamily classified them into several discrete branches with members showing significant sequence homology, and often also related functions. Interestingly, the phylogenetic structure of the superfamily revealed that about 30 % of the members predate metazoans and represent an ancient subset, which is gradually evolving to acquire complex and highly diverse functions.

The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders.