Albertocetus meffordorum, previously only known from the holotype skull. The other is a fragmentary petrosal from the upper Oligocene Belgrade Formation that we refer to Echovenator sp, indicating at least two xenorophids from that unit. Two of the Albertocetus meffordorum specimens are from the lower Oligocene Ashley Formation: 1) a partial skeleton with neurocranium, fragmentary mandible, ribs, vertebrae, and chevrons, and 2) an isolated braincase. The partial vertebral column indicates that Albertocetus retained the ancestral morphology and locomotory capabilities of basilosaurid archaeocetes, toothed mysticetes, and physeteroids, and caudal vertebrae that are as wide as tall suggest that the caudal peduncle, which occurs in all extant Cetacea, was either wide or lacking. CT data from the isolated braincase were used to generate a digital endocast of the cranial cavity. The estimated EQ of this specimen is relatively high for an Oligocene odontocete, and other aspects of the brain, such as its anteroposterior length and relative size of the temporal lobe, are intermediate in morphology between those of extant cetaceans and terrestrial artiodactyls. Ethmoturbinals are also preserved, and are similar in morphology and number to those described for the Miocene odontocete Squalodon. These fossils extend the temporal range of Albertocetus meffordorum into the early Oligocene, its geographic range into South Carolina, and expand our paleobiological understanding of the Xenorophidae.We report five new specimens of xenorophid dolphins from North and South Carolina. Four of the specimens represent the xenorophid  Xenorophus sloanii, Albertocetus meffordorum) or have scant  postcranial material. This study refers new specimens to Albertocetus meffordorum from the upper Oligocene Belgrade Formation and lower Oligocene Ashley Formation . A series of significant discoveries have revealed that the earliest odontocetes could echolocate , possessormation , and forThe partial skeleton CCNHM 303  was mechAlbertocetus meffordorum endocast was segmented using the software Amira for Life Sciences (version 5.4.3). A single endocast segment was created using the “magic wand” and “lasso” tools. The cranial cavity was filled with matrix and at times the density of the matrix and bone were similar. In these cases matrix and bone were differentiated by adjusting the contrast gradient and observing the specimen in multiple planes . The finished endocast was smoothed by using the simplifier button under the SurfaceGen module. The total number of faces were decreased by 30% . Final images were taken using snapshot button while rotating the endocast. The SurfaceGen feature in Amira was used to generate an isosurface on the Albertocetus endocast. The smoothening of the endocast was left unconstrained to ensure a more accurate final value. Next, the SurfaceArea option was applied to calculate the surface area and volume of the specimen.The Albertocetus and other fossil specimens were reduced based on an estimate of non-neural tissue  within the endocast. To make this estimate we took data on brain mass and brain adnexia in cetaceans from a previous study [The endocranial volumes for us study , convertus study . These eus study  ) – 5.345). In order to assess the robustness of our body mass estimate we also calculated the body mass of Albertocetus and other Oligocene fossil odontocetes using two other equations derived from linear regressions of measurements obtained from extant taxa. The first is an equation that estimates total body length (cm) from the bizygomatic width (BIZYG) of the skull in cm [ - 4.464).Mass = 1000 x 10ll in cm : log retention of a narrow sagittal crest deviating approximately 5–15° anterolaterally to the left (unknown in Xenorophus sloanii), 2) triangular apex of occipital shield in dorsal view, 3) nasals rectangular with parallel lateral margins, transverse anterior margin, and dorsally flattened, 4) petrosal with small, circular, pit-like suprameatal fossa . These features indicate that CCNHM 218 and 303 are identifiable as Albertocetus meffordorum and not to other named xenorophids.Specimens CCNHM 218 and 303 possess the following synapomorphies of Xenorophidae: 1) nasal process of premaxilla greatly expanded and underlies ascending process of maxilla, 2) premaxilla with lateral crest adjacent to the bony nares, 3) frontal window posterior to orbit with exposure of premaxilla, and 4) large and laterally projecting lateral tuberosity of petrosal [ex situ from the Wando River with adhering concretionary glauconitic limestone matrix; exposures in this area are mapped as the