Analysis of the physiological function of TNF Receptor I Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD) by using conditional gene targeting in mice

The aim of the project was to apply the conditional gene targeting approach based on the usage of Cre/LoxP system of site-specific DNA recombination to investigate physiological function of two putative mediators of inflammation – TNF Receptor 1 Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD), in a mouse model. TRADD is an adaptor molecule postulated to be essential for signal transduction through TNF Receptor 1 (TNFR1). The in vivo physiological role of TRADD has not been determined so far. CYLD is a tumor suppressor that has been described as a negative regulator of TNFR1-mediated signaling and signaling by Toll like Receptors (TLRs). TLRs are essential components of mammalian innate immunity belonging to a group of sensors that directly recognize bacterial and viral products as well as markers of tissue stress. Upon activation TLRs induce intracellular signaling events leading to production of cytokines, chemokines and other mediators that promote protective responses. Tumor Necrosis Factor (TNF) is a pleiotropic cytokine produced by a variety of cells upon TLR stimulation. It plays a key role in the amplification of the initial immune response. The majority of effects that are induced by TNF are dependant on TNFR1. TLR signaling and TNF signaling through TNFR1 share common mechanism of negative regulation that is based on the the removal of K63-linked polyubiquitin chains from specific key components of receptor-associated complexes. Two de-ubiquitinating enzymes – A20 and CYLD are currently known to be responsible for this process. The physiological function of A20 is well characterized by gene knockout studies while the precise role of CYLD remains enigmatic. We successfully generated mice carrying “floxed” (modified by the insertion of LoxP sites and extra DNA fragments at specific locations) alleles of TRADD and CYLD by using homologous recombination in embryonic stem cells. In case of TRADD the deletion of the LoxP-flanked sequence would generate a null TRADD allele; in case of CYLD the last exon of the gene would be replaced by a mutated copy resulting in the expression of C-terminally truncated form of CYLD that is lacking catalytic activity. We then generated TRADD knockout and CYLD complete mutant mice by crossing the homozygous “floxed” animals to a ubiquitous Cre-Deleter strain. By analyzing TRADD knockout mice we could observe that TNFR1-mediated apoptosis was completely blocked in these mice while TNF-induced pro-inflammatory and anti-bacterial responses were dramatically reduced but still present. We obtained similar results by evaluating the response of TRADD deficient primary cells to TNF. To our surprise we discovered that TRADD knockout mice had impaired immediate responses to stimulation of Toll like receptors 3 and 4. Consistent with this observation TRADD deficient primary cells demonstrated reduced cytokine production as well as impaired activation of NF-kB and MAP kinases upon stimulation with poly(I:C). On the basis of co-expression experiments performed in HEK293T cells we propose that TRADD is recruited to TLR adaptor TRIF via Receptor-Interacting Protein 1 (RIP1) and acts as a mediator of TRIF-dependant TLR signaling. To our surprise CYLD homozygous mutant mice did not survive until the age of weaning. By carefully following the pups we observed that the mutants died within minutes after birth showing signs of cyanosis and respiratory distress. Mutant pups were smaller then control littermates and demonstrated altered morphology of the tail. We then produced mutant mouse embryonic fibroblasts (MEFs) and analyzed the response of these cells to cytokines. Consistent with the role of CYLD as a negative regulator of pro-inflammatory signaling, mutant cells showed elevated activation of NF-kB and JNK cascades upon stimulation with TNF and IL-1bet

Locomotor activity of the Holarctic molluscs Radix auricularia (from Lake Baikal) in various light pollution conditions

Light pollution is a modern environmental problem. The scale of light pollution is increasing yearly and is negatively affecting the functioning of terrestrial and aquatic ecosystems. Of the aquatic ecosystems, marine ecosystems are the most studied, while there is very little information on the effect of artificial lighting on freshwater ecosystems. Among freshwater aquatic organisms, there are relatively little data on the effect of artificial light on crustaceans and fish, while we could find no meaningful data on the effect of artificial light on molluscs are practically absent. Here we test whether different types of artificial lighting, differing in their spectra, affect the activity of the Holarctic mollusc Radix auricularia. For this, we used two light sources (with warm and cold light) and a 1-m long aquarium. We found that both light sources affect individuals of this species, but the effects of this exposure are different. Artificial lighting (depending on the spectral characteristics) can increase the activity of molluscs of this species or reduce it. In the long term, the impact on the ecosystem will depend on the type of water body where light pollution is present, where individuals of this species live, and the type of light sources

Meeting Report: Aging Research and Drug Discovery

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community

The invertebrate lysozyme effector ILYS-3 is systemically activated in response to danger signals and confers antimicrobial protection in C. elegans

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material

DAF-16/FOXO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage

Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature ageing. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with ageing. Here we show that the FOXO transcription factor DAF-16 is activated in response to DNA damage during development, whereas the DNA damage responsiveness of DAF-16 declines with ageing. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA-damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16-mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists

