A framework for the targeted recruitment of crop-beneficial soil taxa based on network analysis of metagenomics data.

BACKGROUND: The design of ecologically sustainable and plant-beneficial soil systems is a key goal in actively manipulating root-associated microbiomes. Community engineering efforts commonly seek to harness the potential of the indigenous microbiome through substrate-mediated recruitment of beneficial members. In most sustainable practices, microbial recruitment mechanisms rely on the application of complex organic mixtures where the resources/metabolites that act as direct stimulants of beneficial groups are not characterized. Outcomes of such indirect amendments are unpredictable regarding engineering the microbiome and achieving a plant-beneficial environment. RESULTS: This study applied network analysis of metagenomics data to explore amendment-derived transformations in the soil microbiome, which lead to the suppression of pathogens affecting apple root systems. Shotgun metagenomic analysis was conducted with data from 'sick' vs 'healthy/recovered' rhizosphere soil microbiomes. The data was then converted into community-level metabolic networks. Simulations examined the functional contribution of treatment-associated taxonomic groups and linked them with specific amendment-induced metabolites. This analysis enabled the selection of specific metabolites that were predicted to amplify or diminish the abundance of targeted microbes functional in the healthy soil system. Many of these predictions were corroborated by experimental evidence from the literature. The potential of two of these metabolites (dopamine and vitamin B12) to either stimulate or suppress targeted microbial groups was evaluated in a follow-up set of soil microcosm experiments. The results corroborated the stimulant's potential (but not the suppressor) to act as a modulator of plant beneficial bacteria, paving the way for future development of knowledge-based (rather than trial and error) metabolic-defined amendments. Our pipeline for generating predictions for the selective targeting of microbial groups based on processing assembled and annotated metagenomics data is available at https://github.com/ot483/NetCom2 . CONCLUSIONS: This research demonstrates how genomic-based algorithms can be used to formulate testable hypotheses for strategically engineering the rhizosphere microbiome by identifying specific compounds, which may act as selective modulators of microbial communities. Applying this framework to reduce unpredictable elements in amendment-based solutions promotes the development of ecologically-sound methods for re-establishing a functional microbiome in agro and other ecosystems. Video Abstract.

De novo mutation rates at the single-mutation resolution in a human HBB gene region associated with adaptation and genetic disease.

Although it is known that the mutation rate varies across the genome, previous estimates were based on averaging across various numbers of positions. Here, we describe a method to measure the origination rates of target mutations at target base positions and apply it to a 6-bp region in the human hemoglobin subunit beta (HBB) gene and to the identical, paralogous hemoglobin subunit delta (HBD) region in sperm cells from both African and European donors. The HBB region of interest (ROI) includes the site of the hemoglobin S (HbS) mutation, which protects against malaria, is common in Africa, and has served as a classic example of adaptation by random mutation and natural selection. We found a significant correspondence between de novo mutation rates and past observations of alleles in carriers, showing that mutation rates vary substantially in a mutation-specific manner that contributes to the site frequency spectrum. We also found that the overall point mutation rate is significantly higher in Africans than in Europeans in the HBB region studied. Finally, the rate of the 20A→T mutation, called the "HbS mutation" when it appears in HBB, is significantly higher than expected from the genome-wide average for this mutation type. Nine instances were observed in the African HBB ROI, where it is of adaptive significance, representing at least three independent originations; no instances were observed elsewhere. Further studies will be needed to examine mutation rates at the single-mutation resolution across these and other loci and organisms and to uncover the molecular mechanisms responsible.

Acute social isolation and regrouping cause short- and long-term molecular changes in the rat medial amygdala.

