Several studies indicate that the PI3K/PTEN/AKT signaling pathways are critical regulators of ovarian function including the formation of the germ cell precursors, termed primordial germ cells, and the follicular pool maintenance. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/AKT pathways during primordial germ cell development and the dynamics of the ovarian primordial follicle reserve and how dysregulation of these signaling pathways may contribute to the development of some types of germ cell tumors and ovarian dysfunctions. Primordial germ cells (PGCs) are embryonic germ cell precursors. They derive from a subset of epiblast cells that migrate to the extraembryonic region shortly before gastrulation, prior to reentering into the embryo proper and moving into the gonadal ridges (GRs). Here, they give rise to oocytes or prospermermatogonia  in the developing fetal ovaries and testes, respectively. This sequence of events occurs in all mammalian species studied including humans, although the involved factors and timing can be different. Nevertheless, from the experimental evidence accumulated during the last thirty years, a correct activation level and regulation of the PI3K/PTEN/AKT signaling pathways are conserved features of the mammalian PGC development. In the first part of the present paper, we review the main data obtained by several laboratories including our own, supporting this notion mainly with regard to PGC survival/proliferation and migration. Later on, in the establishing of the follicle stockpile in the ovary and its dynamics, the PI3K/PTEN/AKT signaling pathways continue to exert a pivotal role in regulating the activation of the primordial follicles and the oocyte growth. The second part of this work is focused on these subjects. In both sections, we report evidence on how dysregulation of the PI3K/PTEN/AKT signaling pathways may contribute to development of some types of germ cell tumors arising from PGCs or ovarian dysfunctions involving the follicle dynamics control.Readers interested in these topics can also refer to the optimal reviews by Makker-vet and colleagues  and MaidPhosphoinositide 3-kinases (PI3Ks) are lipid kinases that play a central role in the regulation of a variety of cell activities including apoptosis, autophagia, cell cycle, differentiation, and motility. They are activated downstream tyrosine kinase receptors (RTKs) and/or G proteins coupled receptors (GPCRs). Four major extracellular signals, growth factors, cytokines, hormones/ chemokines, and integrins, are able to activate PI3Ks. In the cytoplasm, PI3K phosphorylates the 3′-hydroxy position of the inositol ring of phosphatidylinositides, yielding to the production of phosphatidylinositol-3,4,5-trisphosphate (PIP3), a second messenger that recruits protein kinase B (AKT) to the cell membrane by phosphorylation. This step is tightly regulated by the phosphatase and tensin homolog (PTEN), which acts as a negative regulator of the pathways by converting PIP3 back to PIP2. To be fully active, AKT requires an additional phosphorylation by mTORC2, after which it is able to phosphorylate several substrates. To date, at least 13 substrates of AKT have been identified in mammalian cells [It is now largely accepted that in mammalian embryos, the germline is specified from a small cluster of cells of the epiblast, the layer of the blastocyst known to generate all fetal cell lineages, shortly before gastrulation. Under the inductive action of WNT and BMP growth factor signaling, which basically rely on the SMAD-and β-catenin pathways ,5, theseDuring this period, PGCs also express the KIT receptor for the Kit ligand (KL) cytokine and receptors for growth factors such as fibroblast growth factors (FGFs) or other cytokine such as leukemia inhibitory factors (LIF), the LIFR-gp180 complex ,13,14. TKL, known as KITL or stem cell factor (SCF), exists as a membrane-anchored and soluble isoform that arises from alternative RNA splicing and proteolytic processing ,20. It iKIT belongs to the type III receptor tyrosine kinase family, and it is well known that stimulation of KIT by KL leads to dimerization, activation of its intrinsic tyrosine kinase activity, and phosphorylation of key tyrosine residues within the receptor. The resulting phosphotyrosine residues serve as docking sites for molecules containing SH2 and other phosphotyrosine-binding domains. Activated KIT also catalyzes direct phosphorylation of substrate proteins. Among the major signaling pathways ultimately activated by the KL/KIT including RAS-RAF-MEK-ERK, PLCγ, Src, and PI3K/AKT, these two latter are probably the most important to sustain PGC survival and take part in their active migration toward the gonadal anlages ,23,24. IIt can be postulated that in post migratory PGCs as in other cell types, the p85 regulatory subunit of PI3K binds to phosphotyrosine residues Y719 of activated KIT by one or both of its SH2 domains, and p85–p110 interaction leads to the allosteric activation of the catalytic subunit of the enzyme ,27. UponIn the same paper, a novel role for KL as the PGC chemoattractant and for PI3K/AKT and Src kinase, as players involved in the activation of the PGC migratory machinery, were demonstrated. In addition, evidence for two other possibly PI3K-dependent KL/KIT action on PGCs, adhesion to somatic cells , and surAmong more 16 substrates downstream AKT, which ones might be directly or indirectly involved in PGC migration and survival are not known. Some of these are indicated in Germ cell tumors (GCTs) represent neoplasms derived from germ cells that can contain both immature and mature elements differentiating into several tissue types. Gonads are the preferred location for the onset of these tumors; however, they can also be found in extragonadal sites (EGCTs). EGCTs are usually seen in children or young adults and typically arise in midline locations where the cranium and sacrococcygeal region are the most common sites. In testis, GCTs are termed testicular germ cell tumors (TGCTs); in ovaries, ovarian germ cell tumors (OGCTs). Histologically, EGCTs and TGCTs comprise seminomas (SEs), also known as germinoma, and nonseminomatous tumors (NSTs). The latter includes a variety of histological distinct types such as teratoma, embryonal carcinoma, endodermal sinus tumor (yolk sac tumor), choriocarcinoma, and tumors with mixed histology. Nonseminomas containing a pluripotent component known as embryonal carcinoma (EC) cells are known as teratocarcinomas. There are four main types of OGCTs, namely dysgerminomas, comparable to testicular seminomas, and teratoma, yolk sac tumor and choriocarcinoma histologically equivalent to their testicular counterparts. Further information and clinical outcomes of these tumors were not subject of the present review. We refer the interested readers to our recent reviews ,29. HereIn fact, a widely accepted hypothesis suggests that EGCTs and the most part of TGCTs and OGCTs arise from PGCs with altered survival, deregulated cell cycle, and failed complete germline determination, probably acting together. A theory concerning the tumorigenic potential of extragonadal germ cells known as the ‘germ cell theory’ was first proposed by Gunnar Teilum, suggesting that EGCTs originate from stray migratory PGCs . PGCs miIt is likely that the correct development of PGCs moving from the yolk sac toward the GRs will depend on the balance between the cytokine/growth factor signaling pathways that control their cell cycle and survival and the core transcription factors and RNA binding proteins that maintain their intrinsic pluripotency and preserve their germline identity by repressing differentiation toward somatic lineages. As a matter of fact, the latent pluripotency derived from specification and the highly hypomethylated status of the chromatin imposed to PGCs during migration represent a risk for tumorigenic transformation. As reported above, PGC migration is accompanied by a wave of KL expression by the surrounding somatic cells, which through KIT/PI3K/AKT pathways sustain their survival and facilitate migration . SeveralThe latent pluripotency of migratory and gonadal PGCs has been elegantly demonstrated by in vitro transformation of mouse or human PGCs in the presence of a cocktail of growth factors such as KL, LIF, and bFGF ,33,34 toOne of the first mechanisms identified as responsible for PGC transformation in EG cells was the deregulation of cell cycle/survival signals linked to overactive PI3K/AKT pathway or downregulated PTEN ,21,35, fAs reported above, upon colonization of the gonadal, female PGCs give rise to oogonia that after some rounds of mitotic proliferation enter meiosis I as primary oocytes. These are surrounded by ovarian pregranulosa cells and become progressively arrested at the diplotene/dictyate stage of prophase I and singularly enclosed in a structure termed primordial follicle (PMF). During the last period of prenatal development, cohorts of PMFs are recruited to begin growth. After birth, recruitment from the PMF pool continues until it has been depleted, at which point infertility ensues and the female is said to have entered reproductive senescence, or menopause in the case of humans . Although recent evidence supports the possibility that in mammals, as occurs in other species, new follicles can be formed after birth ,32, PMFsAtresia of growing follicles seems to primarily begin in granulosa cells and subsequently encompass the oocyte. Granulosa cell apoptosis mostly depending on gonadotropins and steroids is widely considered as an underlying mechanism of follicular atresia. Conversely, atresia of PMFs is believed to accompany or follow oocyte death. In principle, as reported above, PMFs within an adult ovary should be subjected to mechanisms that keep them dormant, preserve survival, and at the same time, the oocyte quality, for long time. Autophagy and apoptosis, which appear to co-exist both in oocytes and granulosa cells, are likely to regulate oocyte survival and follicle atresia during folliculogenesis and PI3K/AKT signaling pathways to play a major role in either processes.The notion that PI3K/AKT signaling pathways are major regulators of PF survival, mainly of the oocyte, but also of follicle activation as we will discuss later on, began to be uncovered in the early nineties. Similar to PGCs, the KL/KIT system in oocytes seems to be the major upstream activator of PI3K/AKT signaling pathways, although other growth factors and receptors, for example, IGF1/IGFR, are likely to contribute to the pathways ,34. The From the above, it is easy to foresee that defects in PI3K/AKT signaling pathways can result in serious ovarian dysfunctions such as diminished ovarian reserve (DOR), premature ovarian insufficiency (POI), or premature ovarian failure (POF), and poor ovarian response (POR), characterized by reduced PMF/oocyte survival or defects in follicular activation. Summarizing and interpreting the numerous available data, it is possible to envisage that basal activation of PI3K/AKT signaling pathways are important components of the set of factors supporting the survival