Splice Variant of Spalax Heparanase Skipping Exon 12.

The subterranean blind mole rat, Spalax, has evolved significantly over 47 million years to thrive in its underground habitat. A key enzyme in this adaptation is heparanase, which degrades heparan sulfate (HS) in the extracellular matrix (ECM), facilitating angiogenesis and releasing growth factors for endothelial cells. Spalax heparanase has various splice variants influencing tumor growth and metastasis differently. We report a novel splice variant from a hypoxia-exposed kidney sample resulting from exon 12 skipping. This variant maintains the translation frame but lacks enzymatic activity, offering insights into Spalax's unique adaptations.

Damage-Free Shortening of Telomeres Is a Potential Strategy Supporting Blind Mole-Rat Longevity.

Telomere shortening or loss of shelterin components activates DNA damage response (DDR) pathways, leading to a replicative senescence that is usually coupled with a senescence-associated secretory phenotype (SASP). Recent studies suggested that telomere aberration that activates DDR may occur, irrespective of telomere length or loss of shelterin complex. The blind mole-rat (Spalax) is a subterranean rodent with exceptional longevity, and its cells demonstrate an uncoupling of senescence and SASP inflammatory components. Herein, we evaluated Spalax relative telomere length, telomerase activity, and shelterin expression, along with telomere-associated DNA damage foci (TAFs) levels with cell passage. We show that telomeres shorten in Spalax fibroblasts similar to the process in rats, and that the telomerase activity is lower. Moreover, we found lower DNA damage foci at the telomeres and a decline in the mRNA expression of two shelterin proteins, known as ATM/ATR repressors. Although additional studies are required for understanding the underling mechanism, our present results imply that Spalax genome protection strategies include effective telomere maintenance, preventing early cellular senescence induced by persistent DDR, thereby contributing to its longevity and healthy aging.

Senescent Secretome of Blind Mole Rat Spalax Inhibits Malignant Behavior of Human Breast Cancer Cells Triggering Bystander Senescence and Targeting Inflammatory Response.

Subterranean blind mole rat, Spalax, has developed strategies to withstand cancer by maintaining genome stability and suppressing the inflammatory response. Spalax cells undergo senescence without the acquisition of senescence-associated secretory phenotype (SASP) in its canonical form, namely, it lacks the main inflammatory mediators. Since senescence can propagate through paracrine factors, we hypothesize that conditioned medium (CM) from senescent Spalax fibroblasts can transmit the senescent phenotype to cancer cells without inducing an inflammatory response, thereby suppressing malignant behavior. To address this issue, we investigated the effect of CMs of Spalax senescent fibroblasts on the proliferation, migration, and secretory profile in MDA-MB-231 and MCF-7 human breast cancer cells. The results suggest that Spalax CM induced senescence in cancer cells, as evidenced by increased senescence-associated beta-galactosidase (SA-ß-Gal) activity, growth suppression and overexpression of senescence-related p53/p21 genes. Contemporaneously, Spalax CM suppressed the secretion of the main inflammatory factors in cancer cells and decreased their migration. In contrast, human CM, while causing a slight increase in SA-ß-Gal activity in MDA-MB-231 cells, did not decrease proliferation, inflammatory response, and cancer cell migration. Dysregulation of IL-1α under the influence of Spalax CM, especially the decrease in the level of membrane-bound IL1-α, plays an important role in suppressing inflammatory secretion in cancer cells, which in turn leads to inhibition of cancer cell migration. Overcoming of SASP in tumor cells in response to paracrine factors of senescent microenvironment or anti-cancer drugs represents a promising senotherapeutic strategy in cancer treatment.

Perioral secretions enable complex social signaling in African mole-rats (genus Fukomys).