Social isolation poses a severe mental and physiological burden on humans. Most animal models that investigate this effect are based on prolonged isolation, which does not mimic the milder conditions experienced by people in the real world. We show that in adult male rats, acute social isolation causes social memory loss. This memory loss is accompanied by significant changes in the expression of specific mRNAs and proteins in the medial amygdala, a brain structure that is crucial for social memory. These changes particularly involve the neurotrophic signaling and axon guidance pathways that are associated with neuronal network remodeling. Upon regrouping, memory returns, and most molecular changes are reversed within hours. However, the expression of some genes, especially those associated with neurodegenerative diseases remain modified for at least a day longer. These results suggest that acute social isolation and rapid resocialization, as experienced by millions during the COVID-19 pandemic, are sufficient to induce significant changes to neuronal networks, some of which may be pathological.

Cellular pathways during spawning induction in the starlet sea anemone Nematostella vectensis.

In cnidarians, long-term ecological success relies on sexual reproduction. The sea anemone Nematostella vectensis, which has emerged as an important model organism for developmental studies, can be induced for spawning by temperature elevation and light exposure. To uncover molecular mechanisms and pathways underlying spawning, we characterized the transcriptome of Nematostella females before and during spawning induction. We identified an array of processes involving numerous receptors, circadian clock components, cytoskeleton, and extracellular transcripts that are upregulated upon spawning induction. Concurrently, processes related to the cell cycle, fatty acid metabolism, and other housekeeping functions are downregulated. Real-time qPCR revealed that light exposure has a minor effect on expression levels of most examined transcripts, implying that temperature change is a stronger inducer for spawning in Nematostella. Our findings reveal the potential mechanisms that may enable the mesenteries to serve as a gonad-like tissue for the developing oocytes and expand our understanding of sexual reproduction in cnidarians.

Genetic and physiological traits conferring tolerance to ocean acidification in mesophotic corals.

The integrity of coral reefs worldwide is jeopardized by ocean acidification (OA). Most studies conducted so far have focused on the vulnerability to OA of corals inhabiting shallow reefs while nothing is currently known about the response of mesophotic scleractinian corals. In this study, we assessed the susceptibility to OA of corals, together with their algal partners, inhabiting a wide depth range. We exposed fragments of the depth generalist coral Stylophora pistillata collected from either 5 or 45 m to simulated future OA conditions, and assessed key molecular, physiological and photosynthetic processes influenced by the lowered pH. Our comparative analysis reveals that mesophotic and shallow S. pistillata corals are genetically distinct and possess different symbiont types. Under the exposure to acidification conditions, we observed a 50% drop of metabolic rate in shallow corals, whereas mesophotic corals were able to maintain unaltered metabolic rates. Overall, our gene expression and physiological analyses show that mesophotic corals possess a greater capacity to cope with the effects of OA compared to their shallow counterparts. Such capability stems from physiological characteristics (i.e., biomass and lipids energetics), a greater capacity to regulate cellular acid-base parameters, and a higher baseline expression of cell adhesion and extracellular matrix genes. Moreover, our gene expression analysis suggests that the enhanced symbiont photochemical efficiency under high pCO2 levels could prevent acidosis of the host cells and it could support a greater translocation of photosynthates, increasing the energy pool available to the host. With this work, we provide new insights on the response to OA of corals living at mesophotic depths. Our investigation discloses key genetic and physiological traits underlying the potential for corals to cope with future OA conditions.

Combined responses of primary coral polyps and their algal endosymbionts to decreasing seawater pH.

With coral reefs declining globally, resilience of these ecosystems hinges on successful coral recruitment. However, knowledge of the acclimatory and/or adaptive potential in response to environmental challenges such as ocean acidification (OA) in earliest life stages is limited. Our combination of physiological measurements, microscopy, computed tomography techniques and gene expression analysis allowed us to thoroughly elucidate the mechanisms underlying the response of early-life stages of corals, together with their algal partners, to the projected decline in oceanic pH. We observed extensive physiological, morphological and transcriptional changes in surviving recruits, and the transition to a less-skeleton/more-tissue phenotype. We found that decreased pH conditions stimulate photosynthesis and endosymbiont growth, and gene expression potentially linked to photosynthates translocation. Our unique holistic study discloses the previously unseen intricate net of interacting mechanisms that regulate the performance of these organisms in response to OA.

Evolution of Protein-Mediated Biomineralization in Scleractinian Corals.