Runnymede Marl Member of the Ashley Formation [in situ from an exposure of the Givhans Ferry Member of the Ashley Formation which has produced other vertebrates including a skeleton of a large, undescribed species of Xenorophus (CCNHM 168), an incomplete humerus of the giant bony toothed bird Pelagornis sp., cf. P. sandersi (CCNHM 786), and a partial skull of Aglyptorhynchus sp. (CCNHM 1837). Adhering matrix consists of sparsely glauconitic and discontinuously cemented calcarenite; a poorly preserved juvenile odontocete squamosal and fish bones, including a preural vertebra of the blochiid billfish Aglyptorhynchus, were found in association with CCNHM 303. Detailed locality information is available on request from CCNHM. The Givhans Ferry Member of the Ashley Formation is lower Oligocene in age and has yielded 87Sr/86Sr dates of 28.43–28.75 Ma, and the underlying Runnymede Marl Member has yielded a date of 29.0 Ma [Albertocetus meffordorum from the Ashley Formation.The Ashley Formation is the oldest of three Oligocene marine units in the Charleston Embayment of South Carolina . The Ashormation . Partial 29.0 Ma . An age Crassostrea gigantissima) reflecting estuarine or possibly shallow marine deposition , a nurse shark (Nebrius or Ginglymostoma), an angel shark (Squatina sp.), sand tiger sharks , megatoothed sharks (Carcharocles angustidens), snaggletooth sharks (Hemipristis serra), reef and lemon sharks , tiger sharks , sawfish (Anoxypristis sp.), cownose and bat rays , an extinct devil ray (Plinthicus stenodon), a pinfish (Lagodon sp.), indeterminate crocodilians and sea turtles, indeterminate odontocete remains, and xenorophid dolphin specimens USNM 559392 (Albertocetus meffordorum) and CCNHM 1188 (cf. Echovenator) reported in this study. Unfortunately, at Belgrade Quarry collecting is only permitted on spoil piles where specimens are reworked; Pliocene marine vertebrate  and Pleistocene terrestrial mammal (e.g. Equus) remains are found occasionally interspersed. However, within the Oligocene section at the quarry studied by [Albertocetus meffordorum holotype), washed up on Onslow Beach are assumed to have been eroded from submarine exposures of the Belgrade Formation; 87Sr/86Sr dates of mollusks from these cobbles indicate an age of 26.5 Ma [87Sr/86Sr dates from the underlying upper River Bend Formation are 29.9–28.6 Ma [The Belgrade Formation is exposed in various quarries in southeastern North Carolina Figs  and 2C a Member; ). Echino Member;  and Cras7 ± 1 Ma . Belgradudied by , marine  26.5 Ma , whereas 26.5 Ma , 32 At B–28.6 Ma , 32, but–28.6 Ma , 31, latThe posterior end of the premaxilla extends further posterior than the nasals and has wide contact with the frontal posteriorly and ventrally ; Table 1Xenorophus sloanii [Little of the maxilla is preserved in either specimen except for a rostral fragment of the right maxilla and the base of the rostral portion of the left maxilla . SeveraEchovenator and Cotylocara . Ventrally, the frontal forms the anteromedial part of the temporal fossa. The ventral part of the frontoparietal suture is vertical but as it ascends it curves anteriorly to form a sigmoidal curve towards the orbitotemporal crest. Though damaged in CCNHM 303, the infraorbital region is well-preserved in CCNHM 218, where it exhibits a large, deep, and anterolaterally curving frontal groove. The canal emanates from the large orbital fissure; the orbital fissure is possibly taphonomically enlarged in both specimens. The frontal forms the anterior margin of the orbital fissure.A narrow band of frontal is exposed dorsally, forming a trapezoidal shape and extending anteriorly between the nasal processes of the premaxillae to meet the nasals ; Table 1Echovenator , but appears to be absent in Cotylocara macei and Echovenator sandersi. The median parietal suture is incompletely closed in both skulls, particularly in CCNHM 303  is biloThe vomer extends posteriorly almost to the anterior margin of the basioccipital crests, and in the process underlaps the pre- and basisphenoid . AnterioAlbertocetus referred to as middle and inferior meatus are unlikely to be homologous to those structures in humans [Squalodon from the Miocene Calvert Cliffs [Squalodon outside the odontocete crown group , we canAlbertocetus is more elongate and intermediate in morphology between those of the terrestrial artiodactyls, such as Diacodexis ilicis [Prosqualodon davidis [Albertocetus is more anteroposteriorly compressed than a figured, but undescribed, endocast of another species of xenorophid represented by ChM PV4266  and proportionally larger than in Cotylocara (47%). A delicate vertical