Subterranean common mole-rats of the genus Fukomys (family Bathyergidae) live in large, cooperatively-breeding families. Odor cues have been hypothesized to play an important role in mediating social behaviors in the underground ecotope, but only little is known about the role of olfactory signaling in burrowing mammals. Here we characterize the so far neglected perioral glands of Fukomys and other African mole-rats as an important source of olfactory social information. Histology demonstrates these structures to be derived sebaceous glands that are developed regardless of sex and reproductive status. However, gland activity is higher in Fukomys males, leading to sexually dimorphic patterns of stain and clotting of the facial pelage. Behavioral assays revealed that conspecifics prefer male but not female perioral swabs over scent samples from the back fur and that male sebum causes similar attraction as anogenital scent, a known source of social information in Fukomys. Finally, we assessed volatile compounds in the perioral sebum of the giant mole-rat (Fukomys mechowii) via GCxGC-MS-based metabolomic profiling. Volatiles display pronounced sex-specific signatures but also allow to differentiate between intrasexual reproductive status groups. These different lines of evidence suggest that mole-rat perioral glands provide complex odor signals which play a crucial role in social communication.

Phase II testing of Xenon International on Mount Schauinsland, Germany.

Station RN33 on Mount Schauinsland near Freiburg, Germany, is part of the International Monitoring System monitoring radioxenon in air (131mXe, 133Xe, 133mXe, and 135Xe) for verification of the Comprehensive Nuclear Test Ban Treaty. Here, we present data from phase II testing of a new system, Xenon International at RN33, July 14th, 2021 to Jan 22nd, 2022, together with SPALAX data from the same time period. Radioxenon could be detected in 473 of 719 samples, among them many multiple isotope detections. Activity concentrations of spiked and selected environmental samples were verified by laboratory reanalysis. The sensitivity of Xenon International for radioxenons is up to one order of magnitude better for the metastable isotopes than that of the SPALAX, with a shorter sampling duration of 6 h.

Local Adaptation of Bitter Taste and Ecological Speciation in a Wild Mammal.

Sensory systems are attractive evolutionary models to address how organisms adapt to local environments that can cause ecological speciation. However, tests of these evolutionary models have focused on visual, auditory, and olfactory senses. Here, we show local adaptation of bitter taste receptor genes in two neighboring populations of a wild mammal-the blind mole rat Spalax galili-that show ecological speciation in divergent soil environments. We found that basalt-type bitter receptors showed higher response intensity and sensitivity compared with chalk-type ones using both genetic and cell-based functional analyses. Such functional changes could help animals adapted to basalt soil select plants with less bitterness from diverse local foods, whereas a weaker reception to bitter taste may allow consumption of a greater range of plants for animals inhabiting chalk soil with a scarcity of food supply. Our study shows divergent selection on food resources through local adaptation of bitter receptors, and suggests that taste plays an important yet underappreciated role in speciation.

Distinct organization of adaptive immunity in the long-lived rodent Spalax galili.

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

Liver anatomy, intrahepatic vascular and biliary branching system of the mole rat (Spalax leucodon).

BACKGROUND: There are many studies on the morphology of the liver and its blood vessels in experimental animals, but such studies are lacking in the mole rat (Spalax leucodon). The aim of this paper was a detailed basic study on the topography, morphology, vascular and biliary branching systems of the liver in the mole rat. MATERIALS AND METHODS: Coloured gelatine and mixture of coloured lead oxide and linseed oil were injection contrast masses used to obtain vascular and biliary branching pattern in the liver. It was revealed that the liver of the mole rat had five lobes (left, quadrate, right medial, right lateral and caudate lobes). RESULTS: The left, undivided lobe was the largest lobe of the liver. The quadrate lobe was divided into two components by a deep notch. The gallbladder, of cylindrical shape, was present and attached to the quadrate lobe. The common bile duct was formed by the union of the left and right hepatic ducts. The pancreatic duct joined the common bile duct before it entered the duodenum. In the present study, only the right medial lobe and quadrate lobe always showed a single lobar artery, portal and hepatic veins. The left lobe showed four lobar arteries, portal and hepatic veins. The caudate lobe with its two processes and the right lateral and medial lobes had different arterial and portal blood supply as well as hepatic and biliary drainage of these lobes. The intrahepatic branches of the proper hepatic artery ran parallel to the branches of the common portal vein in the same lobes of the liver. CONCLUSIONS: The results of this study are significant for comparative studies among different species of rodents and other experimental animals. Morphology, vasculature and biliary tract of the liver in the mole rat were similar to that of other experimental animals and identified differences may be related to the adaptation to the mode of life and diet of this rodent.