While recent strides have been made in understanding the biological process by which stony corals calcify, much remains to be revealed, including the ubiquity across taxa of specific biomolecules involved. Several proteins associated with this process have been identified through proteomic profiling of the skeletal organic matrix (SOM) extracted from three scleractinian species. However, the evolutionary history of this putative "biomineralization toolkit," including the appearance of these proteins' throughout metazoan evolution, remains to be resolved. Here we used a phylogenetic approach to examine the evolution of the known scleractinians' SOM proteins across the Metazoa. Our analysis reveals an evolutionary process dominated by the co-option of genes that originated before the cnidarian diversification. Each one of the three species appears to express a unique set of the more ancient genes, representing the independent co-option of SOM proteins, as well as a substantial proportion of proteins that evolved independently. In addition, in some instances, the different species expressed multiple orthologous proteins sharing the same evolutionary history. Furthermore, the non-random clustering of multiple SOM proteins within scleractinian-specific branches suggests the conservation of protein function between distinct species for what we posit is part of the scleractinian "core biomineralization toolkit." This "core set" contains proteins that are likely fundamental to the scleractinian biomineralization mechanism. From this analysis, we infer that the scleractinians' ability to calcify was achieved primarily through multiple lineage-specific protein expansions, which resulted in a new functional role that was not present in the parent gene.

Molecular and skeletal fingerprints of scleractinian coral biomineralization: From the sea surface to mesophotic depths.

Reef-building corals, the major producers of biogenic calcium carbonate, form skeletons in a plethora of morphological forms. Here we studied skeletal modifications of Stylophora pistillata (clade 4) colonies that adapt to increasing depths with decreasing ambient light. The coral show characteristic transitions from spherical morphologies (shallow depths, 5 m deep) to flat and branching geometries (mesophotic depths, 60 m deep). Such changes are typically ascribed to the algal photosymbiont physiological feedback with the coral that host them. We find specific fine-scale skeletal variability in accretion of structure at shallow- and mesophotic depth morphotypes that suggest underlying genomic regulation of biomineralization pathways of the coral host. To explain this, we conducted comparative morphology-based analyses, including optical and electron microscopy, tomography and X-ray diffraction analysis coupled with a comprehensive transcriptomic analysis of S. pistillata. The samples originated from Gulf of Eilat in the Red Sea collected along a depth gradient from shallow to mesophotic depths (5 to 60 m). Additional samples were experimentally transplanted from 5 m to 60 m and from 60 m to 5 m. Interestingly, both morphologically and functionally, transplanted corals partly adapt by exhibiting typical depth-specific properties. In mesophotic depths, we find that the organic matrix fraction is enriched in the coralla, well matching the overrepresentation of transcripts encoding biomineralization "tool-kit" structural extracellularproteins that was observed. These results provide insights into the molecular mechanisms of calcification and skeletal adaptation that repeatedly allowed this coral group to adapt to a range of environments presumably with a rich geological past. STATEMENT OF SIGNIFICANCE: Understanding the reef coral physiological plasticity under a rapidly changing climate is of crucial importance for the protection of coral reef ecosystems. Most of the reef corals operate near their upper limit of heat tolerance. A possible rescue for some coral species is migration to deeper, cooler mesophotic depths. However, gradually changing environmental parameters (especially light) along the depth gradient pose new adaptative stress on corals with largely unknown influences on the various biological molecular pathways. This work provides a first comprehensive analysis of changes in gene expression, including biomineralization "tool kit" genes, and reports the fine-scale microstructural and crystallographic skeletal details in S. pistillata collected in the Red Sea along a depth gradient spannign 5 to 60 m.

Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis.

