Spatio-temporal dynamics of early somite segmentation in the chicken embryo.

During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is controlled by the somitogenesis embryo clock (EC), which was first described as gene expression oscillations of hairy1 (hes4) in the presomitic mesoderm of chick embryos with 15-20 somites. Here, the EC displays the same periodicity as somite formation, 90 min, whereas the posterior-most somites (44-52) only arise every 150 minutes, matched by a corresponding slower pace of the EC. Evidence suggests that the rostral-most somites are formed faster, however, their periodicity and the EC expression dynamics in these early stages are unknown. In this study, we used time-lapse imaging of chicken embryos from primitive streak to somitogenesis stages with high temporal resolution (3-minute intervals). We measured the length between the anterior-most and the last formed somitic clefts in each captured frame and developed a simple algorithm to automatically infer both the length and time of formation of each somite. We found that the occipital somites (up to somite 5) form at an average rate of 75 minutes, while somites 6 onwards are formed approximately every 90 minutes. We also assessed the expression dynamics of hairy1 using half-embryo explants cultured for different periods of time. This showed that EC hairy1 expression is highly dynamic prior to somitogenesis and assumes a clear oscillatory behaviour as the first somites are formed. Importantly, using ex ovo culture and live-imaging techniques, we showed that the hairy1 expression pattern recapitulates with the formation of each new pair of somites, indicating that somite segmentation is coupled with EC oscillations since the onset of somitogenesis.

Avian pathogenic Escherichia coli T6SS effector protein Hcp2a causes mitochondrial dysfunction through interaction with LETM1 protein in DF-1 cells.

The type VI secretion system (T6SS) of avian pathogenic Escherichia coli (APEC) can affect the functions of eukaryotic cells by secreting or injecting effectors. Hemolysin co-regulatory protein (Hcp), one of the markers of the T6SS, is both a structural protein and an effector protein of the T6SS. According to previous studies, mitochondria in eukaryotic cells are targeted by pathogenic bacteria. However, little is known about the regulation of mitochondria in eukaryotic host cells by the T6SS effector protein Hcp of APEC. In our study, DF-1 cells co-incubated with Hcp2a protein for 6 h showed decreased mitochondrial membrane potential, increased Ca2+ concentration, and increased cellular reactive oxygen species (ROS) levels. We therefore conclude that Hcp2a protein causes dysfunction to mitochondria in DF-1 cells. To explain the mechanism that causes mitochondrial dysfunction, we reanalyzed the Hcp2a interaction protein dataset in DF-1 cells, and the Leucine zipper EF-hand-containing transmembrane protein 1 (LETM1), which is associated with mitochondria, was screened. The protein and molecular docking results showed that Hcp2a protein and LETM1 protein have better binding. Finally, subcellular localization results showed that Hcp2a was localized to mitochondria. In summary, Hcp2a effector proteins caused dysfunction to DF-1 cellular mitochondria, and we hypothesize that the interaction of Hcp2a protein with LETM1 protein induces mitochondrial dysfunction and promotes mitochondrial localization of Hcp2a in DF-1 cells.

A flowchart for adequate controls in virus-based monosynaptic tracing experiments identified Cre-independent leakage of the TVA receptor in RΦGT mice.

BACKGROUND: A pseudotyped modified rabies virus lacking the rabies glycoprotein (G-protein), which is crucial for transsynaptic spread, can be used for monosynaptic retrograde tracing. By coupling the pseudotyped virus with transgene expression of the G-protein and the avian leukosis and sarcoma virus subgroup A receptor (TVA), which is necessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines, like the RΦGT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. These mouse lines are valuable tools because they reduce the number of viral injections needed compared to when using helper viruses. Since RΦGT mice do not express Cre themselves, introducing the pseudotyped rabies virus into their brain should not result in viral cell entry or spread. RESULTS: We present a straightforward flowchart for adequate controls in tracing experiments, which we employed to demonstrate Cre-independent expression of TVA in RΦGT mice. CONCLUSIONS: Our observations revealed TVA leakage, indicating that RΦGT mice should be used with caution for transgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments can identify the source of potential caveats in virus-based neuronal tracing experiments.