of oocytes within the PFs. The system likely acts by sustaining antiapoptotic pathways and perhaps maintaining the basal level of autophagy. However, the exact AKT substrates responsible for such actions have not been revealed thus far. Since the antiapoptotic myeloid cell leukaemia-1 (MCL1), to a lesser extent BCL-2 and BCL-xL and the proapoptotic BAX, PUMA and NOXA proteins, together with the initiator caspase 8 and the effector caspases 3 and 7, has been identified as major players of oocyte survival/apoptosis at this stage , AKT acIn the last few years, it has become evident that the PI3K/AKT activity downstream KL/KIT that sustains the prolonged survival of the oocyte is only part of the PI3K/AKT signaling pathways that play a major role in the maintenance of the PMF pool within the mammalian ovary including humans. In fact, these pathways are also involved in inducing the activation of dormant follicles, oocyte growth, and further follicular maturation ,56. It sRecent results indicate that the activity of mTOR downstream of the PI3K/AKT pathways in granulosa cells is a major trigger for PF activation . InteresRobust evidence indicates that mTOR is an indirect effector of AKT in regulating oocyte growth and the subsequent follicular development ,63. AKT Follicular activation also requires inactivation and removal from the oocyte nucleus of the transcription factor FOXO3a and the cyclin dependent kinase (CDK) inhibitor 1B (commonly known as p27kip1 or p27); this latter is also inactivated in pre-granulosa cells ,65. FOXOFinally, Liu and colleagues  reportedFor the sake of truth, we must cite that although all results reported above strongly indicate a central role of KL/KIT in PMF activation, by employing a knock-in mutation (KitY719F) that completely abrogates signaling via PI3K, John’s group  observedThe importance of the mTOR/AKT signaling pathway for female fertility was, however, recently demonstrated in women suffering from POI/POF who, after receiving an AKT activator and the activation of Hippo signaling, were shown to achieve follicular maturation, IVF of oocytes, and healthy offspring (reviewed in ). Under stress conditions, the apoptosis process of oocytes within PMFs is mediated by a distinct cell surveillance mechanism involving N-terminal transactivation domain p63 (TAp63α) ,75, a p5Remarkably, several recent studies have also implicated AKT in modulating DNA damage responses and genome stability . HigPTEN/PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes of female gametogenesis from PGC development to oocyte growth and folliculogenesis. In a number of cell types including PGCs as highlighted here, components of these pathways have been described as causal forces in tumors. Robust evidence sustains the notion that dysregulation of the activity of these components in PGCs can alter survival, proliferation, and differentiation of PGC, providing an important contribution to their transformation in tumorigenic cells. In addition, several data indicate that elements of these signaling pathways play essential roles in preserving the normal female fertility and reproductive lifespan. This provides a rationale for exploring the possible use of targeted therapies for the inhibition of the PI3K/PTEN/Akt and TSC/mTOR signaling cascades in certain ovarian dysfunctions including infertility and POF. Notably, the results of these studies represent the basis for improving current assisted reproductive methods including in vitro maturation of immature oocytes, and for the preservation of fertility by in vitro activation of follicles in cryopreserved ovarian tissue obtained from cancer patients or from women with reduced ovarian reserves.


The large volumes of rapidly emplaced individual eruptions present a case study for examining the tempo of voluminous silicic magma generation and emplacement. Here were report high-precision 206Pb/238U zircon ages and show that the largest sequentially dated eruptions occurred within 48 ± 34 kyr (29.755 ± 0.023 Ma to 29.707 ± 0.025 Ma), yielding the highest known long-term volumetric extrusive rate of silicic volcanism on Earth. While these are the largest known sequential silicic supereruptions, they did not cause major global environmental change. We also provide a robust tie-point for calibration of the geomagnetic polarity timescale by integrating 40Ar/39Ar data with our 206Pb/238U ages to yield new constraints on the duration of the C11n.1r Subchron.The main phase of silicic volcanism from the Afro-Arabian large igneous province preserves some of the largest volcanic eruptions on Earth, with six units totaling >8,600 km 3, occurred within 48 ± 34 kyr and yield the highest known long term volumetric extrusive rate of silicic volcanism on Earth.Quantifying the tempo of large-volume silicic magma generation and eruption is a long-standing but elusive task. Here we show that the three largest sequentially dated eruptions, totaling >4,300 km Although LIPs are generally considered to represent the most productive magmatic systems on Earth4, uncertainty about volume estimates and imprecise or inaccurate age data for individual events preclude robust estimates of magma flux and volcanic output5. The silicic component of LIPs is largely understudied relative to their mafic counterpart and long-term rates of silicic magma generation have important implications for the energy budget and thermal evolution of the Earth. The Northern Yemen section of the Afro-Arabian LIP is an ideal testbed for using high-precision 206Pb/238U zircon dating to quantify the long-term volumetric extrusive rate of a series of flood volcanic eruptions, with three silicic supereruptions (1015 kg or ~450 km3 of magma7) occurring within a 70–310 kyr timeframe at ca. 29.7 Ma10.Many of the largest silicic eruptions on Earth occur in large igneous provinces (LIPs), with total eruptive volumes often exceeding 1000 km40Ar/39Ar dating10: Main Basalts (31–29.7 Ma), Main Silicics (29.7–29.5 Ma), and Upper Bimodal (29.6–27.7 Ma). The Main Basalts phase is characterized by effusive basaltic volcanism and volumetrically represents 60–70% of the total erupted volume of Afro-Arabian lavas11. The Main Silicics phase saw the rapid emplacement of seven silicic pyroclastic units and the Upper Bimodal phase includes small-volume basaltic and rhyolitic eruptions9. The Northern Yemen section has excellent exposure and well-characterized stratigraphic relationships from field mapping, paleomagnetic studies, and correlations with distal deep sea co-ignimbrite ash deposits12, while the Ethiopian section has been extensively faulted from active rifting with significant erosion around the volcanic plateau margin13.Oligocene volcanism in Northern Yemen Fig.  has been3 DRE emplaced in present-day Yemen and Ethiopia over a period from 29.7 to 29.5 Ma9. Volcano–stratigraphic correlations in Yemen9 suggest the emplacement of the Jabal Kura’a Ignimbrite  and Escarpment Ignimbrite  was followed by a brief period of subsidence and erosion and then the rapid emplacement of the Green Tuff  of Cande and Kent14 and Huestis and Acton15. While there are several cooling events identified in the Oligocene δ18O and δ13C chemostratigraphy17, the uncertainties of these ages also hinder the correlation of the Afro-Arabian silicic eruptions to any isotopic perturbations. In contrast to existing 40Ar/39Ar ages, the 0.1% precision of state-of-the-art chemical abrasion thermal ionization mass spectroscopy (CA-TIMS) U-Pb ages of zircons18 can distinguish between the ages of these units outside analytical uncertainty. These new high-precision 206Pb/238U zircon ages are crucial to quantifying the rapid emplacement of voluminous Afro-Arabian silicic magmas in order to understand the transient nature of silicic supereruptions, demonstrating that these eruptions had little to no observed impact on long-term climate change, and constraining the duration of the C11n.1r Subchron.Previous paleomagnetism and Zircon crystals from the Escarpment, SAM and Sana’a Ignimbrites, and Iftar Alkalb were analyzed by cathodoluminescence (CL) imaging and laser ablation inductively coupled mass spectrometry (LA-ICP-MS) in order to distinguish petrochemical populations prior to CA-TIMS dating. The Escarpment Ignimbrite contains elongate prismatic crystals  and smaller equant crystals (50–75 μm in length). Some prismatic crystals have oscillatory zoning with U-rich non-luminescent cores (CL dark). The SAM Ignimbrite contains elongate prismatic crystals that are both smaller  and less numerous than those found in the Escarpment Ignimbrite. Few crystals have subtle oscillatory zoning and one larger crystal ~120 μm in length has a non-luminescent, oscillatory zoned core with a lighter overgrowth rim. Crystals in the SAM Ignimbrite have a weakly paramagnetic behavior, likely due to abundant Fe-Ti oxide and apatite inclusions. The Sana’a Ignimbrite contains small elongate prismatic crystals (30–75 μm) with subtle to no oscillatory zoning. Zircon is abundant in Iftar Alkalb as anhedral to euhedral elongate prismatic and equant crystals that range in length from 30 to 120 μm. Internal morphologies are variable in Iftar Alkalb with populations of non-luminescent and luminescent zircon crystals with no oscillatory zoning, crystals with non-luminescent cores and lighter rims, and a few crystals with strong oscillatory zoning (see 19) and antecrysts . The median uncertainty of a single LA-ICP-MS 206Pb/238U spot analysis is 3 Ma, too imprecise to distinguish antecryst populations for this magmatic system but adequate to determine older xenocrystic zircon crystals. Every unit except the Escarpment Ignimbrite contains >10% zircon crystals with LA-ICP-MS 206Pb/238U ages >33 Ma. The Sana’a Ignimbrite and Iftar Alkalb contain significant proportions of older zircons , although in the Sana’a Ignimbrite this may be due to the low sample number (n = 31). There is no correlation between age and trace element  concentrations. CL dark zircon crystals in the Escarpment Ignimbrite and Iftar Alkalb have among the highest HREE concentrations and europium anomalies (Eu/Eu*) in each respective unit and the ages of the cores and rims of the few zircon crystals with clear zonation were indistinguishable outside uncertainty  = 0.62; Fig. 206Pb/238U age of the underlying unit and inferred to be an antecryst), the remaining eight zircon crystals yielded a weighted mean date of 29.728 ± 0.017 Ma (MSWD = 0.34). Six zircon crystals from the Sana’a Ignimbrite yielded a weighted mean date of 29.707 ± 0.025 Ma  and 29.793 ± 0.042 (MSWD = 8.96), respectively.Thirty-two grains that showed no sign of inclusions and yielded consistent U-Pb laser ablation dates were plucked from their respective grain mounts for high-precision CA-ID-TIMS geochronology . PrefereAlthough Iftar Alkalb is the stratigraphically youngest unit dated, nine zircon crystals were consistently older , Kura’a Basalt (30.22 ± 0.26 Ma), Akraban Andesite (29.80 ± 0.08 Ma), an overlying small-volume rhyolitic tuff (28.58 ± 0.14 Ma) and ignimbrite (28.18 ± 0.10 Ma), and the Bayt Mawjan Ignimbrite (27.85 ± 0.12 Ma) have