The metabotropic gamma-aminobutyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. While GABABR-mediated signalling has been suggested to play central roles in neuronal differentiation and proliferation across evolution, it has mostly been studied in the mammalian brain. Here, we demonstrate that ectopic activation of GABABR signalling affects neurogenic functions in the sea anemone Nematostella vectensis. We identified four putative Nematostella GABABR homologues presenting conserved three-dimensional extracellular domains and residues needed for binding GABA and the GABABR agonist baclofen. Moreover, sustained activation of GABABR signalling reversibly arrests the critical metamorphosis transition from planktonic larva to sessile polyp life stage. To understand the processes that underlie the developmental arrest, we combined transcriptomic and spatial analyses of control and baclofen-treated larvae. Our findings reveal that the cnidarian neurogenic programme is arrested following the addition of baclofen to developing larvae. Specifically, neuron development and neurite extension were inhibited, resulting in an underdeveloped and less organized nervous system and downregulation of proneural factors including NvSoxB(2), NvNeuroD1 and NvElav1. Our results thus point to an evolutionarily conserved function of GABABR in neurogenesis regulation and shed light on early cnidarian development.

A tentacle for every occasion: comparing the hunting tentacles and sweeper tentacles, used for territorial competition, in the coral Galaxea fascicularis.

BACKGROUND: Coral reefs are among the most diverse, complex and densely populated marine ecosystems. To survive, morphologically simple and sessile cnidarians have developed mechanisms to catch prey, deter predators and compete with adjacent corals for space, yet the mechanisms underlying these functions are largely unknown. Here, we characterize the histology, toxic activity and gene expression patterns in two different types of tentacles from the scleractinian coral Galaxea fascilcularis - catch tentacles (CTs), used to catch prey and deter predators, and sweeper tentacles (STs), specialized tentacles used for territorial aggression. RESULTS: STs exhibit more mucocytes and higher expression of mucin genes than CTs, and lack the ectodermal cilia used to deliver food to the mouth and remove debris. STs and CTs also express different sensory rhodopsin-like g-protein coupled receptors, suggesting they may employ different sensory pathways. Each tentacle type has a different complement of stinging cells (nematocytes), and the expression in the two tentacles of genes encoding structural nematocyte proteins suggests the stinging cells develop within the tentacles. CTs have higher neurotoxicity to blowfly larvae and hemolytic activity compared to the STs, consistent with a role in prey capture. In contrast, STs have higher phospholipase A2 activity, which we speculate may have a role in inducing tissue damage during territorial aggression. The expression of genes encoding cytolytic toxins (actinoporins) and phospholipases also differs between the tentacle types. CONCLUSIONS: These results show that the same organism utilizes two distinct tentacle types, each equipped with a different venom apparatus and toxin composition, for prey capture and defense and for territorial aggression.

Optimization of skeletal protein preparation for LC-MS/MS sequencing yields additional coral skeletal proteins in Stylophora pistillata.

Stony corals generate their calcium carbonate exoskeleton in a highly controlled biomineralization process mediated by a variety of macromolecules including proteins. Fully identifying and classifying these proteins is crucial to understanding their role in exoskeleton formation, yet no optimal method to purify and characterize the full suite of extracted coral skeletal proteins has been established and hence their complete composition remains obscure. Here, we tested four skeletal protein purification protocols using acetone precipitation and ultrafiltration dialysis filters to present a comprehensive scleractinian coral skeletal proteome. We identified a total of 60 proteins in the coral skeleton, 44 of which were not present in previously published stony coral skeletal proteomes. Extracted protein purification protocols carried out in this study revealed that no one method captures all proteins and each protocol revealed a unique set of method-exclusive proteins. To better understand the general mechanism of skeletal protein transportation, we further examined the proteins' gene ontology, transmembrane domains, and signal peptides. We found that transmembrane domain proteins and signal peptide secretion pathways, by themselves, could not explain the transportation of proteins to the skeleton. We therefore propose that some proteins are transported to the skeleton via non-traditional secretion pathways.

The Role of Land Use Types and Water Chemical Properties in Structuring the Microbiomes of a Connected Lake System.

