Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

Will the Antarctic tardigrade Acutuncus antarcticus be able to withstand environmental stresses related to global climate change?

Because conditions in continental Antarctica are highly selective and extremely hostile to life, its biota is depauperate, but well adapted to live in this region. Global climate change has the potential to impact continental Antarctic organisms because of increasing temperatures and ultraviolet radiation. This research evaluates how ongoing climate changes will affect Antarctic species, and whether Antarctic organisms will be able to adapt to the new environmental conditions. Tardigrades represent one of the main terrestrial components of Antarctic meiofauna; therefore, the pan-Antarctic tardigrade Acutuncus antarcticus was used as model to predict the fate of Antarctic meiofauna threatened by climate change. Acutuncus antarcticus individuals tolerate events of desiccation, increased temperature and UV radiation. Both hydrated and desiccated animals tolerate increases in UV radiation, even though the desiccated animals are more resistant. Nevertheless, the survivorship of hydrated and desiccated animals is negatively affected by the combination of temperature and UV radiation, with the hydrated animals being more tolerant than desiccated animals. Finally, UV radiation has a negative impact on the life history traits of successive generations of A. antarcticus, causing an increase in egg reabsorption and teratological events. In the long run, A. antarcticus could be at risk of population reductions or even extinction. Nevertheless, because the changes in global climate will proceed gradually and an overlapping of temperature and UV increase could be limited in time, A. antarcticus, as well as many other Antarctic organisms, could have the potential to overcome global warming stresses, and/or the time and capability to adapt to the new environmental conditions.

Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

Increasing temperature-driven changes in life history traits and gene expression of an Antarctic tardigrade species.

The Antarctic region has been experiencing some of the planet's strongest climatic changes, including an expected increase of the land temperature. The potential effects of this warming trend will lead ecosystems to a risk of losing biodiversity. Antarctic mosses and lichens host different microbial groups, micro-arthropods and meiofaunal organisms (e.g., tardigrades, rotifers). The eutardigrade Acutuncus antarcticus is considered a model animal to study the effect of increasing temperature due to global warming on Antarctic terrestrial communities. In this study, life history traits and fitness of this species are analyzed by rearing specimens at two different and increasing temperatures (5°C vs. 15°C). Moreover, the first transcriptome analysis on A. antarcticus is performed, exposing adult animals to a gradual increase of temperature (5°C, 10°C, 15°C, and 20°C) to find differentially expressed genes under short- (1 day) and long-term (15 days) heat stress. Acutuncus antarcticus specimens reared at 5°C live longer (maximum life span: 686 days), reach sexual maturity later, lay more eggs (which hatch in longer time and in lower percentage) compared with animals reared at 15°C. The fitness decreases in animals belonging to the second generation at both rearing temperatures. The short-term heat exposure leads to significant changes at transcriptomic level, with 67 differentially expressed genes. Of these, 23 upregulated genes suggest alterations of mitochondrial activity and oxido-reductive processes, and two intrinsically disordered protein genes confirm their role to cope with heat stress. The long-term exposure induces alterations limited to 14 genes, and only one annotated gene is upregulated in response to both heat stresses. The decline in transcriptomic response after a long-term exposure indicates that the changes observed in the short-term are likely due to an acclimation response. Therefore, A. antarcticus could be able to cope with increasing temperature over time, including the future conditions imposed by global climate change.

Resistance to Extreme Stresses by a Newly Discovered Japanese Tardigrade Species, Macrobiotus kyoukenus (Eutardigrada, Macrobiotidae).