also been recalculated. The 206Pb/238U zircon ages are in agreement with the recalculated 40Ar/39Ar ages and are compiled and presented here as a revised chronostratigraphy of the Northern Yemen flood volcanics  and the europium anomaly (Eu/Eu*) in rare earth element patterns resulting from feldspar fractionation are useful indicators of magma differentiation. Assuming that both elements remain incompatible, more differentiated rhyolites will evolve towards higher Th/Y ratios while Eu/Eu* will decrease with continued feldspar crystallizationite Fig. . Thus, a3/yr) were calculated for 100 and 400 kyr of residence for the Escarpment, SAM, and Sana’a Ignimbrites based on the age difference between the most and least evolved zircon crystals in each unit. For 100 kyr residence, magma flux rates are 3.6 × 10−3 km3/yr, 2.4 × 10−2 km3/yr, and 1.6 × 10−2 km3/yr for the Escarpment, SAM, and Sana’a Ignimbrites, respectively. For 400 kyr residence, magma flux rates are 9.0 × 10−4 km3/yr, 6.0 × 10−3 km3/yr, and 4.0 × 10−3 km3/yr for the Escarpment, SAM, and Sana’a Ignimbrites, respectively. Upper estimates of 3.6 × 10−3–2.4 × 10−2 km3/yr for 100 kyr residence are similar to those calculated for other rapidly assembled large-volume silicic systems . The most conservative estimates using 400 kyr residence (9.0 × 10−4–6.0 × 10−4 km3/yr) are similar to but lower than the minimum calculated magma flux from Yellowstone  and significantly lower than that of Taupo (>0.33 km3/yr for the 530 km3 Oruanui eruption26).Magma flux rates , yielding a long-term volumetric extrusive rate of 5.27 × 104. Average extrusive rates in silicic systems are calculated to be highest for continental arcs (4.90 ± 0.15 × 10−3 km3/yr) followed by oceanic arcs (4.50 ± 0.79 × 10−3 km3/yr), continental rifts (4.48 ± 0.86 × 10−3 km3/yr), continental hotspots (1.29 ± 0.25 × 10−3 km3/yr), and continental volcanic fields (6.47 ± 1.96 × 10−4 km3/yr). The extrusive rate of the Main Silicics phase of the Northern Yemen section of the Afro-Arabian province is most similar to—but notably higher than—the extrusive rates of the central Taupo volcanic zone , the silicic portion of Kamchatka , and Quaternary phonolites from the Kenya rift valley . Our findings are consistent with observations at other large-volume silicic systems that record rapid periods of differentiation and magma reservoir assembly superimposed on lower background fluxes. While some silicic systems have produced more voluminous individual eruptions  and larger cumulative eruptive volumes over longer time intervals , the eruptions of the Main Silicics phase in Northern Yemen represent the largest long-term volumetric extrusive rate of silicic volcanism on Earth.Long-term volumetric extrusive rates of basaltic and andesitic systems are thought to be higher than those of silicic systems by up to two orders of magnitude35. Models for volcanism-driven environmental change predict years of cooling from SO2 injection into the stratosphere from a single eruption and/or tens of thousands of years of warming from CO2 emissions36. Several of the Afro-Arabian silicic supereruptions have been correlated to 10–15 cm-thick tephra layers located >2700 km away in the Indian Ocean12  and C10 (Oi2* and Oi2a38) indicate that the perturbations in δ18O and δ13C pre-date the eruptions17 16, and the sensitivity of the climate proxies to the effects of individual volcanic eruptions. While the Afro-Arabian Main Silicics phase eruptions represent the largest known long-term volumetric extrusive rate of silicic volcanism, they did not cause major global climate change at the current resolution of these data. Challenges remain in discerning the various roles of the tempo, volatile budget, eruption mechanism, and volume of magma extruded from LIPs and their effect on global environmental change. However, robust temporal constraints continue to provide critical insight into this relationship.Some volcanic provinces appear to coincide with major global environmental change and mass extinctions , yet others, even those with silicic supereruptions , do not39 but those were unable to unambiguously distinguish between the GPTS of Cande and Kent14 and Huestis and Acton15. Recent studies on the Oligocene magnetic polarity sequence have utilized astronomical age models38, radio-isotope age models37, recalculations of the Cande and Kent14 GPTS using updated 40Ar/39Ar flux monitor ages40, and a combination of all three37. One of the lingering issues with distinguishing between an appropriate method for determining the Rupelian age (33.9–28.1 Ma) is the lack of tie points from radio-isotopic dates. The Rupelian/Chattian boundary Global Boundary Stratotype Section and Point records a nearly continuous record of astronomically tuned magnetostratigraphy for the Oligocene but only provides one tie point for the Rupelian for the uppermost Chron C12r with a gap between 31.8 ± 0.2 and 27.0 ± 0.1 Ma41. The 2012 Geologic Time Scale for the Paleogene37 favored an integrated radio-isotope, GPTS, and cyclostratigraphy model with sixth-order polynomial fit to produce a complete C-sequence. The C11n.1r Subchron is estimated to have a duration of 0.050 Ma with a −0.654 Ma discrepancy between radio-isotopic and astronomic age models37. The only discrepancy between the combined age model of the 2012 Geologic Time Scale and new 2020 Geologic Time Scale for the time range of interest is a shift of the base of Chron C12n to 30.977 from 31.034 Ma42.Previous efforts have been made to correlate Oligocene Afro-Arabian volcanic deposits with the GPTS206Pb/238U zircon age of the SAM Ignimbrite and 29.67 ± 0.13 Ma 40Ar/39Ar sanidine age of Iftar Alkalb—further constrained to 29.67 ± 0.13 Ma by the 29.707 ± 0.025 Ma 206Pb/238U age of the Sana’a Ignimbrite—can be used as tie points for the GPTS. Our chronostratigraphy and magnetostratigraphy are definitively in agreement with the Cande and Kent14 GPTS  at 180–200 °C for 12 h and then rinsed in 3.5 M HNO3 in an ultrasonic bath for 60 min. The residual crystals were rinsed twice in ultrapure H2O and transferred to Teflon PFA microcapsules and spiked with ET535 mixed U-Pb isotope tracer solution45. The spiked residual crystals were dissolved in Parr vessels in 120 μL of 29 M HF at 220 °C for 48 h, dried, and redissolved in 6 M HCl at 180 °C overnight46. Pb and U were purified from the chloride matrix using HCl-based anion-exchange chromatography and dried with 2 μL of 0.05 N H3PO4. High-precision isotope dilution U and Pb isotope ratio measurements were made using a single Re filament silica gel technique on an Isotopx Isoprobe-T multi-collector TIMS equipped with an ion-counting Daly detector (47. Analytical uncertainties on dates are reported to be 2σ and propagated using the algorithms of Schmitz and Schoene48.A total of 32 crystals from the 4 units were selected for CA-TIMS analysis on the basis of morphology, zoning, chemistry, and preliminary detector . Dates aSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Tumors exist in a complex milieu where interaction with their associated microenvironment significantly contributes to disease progression. Cancer-associated fibroblasts (CAFs) are the primary component of the tumor microenvironment and participate in complex bidirectional communication with tumor cells. CAFs support the development of various hallmarks of cancer through diverse processes, including direct cell–cell contact, paracrine signaling, and remodeling and deposition of the extracellular matrix. Calcium signaling is a key second messenger in intra- and inter-cellular signaling pathways that contributes to cancer progression; however, the links between calcium signaling and CAFs are less well-explored. In this review, we put into context the role of calcium signaling in interactions between cancer cells and CAFs, with a focus on migration, proliferation, chemoresistance, and genetic instability. I. I2+ inf2+-dependent manner  and a calcium channel (TRPC3). PDAC-conditioned media significantly increase migration and Ca2+ influx in PSCs, and either pharmacological inhibition of KCa3.1 or siRNA knockdown of TRPC3 blocked this migration, indicating that both TRPC3 and KCa3.1 channels and Ca2+ influx were required for PDAC-mediated migration in PSCs (Pancreatic stellate cells (PSCs) are the most common CAF subtype in pancreatic ductal adenocarcinomas (PDAC), and their presence correlates with poor survival . Like ot in PSCs B 57]..Ca3.1) a2+/RHO/ROCK signaling axis, facilitating colorectal cancer cell migration into the stroma and surrounding regions -HETE , facilit regions C 7]. Th. Th2+/RH2+ and increased proliferation and migration through GPER/EGFR/ERK signaling. This provides circumstantial evidence for a link between GPER-mediated Ca2+ signaling and proliferation and migration in CAFs  formation is a type of aberrant wound healing that is heavily dependent on fibroblast activation ,78. A re2+ signaling in promoting tumor growth, as well as in the development of CAF features. Diverse mechanisms have been revealed in CAF–cancer Ca2+ signaling; however, TRPA1-mediated VEGF secretion has emerged as a consistent contributor to disease progression. Given the availability of pharmacological inhibitors, this channel is a potential therapeutic target, particularly in the treatment of prostate cancer. We also presented evidence supporting the transition from monoculture to co-culture models that more closely replicate the in vivo setting. There remain many unexplored avenues for Ca2+ signaling in CAF–cancer interactions that will continue to be revealed to the extent that CAFs remain at the forefront of TME research.Cancer cells and CAFs exist in a complex and reciprocal signaling environment where cues from one cell compartment have dramatic effects on the growth and survival of the niche as a whole. The studies highlighted in this review demonstrate diverse roles for Ca