Lakes and other freshwater bodies are intimately connected to the surrounding land, yet to what extent land-use affects the quality of freshwater and the microbial communities living in various freshwater environments is largely unknown. We address this question through an analysis of the land use surrounding 46 inter-connected lakes located within seven different drainage basins in northern Germany, and the microbiomes of these lakes during early summer. Lake microbiome structure was not correlated with the specific drainage basin or by basin size, and bacterial distribution did not seem to be limited by distance. Instead, land use within the drainage basin could predict, to some extent, NO2 + NO3 concentrations in the water, which (together with temperature, chlorophyll a and total phosphorus) correlated to some extent with the water microbiome structure. Land use directly surrounding the water bodies, however, had little observable effects on water quality or the microbiome. Several microbial lineages, including Cyanobacteria and Verrucomicrobia, were differentially partitioned between the lakes. Significantly more data, including time-series measurements of land use and water chemical properties, are needed to fully understand the interaction between the environment and the organization of microbial communities.

Possible cooption of a VEGF-driven tubulogenesis program for biomineralization in echinoderms.

Biomineralization is the process by which living organisms use minerals to form hard structures that protect and support them. Biomineralization is believed to have evolved rapidly and independently in different phyla utilizing preexisting components. The mechanistic understanding of the regulatory networks that drive biomineralization and their evolution is far from clear. Sea urchin skeletogenesis is an excellent model system for studying both gene regulation and mineral uptake and deposition. The sea urchin calcite spicules are formed within a tubular cavity generated by the skeletogenic cells controlled by vascular endothelial growth factor (VEGF) signaling. The VEGF pathway is essential for biomineralization in echinoderms, while in many other phyla, across metazoans, it controls tubulogenesis and vascularization. Despite the critical role of VEGF signaling in sea urchin spiculogenesis, the downstream program it activates was largely unknown. Here we study the cellular and molecular machinery activated by the VEGF pathway during sea urchin spiculogenesis and reveal multiple parallels to the regulation of vertebrate vascularization. Human VEGF rescues sea urchin VEGF knockdown, vesicle deposition into an internal cavity plays a significant role in both systems, and sea urchin VEGF signaling activates hundreds of genes, including biomineralization and interestingly, vascularization genes. Moreover, five upstream transcription factors and three signaling genes that drive spiculogenesis are homologous to vertebrate factors that control vascularization. Overall, our findings suggest that sea urchin spiculogenesis and vertebrate vascularization diverged from a common ancestral tubulogenesis program, broadly adapted for vascularization and specifically coopted for biomineralization in the echinoderm phylum.

A-to-I RNA editing in the rat brain is age-dependent, region-specific and sensitive to environmental stress across generations.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification catalyzed by adenosine deaminases acting on RNA (ADARs), and is especially prevalent in the brain. We used the highly accurate microfluidics-based multiplex PCR sequencing (mmPCR-seq) technique to assess the effects of development and environmental stress on A-to-I editing at 146 pre-selected, conserved sites in the rat prefrontal cortex and amygdala. Furthermore, we asked whether changes in editing can be observed in offspring of stress-exposed rats. In parallel, we assessed changes in ADARs expression levels. RESULTS: In agreement with previous studies, we found editing to be generally higher in adult compared to neonatal rat brain. At birth, editing was generally lower in prefrontal cortex than in amygdala. Stress affected editing at the serotonin receptor 2c (Htr2c), and editing at this site was significantly altered in offspring of rats exposed to prereproductive stress across two generations. Stress-induced changes in Htr2c editing measured with mmPCR-seq were comparable to changes measured with Sanger and Illumina sequencing. Developmental and stress-induced changes in Adar and Adarb1 mRNA expression were observed but did not correlate with editing changes. CONCLUSIONS: Our findings indicate that mmPCR-seq can accurately detect A-to-I RNA editing in rat brain samples, and confirm previous accounts of a developmental increase in RNA editing rates. Our findings also point to stress in adolescence as an environmental factor that alters RNA editing patterns several generations forward, joining a growing body of literature describing the transgenerational effects of stress.

Hypoxia tolerance, longevity and cancer-resistance in the mole rat Spalax - a liver transcriptomics approach.