Tardigrades are small micrometazoans able to resist several environmental stresses in any stage of their life cycle. An integrated analysis of tardigrade specimens collected in Tsukuba (Japan) revealed a peculiar morphology and a new sensory field in the cloaca. Molecular taxonomy and phylogenetic analysis on different genes (COI, ITS2, 18S and 28S) confirmed that this population is a new species, Macrobiotus kyoukenus sp. nov., belonging to the widespread Macrobiotus hufelandi group. The stress resistance capabilities of M. kyoukenus sp. nov. have been tested by submitting animals to extreme desiccation, rapid freezing, and high levels of ultraviolet radiations (UVB and UVC). Animals were able to survive desiccation (survivorship 95.71 ± 7.07%) and freezing up to -80 °C (82.33 ± 17.11%). Both hydrated and desiccated animals showed a high tolerance to increasing UV radiations: hydrated animals survived to doses up to 152.22 kJ m-2 (UVB) and up to 15.00 kJ m-2 (UVC), while desiccated specimens persisted to radiations up to 165.12 kJ m-2 (UVB) and up to 35.00 kJ m-2 (UVC). Present data contribute to the discovery of a larger tardigrade biodiversity in Japan, and the tolerance capabilities of M. kyoukenus sp. nov. show that it could become a new emerging model for stress resistance studies.

Antioxidant Response during the Kinetics of Anhydrobiosis in Two Eutardigrade Species.

Anhydrobiosis, a peculiar adaptive strategy existing in nature, is a reversible capability of organisms to tolerate a severe loss of their body water when their surrounding habitat is drying out. In the anhydrobiotic state, an organism lacks all dynamic features of living beings since an ongoing metabolism is absent. The depletion of water in the anhydrobiotic state increases the ionic concentration and the production of reactive oxygen species (ROS). An imbalance between the increased production of ROS and the limited action of antioxidant defences is a source of biomolecular damage and can lead to oxidative stress. The deleterious effects of oxidative stress were demonstrated in anhydrobiotic unicellular and multicellular organisms, which counteract the effects using efficient antioxidant machinery, mainly represented by ROS scavenger enzymes. To gain insights into the dynamics of antioxidant patterns during the kinetics of the anhydrobiosis of two tardigrade species, Paramacrobiotus spatialis and Acutuncus antarcticus, we investigated the activity of enzymatic antioxidants (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) and the amount of non-enzymatic antioxidants (glutathione) in the course of rehydration. In P. spatialis, the activity of catalase increases during dehydration and decreases during rehydration, whereas in A. antarcticus, the activity of superoxide dismutase decreases during desiccation and increases during rehydration. Genomic varieties, different habitats and geographical regions, different diets, and diverse evolutionary lineages may have led to the specialization of antioxidant strategies in the two species.

Production of reactive oxygen species and involvement of bioprotectants during anhydrobiosis in the tardigrade Paramacrobiotus spatialis.

Water unavailability is an abiotic stress causing unfavourable conditions for life. Nevertheless, some animals evolved anhydrobiosis, a strategy allowing for the reversible organism dehydration and suspension of metabolism as a direct response to habitat desiccation. Anhydrobiotic animals undergo biochemical changes synthesizing bioprotectants to help combat desiccation stresses. One stress is the generation of reactive oxygen species (ROS). In this study, the eutardigrade Paramacrobiotus spatialis was used to investigate the occurrence of ROS associated with the desiccation process. We observed that the production of ROS significantly increases as a function of time spent in anhydrobiosis and represents a direct demonstration of oxidative stress in tardigrades. The degree of involvement of bioprotectants, including those combating ROS, in the P. spatialis was evaluated by perturbing their gene functions using RNA interference and assessing the successful recovery of animals after desiccation/rehydration. Targeting the glutathione peroxidase gene compromised survival during drying and rehydration, providing evidence for the role of the gene in desiccation tolerance. Targeting genes encoding glutathione reductase and catalase indicated that these molecules play roles during rehydration. Our study also confirms the involvement of aquaporins 3 and 10 during rehydration. Therefore, desiccation tolerance depends on the synergistic action of many different molecules working together.

Distinguishing affective depersonalization from anhedonia in major depression and bipolar disorder.