ridge extends dorsal to the posterior bullar facet and clearly separates the stylomastoid fossa from the facial sulcus.The petrosal is compact with a very large pars cochlearis, short posterior process, a long hatchet-shaped anterior process, and an enlarged and triangular lateral tuberosity ; Table 2ly small . RelativThe dorsal apertures for the facial canal, foramen singulare, and spiral cribriform tract are recessed into an ovoid internal acoustic meatus . The apeAlbertocetus meffordorum on the basis of several shared features with CCNHM 303, including a small, circular, pit-like suprameatal fossa, bulbous posterodorsal angle with radially oriented sulci, and an equidimensional, quadrate posterior bullar facet; none of these features are visible in the holotype petrosal. USNM 559392 differs from CCNHM 303 in a few minor ways, including possessing a deeper groove for the tensor tympani, a more shallowly incised crease anterolateral to the posterior bullar facet, and a dorsoventrally lower and transversely less inflated superior process and posterodorsal angle. Other differences  are attributable to damage in these specimens.An isolated petrosal , but differ from Xenorophus sp. in their smaller size, proportionally smaller pars cochlearis, longer anterior process, and absence of a longitudinal ridge on the ventral surface of the pars cochlearis. In Cotylocara and Echovenator, the suprameatal fossa is transversely wider and anteroposteriorly longer than in CCNHM 303, and the fossa is relatively larger than the internal acoustic meatus; faint sulci radiating from the suprameatal fossa are also present in Cotylocara and Echovenator.These petrosals of The tympanic bulla is subrectangular in dorsal and ventral views, and the bulla anterior to the lateral furrow (= anterior lobe of ) is muchThe posterior part of a fragmentary right mandible is preserved but lacks the coronoid process and mandibular condyle; the mandibular walls are thin and a large mandibular foramen was present  No margiExcluding the transverse process and hypapophysis, the atlas is oval in anterior view and slightly wider than high Figs ; Table 4The axis is narrower than the atlas and dorsoventrally higher; the neural spine is tall with a bifurcated apex and sagittal groove on its posterior side Figs ; Table 4Mirocetus riabinini , as well as the complete cervical series of Xenorophus sp. (CCNHM 168). The vertebra considered to be C4 is an anterior cervical vertebra based on the presence of a hypapophysis, but its anterior epiphysis does not match the posterior epiphysis of the axis, meaning it is the C4 not the C3. The vertebrae considered to be C5 lacks a hypapophysis and is thus posterior to C4, but it lacks the ventrolaterally projecting transverse process of the C6. The C6 is identifiable based on the elongate, ventrolaterally projecting transverse process as well as the absence of a hypapophysis. These vertebrae differ most strongly in the structure of the transverse process; but possess centra similar in size and shape. All share neural arches with delicate lamina, dorsoventrally short and narrow neural spines, and bear anteroposteriorly flattened, transversely wide pedicles. The C6 has an anteroposteriorly longer centrum, but in lateral view, the centrum is distinctly wedge-shaped and narrows dorsally, perhaps contributing to a dorsally concave bend in the cervical series. The C4 and C5 had large transverse foramina between the delicate dorsal and larger ventral transverse processes, and appear to have been laterally open given that the ventral transverse processes are complete. In C4 the transverse process is twisted, subtriangular, and oriented anteroventrally to dorsoposteriorly in lateral view. That process widens laterally and exhibits a concave lateral margin in dorsal view with anterior and posterior apices. Several epiphyses C4-C6are fully fused, smooth, and lack a punctate texture indicative of immaturity; in C5, the anterior epiphysis is lost and the posterior epiphysis is partially fused. Both epiphyses in C6 are incompletely fused, with a fine line crease remaining along the margin. Faint annular sulci are present, but notochordal pits are absent.Three other cervical vertebrae are present, including C4, C5, and C6 Figs ; Table 5Mirocetus riabinini [Mirocetus; [Four thoracic vertebrae are preserved Figs ; Table 5iabinini , TA and rocetus; ). All shSeven lumbar vertebrae are preserved, and similar to the thoracic vertebrae, cannot be precisely identified to position and instead are identified as LA, LB, LC, LD, LE, LF, and LG Figs ; Table 5sensu [Kogia [Kogia [Six caudal vertebrae are