The Hedgehog/GLI (Hh/GLI) pathway plays a major role during development and it is commonly dysregulated in many diseases, including cancer. This highly concerted series of ligands, receptors, cytoplasmic signaling molecules, transcription factors, and co-regulators is involved in regulating the biological functions controlled by this pathway. Activation of Hh/GLI in cancer is most often through a non-canonical method of activation, independent of ligand binding. This review is intended to summarize our current understanding of the Hh/GLI signaling, non-canonical mechanisms of pathway activation, its implication in disease, and the current therapeutic strategies targeting this cascade.Drosophila as a major regulator of segment patterning in development. This pathway consists of a series of ligands , transmembrane receptors (Ptch1 and Ptch2), transcription factors (GLI1–3), and signaling regulators  that work in concert to repress  or activate  the signaling cascade. Not long after the initial discovery, dysregulation of the Hh/GLI signaling pathway was implicated in human disease. Activation of this signaling pathway is observed in many types of cancer, including basal cell carcinoma, medulloblastoma, colorectal, prostate, pancreatic, and many more. Most often, the activation of the Hh/GLI pathway in cancer occurs through a ligand-independent mechanism. However, in benign disease, this activation is mostly ligand-dependent. The upstream signaling component of the receptor complex, SMO, is bypassed, and the GLI family of transcription factors can be activated regardless of ligand binding. Additional mechanisms of pathway activation exist whereby the entirety of the downstream signaling pathway is bypassed, and PTCH1 promotes cell cycle progression and prevents caspase-mediated apoptosis. Throughout this review, we summarize each component of the signaling cascade, non-canonical modes of pathway activation, and the implications in human disease, including cancer. The Hh/GLI signaling pathway was originally discovered in  Drosophila homologue of the GLI family of transcription factors [Drosophila. The pair discovered 15 genetic loci implicated in segment patterning in Drosophila. When mutated, each locus produced distinct alterations in larvae patterning. The mutation in the Hh locus produced denticles on the body of the fly that were oriented in the opposite direction of non-mutants, indicating the importance of Hh in polarity and development in larvae. The resemblance of these mutants to a hedgehog inspired the name of this genetic locus. This study by Nusslein-Volhard and Wieschaus was foundational for the later characterization of the Hh signaling pathway, as they not only identified the Hh ligand but also identified Ptch1 as another Hh-related locus crucial for development [The first identified components of the Hh/GLI signaling pathway were fused and cubitus interruptus, the  factors ,2. In 19elopment .Drosophila [Drosophila Hh gene and that of vertebrates. First, the Sonic Hedgehog (Shh) ligand was characterized in chick embryos, and not long after, the three vertebral homologues, Shh, Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh), were discovered [Drosophila have alterations in segment patterns and often have ovarian tumors, but overexpression of SUFU abrogated these phenotypes [Within the next two decades, scientists had thoroughly detailed the full DNA sequence of the Hh gene in osophila ,5,6. Froscovered ,8. It wascovered ,11,12,13enotypes . By the Drosophila caused by aberrant Hh/GLI signaling indicated a significant role of the Hh/GLI pathway in development [Along with the increasing understanding of the Hh/GLI signaling pathway components came insights into its role in physiology. From the outset, alterations in segment patterning in elopment ,2,3,14. elopment ,16,17,18elopment .Evidence for a role of the Hh/GLI signaling pathway in human disease was not defined until the late 1990s. The first implication was in basal cell nevus syndrome in 1996 ,21. CharDrosophila, while there are 56 Hh/GLI signaling pathway genes in mammalians [By the early 2000s, researchers were beginning to appreciate the complexity of the Hh/GLI signaling pathway, not only on a molecular level, but also in terms of disease relevance. The pathway consists of a highly concerted series of interactions between ligands, receptors, transducers, co-regulators, and transcription factors, with the potential to function both as an autocrine and paracrine signaling cascade. Further, the vast network of target genes suggested a degree of responsiveness to cellular context inherent to this signaling pathway. From the outset, there was a considerable degree of complexity to the Hh/GLI signaling pathway that left some wondering whether this cascade should be considered as part of a network instead of an isolated signaling pathway ,37. To dmmalians . PathwayThe Hh/GLI signaling pathway is a highly regulated, concerted cascade of extracellular ligands, receptor proteins, cytoplasmic signaling molecules, transcription factors, co-regulators, and target genes . The intIn vertebrates, the Hh/GLI signaling pathway can be activated by Shh, Ihh, or Dhh. These extracellular ligands are lipid-modified proteins which, although they share high N-terminal sequence identity (76–91%), carry out different developmental functions . Dhh hadPerhaps the most unique characteristic of the Hh ligands is the post-translational modifications, including the addition of a cholesterol molecule to the N-terminal domain which is required for activation and transport of the ligand between cells ,59,60. TThough the fully modified ligand has been secreted, the simple release of the tethered ligand is not enough to create the highly regulated morphogen gradient that is required for N-Hh to regulate development. For this, additional proteins and enzymes are required to chaperone the diffusion of the ligand. The heparan-sulfate-synthesizing enzyme, EXT, is required for the movement of N-Hh ,73,74,75The Hh/GLI signaling pathway receptors are localized to the primary cilia (PC) in most tissues. This microtubule-based organelle emerges from the cell surface and is implicated in mediating and interpreting mechanical, chemical, and thermal signaling ,80,81. Ti family of G proteins, which, in some cellular contexts, is required for the activation of GLI transcription factors [In the absence of PTCH1, SMO is phosphorylated by CK1α and G protein-coupled receptor kinase 2 (GRK2). This activates SMO and promotes its translocation into the PC ,88. SMO  factors ,98,99. T factors ,101. It PTCH1 is primarily expressed in mesenchymal cells, with Shh produced in neighboring epithelial cells, while Ptch2 is expressed mainly in testicular and skin epithelial cells ,103. ThoTriggering of the Hh/GLI signaling pathway results in the activation of the GLI family of transcription factors to translate the extracellular ligand binding into a gene expression response. The GLI family of transcription factors can interact with each other and a variety of co-regulators to regulate specific subsets of gene targets based on those interactions. Though GLI1–3 have high sequence homology, they exhibit differences in tissue expression, transcriptional regulation, and binding partners. Not only does this increase the complexity of the signaling cascade, it also helps to explain how a single signaling cascade can have such a wide variety of transcriptional responses based on cellular and tissue context.The GLI family of transcription factors are a subfamily of the Krüppel family of transcription factors, which share a highly conserved zinc-finger domain for DNA binding ,110,111.GLI protein activity is regulated through various mechanisms. Internal regulatory sequences, post-translational modifications, and co-regulatory proteins all have a role in modulating GLI activity . GLI2/3 In addition to proteasomal degradation, GLI proteins can also be regulated through protein–protein interactions . GLI1–3 Drosophila SUFU protein, indicating that this behavior is important for SUFU function. The N- and C-terminal domains of SUFU will come together to “sandwich” GLI and promote a stable interaction. It is suggested that GLI might mediate SUFU’s conformational change from the open to closed state, interacting with both the N- and C-terminal domains and dragging them together. The closed state predominates when SUFU is negatively repressing GLI activity, and this conformation is inhibited when Hh/GLI signaling is active. GLI dissociation is promoted by Hh signaling, and SUFU takes on the open conformation, yet the mechanistic details of this dissociation are still unknown [The major negative regulator of the GLI family of transcription factors, SUFU, is a 484 amino acid protein that acts as a negative regulator of GLI1–3 . SUFU ca unknown .SUFU is an adaptor protein and interacts with both cytoplasmic and nuclear proteins. Most of the SUFU protein–protein interactions are with other negative regulators of transcription, however, there have been a few reports of SUFU interacting with positive regulators of transcription, though the nature of those interactions has not been fully characterized. SUFU can interact with SAP18, a component of the Sin3 co-repressor complex that recruits HDACs to remodel chromatin in promoter regions and decrease gene expression. SUFU, SAP18, and Sin3 cannot bind the DNA, but require the SUFU–GLI interaction to be recruited to GLI-responsive promoters ,155,156.The various degrees of cooperation between GLI1, 2, and 3 create a network of interaction commonly referred to as the GLI code. The interactions between these proteins are largely dictated by cellular context, and the repressor and activators can work together to create a spectrum of transcriptional responses ,127. IsoAnother subfamily of the Krüppel family of proteins is the GLI-similar (GLIS) transcription factors. GLIS1–3 contain zinc-finger domains and are closely related to GLI1–3 ,164,165.The Hh/GLI signaling pathway is implicated in development, and typically, the target genes of this pathway are cell-type- and context-dependent. Many of the general gene targets of this pathway are developmental regulators, such as FGF4, Pax6-9, ABCG2, and Hhip, cell cycle regulators such as CCND2 and CCNE1, apoptosis regulator, BCL2, transcription factors FOXM1 and N-myc, and Wnt pathway proteins, JAG1, SFRP1, and Wnt ,175,176.Typically, canonical pathway activation refers to extracellular ligand (Shh/Dhh/Ihh) binding PTCH1 and relieving repression of SMO to activate the downstream signaling cascade ,180. Thoi proteins, through which it carries out this type II non-canonical pathway activation [i proteins to activate phosphoinositide 3-kinase (PI3K) kinase, small GTPases Ras homologous family member A (RhoA), and Ras-related C3 botulinum toxin substrate 1 (Rac1) [i proteins also promote Ca2+ influx into the cytosol. The Shh-SMO-Gi protein cascade activates phospholipase C (PLC) production, which increases PI3K and promotes opening of calcium channels in the smooth endoplasmic reticulum membrane, leading to a cytosolic spike in Ca2+ [2+ influx affects differentiation, proliferation, apoptosis, and the migration of both neural and neuronal precursor cells [2+ signaling axis from the Hh/GLI signaling pathway and drive metabolic changes favoring the Warburg effect [In type I non-canonical Hh/GLI pathway activation, PTCH1 does not interact with the SMO receptor. Instead, PTCH1 is involved in the regulation of cell cycle and apoptosis through the caspase protein, and Shh ligand binding to the extracellular domain of PTCH1 can influence these interactions. In the unbound state, PTCH1 interacts with phosphorylated cyclin B1 ,182. PTCtivation ,182. Whe1 (Rac1) . The SMO1 (Rac1) . In addi in Ca2+ . This Caor cells ,187. Addg effect . TogetheCrosstalk between other signaling pathways is another way in which the Hh/GLI pathway can be activated independent of ligand binding. There are many reports of the TGFβ, KRAS, and Wnt/β-catenin pathways activating the downstream constituents of the Hh/GLI pathway  189,190,200,201.The KRAS signaling pathway is another pathway that exhibits crosstalk with and activation of the Hh/GLI signaling cascade ,200. KRAThe Wnt/β-catenin signaling pathway is a non-canonical activator of the Hh/GLI