BACKGROUND: Affective depersonalization has received limited attention in the literature, although its conceptualization may have implications in terms of identification of clinical endophenotypes of mood disorders. Thus, this study aims to test the hypothesis that anhedonia and affective depersonalization represent 2 distinct psychopathological dimensions and to investigate their clinical correlates in patients with major depressive disorder (MDD) and bipolar disorder (BD). METHODS: Using a data pool of 258 patients with mood and anxiety disorders, an item response theory-based factor analysis approach was carried out on 16 items derived from 2 clinical instruments developed in the Spectrum Project (the Structured Clinical Interview for Mood Spectrum and the Structured Clinical Interview for Derealization-Depersonalization Spectrum). Clinical correlates of these psychometrically derived dimensions were subsequently investigated in patients with BD or MDD. RESULTS: Using an item response theory-based factor analysis, a 2-factor solution was identified, accounting overall for the 47.0% of the variance. Patients with BD showed statistically significant higher affective depersonalization factor scores than those with MDD (Z = 2.215, P = .027), whereas there was no between-groups difference in anhedonia scores (Z = 0.825 P = .411). In patients with BD, age of onset of the disease correlated with affective depersonalization factor scores (rho = -0.330, P = .001) but not with anhedonia factor scores (rho = -0.097, P = .361). CONCLUSIONS: Affective depersonalization and anhedonia seem to be 2 distinct psychopathological dimensions, although closely related, bearing the opportunity to identify patients with a specific profile for a better clinical and neurobiological definition.

The Compact Body Plan of Tardigrades Evolved by the Loss of a Large Body Region.

The superphylum Panarthropoda (Arthropoda, Onychophora, and Tardigrada) exhibits a remarkable diversity of segment morphologies, enabling these animals to occupy diverse ecological niches. The molecular identities of these segments are specified by Hox genes and other axis patterning genes during development [1, 2]. Comparisons of molecular segment identities between arthropod and onychophoran species have yielded important insights into the origins and diversification of their body plans [3-9]. However, the relationship of the segments of tardigrades to those of arthropods and onychophorans has remained enigmatic [10, 11], limiting our understanding of early panarthropod body plan diversification. Here, we reveal molecular identities for all of the segments of a tardigrade. Based on our analysis, we conclude that tardigrades have lost a large intermediate region of the body axis-a region corresponding to the entire thorax and most of the abdomen of insects-and that they have lost the Hox genes that originally specified this region. Our data suggest that nearly the entire tardigrade body axis is homologous to just the head region of arthropods. Based on our results, we reconstruct a last common ancestor of Panarthropoda that had a relatively elongate body plan like most arthropods and onychophorans, rather than a compact, tardigrade-like body plan. These results demonstrate that the body plan of an animal phylum can originate by the loss of a large part of the body.

Superoxide Anion Radical Production in the Tardigrade Paramacrobiotus richtersi, the First Electron Paramagnetic Resonance Spin-Trapping Study.

Anhydrobiosis is an adaptive strategy that allows withstanding almost complete body water loss. It has been developed independently by many organisms belonging to different evolutionary lines, including tardigrades. The loss of water during anhydrobiotic processes leads to oxidative stress. To date, the metabolism of free radicals in tardigrades remained unclear. We present a method for in vivo monitoring of free radical production in tardigrades, based on electron paramagnetic resonance and spin-trap DEPMPO, which provides simultaneous identification of various spin adducts (i.e., different types of free radicals). The spin trap can be easily absorbed in animals, and tardigrades stay alive during the measurements and during 24-h monitoring after the treatment. The results show that hydrated specimens of the tardigrade Paramacrobiotus richtersi produce the pure superoxide anion radical ((•)O2(-)). This is an unexpected result, as all previously examined animals and plants produce both superoxide anion radical and hydroxyl radical ((•)OH) or exclusively hydroxyl radical.

Decoy oligodeoxynucleotides targeting NF-kappaB transcription factors: induction of apoptosis in human primary osteoclasts.

