Insect prophenoloxidases (PPOs) are important immunity proteins for defending against the invading pathogens and parasites. As a Type-Ⅲ copper-containing proteins, unlike Homo sapiens tyrosinases, the insect PPOs and most bacterial tyrosinases contain no signal peptides for unknown reason, however they can still be released. To this end, we fused different signal peptides to Drosophila melanogaster PPOs for in vitro and in vivo expression, respectively. We demonstrate that an artificial signal peptide can help PPO secretion in vitro. The secreted PPO appeared larger than wild-type PPO on molecular weight sizes due to glycosylation when expressed in S2 cells. Two asparagine residues for potential glycosylation in PPO1 were identified when a signal peptide was fused. After purification, the glycosylated PPO1 lost zymogen activity. When PPO1 containing a signal peptide was over-expressed in Drosophila larvae, the glycosylation and secretion of PPO1 was detected in vivo. Unlike insect PPO, human tyrosinase needs a signal peptide for protein expression and maintaining enzyme activity. An artificial signal peptide fused to bacterial tyrosinase had no influence on the protein expression and enzyme activity. These Type-Ⅲ copper-containing proteins from different organisms may evolve to perform their specific functions. Intriguingly, our study revealed that the addition of calcium inhibits PPO secretion from the transiently cultured larval hindguts in vitro, indicating that the calcium concentration may regulate PPO secretion. Taken together, insect PPOs can maintain enzyme activities without any signal peptide.

• Klíčová slova
• Enzyme activity, Glycosylation, Insect, Prophenoloxidase, Signal peptide,
• MeSH
• buněčné linie MeSH
• Drosophila melanogaster * imunologie   metabolismus   MeSH
• glykosylace MeSH
• hmyzí proteiny metabolismus   genetika   MeSH
• katecholoxidasa * metabolismus   MeSH
• larva metabolismus   MeSH
• lidé MeSH
• prekurzory enzymů * metabolismus   MeSH
• proteinové prekurzory metabolismus   MeSH
• proteiny - lokalizační signály * MeSH
• proteiny Drosophily metabolismus   genetika   MeSH
• tyrosinasa metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hmyzí proteiny MeSH
• katecholoxidasa * MeSH
• prekurzory enzymů * MeSH
• pro-phenoloxidase MeSH Prohlížeč
• proteinové prekurzory MeSH
• proteiny - lokalizační signály * MeSH
• proteiny Drosophily MeSH
• tyrosinasa MeSH
• vápník MeSH

Regulation of neuroimmune interactions varies across avian species. Little is presently known about the interplay between periphery and central nervous system (CNS) in parrots, birds sensitive to neuroinflammation. Here we investigated the systemic and CNS responses to dextran sulphate sodium (DSS)- and lipopolysaccharide (LPS)-induced subclinical acute peripheral inflammation in budgerigar (Melopsittacus undulatus). Three experimental treatment groups differing in DSS and LPS stimulation were compared to controls. Individuals treated with DSS showed significant histological intestinal damage. Through quantitative proteomics we described changes in plasma (PL) and cerebrospinal fluid (CSF) composition. In total, we identified 180 proteins in PL and 978 proteins in CSF, with moderate co-structure between the proteomes. Between treatments we detected differences in immune, coagulation and metabolic pathways. Proteomic variation was associated with the levels of pro-inflammatory cytokine mRNA expression in intestine and brain. Our findings shed light on systemic impacts of peripheral low-grade inflammation in birds.