pathway through transcriptional regulation and protein–protein interactions. When Wnt signaling is off, cytoplasmic β-catenin is degraded through a similar mechanism as the GLI family of transcription factors. The Axin complex, which contains CK1 and GSK3β, will phosphorylate β-catenin in the absence of Wnt signaling, promoting the proteasomal degradation of β-catenin. The turnover of β-catenin in the cytoplasm prevents nuclear translocation, inhibiting the transcription of Wnt target genes. When Wnt binds to the Frizzled receptor, the receptor complexes with LRP6, and together, the complex recruits and sequesters the Axin complex, inhibiting its kinase activity. β-catenin can then accumulate in the cytoplasm, translocate into the nucleus, and bind either TCF or LEF to activate target genes  226,227227,228. The Hh/GLI signaling pathway is essential for regulating many developmental processes. It comes as no surprise that mutations, loss of function, or aberrant activation of the pathway can lead to developmental defects and disease ,180,194.While mutations play a crucial role in the aberrant Hh pathway leading to a plethora of developmental malformations, they can also lead to cancer. The pathway activators  are considered proto-oncogenes due to their ability to upregulate the pathway and promote tumorigenesis. There are different mechanisms whereby the canonical Hh/GLI pathway can by dysregulated, leading to unwarranted activation. Ligand-independent activation of the signaling cascade increases the activity of GLI1–3, upregulating the expression of Hh/GLI pathway target genes. This type of pathway activation is commonly seen in Gorlin Syndrome, which is associated with the development of medulloblastomas, basal cell carcinomas, and rhabdomyosarcomas ,235. AddThe repressors of the pathway (SUFU and PTCH1) are considered tumor suppressors, as their loss of function upregulates the Hh/GLI signaling activity to drive tumorigenesis ,244,245.A study of patients with multiple hereditary infundibulocystic basal cell carcinoma syndrome was conducted to determine the role of SUFU mutations in disease progression. Individuals with the germline splice mutation resulted in not only the abolishment of SUFU protein, but it was also enough to place the individuals at higher risk for hereditary infundibulocystic basal cell carcinoma syndrome . AnotherIn addition to the canonical activation of the Hh/GLI signaling pathway, non-canonical mechanisms of pathway activation have been implicated in disease. As previously described, the KRAS signaling pathway can result in overexpression of Hh/GLI target genes through interaction with and activation of GLI transcription factors. This non-canonical Hh/GLI pathway activation has been observed in many types of disease, including pancreatic ductal adenocarcinoma, melanoma, gastric cancer, colon cancer, lung cancers, multiple myelomas, and many more ,253,254.This profound effect in tumor biology has triggered the development of Hh/GLI inhibitors for cancer treatment. Presently, most therapies in targeting the Hh pathway have been focused on developing SMO inhibitors (SMOi). Some of the therapeutics include sonidegib, vismodegib, saridegib, BMS-833923, taladegib, and glasdegib ,260. TheDrosophila provided the first insights into its role in regulating development. Aberrations in the Hh/GLI signaling pathway can lead to developmental defects and disease, including cancer. Further studies into the pathway have demonstrated that not only mutations lead to increased Hh/GLI signaling observed in disease, but there is also an additional subset of pathway interactions and non-canonical mechanisms of pathway activation that are significant contributors to the altered activity observed in disease.The Hh/GLI signaling pathway is a highly regulated complex cascade of ligands, receptors, transcriptional effectors, and regulatory proteins. Its initial discovery in


The formal conclusion of the Comprehensive Agreement on Investments (CAI) has drawn much criticism. Criticisms for member states did not always recognise the same critical points. The case of Italy presents an instance in which the issue rather than laying in the content of the agreement was identified in the process. Not only had Italy been marginalised in the process of negotiation that led to the conclusion of the CAI, but also exponents from the government claim that leading negotiators, amongst which France and Germany, ignored Italy’s doubts about the deal when these were raised. Beyond politics, the paper shows that the Italian business community displays a response to the agreement in line with that of the rest of the EU in its positive assessment of the outcome. The Comprehensive Agreement on Investments’ (CAI) formal conclusion between the European Union and the People’s Republic of China came at the very end of December 2020, after seven years of negotiations. Usually, in Brussels, the end of December is a relatively quiet time, as most people go on holiday and the institutions work at a reduced regime. However, 2020 demonstrated to be an exceptional year as the pandemic of Cov-Sars-2 disrupted the usual business until the very end; and the conclusion of the CAI fed into this exceptionalism. Admittedly, very few had predicted the achievement of the deal by the end of the year, if not at all. After the online EU-China summit held in September, even European Commission’s President Ursula Von der Leyen cautioned that plenty remained to be done for the deal to be concluded Tiezzi, .33The agreement’s formal finalisation was not met with few criticisms, notably in reaction to China’s behaviour in 2020. Criticisms spanned from the suppression of voices that could have launched an early alarm of the pandemic, to the delaying of an international investigation on the origins of the virus, from the introduction of the national security law in Hong Kong to the (re)emergence of information regarding the situation in Xinjiang, from the so-called “wolf warrior” diplomacy to increased military exercises in the South China Sea and near Taiwan. These are only a few of the elements that saw China’s assertiveness further growing throughout 2020. Finally, most believed that following the election of a more like-minded US president, the EU would have waited and consulted with the new Biden administration before concluding such a deal with China.Despite the formality, the deal is far from being concluded. The European Commission might have formally concluded the agreement, but it will need the European Parliament and the Council’s approval to make it effective. Considering the comments that followed the CAI announcement, it is not difficult to foresee lengthy negotiations with the European Parliament; the deal will not go through without intense scrutiny and negotiations. However interesting the European Parliament’s endeavours might be, it is the happenings within the Council that offer the perfect springboard to look at the focus of this brief paper, namely, how Italy positioned itself and reacted to the CAI. Officially, when asked, some Council members raised doubts about the deal, but none of them attempted to block it.Interestingly, of all the contentious elements mentioned above and others raised regarding China and the striking of the agreement, Italy was most concerned with how the deal came to be and how it was finalised. The two, the process and the conclusion, have in common that Italy’s role was marginalised. These were the main elements lamented by Ivan Scalfarotto, at the time, Italy’s undersecretary to foreign affairs Fubini, .44To better understand Italy’s disappointment, we must take a step back to 2016/2017, when it looked as if Italy could join France and Germany and occupy a leading position within the EU. This fortunate situation for Italy resulted from two elements: the outcome of the Brexit referendum and the governmental setting of Italy. The announcement of the UK’s exit from the EU meant that the three big powers that for better or for worse had been balancing and shaping EU’s foreign policy, France, Germany, and the UK, suddenly found themselves scarce of one actor. The new setting was not a problem in se, but it was an opportunity for a country, such as Italy, which had been attempting to occupy a more important role at the EU level. In those years, Italy was governed by the Democratic Party (PD), a very pro-European party, and up to the end of 2016, the then Prime Minister Matteo Renzi had been trying to carve out a more prominent position for Italy in Europe and, arguably, in the world. Confidence in the success of the attempt was the result not only of the reformative and progressive spirit of the government with a relatively young and ambitious leader but also of the impressive victory PD led by Renzi brought home at the European elections of 2014 (40.8% of votes) , Marco Marazzi, a partner at Baker and McKenzie, expressed his positive account of new possibilities for European and Italian businesses about the conclusion of the deal. According to Marazzi, Italy could obtain particular benefits from the CAI’s openings in the health sector (Marazzi, 17It is precisely the Italian business sector that expressed a more positive assessment of the CAI. In line with most of the business’s voices around Europe, Italian companies welcomed China’s agreement as an opportunity. In the newspaper Marazzi, .14 LaterMarazzi, .15 ThereMarazzi, .16 HowevMarazzi, .17This short analysis proposed by Marazzi and Boselli touches upon a fundamental yet often overlooked issue with the CAI. The EU can make a superb deal, but it is up to member states and businesses to eventually grasp these opportunities. In Italy, this is far from being an assured outcome. In a country where 92%20Italy's disappointment about how the negotiation and conclusion of the deal were held might have been stronger than that of other EU countries. Its recent history had fed the country with tangible hopes of a more proactive role on the European scene. However, internal political endeavours had made those hopes disappear. Furthermore, the attempt to gain a more prominent role by striking unusual partnerships did not pay off, not even when that same unusual partner, China, was on the other side of Brussels' negotiation table. The CAI conclusion showed to Italy, once more, that for the better, or the worse, when it comes to EU’s foreign policy, its voice is not more relevant than that of any other member state, exception made for France and Germany.Nonetheless, if we leave the process aside and we look at content, the positive assessment made both by Ivan Scalfarotto and exponents of the Italian business word might have to be reconsidered or sized down. In reality, the grand majority of small and medium Italian enterprises is unlikely to benefit from CAI. Therefore, in theory, CAI could be an excellent opportunity for Italian and European enterprises. However, in reality, the number of enterprises that will benefit from this agreement might be significantly lower and focused on larger economic actors, which often do not include Italian companies.