Proteins belonging to the nuclear factor kappaB (NF-kappaB) superfamily are involved in osteoclast formation, playing a very important role for both differentiation of osteoclast precursors and survival of mature osteoclasts. Several drugs used to fight bone loss in a variety of human pathologies, including osteoporosis, act by increasing the frequency of osteoclast apoptosis, since it was demonstrated that small changes in osteoclast apoptosis can result in large changes in bone formation. In this respect, targeting of NF-kappaB transcription factor could be of great interest. Among nonviral gene therapy strategies recently proposed to inhibit or even block NF-kappaB activity, the transcription factor decoy (TFD) should be taken in great consideration. The main issue of the present study was to examine the effects of decoy DNA/DNA molecules targeting NF-kappaB on apoptosis of human osteoclasts (OCs), with the aim to interfere with the pathway regulating osteoclast differentiation and programmed cell death. To this aim, we used a mixture of receptor activator of NF-kappaB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and parathyroid hormone (PTH) to prepare human OCs from peripheral blood cells. Then, transfection with the decoy molecules targeting NF-kappaB was performed. The results obtained demonstrate that in primary cells expressing typical osteoclast markers such as TRAP and MMP9, NF-kappaB decoy significantly stimulated apoptosis. Inhibition of IL-6 expression and induction of Caspase 3 were found in OCs treated with NF-kappaB DNA/DNA decoys. We consider these data as the basis for setting up experimental conditions allowing nonviral gene therapy of several bone disorders.

Clinical correlates of depersonalization symptoms in patients with bipolar disorder.

BACKGROUND: Prevalence and clinical correlates of dissociative symptoms in general, and depersonalization (DP) in particular, in patients with mood disorders have received limited attention in the literature. Nevertheless, the identification of these symptoms may have important implications in terms of a better definition of clinical endophenotypes. Thus, this study aimed at investigating frequency and clinical correlates of dissociative symptoms, with special attention to DP symptoms, in patients with bipolar disorder (BD) looking specifically at differences between BD-I and BD-II and the comorbidity with panic disorder. METHODS: The study sample included 91 adult patients with BD (BD-I=43; BD-II=48) assessed with the Semi-structured Clinical Interview for Temperament (TEMPS-I), the Dissociative Experiences Scale (DES) and the Structured Clinical Interview for Depersonalization-Derealization Spectrum (SCI-DER). RESULTS: There was no difference in lifetime dissociative experiences or DP symptoms between BD-I and BD-II patients. There was no difference in relation to temperament characteristics. Lifetime DP symptoms, as assessed with the SCI-DER, were associated to an early onset of the BD (beta=-0.436, t=-4.572, p<0.001). Derealization symptoms correlated with panic disorder comorbidity (OR=1.22; 95%CI=1.03-1.46, Wald=5.177, p=0.023). CONCLUSIONS: Our study suggests that lifetime DP symptoms are correlated with an early onset of the BD and derealization symptoms with panic disorder comorbidity, bearing the opportunity to identify patients with a specific profile for a better clinical and neurobiological definition.

Validity and reliability of the Structured Clinical Interview for Depersonalization-Derealization Spectrum (SCI-DER).

This study evaluates the validity and reliability of a new instrument developed to assess symptoms of depresonalization: the Structured Clinical Interview for the Depersonalization-Derealization Spectrum (SCI-DER). The instrument is based on a spectrum model that emphasizes soft-signs, sub-threshold syndromes as well as clinical and subsyndromal manifestations. Items of the interview include, in addition to DSM-IV criteria for depersonalization, a number of features derived from clinical experience and from a review of phenomenological descriptions. Study participants included 258 consecutive patients with mood and anxiety disorders, 16.7% bipolar I disorder, 18.6% bipolar II disorder, 32.9% major depression, 22.1% panic disorder, 4.7% obsessive compulsive disorder, and 1.5% generalized anxiety disorder; 2.7% patients were also diagnosed with depersonalization disorder. A comparison group of 42 unselected controls was enrolled at the same site. The SCI-DER showed excellent reliability and good concurrent validity with the Dissociative Experiences Scale. It significantly discriminated subjects with any diagnosis of mood and anxiety disorders from controls and subjects with depersonalization disorder from controls. The hypothesized structure of the instrument was confirmed empirically.