SMPD3 expression is spatially regulated in the developing embryo by SOXE factors.

During epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression of SMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide and necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of the SMPD3 locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenous SMPD3 within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SOXE-family transcription factors SOX9 and SOX10 regulate the expression of SMPD3 in migrating neural crest cells. Further, ChIP-seq and nascent transcription analysis reveal that SOX10 directly regulates expression of an SMPD3 enhancer specific to migratory neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.

Avian coronavirus infectious bronchitis virus Beaudette strain NSP9 interacts with STAT1 and inhibits its phosphorylation to facilitate viral replication.

Avian coronavirus, known as infectious bronchitis virus (IBV), is the causative agent of infectious bronchitis (IB). Viral nonstructural proteins play important roles in viral replication and immune modulation. IBV NSP9 is a component of the RNA replication complex for viral replication. In this study, we uncovered a function of NSP9 in immune regulation. First, the host proteins that interacted with NSP9 were screened. The immune-related protein signal transducer and activator of transcription 1 (STAT1) was identified and the interaction between NSP9 and STAT1 was further confirmed. Furthermore, IBV replication was inhibited in STAT1-overexpressing cells but inversely affected in STAT1 knock-down cells. Importantly, NSP9 inhibited STAT1 phosphorylation. Finally, the expression of JAK/STAT pathway downstream genes IRF7 and ISG20 was significantly decreased in NSP9-overexpressing cells. These results showed the important role of IBV NSP9 in immunosuppression.

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

Birds of a feather flock together: a dataset for Clock and Adcyap1 genes from migration genetics studies.

Birds in seasonal habitats rely on intricate strategies for optimal timing of migrations. This is governed by environmental cues, including photoperiod. Genetic factors affecting intrinsic timekeeping mechanisms, such as circadian clock genes, have been explored, yielding inconsistent findings with potential lineage-dependency. To clarify this evidence, a systematic review and phylogenetic reanalysis was done. This descriptor outlines the methodology for sourcing, screening, and processing relevant literature and data. PRISMA guidelines were followed, ultimately including 66 studies, with 34 focusing on candidate genes at the genotype-phenotype interface. Studies were clustered using bibliographic coupling and citation network analysis, alongside scientometric analyses by publication year and location. Data was retrieved for allele data from databases, article supplements, and direct author communications. The dataset, version 1.0.2, encompasses data from 52 species, with 46 species for the Clock gene and 43 for the Adcyap1 gene. This dataset, featuring data from over 8000 birds, constitutes the most extensive cross-species collection for these candidate genes, used in studies investigating gene polymorphisms and seasonal bird migration.

Dietary proteins modulate high-density lipoprotein characteristics in a sex-specific way in Apoe-deficient mice.

OBJECTIVES: The type and amount of dietary protein have become a topic of renewed interest, considering their involvement in several diseases. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. In a previous study, we saw that compared with soybean protein, the consumption of avian proteins, depending on sex, resulted in similar or lower atherosclerosis with a higher paraoxonase 1 activity, an antioxidant enzyme carried by high-density lipoproteins (HDL). This suggests that under these conditions, the HDL lipoproteins may undergo important changes. The aim of this research was to study the influence of soybean, chicken, and turkey proteins on the characteristics of HDL. METHODS: Male and female Apoe-deficient mice were fed purified Western diets based on the AIN-93 diet, differing only in the protein source, for 12 wk. After this period, blood and liver samples were taken for analysis of HDL composition and hepatic expression of genes related to HDL metabolism (Abca1, Lcat, Pltp, Pon1, and Scarb1). Depending on sex, these genes define a different network of interactions. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, which can be due to larger very-low-density lipoproteins (VLDLs) calculated by molar ratio triacylglycerols/VLDL cholesterol and higher expression of Lcat. In contrast, in males, a higher ratio of paraoxonase1 to apolipoprotein A1 decreased the oxidative status of the different lipoproteins, and augmented Abca1 expression was observed. CONCLUSIONS: The source of protein has an effect on the development of atherosclerosis depending on sex by modifying HDL characteristics and the expression of genes involved in their properties.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Singlet-triplet dephasing in radical pairs in avian cryptochromes leads to time-dependent magnetic field effects.