Genome evolution of blind subterranean mole rats: Adaptive peripatric versus sympatric speciation.

Speciation mechanisms remain controversial. Two speciation models occur in Israeli subterranean mole rats, genus Spalax: a regional speciation cline southward of four peripatric climatic chromosomal species and a local, geologic-edaphic, genic, and sympatric speciation. Here we highlight their genome evolution. The five species were separated into five genetic clusters by single nucleotide polymorphisms, copy number variations (CNVs), repeatome, and methylome in sympatry. The regional interspecific divergence correspond to Pleistocene climatic cycles. Climate warmings caused chromosomal speciation. Triple effective population size, Ne , declines match glacial cold cycles. Adaptive genes evolved under positive selection to underground stresses and to divergent climates, involving interspecies reproductive isolation. Genomic islands evolved mainly due to adaptive evolution involving ancient polymorphisms. Repeatome, including both CNV and LINE1 repetitive elements, separated the five species. Methylation in sympatry identified geologically chalk-basalt species that differentially affect thermoregulation, hypoxia, DNA repair, P53, and other pathways. Genome adaptive evolution highlights climatic and geologic-edaphic stress evolution and the two speciation models, peripatric and sympatric.

Microbiome and Longevity: High Abundance of Longevity-Linked Muribaculaceae in the Gut of the Long-Living Rodent Spalax leucodon.

With a world population living longer as well as marked disparities in life expectancy, understanding the determinants of longevity is one of the priority research agendas in 21st century life sciences. To this end, the blind mole-rat (Spalax leucodon), a subterranean mammalian, has emerged as an exceptional model organism due to its astonishing features such as remarkable longevity, hypoxia and hypercapnia tolerance, and cancer resistance. The microbiome has been found to be a vital parameter for cellular physiology and it is safe to assume that it has an impact on life expectancy. Although the unique characteristics of Spalax make it an ideal experimental model for longevity research, there is limited knowledge of the bacterial composition of Spalax microbiome, which limits its in-depth utilization. In this study, using 16S rRNA amplicon sequencing, we report the gut and skin bacterial structure of Spalax for the first time. The diversity between fecal and skin samples was manifested in the distant clustering, as revealed by beta diversity analysis. Importantly, the longevity-linked Muribaculaceae bacterial family was found to be the dominating bacterial taxa in Spalax fecal samples. These new findings contribute toward further development of Spalax as a model for longevity research and potential linkages between microbiome composition and longevity.

Cloning of two splice variants of Spalax heparanase encoding for truncated proteins.

Heparanase is an endoglycosidase that degrades heparan sulfate side chains of heparan sulfate-proteoglycans. It liberates heparan sulfate-bound growth factors and thereby promotes blood vessel sprouting and angiogenesis. The subterranean blind mole rat, Spalax, is a wild mammal that lives most of its life in underground tunnels where it experiences sharp fluctuations in oxygen and carbon dioxide levels. We described two splice variants of heparanase from Spalax, Splice 7 and splice 36, both devoid of heparanase enzymatic activity. Splice 7 increases tumor growth, while splice 36 functions as a dominant negative to wild-type heparanase and decreases tumor growth and metastasis. Here, we describe two novel splice variants of Spalax heparanase, splice 67 and splice 612. These splice variants result in production of a shorter heparanase proteins that are similar to the wild-type native heparanase in their N-terminal but have unique C-terminals. Both splice 67 and 612 lack heparan sulfate degradation activity.

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long-lived and cancer-resistant subterranean wild rodent, Spalax.

The blind mole rat (Spalax) is a wild, long-lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence-associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide-induced senescence (EIS), evidenced by an increased activity of senescence-associated beta-galactosidase (SA-ß-Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well-known SASP factors: interleukin-6 (IL6), IL8, IL1α, growth-related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM-1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro-inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA-MB-231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF-κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory-related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age-related chronic inflammation supporting healthy aging and longevity.