• Klíčová slova
• Cerebrospinal fluid, Dextran sulphate sodium, Endotoxin, Parrot, Plasma, Proteomics,
• MeSH
• centrální nervový systém * metabolismus   imunologie   MeSH
• cytokiny metabolismus   MeSH
• lipopolysacharidy * imunologie   MeSH
• Melopsittacus * imunologie   MeSH
• mozek metabolismus   imunologie   MeSH
• nemoci ptáků imunologie   metabolismus   MeSH
• neuroimunomodulace MeSH
• neurozánětlivé nemoci imunologie   MeSH
• proteom * metabolismus   MeSH
• proteomika metody   MeSH
• ptačí proteiny metabolismus   genetika   MeSH
• síran dextranu * MeSH
• střeva imunologie   MeSH
• zánět * imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny MeSH
• lipopolysacharidy * MeSH
• proteom * MeSH
• ptačí proteiny MeSH
• síran dextranu * MeSH

Diseases caused by pathogens contribute to molecular adaptations in host immunity. Variety of viral pathogens challenging animal immunity can drive positive selection diversifying receptors recognising the infections. However, whether distinct virus sensing systems differ across animals in their evolutionary modes remains unclear. Our review provides a comparative overview of natural selection shaping molecular evolution in vertebrate viral-binding pattern recognition receptors (PRRs). Despite prevailing negative selection arising from the functional constraints, multiple lines of evidence now suggest diversifying selection in the Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) and oligoadenylate synthetases (OASs). In several cases, location of the positively selected sites in the ligand-binding regions suggests effects on viral detection although experimental support is lacking. Unfortunately, in most other PRR families including the AIM2-like receptor family, C-type lectin receptors (CLRs), and cyclic GMP-AMP synthetase studies characterising their molecular evolution are rare, preventing comparative insight. We indicate shared characteristics of the viral sensor evolution and highlight priorities for future research.

• Klíčová slova
• Evolutionary adaptation, Innate immunity, Molecular evolution, Pattern recognition receptor, Positive selection, Virus detection,
• MeSH
• molekulární evoluce MeSH
• obratlovci MeSH
• přirozená imunita * MeSH
• receptory rozpoznávající vzory * genetika   MeSH
• selekce (genetika) MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• receptory rozpoznávající vzory * MeSH

Species-specific neural inflammation can be induced by profound immune signalling from periphery to brain. Recent advances in transcriptomics offer cost-effective approaches to study this regulation. In a population of captive zebra finch (Taeniopygia guttata), we compare the differential gene expression patterns in lipopolysaccharide (LPS)-triggered peripheral inflammation revealed by RNA-seq and QuantSeq. The RNA-seq approach identified more differentially expressed genes but failed to detect any inflammatory markers. In contrast, QuantSeq results identified specific expression changes in the genes regulating inflammation. Next, we adopted QuantSeq to relate peripheral and brain transcriptomes. We identified subtle changes in the brain gene expression during the peripheral inflammation (e.g. up-regulation in AVD-like and ACOD1 expression) and detected co-structure between the peripheral and brain inflammation. Our results suggest benefits of the 3'end transcriptomics for association studies between peripheral and neural inflammation in genetically heterogeneous models and identify potential targets for the future brain research in birds.

• Klíčová slova
• Avian cytokine, Differential gene expression, Neurogenic inflammation, Neuroimmune interaction, Peripheral immunity, Transcriptome,
• MeSH
• messenger RNA metabolismus   MeSH
• mozek metabolismus   MeSH
• pěnkavovití * genetika   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• zánět genetika   metabolismus   MeSH
• zpěvní ptáci * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH

Hemolymph is the circulatory fluid that fills the body cavity of crustaceans, analogous to blood in vertebrates. Hemolymph coagulation, similar to blood clotting in vertebrates, plays a crucial role in wound healing and innate immune responses. Despite extensive studies on the clotting process in crustaceans, no comparative quantitative analysis of the protein composition of non-clotted and clotted hemolymph in any decapod has been reported. In this study, we used label-free protein quantification with high-resolution mass spectrometry to identify the proteomic profile of hemolymph in crayfish and quantify significant changes in protein abundances between non-clotted and clotted hemolymph. Our analysis identified a total of two-hundred and nineteen proteins in both hemolymph groups. Furthermore, we discussed the potential functions of the top most high and low-abundant proteins in hemolymph proteomic profile. The quantity of most of the proteins was not significantly changed during coagulation between non-clotted and clotted hemolymph, which may indicate that clotting proteins are likely pre-synthesized, allowing for a swift coagulation response to injury. Four proteins still showed abundance differences (p < 0.05, fold change>2), including C-type lectin domain-containing proteins, Laminin A chain, Tropomyosin, and Reverse transcriptase domain-containing proteins. While the first three proteins were down-regulated, the last one was up-regulated. The down-regulation of structural and cytoskeletal proteins may affect the process of hemocyte degranulation needed for coagulation, while the up-regulation of an immune-related protein might be attributed to the phagocytosis ability of viable hemocytes during coagulation.