Cryptochrome 4a (Cry4a) has been proposed as the sensor at the heart of the magnetic compass in migratory songbirds. Blue-light excitation of this protein produces magnetically sensitive flavin-tryptophan radical pairs whose properties suggest that Cry4a could indeed be suitable as a magnetoreceptor. Here, we use cavity ring-down spectroscopy to measure magnetic field effects on the kinetics of these radical pairs in modified Cry4a proteins from the migratory European robin and from nonmigratory pigeon and chicken. B1/2, a parameter that characterizes the magnetic field-dependence of the reactions, was found to be larger than expected on the basis of hyperfine interactions and to increase with the delay between pump and probe laser pulses. Semiclassical spin dynamics simulations show that this behavior is consistent with a singlet-triplet dephasing (STD) relaxation mechanism. Analysis of the experimental data gives dephasing rate constants, rSTD, in the range 3-6 × 107 s-1. A simple "toy" model due to Maeda, Miura, and Arai [Mol. Phys. 104, 1779-1788 (2006)] is used to shed light on the origin of the time-dependence and the nature of the STD mechanism. Under the conditions of the experiments, STD results in an exponential approach to spin equilibrium at a rate considerably slower than rSTD. We attribute the loss of singlet-triplet coherence to electron hopping between the second and third tryptophans of the electron transfer chain and comment on whether this process could explain differences in the magnetic sensitivity of robin, chicken, and pigeon Cry4a's.

Mitochondrial DNA release mediated by TFAM deficiency promotes copper-induced mitochondrial innate immune response via cGAS-STING signalling in chicken hepatocytes.

Copper (Cu) is pollution metal that is a global concern due to its toxic effects. A recent study found that the release of mitochondrial DNA (mtDNA) into the cytoplasm can activate the innate immune response, but the exact mechanisms underlying the effect of Cu exposure remains unknown. In this study, we identified that the reduction in transcription Factor A (TFAM) led to mtDNA leakage into the cytoplasm under Cu exposure in hepatocytes, accompanied by the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway-mediated innate immunity (increased expression of cGAS, STING, TANK-binding kinase-1 (TBK1), and interferon regulatory factor-3 (IRF3)) genes and proteins, and enhanced phosphorylation levels of TBK1 and IRF3). Subsequently, silencing TFAM (siTFAM) significantly aggravated mtDNA release and the innate immune response under Cu treatment. Mitochondrial DNA depletion alleviated Cu-induced innate immunity in hepatocytes, while mtDNA transfection further enhanced the innate immune response. Notably, the inhibition of STING effectively alleviated the phosphorylation levels of the TBK1 and IRF3 proteins induced by Cu, while the upregulation of STING aggravated the Cu-induced innate immunity. Furthermore, EtBr and H-151(a STING inhibitor) treatment dramatically reversed the effect of TFAM depletion on the sharpened innate immune response induced by Cu via the cGAS-STING pathway. In general, these findings demonstrated the TFAM deficiency promotes innate immunity by activating the mtDNA-cGAS-STING signalling pathway under Cu exposure in hepatocytes, providing new insight into Cu toxicology.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins.

Jun-mediated lncRNA-IMS promotes the meiosis of chicken spermatogonial stem cells via gga-miR-31-5p/stra8.

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs) are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis (lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1 mRNAs, in the chick inner ear.

Prosaposin is a glycoprotein that is widely conserved in vertebrates. It serves as a precursor for saposins A, B, C, and D, which are necessary activators of lysosomal sphingolipid hydrolases. It can also act as a neurotrophic factor. Prosaposin plays a crucial role in the mammalian vestibuloauditory system because it prevents progressive deafness and severe vestibular dysfunction. Prosaposin can exhibit a neurotrophic effect through the G protein-coupled receptor (GPR), and GPR37 and GPR37L1 are its candidate receptors. In this study, we examined the expression patterns of prosaposin, GPR37, and GPR37L1 mRNAs in postnatal day 0 chick vestibuloauditory organs by in situ hybridization. Prosaposin mRNA expression was observed in all vestibular end organs, the vestibular and spiral ganglions, whereas no hybridization signal was observed in the auditory organ, namely basilar papilla. While GPR37L1 mRNA expression was observed in the oligodendrocytes/Schwann cells in the vestibular ganglion, GPR37 mRNA expression was observed in the crista ampullaris base region. These findings suggest that prosaposin expression in the auditory hair cells is acquired uniquely in mammals partly due to the loss of regeneration upon maturation and improved autophagic activity in mammalian auditory hair cells. In addition, as GPR37L1 expression in the chick glial cells differed from GPR37 expression in mammalian glial cells, the roles of GPR37 and GPR37L1 for prosaposin may differ between birds and mammals.