Disease progression in infected individuals is the result of HTLV-1-driven clonal expansion of CD4+ T-cells and is generally associated with the activities of the viral oncoproteins Tax and Hbz. A closely related virus, HTLV-2, exhibits similar genomic features and the capacity to transform T-cells, but is non-pathogenic. In vitro, HTLV-1 primarily immortalizes or transforms CD4+ T-cells, while HTLV-2 displays a transformation tropism for CD8+ T-cells. This distinct tropism is recapitulated in infected people. Through comparative studies, the genetic determinant for this divergent tropism of HTLV-1/2 has been mapped to the viral envelope (Env). In this review, we explore the emerging roles for Env beyond initial viral entry and examine current perspectives on its contributions to HTLV-1-mediated disease development.Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4 Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered . While t+ T-cell malignancy [+ T-cell infiltrates in the central nervous system [+ T-cells [HTLV-1 is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4lignancy ,25 that lignancy . Viral ilignancy ,28, a nes system . Other d T-cells . ATL is  T-cells  that dic T-cells . For the T-cells . Factors T-cells . A natio T-cells . A featu T-cells ,33.gag, pro, pol, and env), as well as regulatory and accessory genes from a unique region termed ‘pX’, located 3′ to the env gene [hbz. In newly infected cells, viral RNA is reverse transcribed into double-stranded DNA, which randomly integrates into the cellular genome. After integration, tax/rex is the initial dominant transcript that is produced through splicing of viral mRNA. Of the HTLV-1 gene products, at least two, tax and hbz, have been strongly implicated in T-cell transformation and ATL pathogenesis [hbz mRNA and protein support the proliferation of infected cells and ATL cells in vitro [HTLV-1 is classified within the deltaretrovirus genus. This genus encompasses the primate T-lymphotropic viruses, including the strains that infect humans (HTLVs) and bovine leukemia virus . The sinenv gene . HTLV-1 ogenesis ,37. Tax ogenesis . Tax iniogenesis . In contogenesis ,40,41. Iin vitro , and thain vitro ,44. Althin vitro , and thu+ T-cell origin, and a few cases of neurological conditions with HTLV-2 infection have been reported [While Tax and Hbz are key contributors to HTLV-1-mediated disease development, a potential pathogenic role for the viral envelope (Env) has emerged from comparative studies between HTLV-1 and HTLV-2. These viruses share high genomic sequence homology and functional similarity of their gene products ,47, but reported ,49,50. Creported . The disHTLV-1 and HTLV-2 are transmitted mainly through cell-to-cell contact ,53,54,55Env is synthesized as a precursor protein (gp62) that undergoes a maturation process in the endoplasmic reticulum, involving protein folding, oligomerization, and glycosylation . When th+ T-cells, whereas HTLV-2 targets CD8+ T-cells [+ T-cells—both in asymptomatic carriers as well as in patients with leukemia or neurological conditions [+ compared to CD8+ T-cells, suggesting that amplified viral transcription drives cell tropism and leukemogenesis mediated by HTLV-1 [+ T-cells in the cerebrospinal fluid, demonstrating that an additional HTLV-1 reservoir can be established in CD8+ T-cells in vivo [As its entry receptors are ubiquitously expressed, HTLV is capable of establishing infection in various cell types in vitro. Cells of the nervous system ,76, endo T-cells . These inditions ,83,84. Iy HTLV-1 . Nonethe in vivo .+ and CD8+ T-cell subsets in vivo [+ T-cells [+ or CD8+ T-cells in an established HTLV experimental system [+ or CD8+ T-cells isolated from healthy donor peripheral blood mononuclear cells (PBMCs). HTLV-2 was shown to equally infect both purified CD4+ and CD8+ T-cells, and there were no observed differences between the cell types in terms of viral transcription or viral particle production, as measured by the detection of p19gag antigen in the culture supernatant. However, CD8+ T-cells were preferentially transformed when the HTLV-2 producer cells were co-cultured with a mixed population of PBMCs or purified T-cell subsets. These early experiments indicated that further study of the genetic differences between HTLV-1 and HTLV-2 could reveal the viral determinant of transformation tropism and suggested that these preferences could be driven by interactions between viral proteins and cellular proteins specific to each T-cell type.HTLV-2 has been found in both CD4 in vivo ,88,89, al system . In the l system . As HTLVl system ,53,54,55l system . To assetax gene is located in a reading frame that is separate from, but overlaps with, the reading frame carrying the rex gene, whose product regulates the transport of unspliced and incompletely spliced viral mRNAs into the cytoplasm [tax and rex genes between the viruses to determine the effect on transformation tropism  and wild-type (wt)HTLV-1 transformed CD4+ T-cells, while HTLV-1/Env-2 and wtHTLV-2 transformed CD8+ T-cells. This was the first study to highlight a potential role for Env in the pathogenic differences between HTLV-1 and HTLV-2. The observed preferences for T-cell type in vitro also have clinical relevance. HTLV-1-associated ATL is a CD4+ T-cell malignancy, while a higher proviral burden in CD8+ T-cells has been found in HAM/TSP patients and asymptomatic HTLV-1 carriers [+ T-cells [+ and CD8+ T-cells are key players in the mechanisms underlying HTLV-mediated disease development.Experiments with HTLV-1 and HTLV-2 equences . The envcarriers ,96,97. H+ and CD8+ T-cells isolated from blood. The data showed that both cell types are infected as early as week 1 post-inoculation with HTLV-1 or HTLV-2, and there is no T-cell preference exhibited by either virus at the initial stages of infection in vivo [+ and CD8+ phenotype of HTLV-transformed T-cells longitudinally, the in vitro immortalization assay was conducted over a 9-week period. In the early weeks, CD4+ and CD8+ T-cells grew proportionally in HTLV-1 or HTLV-2 co-culture conditions; however, over time, the preferred subset emerged as a dominant population [To determine whether the distinct transformation tropism conferred by viral Env is at the level of entry or occurs later during infection, Kannian et al. investigated preference for T-cell type in an immune competent New Zealand white (NZW) rabbit model of HTLV infection and persistence ,102,103. in vivo . This in in vivo . To evalpulation . These dpulation .+:HTLV-1 and CD8+:HTLV-2). Recombinant HTLV-2/Env-1 drove proliferation of CD4+ T-cells, while HTLV-1/Env-2 drove proliferation of both CD4+ and CD8+ T-cells. In contrast to their distinct pathogenic outcomes in humans, both HTLV-1 and HTLV-2 induced leukemia/lymphoma in mice, emphasizing the importance of immune function in limiting infection and disease progression [env gene, this study demonstrated the utility of humanized mice as a model for HTLV T-cell preference and lymphoproliferative disease, and supports the investigation of how other viral proteins, such as Tax and Hbz, function together with Env to drive cellular transformation and oncogenesis.While NZW rabbits mimic asymptomatic infection in humans and enable the study of early viral replication patterns, they are not a suitable model for HTLV-mediated disease development. Huey et al. examined the contribution of Env to T-cell transformation in humanized mice used to model lymphoproliferative disease induced by HTLV infection . Humanizgression . Althoug+ T-cells, favored by HTLV-1, express higher levels of HSPGs (utilized by Env-1 for attachment to target cells) compared to CD8+ T-cells [+ T-cells, favored by HTLV-2, express more GLUT-1 compared to CD4+ T-cells [+ and CD8+ T-cells, respectively. In transfection experiments to alter the levels of these receptors on the cell surface, GLUT-1 overexpression enhanced the entry of HTLV-2 virions into CD4+ T-cells, and HSPG expression on CD8+ T-cells increased HTLV-1 entry [+ and CD8+ T-cells proliferate comparably, until one population is selected for predominant clonal expansion [It has been posited that differences in the in vitro and in vivo T-cell tropism of HTLV-1 and HTLV-2 can be ascribed to different receptor usage by these viruses. CD4 T-cells ,72,74. C-1 entry ,77,78,79xpansion . This ev+ to CD8+ T-cells in culture [+ T-cells. The predominance of CD8+ T-cells developed more slowly compared to wtHTLV-2, suggesting that the transformation tropism of HTLV results from the cooperation of other domains or residues [Studies of other oncogenic retroviruses have shown that Env can contribute to viral transforming activities. Env of Friend spleen focus-forming virus (SFFV) interacts with the erythropoietin receptor to induce the proliferation and differentiation of erythroid cells in the absence of erythropoietin . This wa culture . Consistresidues .HTLV-1 infection elicits both humoral and cell-mediated immune responses. Infants born to HTLV-1-positive mothers are protected from infection in their first months of life through transplacental acquisition of anti-HTLV-1 maternal antibodies . Anti-HTenv gene and peptide constructs containing a B-cell epitope from the Env region conferred protection to immunized non-human primates challenged with HTLV-1 [Alongside Gag and Tax, Env is one of the immunodominant viral proteins , as it ih HTLV-1 ,125.