• Klíčová slova
• Clot proteomics, Decapods, Innate immunity, Protein,
• MeSH
• hemocyty MeSH
• hemokoagulace fyziologie   MeSH
• hemolymfa * metabolismus   MeSH
• koagulační faktory metabolismus   MeSH
• proteomika MeSH
• severní raci * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• koagulační faktory MeSH

Toll-like receptors (TLRs) form a key component of animal innate immunity, being responsible for recognition of conserved microbial structures. As such, TLRs may be subject to diversifying and balancing selection, which maintains allelic variation both within and between populations. However, most research on TLRs in non-model avian species is focused on bottlenecked populations with depleted genetic variation. Here, we assessed variation at the extracellular domains of three TLR genes (TLR1LA, TLR3, TLR4) across eleven species from two passerine families of buntings (Emberizidae) and finches (Fringillidae), all having large breeding population sizes (millions of individuals). We found extraordinary TLR polymorphism in our study taxa, with >100 alleles detected at TLR1LA and TLR4 across species and high haplotype diversity (>0.75) in several species. Despite recent species divergence, no nucleotide allelic variants were shared between species, suggesting rapid TLR evolution. Higher variation at TLR1LA and TLR4 than TLR3 was associated with a stronger signal of diversifying selection, as measured with nucleotide substitutions rates and the number of positively selected sites (PSS). Structural protein modelling of TLRs showed that some PSS detected within TLR1LA and TLR4 were previously recognized as functionally important sites or were located in their proximity, possibly affecting ligand recognition. Furthermore, we identified PSS responsible for major surface electrostatic charge clustering, which may indicate their adaptive importance. Our study provides compelling evidence for the divergent evolution of TLR genes in buntings and finches and indicates that high TLR variation may be adaptively maintained via diversifying selection acting on functional ligand binding sites.

• Klíčová slova
• Allele diversity, Birds, Divergent evolution, Polymorphism, Positive selection, Toll-like receptors,
• MeSH
• ligandy MeSH
• molekulární evoluce MeSH
• Passeriformes * genetika   MeSH
• pěnkavovití * genetika   MeSH
• toll-like receptor 3 genetika   MeSH
• toll-like receptor 4 genetika   MeSH
• toll-like receptory genetika   chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ligandy MeSH
• toll-like receptor 3 MeSH
• toll-like receptor 4 MeSH
• toll-like receptory MeSH

Crustacean hemocytes are important mediators of immune functions such as coagulation and phagocytosis. We employed an in situ approach to investigate the ultrastructural behavior of hemocytes during coagulation and phagocytosis in the early stages after injury caused by leg amputation, using transmission electron microscopy technique in marbled crayfish Procambarus virginalis. Hemocytes underwent drastic morphological changes during coagulation. The morphology of the cytoplasmic granules changed from electron-dense to electron-lucent forms in an expanding manner. The transformed granules containing amorphous electron-lucent material were observed to merge and discharge their contents into extracellular space for coagulation. We also observed that the contents of the nucleus participate in the process of coagulation. In addition, leg amputation induced extensive muscle degeneration and necrotic tissues were avidly taken up by the phagocytic hemocytes containing distinct phagosomes. Interestingly, we observed for the first time how the digested contents of phagocytized necrotic tissues are incorporated into granules and other cellular components that change the cell morphology by increasing the granularity of the hemocytes. Nevertheless, the degranulation of hemocytes during coagulation can also reduce their granularity. Given that morphological traits are important criteria for hemocyte classification, these morphological changes that occur during coagulation and phagocytosis must be taken into account.