Convergent reduction of V1R genes in subterranean rodents.

BACKGROUND: Vomeronasal type 1 receptor genes (V1Rs) are expected to detect intraspecific pheromones. It is believed that rodents rely heavily on pheromonal communication mediated by V1Rs, but pheromonal signals are thought to be confined in subterranean rodents that live in underground burrows. Thus, subterranean rodents may show a contrasting mode of V1R evolution compared with their superterranean relatives. RESULTS: We examined the V1R evolution in subterranean rodents by analyzing currently available genomes of 24 rodents, including 19 superterranean and 5 subterranean species from three independent lineages. We identified a lower number of putatively functional V1R genes in each subterranean rodent (a range of 22-40) compared with superterranean species (a range of 63-221). After correcting phylogenetic inertia, the positive correlation remains significant between the small V1R repertoire size and the subterranean lifestyle. To test whether V1Rs have been relaxed from functional constraints in subterranean rodents, we sequenced 22 intact V1Rs in 29 individuals of one subterranean rodent (Spalax galili) from two soil populations, which have been proposed to undergo incipient speciation. We found 12 of the 22 V1Rs to show significant genetic differentiations between the two natural populations, indicative of diversifying selection. CONCLUSION: Our study demonstrates convergent reduction of V1Rs in subterranean rodents from three independent lineages. Meanwhile, it is noteworthy that most V1Rs in the two Spalax populations are under diversifying selection rather than relaxed selection, suggesting that functional constraints on these genes may have retained in some subterranean species.

Telomeres and Longevity: A Cause or an Effect?

Telomere dynamics have been found to be better predictors of survival and mortality than chronological age. Telomeres, the caps that protect the end of linear chromosomes, are known to shorten with age, inducing cell senescence and aging. Furthermore, differences in age-related telomere attrition were established between short-lived and long-lived organisms. However, whether telomere length is a "biological thermometer" that reflects the biological state at a certain point in life or a biomarker that can influence biological conditions, delay senescence and promote longevity is still an ongoing debate. We cross-sectionally tested telomere length in different tissues of two long-lived (naked mole-rat and Spalax) and two short-lived (rat and mice) species to tease out this enigma. While blood telomere length of the naked mole-rat (NMR) did not shorten with age but rather showed a mild elongation, telomere length in three tissues tested in the Spalax declined with age, just like in short-lived rodents. These findings in the NMR, suggest an age buffering mechanism, while in Spalax tissues the shortening of the telomeres are in spite of its extreme longevity traits. Therefore, using long-lived species as models for understanding the role of telomeres in longevity is of great importance since they may encompass mechanisms that postpone aging.

The transcriptome landscape of the carcinogenic treatment response in the blind mole rat: insights into cancer resistance mechanisms.

BACKGROUND: Spalax, the blind mole rat, developed an extraordinary cancer resistance during 40 million years of evolution in a subterranean, hypoxic, thus DNA damaging, habitat. In 50 years of Spalax research, no spontaneous cancer development has been observed. The mechanisms underlying this resistance are still not clarified. We investigated the genetic difference between Spalax and mice that might enable the Spalax relative resistance to cancer development. We compared Spalax and mice responses to a treatment with the carcinogen 3-Methylcholantrene, as a model to assess Spalax' cancer-resistance. RESULTS: We compared RNA-Seq data of untreated Spalax to Spalax with a tumor and identified a high number of differentially expressed genes. We filtered these genes by their expression in tolerant Spalax that resisted the 3MCA, and in mice, and found 25 genes with a consistent expression pattern in the samples susceptible to cancer among species. Contrasting the expressed genes in Spalax with benign granulomas to those in Spalax with malignant fibrosarcomas elucidated significant differences in several pathways, mainly related to the extracellular matrix and the immune system. We found a central cluster of ECM genes that differ greatly between conditions. Further analysis of these genes revealed potential microRNA targets. We also found higher levels of gene expression of some DNA repair pathways in Spalax than in other murines, like the majority of Fanconi Anemia pathway. CONCLUSION: The comparison of the treated with the untreated tissue revealed a regulatory complex that might give an answer how Spalax is able to restrict the tumor growth. By remodeling the extracellular matrix, the possible growth is limited, and the proliferation of cancer cells was potentially prevented. We hypothesize that this regulatory cluster plays a major role in the cancer resistance of Spalax. Furthermore, we identified 25 additional candidate genes that showed a distinct expression pattern in untreated or tolerant Spalax compared to animals that developed a developed either a benign or malignant tumor. While further study is necessary, we believe that these genes may serve as candidate markers in cancer detection.