The blind subterranean mole rat Spalax shows a remarkable tolerance to hypoxia, cancer-resistance and longevity. Unravelling the genomic basis of these adaptations will be important for biomedical applications. RNA-Seq gene expression data were obtained from normoxic and hypoxic Spalax and rat liver tissue. Hypoxic Spalax broadly downregulates genes from major liver function pathways. This energy-saving response is likely a crucial adaptation to low oxygen levels. In contrast, the hypoxia-sensitive rat shows massive upregulation of energy metabolism genes. Candidate genes with plausible connections to the mole rat's phenotype, such as important key genes related to hypoxia-tolerance, DNA damage repair, tumourigenesis and ageing, are substantially higher expressed in Spalax than in rat. Comparative liver transcriptomics highlights the importance of molecular adaptations at the gene regulatory level in Spalax and pinpoints a variety of starting points for subsequent functional studies.

Parallel embryonic transcriptional programs evolve under distinct constraints and may enable morphological conservation amidst adaptation.

Embryonic development evolves by balancing stringent morphological constraints with genetic and environmental variation. The design principle that allows developmental transcriptional programs to conserve embryonic morphology while adapting to environmental changes is still not fully understood. To address this fundamental challenge, we compare developmental transcriptomes of two sea urchin species, Paracentrotus lividus and Strongylocentrotus purpuratus, that shared a common ancestor about 40 million years ago and are geographically distant yet show similar morphology. We find that both developmental and housekeeping genes show highly dynamic and strongly conserved temporal expression patterns. The expression of other gene sets, including homeostasis and response genes, show divergent expression which could result from either evolutionary drift or adaptation to local environmental conditions. The interspecies correlations of developmental gene expressions are highest between morphologically similar developmental time points whereas the interspecies correlations of housekeeping gene expression are high between all the late zygotic time points. Relatedly, the position of the phylotypic stage varies between these two groups of genes: developmental gene expression shows highest conservation at mid-developmental stage, in agreement with the hourglass model while the conservation of housekeeping genes keeps increasing with developmental time. When all genes are combined, the relationship between conservation of gene expression and morphological similarity is partially masked by housekeeping genes and genes with diverged expression. Our study illustrates various transcriptional programs that coexist in the developing embryo and evolve under different constraints. Apparently, morphological constraints underlie the conservation of developmental gene expression while embryonic fitness requires the conservation of housekeeping gene expression and the species-specific adjustments of homeostasis gene expression. The distinct evolutionary forces acting on these transcriptional programs enable the conservation of similar body plans while allowing adaption.

An exceptional family: Ophiocordyceps-allied fungus dominates the microbiome of soft scale insects (Hemiptera: Sternorrhyncha: Coccidae).

Hemipteran insects of the suborder Sternorrhyncha are plant sap feeders, where each family is obligately associated with a specific bacterial endosymbiont that produces essential nutrients lacking in the sap. Coccidae (soft scale insects) is the only major sternorrhynchan family in which obligate symbiont(s) have not been identified. We studied the microbiota in seven species from this family from Israel, Spain and Cyprus, by high-throughput sequencing of ribosomal genes, and found that no specific bacterium was prevalent and abundant in all the tested species. In contrast, an Ophiocordyceps-allied fungus sp.-a lineage widely known as entomopathogenic-was highly prevalent. All individuals of all the tested species carried this fungus. Phylogenetic analyses showed that the Ophiocordyceps-allied fungus from the coccids is closely related to fungi described from other hemipterans, and they appear to be monophyletic, although the phylogenies of the Ophiocordyceps-allied fungi and their hosts do not appear to be congruent. Microscopic observations show that the fungal cells are lemon-shaped, are distributed throughout the host's body and are present in the eggs, suggesting vertical transmission. Taken together, the results suggest that the Ophiocordyceps-allied fungus may be a primary symbiont of Coccidae-a major evolutionary shift from bacteria to fungi in the Sternorrhyncha, and an important example of fungal evolutionary lifestyle switch.

Metagenomic analysis reveals unusually high incidence of proteorhodopsin genes in the ultraoligotrophic Eastern Mediterranean Sea.