• Klíčová slova
• Degranulation, Hemolymph coagulation, Muscle degeneration, Phagocytosis,
• MeSH
• členovci * MeSH
• fagocytóza MeSH
• fagozomy MeSH
• hemocyty * MeSH
• severní raci MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the κ light chain is replaced by λ before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3' V and 5' J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5' IGLV and 3' IGLJ genes are used. A similar shift toward the use of more 5' IGKV and 3' IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most λ+ B cells are generated earlier, while κ+ B cells develop from late precursors that already contain the λ rearrangement. The initial λ rearrangement is retained in both λ+ and κ+ B lymphocytes, and multiple λ transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The κ repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced λ transcripts in κ+ B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.

• Klíčová slova
• B cell development, B cell receptors, Immunoglobulin light chains, Immunoglobulin rearrangement, Lymphocyte differentiation, Porcine immune system,
• MeSH
• B-lymfocyty MeSH
• geny pro imunoglobuliny MeSH
• imunoglobuliny - kappa-řetězce * genetika   MeSH
• imunoglobuliny - lambda-řetězce genetika   MeSH
• lehké řetězce imunoglobulinů * genetika   MeSH
• lymfoidní tkáň MeSH
• prasata MeSH
• savci genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - kappa-řetězce * MeSH
• imunoglobuliny - lambda-řetězce MeSH
• lehké řetězce imunoglobulinů * MeSH

Entomopathogenic fungi (EPF) have been widely explored for their potential in the biological control of insect pests and as an environmentally friendly alternative to acaricides for limiting tick infestation in the field. The arthropod cuticle is the main barrier against fungal infection, however, an understanding of internal defense mechanisms after EPF intrusion into the invertebrate hemocoel is still rather limited. Using an infection model of the European Lyme borreliosis vector Ixodes ricinus with the EPF Metarhizium robertsii, we demonstrated that ticks are capable of protecting themselves to a certain extent against mild fungal infections. However, tick mortality dramatically increases when the capability of tick hemocytes to phagocytose fungal conidia is impaired. Using RNAi-mediated silencing of tick thioester-containing proteins (TEPs), followed by in vitro and/or in vivo phagocytic assays, we found that C3-like complement components and α2-macroglobulin pan-protease inhibitors secreted to the hemolymph play pivotal roles in M. robertsii phagocytosis.

• Klíčová slova
• Biological control, Entomopathogenic fungi, Hemocytes, Phagocytosis, Thioester-containing proteins, Tick,
• MeSH
• hemocyty MeSH
• klíště * MeSH
• lymeská nemoc * MeSH
• Metarhizium * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Swine use a reverse order of immunoglobulin chain rearrangement compared to humans and mice, and this altered and modified order should have measurable consequences. Here we perform new and defining experiments with developing and mature B cells, characterizing the B cell populations that do not exist in other species. First, we have finally confirmed that light chains κ and λ are rearranged and expressed on the surface before any heavy chain rearrangements using western-blot. And second, we have analyzed a pool of mature B cells on the single-cell level to demonstrate that many κ+ mature B cells carry λ transcripts. According to these findings, we believe that there may be more groups of mammals; one of which uses a pre-BCR-driven developmental pathway for B cell generation (like mice and humans), the second group uses a pre-BCR-independent one (like swine), and some may be even intermediate.

• Klíčová slova
• B cell development, B cell receptors, Cell differentiation, Immunoglobulin rearrangement, Other animals, Porcine immune system,
• MeSH
• B-lymfocyty MeSH
• geny pro imunoglobuliny * MeSH
• imunoglobuliny - kappa-řetězce * genetika   MeSH
• imunoglobuliny - lambda-řetězce genetika   MeSH
• lehké řetězce imunoglobulinů genetika   MeSH
• lidé MeSH
• myši MeSH
• prasata genetika   MeSH
• savci genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - kappa-řetězce * MeSH
• imunoglobuliny - lambda-řetězce MeSH
• lehké řetězce imunoglobulinů MeSH