+ compared to CD8+ T-cells, which likely potentiates leukemogenesis by HTLV-1 [+ and CD8+ T-cells are required. While Env is detectable during early immortalization in vitro, viral sense transcription is silenced in vitro and in vivo through mechanisms such as 5′ LTR hypermethylation or deletion [Tax is the key regulator of HTLV gene expression and the primary driver of the cellular transformation process. Tax functions in transcriptional activation involving major players such as NF-κB, SRF, and CREB, and influences signal transduction, apoptosis inhibition, dysregulation of the cell cycle, and disruption of tumor suppressors ,126. They HTLV-1 . Due to y HTLV-1 ,45, howey HTLV-1 . Howevery HTLV-1 . This isy HTLV-1 . Additioy HTLV-1 . ATL casy HTLV-1 . This iny HTLV-1 , it is cy HTLV-1 . Moreovey HTLV-1 . These adeletion ,138,139;hbz mRNA promotes T-cell proliferation and Hbz protein inhibits viral transcription by Tax [hbz mRNA and Hbz protein expression in established HTLV-1 T-cell lines and newly immortalized HTLV-1 T-cells significantly reduced their proliferation in culture [γc−/− (NOG) transplant mice resulted in significant decreases in tumor formation and infiltration into organs compared to that observed with scramble SLB-1 cells [hbz mRNA does not trigger a CTL response, revealing another key HTLV immune evasion mechanism [In contrast to Tax, Hbz expression is continuous in HTLV-1-infected individuals . It has n by Tax . Althougn by Tax . Rabbits culture . Engraft-1 cells . These e-1 cells . Notablyechanism . The HTLechanism . Inoculaechanism . Differeechanism .+ and the CD8+ T-cell environments, which may contribute to the distinct transformation preferences of HTLV-1 and HTLV-2. These studies will uncover Env-driven post-entry events which mediate the selection of a particular T-cell type for predominant clonal expansion or define specific regions or residues of Env protein that confer these activities. They may also identify synergistic effects with other viral proteins. Does Env set the stage for Tax and Hbz in the immortalization of HTLV-1-infected cells? Does Env, in concert with Hbz, help drive cellular proliferation and mitotic viral replication in the absence of Tax expression? The phase of the immortalization/transformation process in which Env takes part is yet to be elucidated. Additionally, it is possible that further study of Env-2 or other HTLV-2 proteins will provide clues to the long clinical latency period of HTLV-1 infection. Dissecting similarities and differences in the timing and level of gene expression or protein function and interactions with the cellular environment that allow HTLV-2 to persist, but not progress to disease, may illuminate parallels to HTLV-1 latency.Findings from HTLV transformation tropism studies hold clinical relevance, as the in vitro immortalization assay mimics natural cell-to-cell transmission and recapitulates in vivo tropism observed in patients. This experimental system continues to be a useful tool to examine nuances in the molecular biology of HTLV-1 and HTLV-2—two closely related, yet pathologically distinct viruses. Future studies are needed to address the functional differences in Env-1 and Env-2 beyond viral entry, in the context of CD4


The primary aim was to determine differences in talocrural and subtalar joint(STJ) articular cartilage composition, using T1ρ magnetic resonance imaging(MRI) relaxation times, between limbs in individuals with unilateral chronicankle instability (CAI) and compare with an uninjured control. Our secondarypurpose was to determine the association between talocrural and STJcomposition in limbs with and without CAI.2) and 15uninjured control individuals . Talocrural cartilage was segmented manually toidentify the overall talar dome. The SJT cartilage was segmented manually toidentify the anterior, medial, and posterior regions of interest consistentwith STJ anatomical articulations. For each segmented area, a T1ρ relaxationtime mean and variability value was calculated. Greater T1ρ relaxation timeswere interpreted as decreased proteoglycan content.T1ρ MRI relaxation times were collected on 15 CAI  and the healthy control limb (P < 0.05). TheCAI-involved limb also had a higher posterior STJ T1ρ mean relative to thehealthy control limb (P < 0.05). In healthy controls(P < 0.05), but not the CAI-involved orcontralateral limbs (p>0.05), talocrural and posterior STJ compositionmeasures were positively associated.Individuals with CAI demonstrated a higher involved limb talocrural T1ρ meanand variability relative to their contralateral limb (Individuals with CAI have lower proteoglycan content in both the talocruraland posterior STJ in their involved limbs relative to the contralateral anda healthy control limb. Cartilage composition findings may be consistentwith the early development of posttraumatic osteoarthritis. The existing ankle literature has focused almostexclusively on a T2-mapping compositional magnetic resonance (MR) scan of the talocruraljoint  in a CAI-involved limb relative to a healthy controllimb.810 However, the earliestcompositional change of PTOA is a loss of proteoglycan content which is associated withearly PTOA development and can be captured using a T1ρ compositional MR scan. Those with CAI have been shown to have a higher T1ρ mean  as well as higher T1ρ variability across the talar dome of theinvolved limb relative to a healthy control limb. However, no between-limb investigation  has quantified proteoglycan content in those with unilateral CAI and comparedthese values with a healthy control limb, despite asymmetrical movement patterns havingbeen observed in those with CAI.Chronic ankle instability (CAI) is a common sequela following acute lateral anklesprains and evidence indicates that STJ instability increases with lateral ankle ligament sectioning and laxity in those with a history of a lateral ankle sprain. Thus, STJ instability is likely present in those with CAI. If untreated, STJinstability impairs mobility and could contribute to abnormal biomechanics and/ortalocrural degeneration in those with CAI. Given the role that the STJ plays in shock absorption, impaired STJ mobility may also contribute to STJ degeneration over time. However, only a single investigation has quantified STJ cartilage composition,via T2-mapping, in those with CAI. In healthy individuals, the STJ and shank are thought to be kinematically coupledas a result of ankle joint complex anatomy. Thus CAI appears to disrupt the normal shank-rearfoot coupling seen in healthyindividuals, likely because of STJ instability .2123 As a result of a kinematicdecoupling in those with CAI, the talocrural and STJ likely respond to mechanical loadsdifferently. If true, cartilage composition between the talocrural and STJ joints couldalso differ and result in accelerated proteoglycan content changes in one joint relativeto the other. To address this knowledge gap, a concurrent assessment of talocrural andSTJ proteoglycan content in CAI and healthy cohorts is needed.The subtalar joint (STJ) ligaments are often concurrently damaged during a lateral ankle sprain,Therefore, the primary purpose of this investigation was to determine differences intalocrural and STJ proteoglycan content among a CAI-involved, a CAI-contralateral, and ahealthy control limb. Our secondary purpose was to determine the association betweentalocrural and STJ proteoglycan content in a CAI-involved and a healthy control limb. Toachieve these objectives, we will evaluate T1ρ relaxation time means and variability inthe talocrural and STJ. We hypothesize that both the CAI-involved and -contralaterallimbs would have higher T1ρ relaxation times  andgreater T1ρ variability relative to the healthy control limb. We also hypothesized thatT1ρ relaxation time means and T1ρ variability of the talocrural and STJ would associatein a healthy control limb but not in the CAI-involved limb. All participants were between 18 and 35 years of age and free from acutelower extremity and head injuries for at least 3 months prior to testing. Anyonewith equilibrium disorders or symptoms from musculoskeletal or head injuriessustained at any time was excluded. Controls demonstrated an Identification ofFunctional Ankle Instability (IdFAI) score of <11, a Foot & Ankle AbilityMeasure Activities of Daily Living subscale (FAAM-ADL) score of ≥98%, and a Foot& Ankle Ability Measure Sport subscale (FAAM-S) score of ≥98%. Individualswith CAI had a history of at least 1 lateral ankle sprain and at least 2episodes of giving way within the past 6 months; an IdFAI > 11, a FAAM-ADL< 90%, and a FAAM-S < 80% per the International Ankle Consortium guidelines. International Ankle Consortium recommendations for exclusion criteriawere also followed. Rearfoot alignment was not quantified in the sample. Three participantspresented with bilateral CAI and were excluded from the CAI between limbcomparison. For the remaining analyses, the limb with more ankle sprains andworse self-reported function was used as the involved limb. Written informedconsent was provided prior to participation and all procedures were approved bythe university’s biomedical ethics review board. Published STJ T2 valuesindicate a large bias corrected effect size of 1.36 between CAI and control groups. However, we planned for a more conservative effect (0.54) in line withtalocrural T2 values between CAI and control groups. Coupled with an alpha level of 0.05 and 1 − β of 0.80, the total neededsample size was 30 was needed to detect differences.A cross-sectional design was used to address the stated research questions and weused the STROBE  cross-sectional reporting guidelines. A T1ρ prepared 3-dimensional fast low angle shot (FLASH) sequence with aspin lock power at 500 Hz and a bandwidth of 350 Hz/Px was used. Spin lock durations included 40, 30, 20, 10, and 0 ms. Voxel size was 0.8mm × 0.4 mm × 3 mm . T1ρacquisition time for each limb was approximately 12 minutes.A Siemens Magnetom TIM Prisma 3T scanner and an 8-channel large flex coil  acquired anatomical (PD space) and T1ρ MRimages. MR was performed with the foot/ankle complex in neutral (90° to theshank) and the participant supine after a 30-minute unloading  period prior to the scan.0 exp(−TSL/T1ρ). The S corresponds to signal, TSL is the length of the spin-lock time,S0 is the signal intensity when TSL equals 0, and T1ρ is the T1relaxation time in the rotating frame. The program used a 2-parameter fit andexcluded low-signal regions that fit poorly with the model. The talocrural andSTJ were manually segmented using ITK-SNAP version 3.2 software. The talocrural and STJ cartilage for each limb was segmented so that T1ρmean and variability  were calculated. For this investigation, a laminar analysis of the talar dome was notconducted because previous research demonstrated that those with CAI hadconsistently worse composition in both the superficial and deep layer