Sunlight can be directly harvested by photoheterotrophic bacteria to create a pH gradient across the membrane, which can then be utilized to produce ATP. Despite the potential importance of this trophic strategy, when and where such organisms are found in the seas and oceans is poorly described. Here, we describe the abundance and taxonomy of bacteria with different trophic strategies (heterotrophs, phototrophs and photoheterotrophs) in contrasting water masses of the ultra-oligotrophic eastern Mediterranean Sea. These water bodies, an anticyclonic eddy and a high-chlorophyll patch resulting from transport of nutrient-rich coastal waters into offshore oligotrophic waters, each supported different microbial populations in surface waters. Based on infrared microscopy and metagenomics, aerobic anoxygenic photoheterotrophic (AAP) bacteria represented up to 10.4% of the microbial community. In contrast, the proteorhodopsin (PR) gene was found in 78.6%-118.8% of the bacterial genome equivalents, the highest abundance reported to date. These results suggest that PR-mediated photoheterotrophy may be especially important in oligotrophic, potentially phosphate-limited conditions.

Genome maintenance and bioenergetics of the long-lived hypoxia-tolerant and cancer-resistant blind mole rat, Spalax: a cross-species analysis of brain transcriptome.

The subterranean blind mole rat, Spalax, experiences acute hypoxia-reoxygenation cycles in its natural subterranean habitat. At the cellular level, these conditions are known to promote genomic instability, which underlies both cancer and aging. However, Spalax is a long-lived animal and is resistant to both spontaneous and induced cancers. To study this apparent paradox we utilized a computational procedure that allows detecting differences in transcript abundance between Spalax and the closely related above-ground Rattus norvegicus in individuals of different ages. Functional enrichment analysis showed that Spalax whole brain tissues maintain significantly higher normoxic mRNA levels of genes associated with DNA damage repair and DNA metabolism, yet keep significantly lower mRNA levels of genes involved in bioenergetics. Many of the genes that showed higher transcript abundance in Spalax are involved in DNA repair and metabolic pathways that, in other species, were shown to be downregulated under hypoxia, yet are required for overcoming replication- and oxidative-stress during the subsequent reoxygenation. We suggest that these differentially expressed genes may prevent the accumulation of DNA damage in mitotic and post-mitotic cells and defective resumption of replication in mitotic cells, thus maintaining genome integrity as an adaptation to acute hypoxia-reoxygenation cycles.

Adaptive patterns in the p53 protein sequence of the hypoxia- and cancer-tolerant blind mole rat Spalax.

BACKGROUND: The subterranean blind mole rat, Spalax (genus Nannospalax) endures extreme hypoxic conditions and fluctuations in oxygen levels that threaten DNA integrity. Nevertheless, Spalax is long-lived, does not develop spontaneous cancer, and exhibits an outstanding resistance to carcinogenesis in vivo, as well as anti-cancer capabilities in vitro. We hypothesized that adaptations to similar extreme environmental conditions involve common mechanisms for overcoming stress-induced DNA damage. Therefore, we aimed to identify shared features among species that are adapted to hypoxic stress in the sequence of the tumor-suppressor protein p53, a master regulator of the DNA-damage response (DDR). RESULTS: We found that the sequences of p53 transactivation subdomain 2 (TAD2) and tetramerization and regulatory domains (TD and RD) are more similar among hypoxia-tolerant species than expected from phylogeny. Specific positions in these domains composed patterns that are more frequent in hypoxia-tolerant species and have proven to be good predictors of species' classification into stress-related categories. Some of these positions, which are known to be involved in the interactions between p53 and critical DDR proteins, were identified as positively selected. By 3D modeling of p53 interactions with the coactivator p300 and the DNA repair protein RPA70, we demonstrated that, compared to humans, these substitutions potentially reduce the binding of these proteins to Spalax p53. CONCLUSIONS: We conclude that extreme hypoxic conditions may have led to convergent evolutionary adaptations of the DDR via TAD2 and TD/RD domains of p53.