PNX14 but not PNX20 as a novel regulator of preadipocyte differentiation via activating Epac-ERK signaling pathway in Gallus gallus.

Small integral membrane protein 20 (SMIM20) could generate two main peptides, PNX14 and PNX20, which participate in multiple biological roles such as reproduction, inflammation and energy metabolism in mammals. However, little is known about their physiological functions in non-mammalian vertebrates. Using chicken (c-) as an animal model, we found cSMIM20 was moderately expressed in adipose tissues, and its expression was gradually increased during the differentiation of chicken preadipocytes, suggesting that it may play an important role in chicken adipogenesis. Further research showed cPNX14 could facilitate the differentiation of chicken preadipocytes into mature adipocytes by enhancing expression of adipogenic genes including PPARγ, CEBPα and FABP4, and promoting the formation of lipid droplets. This pro-adipogenic effect of cPNX14 was completely attenuated by Epac-specific and ERK inhibitor. Interestingly, cPNX20 failed to regulate the adipogenic genes and lipid droplet content. Collectively, our findings reveal that cPNX14 but not cPNX20 can serve as a novel adipogenesis mediator by activating the Epac-ERK signaling pathway in chickens.

Comparison of PB1-F2 Proximity Interactomes Reveals Functional Differences between a Human and an Avian Influenza Virus.

Most influenza viruses express the PB1-F2 protein which is regarded as a virulence factor. However, PB1-F2 behaves differently in avian and mammalian hosts, suggesting that this protein may be involved in the species barrier crossings regularly observed in influenza viruses. To better understand the functions associated with this viral protein, we decided to compare the BioID2-derived proximity interactome of a human PB1-F2 from an H3N2 virus with that of an avian PB1-F2 from an H7N1 strain. The results obtained reveal that the two proteins share only a few interactors and thus common functions. The human virus protein is mainly involved in signaling by Rho GTPases while the avian virus protein is mainly involved in ribonucleoprotein complex biogenesis. PB1-F2 H3N2 interactors include several members of the 14-3-3 protein family, a family of regulatory proteins involved in many signaling pathways. We then validated the interaction with 14-3-3 proteins and were able to show that the association of H3N2-PB1-F2 with YWHAH increased the activity of the antiviral sensor MDA5, while H7N1-PB1-F2 had no effect. Collectively, these results show that PB1-F2 can associate with a large range of protein complexes and exert a wide variety of functions. Furthermore, PB1-F2 interactome differs according to the avian or human origin of the protein.

Nonspecific Amyloid Aggregation of Chicken Smooth-Muscle Titin: In Vitro Investigations.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing ß-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-ß structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-ß structure-unlike other amyloidogenic proteins-formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

Role and function of the Hintw in early sex differentiation in chicken (Gallus gallus) embryo.

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, Hintw was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress Hintw in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of Hintw on gonadal development of chicken embryos. Results showed that Hintw exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that Foxl2, Cyp19a1 in females were upregulated under the overexpression of Hintw, while Sox9 and Dmrt1 were downregulated Hintw. Overexpression of Hintw can promote the development of gonadal cortex, while interference with Hintw show the opposite result. Additionally, we found that overexpression of the Hintw in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with Hintw in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.

Growth Hormone (GH) Crosses the Blood-Brain Barrier (BBB) and Induces Neuroprotective Effects in the Embryonic Chicken Cerebellum after a Hypoxic Injury.