Blind mole rat (Spalax leucodon) masseter muscle: structure, homology, diversification and nomenclature.

BACKGROUND: It is well known that rodents are defined by a unique masticatory apparatus. The present study describes the design and structure of the masseter muscle of the blind mole rat (Spalax leucodon). The blind mole rat, which emer- ged 5.3-3.4 million years ago during the Late Pliocene period, is a subterranean, hypoxia-tolerant and cancer-resistant rodent. Yet, despite these impressive cha- racteristics, no information exists on their masticatory musculature. MATERIALS AND METHODS: Fifteen adult blind mole rats were used in this study. Dissections were performed to investigate the anatomical characteristics of the masseter muscle. RESULTS: The muscle was comprised of three different parts: the superficial mas- seter, the deep masseter and the zygomaticomandibularis muscle. The superficial masseter originated from the facial fossa at the ventral side of the infraorbital foramen. The deep masseter was separated into anterior and posterior parts. The anterior part of the zygomaticomandibularis muscle arose from the snout and passed through the infraorbital foramen to connect on the mandible. CONCLUSIONS: The construction of the deep masseter and zygomaticomandibularis muscles were of the Myomorpha type. Further studies are needed to reveal features such as muscle biomechanics, muscle types.

Reduced calcium influx in the hypoxia-tolerant Spalax: The role of the erythropoietin receptor.

Tissue hypoxia is a condition that induces calcium influx into living cells. Calcium is a major player in maintaining cell signaling and homeostasis, and mediates the regulation of gene transcription and cell proliferation; however, acute and aggressive calcium influx induced by hypoxia eventually leads to programmed cell death. The blind mole rat, Spalax, is a wild-spread burrowing mammal adapted to hypoxic environments. A tyrosine -to- phenylalanine (F481 in Spalax corresponding to Y485 in human full-length receptor; Y460 in human mature form) substitution is found in the erythropoietin receptor of Spalax and other species, which was previously shown to be strongly involved in the calcium channels activation and subsequent calcium influx. The current work aimed to explore the dynamics of calcium transport across Spalax nonhematopoietic cells' membrane compared to above ground rat and mouse, and the role of the erythropoietin receptor of Spalax in the regulation of calcium influx under hypoxia. We show here that Epo-induced calcium influx in HEK293 cells transfected with Spalax EpoR is significantly lower than that of mouse; in hypoxia this difference was even more pronounced. Western blots confirmed a significant increase of Erk phosphorylation after stimulation with erythropoietin under hypoxia in cells transfected with mouse full length erythropoietin receptor compared to Spalax. Native primary fibroblasts showed lower cytosolic calcium concentrations in Spalax cells when compared to those of rats under normoxic and hypoxic conditions. Spalax EpoR appears to play an important role in preventing deleterious consequences of hypoxia and maintaining cellular homeostasis under stress.

Cancer-free aging: Insights from Spalax ehrenbergi superspecies.

Cancer and ageing can be regarded as two different manifestations of the same underlying process-accumulation of cellular damage-and therefore both are closely linked. Nowadays, the ageing of populations worldwide is leading to an unprecedented increase in cancer cases and fatalities, and therefore the understanding of links between cancer and ageing is more important than ever. Spalax is considered an excellent model for ageing and, additionally, for cancer research, due to not show clear age-related phenotypic changes and not develop spontaneous tumours, despite its relatively long lifespan (∼20 years in captivity). Thereby, the purpose of this review is to summarize the recent knowledge on Spalax, with a particular emphasis on the molecular mechanisms associated with their longevity and cancer resistance.