relativeto uninjured controls. The STJ segmentation created an anterior, medial, and posterior region ofinterest consistent with STJ anatomical articulations. Overall STJ values were not calculated as they are heavily influenced bythe size of the posterior STJ articulation. For this investigation, STJcartilage was defined as the full thickness of the talocalcaneal cartilagebecause of concerns about talocalcaneal thickness (range: 0.55-1.00mm) andhaving enough voxel rows per cartilage layer if it were to be divided.2628A five image series created with a custom MatLab program  calculated Voxel by voxel T1ρ relaxation times usingthe following equation: S(TSL) = S12 T1ρ variability wasdefined as the standard deviation of T1ρ relaxation times across all pixels ofan articulation and higher T1ρ variability was interpreted as a more diffusedistribution of degenerative changes within the cartilage of interest. Secondary measures included T1ρ mean and variability for the anterior andmedial STJ articulations. The CAI-involved and -contralateral limbs as well asthe right and left controls limbs were not registered  because wefocused on an entire joint surface as opposed to a specific region of interest.Total cartilage volume of the talocrural and STJ articulation was calculated toaccount for potential differences in ankle size and/or usable slices.Additionally, a fellowship trained foot and ankle surgeon and 2 orthopaedicresidents evaluated the anatomical sequences for degenerative changes. While astandardized scoring regimen was not used, differences in findings werediscussed until consensus was reached.Following segmentation, the primary and secondary T1ρ outcomes were extracted foreach joint and used for further analysis. The T1ρ relaxation time mean andvariability for the overall talocrural and posterior STJ articulation served asthe primary outcome measures. Greater T1ρ relaxation times were interpreted asgreater cartilage degeneration.t tests. To quantify the relationship between morphologyand the primary T1ρ outcome, several analyses were conducted. Talocrural and STJcartilage volume was compared among the limbs using separate 1-way analyses ofvariance. Associations between talocrural and STJ cartilage volume and theprimary outcome measures were established, for each limb group independently,via Pearson correlations. Finally, the frequency of degenerative observationswas compared between the groups using a chi-square analysis.Participant demographics were compared between groups using independentP ≥ 0.209). Thus, a between-limb average was calculatedfor the primary and secondary variables for the healthy control limb. Theprimary variables  were then compared between the CAI limbs  using paired t tests and betweeneach CAI limb and the healthy control limb using independent ttests. Secondary STJ variables  were submitted to identical analyses. TheCAI-contralateral limb group consisted of contralateral limbs(n = 12) that did not meet our a priori definition of CAI.Bias corrected Hedge’s g effect sizes with their corresponding95% confidence intervals (95% CIs) were also calculated for all between-limbcomparisons.A preliminary analysis compared the dominant and nondominant limbs of the controlgroup and noted no differences in the primary outcome measures was used and an alpha levelof 0.05 was used to determine statistical significance for all analyses.Associations between the overall talocrural and posterior STJ T1ρ relaxation timemeans and variability (primary variables) within the CAI-involved,CAI-contralateral, and healthy control limb data were determined using separatePearson correlations. Correlations were interpreted as weak with a correlationcoefficient from 0.01 to 0.39, moderate from 0.40 to 0.69, and strong from 0.70to 1.00.P > 0.05)but injury history characteristics differed among the CAI-involved,CAI-contralateral, and control limbs (P < 0.05).Fifteen uninjured controls and 15 individuals with CAI participated. Demographics andinjury history means and standard deviations can be seen in 3; CAI-contralateral, 1466.4 ± 245.54 mm3; control,1446.37 ± 294.31 mm3; P = 0.403) and posterior STJcartilage volume  did not differ between the groups. Talocral and STJcartilage volume of the CAI-involved, CAI-uninvolved, and control limbs demonstratednonsignificant (P > 0.05) and weak associations (range: −0.300to 0.093) for all primary outcome measures. Similarly, the frequency of visiblemorphologic changes was not different between groups (P = 0.256). Atotal of 4 (27%) of 15 uninjured controls and 7 (47%) of 15 individuals with CAIwere observed to have degenerative changes. Individuals within the control group hadthe following morphology findings: marrow edema (n = 1), asubchondral cyst (n = 1), a local osteochondral defect(n = 1), and talar osteophytes (n = 1).Individuals with CAI were found to have the following morphologic changes: asubchondral cyst (n = 3), talar osteophytes (n =2), chondral delamination (n = 1), and anterolateral cartilagethinning (n = 1). The changes are thought to be the result oftraumatic chondral shear forces.No differences in cartilage morphology were observed among the CAI and controlgroups. Overall talar cartilage volume  and more T1ρvariability (P ≤ 0.002) relative to the CAI-contralateral limb andthe healthy control limb (P = 0.034) relative to the healthy control limb(P = 0.055) but were associated with a 95%CI that did not cross zero. Among the secondary STJ variables, only the medial STJT1ρ relaxation time variability differed between the CAI-involved and the healthycontrol group (P = 0.043). This difference was associated with alarge effect size and a 95% CI that did not cross zero. No other differences werenoted among the primary or secondary variables   in healthy individuals  and CAI-contralateral limb , weak associations that were not statisticallysignificant were observed between the overall talocrural and posterior STJ T1ρrelaxation time means  in healthy controls  and CAI-contralateral limb , weak nonsignificant relationships were noted between the overall talocruraland posterior STJ T1ρ variability  seeking medical care. greater structural damage  at the talocrural joint duringthe injurious event, and/or preinjury profiles  present in the STJ but not in the talocrural cartilage of thosethat go on to sustain a lateral ankle sprain and/or develop CAI. For example,there is an association between baseline measures of cartilage synthesis anddegradation and a subsequent likelihood of anterior cruciate ligament injury. Further research is warranted to examine how these proposed factors couldinfluence prospective talocrural and STJ cartilage composition changes in bothlimbs following acute lateral ankle sprains. Additionally, the standarddeviation around the talocrural mean was higher in the CAI-uninvolved limbrelative to the CAI-involved limb. This is likely due to the fact that theCAI-uninvolved limb was not required to be injury free and the standarddeviation represents the variance among the ankles that made up theCAI-uninvolved limb mean. This does not imply a homogeneous injury history amongthe CAI-involved limbs but rather the established minimum  may have reduced the variance among the group.To our knowledge, this is the first investigation that has quantified cartilagecomposition of the talocrural or STJ in a CAI-contralateral limb. While thismakes the CAI-contralateral results difficult to contextualize, our results seemto be articulation specific. For example, the CAI-contralateral talocrural T1ρmean more closely resembles the control limb than the CAI-involved limb , it appears that posterior STJ compositional declines are more uniformin nature. Further research is needed to test this hypothesis and determine ifimpact trauma and its associated sequela  or prolongedalterations in joint loading or both play a role in the uniformity ofcompositional changes  at both thetalocrural and STJ using compositional variability  oranother tool such as texture analysis.Talocrural T1ρ variability is elevated in the CAI-involved limb compared with ahealthy control limb as previously reported.1437 Yet, thisis the first investigation to examine cartilage composition associations betweenthe talar dome and posterior STJ articulation. Our most important finding wasthat the posterior STJ T1ρ relaxation time mean and variability demonstratedmoderate to strong positive associations with the T1ρ mean and variability ofthe talar dome in a healthy control limb . Our CAI-contralateral limb was required to befree from CAI but not required to be free from a previous lateral ankle sprain.As a result, the CAI-contralateral limb data may be driven by the initial anklesprain trauma and not asymmetrical movement patterns as hypothesized. Rearfootalignment, which could affect load patterns within the talocrural and STJarticulations, was not quantified in this sample. It is also important to notethat this investigation is limited to T1ρ relaxation times and future researchshould evaluate the differences in cartilage volume and T2-mapping concurrentlywith T1ρ to elucidate the interactions among proteoglycan content, type-IIcollagen orientation, and morphology. Finally, the current design does notprovide insights into how quickly, post index injury, these declines began orhow such declines could be slowed via therapeutic interventions. Therefore,future longitudinal studies are needed to address these knowledge gaps.A CAI-involved limb has a higher T1ρ mean value  atthe talocrural and STJ than a CAI-contralateral and healthy control limb. Similarly,a CAI-involved limb has higher T1ρ variability at the talocrural joint relative to ahealthy control but not the CAI-contralateral limb. A CAI-contralateral limb alsohad a higher T1ρ mean value  at the posterior STJrelative to a healthy control limb. The talocrural and posterior STJ T1ρ means andvariability have a strong positive association in a healthy control limb, but not ina CAI-involved limb. We conclude that CAI creates deleterious and MRI-measurablechanges in ankle and subtalar joint cartilage composition that has implications forfuture models of clinical care, prevention, and research.