Several motor, sensory, cognitive, and behavioral dysfunctions are associated with neural lesions occurring after a hypoxic injury (HI) in preterm infants. Growth hormone (GH) expression is upregulated in several brain areas when exposed to HI conditions, suggesting actions as a local neurotrophic factor. It is known that GH, either exogenous and/or locally expressed, exerts neuroprotective and regenerative actions in cerebellar neurons in response to HI. However, it is still controversial whether GH can cross the blood-brain barrier (BBB), and if its effects are exerted directly or if they are mediated by other neurotrophic factors. Here, we found that in ovo microinjection of Cy3-labeled chicken GH resulted in a wide distribution of fluorescence within several brain areas in the chicken embryo (choroid plexus, cortex, hypothalamus, periventricular areas, hippocampus, and cerebellum) in both normoxic and hypoxic conditions. In the cerebellum, Cy3-GH and GH receptor (GHR) co-localized in the granular and Purkinje layers and in deep cerebellar nuclei under hypoxic conditions, suggesting direct actions. Histological analysis showed that hypoxia provoked a significant modification in the size and organization of cerebellar layers; however, GH administration restored the width of external granular layer (EGL) and molecular layer (ML) and improved the Purkinje and granular neurons survival. Additionally, GH treatment provoked a significant reduction in apoptosis and lipoperoxidation; decreased the mRNA expression of the inflammatory mediators (TNFα, IL-6, IL-1ß, and iNOS); and upregulated the expression of several neurotrophic factors (IGF-1, VEGF, and BDNF). Interestingly, we also found an upregulation of cerebellar GH and GHR mRNA expression, which suggests the existence of an endogenous protective mechanism in response to hypoxia. Overall, the results demonstrate that, in the chicken embryo exposed to hypoxia, GH crosses the BBB and reaches the cerebellum, where it exerts antiapoptotic, antioxidative, anti-inflammatory, neuroprotective, and neuroregenerative actions.

Avian Influenza NS1 Proteins Inhibit Human, but Not Duck, RIG-I Ubiquitination and Interferon Signaling.

The nonstructural protein 1 (NS1) of influenza A viruses is an important virulence factor that controls host cell immune responses. In human cells, NS1 proteins inhibit the induction of type I interferon by several mechanisms, including potentially, by preventing the activation of the retinoic acid-inducible gene I (RIG-I) receptor by the ubiquitin ligase tripartite motif-containing protein 25 (TRIM25). It is unclear whether the inhibition of human TRIM25 is a universal function of all influenza A NS1 proteins or is strain dependent. It is also unclear if NS1 proteins similarly target the TRIM25 of mallard ducks, a natural reservoir host of avian influenza viruses with a long coevolutionary history and unique disease dynamics. To answer these questions, we compared the ability of five different NS1 proteins to interact with human and duck TRIM25 using coimmunoprecipitation and microscopy and assessed the consequence of this on RIG-I ubiquitination and signaling in both species. We show that NS1 proteins from low-pathogenic and highly pathogenic avian influenza viruses potently inhibit RIG-I ubiquitination and reduce interferon promoter activity and interferon-beta protein secretion in transfected human cells, while the NS1 of the mouse-adapted PR8 strain does not. However, all the NS1 proteins, when cloned into recombinant viruses, suppress interferon in infected alveolar cells. In contrast, avian NS1 proteins do not suppress duck RIG-I ubiquitination and interferon promoter activity, despite interacting with duck TRIM25. IMPORTANCE Influenza A viruses are a major cause of human and animal disease. Periodically, avian influenza viruses from wild waterfowl, such as ducks, pass through intermediate agricultural hosts and emerge into the human population as zoonotic diseases with high mortality rates and epidemic potential. Because of their coevolution with influenza A viruses, ducks are uniquely resistant to influenza disease compared to other birds, animals, and humans. Here, we investigate a mechanism of influenza A virus interference in an important antiviral signaling pathway that is orthologous in humans and ducks. We show that NS1 proteins from four avian influenza strains can block the coactivation and signaling of the human RIG-I antiviral receptor, while none block the coactivation and signaling of duck RIG-I. Understanding host-pathogen dynamics in the natural reservoir will contribute to our understanding of viral disease mechanisms, viral evolution, and the pressures that drive it, which benefits global surveillance and outbreak prevention.