Insights from the worm: The C. elegans model for innate immunity

The nematode worm Caenorhabditis elegans comprises an ancestral immune system. C elegans recognizes and responds to viral, bacterial, and fungal infections. Components of the RNA interference machinery respond to viral infection, while highly conserved MAPK signaling pathways activate the innate immune response to bacterial infection. C. elegans has been particularly important for exploring the role of innate immunity in organismal stress resistance and the regulation of longevity. Also functions of neuronal sensing of infectious bacteria have recently been uncovered. Studies on nematode immunity can be instructive in exploring innate immune signaling in the absence of specialized immune cells and adaptive immunity. (C) 2014 Elsevier Ltd. All rights reserved

Systemic DNA damage responses: organismal adapatations to genome instability

DNA damage checkpoints are important tumor-suppressor mechanisms that halt cell cycle progression to allow time for DNA repair, or induce senescence and apoptosis to remove damaged cells permanently. Non-cell-autonomous DNA damage responses activate the innate immune system in multiple metazoan species. These responses not only enable clearance of damaged cells and contribute to tissue remodeling and regeneration but can also result in chronic inflammation and tissue damage. Germline DNA damage-induced systemic stress resistance (GDISR) is mediated by an ancestral innate immune response and results in organismal adjustments to the presence of damaged cells. We discuss GDISR as an organismal DNA damage checkpoint mechanism through which elevated somatic endurance can extend reproductive lifespan when germ cells require extended time for restoring genome stability

Quality control mechanisms in cellular and systemic DNA damage responses

The maintenance of the genome is of pivotal importance for the functional integrity of cells and tissues. The gradual accumulation of DNA damage is thought to contribute to the functional decline of tissues and organs with ageing. Defects in multiple genome maintenance systems cause human disorders characterized by cancer susceptibility, developmental failure, and premature ageing. The complex pathological consequences of genome instability are insufficiently explained by cell-autonomous DNA damage responses (DDR) alone. Quality control pathways play an important role in DNA repair and cellular DDR pathways. Recent years have revealed non-cell autonomous effects of DNA damage that impact the physiological adaptations during ageing. We will discuss the role of quality assurance pathways in cell-autonomous and systemic responses to genome instability. (C) 2015 Elsevier B.V. All rights reserved

Phototransduction in Anuran Green Rods: Origins of Extra-Sensitivity

Green rods (GRs) represent a unique type of photoreceptor to be found in the retinas of anuran amphibians. These cells harbor a cone-specific blue-sensitive visual pigment but exhibit morphology of the outer segment typical for classic red rods (RRs), which makes them a perspective model object for studying cone–rod transmutation. In the present study, we performed detailed electrophysiological examination of the light sensitivity, response kinetics and parameters of discrete and continuous dark noise in GRs of the two anuran species: cane toad and marsh frog. Our results confirm that anuran GRs are highly specialized nocturnal vision receptors. Moreover, their rate of phototransduction quenching appeared to be about two-times slower than in RRs, which makes them even more efficient single photon detectors. The operating intensity ranges for two rod types widely overlap supposedly allowing amphibians to discriminate colors in the scotopic region. Unexpectedly for typical cone pigments but in line with some previous reports, the spontaneous isomerization rate of the GR visual pigment was found to be the same as for rhodopsin of RRs. Thus, our results expand the knowledge on anuran GRs and show that these are even more specialized single photon catchers than RRs, which allows us to assign them a status of “super-rods”

Vázquez-Boland, “Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor

Summary Virulence genes from the facultative intracellular pathogen Listeria mono cytogenes are controlled by the transcriptional regulator PrfA. Although PrfA synthesis is activated at 37 ∞ ∞ ∞ ∞ C, PrfA-dependent expression remains low in rich medium. However, a strong induction of the PrfA regulon is observed when L. monocytogenes is cultured in the presence of activated charcoal. Here, we show that the 'charcoal effect' results from the adsorption of a diffusible autorepressor substance released by L. monocytogenes during exponential growth. Analyses using an L. monocytogenes strain in which the prfA gene is expressed constitutively at 37 ∞ ∞ ∞ ∞ C from a plasmid indicate that the autoregulatory substance represses PrfA-dependent expression by inhibiting PrfA activity. PrfA presumably functions via an allosteric activation mechanism. The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA. Our data indicate that the listerial autorepressor is a low-molecular-weight hydrophobic substance. We suggest that this diffusible substance mediates a quorum-sensing mechanism by which L. monocytogenes restricts the expression of its PrfA virulence regulon. This autoregulatory pathway could serve L. monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37 ∞ ∞ ∞ ∞ C. It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA-dependent virulence factors in the PrfA-activating cytosolic compartment