preserved, identified as CaA-CaF Figs ; Table 5sensu ) is inciu [Kogia , CaC anda [Kogia .Parts of at least nine ribs are preserved . JudgingDorudon atrox and Cynthiacetus peruvianus, the chevrons are smaller relative to the centrum height, further suggesting an anterior position for these chevrons.Parts of two similarly sized chevrons are preserved that include articular tubercles dorsally and a V-shaped body that tapers ventrally Figs –13; theyLinichnus but differ from Linichnus serratus in having smooth (rather than serrated) margins [Linichnus traces, typically inferred to reflect the tips of shark teeth scraping into bone [Waharoa ruwhenua [Vertebrae LD, LE, and the more completely preserved chevron preserve several bone modifications . Two typ margins ; this manto bone . Type 2 nto bone ; similarruwhenua .The finished endocast was shown to have a volume of 478.797cc. If brain density is assumed to be 1.04 g/cc , then thAlbertocetus, the corrected brain mass would be 443.3 g. The body mass of Albertocetus, as inferred by breadth across the occipital condyles [Although the digitally rendered endocast closely resembles an isolated brain, endocasts also contain circulatory structures and meninges, even if these structures are not particularly visible. As described in the methods we estimated the volume of adnexia by calculating a linear regression of log(adnexia volume) and log based on data from a recent study . The rescondyles , is esticondyles  were recAlbertocetus of 1.82 m and a mass of 72.9 kg, about 71% larger than that estimated from breadth across occipital condyles. Similar discrepancies occur for three other Oligocene odontocetes for which we have bizygomatic width data for, whereas alternative body mass estimates for larger specimens are fairly similar  was collected ex situ within a limestone concretion found on Onslow Beach, North Carolina, and presumed to originate from offshore deposits of the upper Oligocene Belgrade Formation [A. meffordorum. The discovery of a new specimen of Albertocetus with a complete petrosal removed from the cranium . Owing to these features, USNM 534010 and CCNHM 1188 are identifiable as Echovenator sp., and in concert with other specimens reported herein, indicate the presence of two xenorophids in the Belgrade Formation of North Carolina. Given that many additional xenorophids have yet to be named from the Ashley and Chandler Bridge formations of South Carolina . FurtheAlbertocetus is within the range of Oligocene odontocetes reported by [Fucaia buelli  and near the higher end of values reported for Chattian odontocetes  and another specimen of uncertain affinities . A similar finding occurs if we compare the EQ values inferred based on model based methods in a phylogenetic context. Montgomery et al. [Xenorophus to a clade that includes all extant odontocetes had an EQ of 1.6804. Thus on the branch leading to Xenorophus there was an inferred change in EQ from 1.6804 to 3.2759. A value of 2.586 for Albertocetus, a member of the Xenorophidae, is consistent with this inferred evolutionary change.An EQ value of 2.586 in orted by , but a morted by  includedorted by ; further buelli ; Fig 16)Albertocey et al.  calculatOne unexpected finding is the difference we found in EQ when using an alternative measures of body mass; specifically bizygomatic width and inferred body length , 23. AmoAlbertocetus meffordorum is small (n = 3 specimens with crania), yet informative for the earliest diverging odontocetes.Ontogenetic studies of extinct cetaceans are rare as they require samples including multiple referred specimens of different ontogenetic stages—whereas many studies emphasizing new taxa and phylogenetic insights have focused on taxa based on single individuals , but comaturity , perhapsmaturity  by recormaturity , 59). A Sr dates .Studies of locomotor evolution in Cetacea have generally focused on the land-to-sea transition, as superbly documented by archaeocete skeletons from Indo-Pakistan, Egypt, and the southeastern United States , 62–67. Kogia, Physeter) are proportionally similar, permitting dorsoventral undulation along most of the column ; these ern 1 of ) or throern 1 of ). Vertebern 1 of , with exPhyseter and Kogia) further express anteroposteriorly foreshortened anterior caudal vertebrae, serving to stiffen the peduncle  possesses the first postcranial skeleton for a xenorophid dolphin . Lack ouchholtz . This isontoceti  stronglyphiidae; ).Albertocetus meffordorum—and likely other early diverging odontocetes—primitively lacked a transversely narrow caudal peduncle, similar to the basilosaurid Dorudon atrox [Aetiocetus cotylalveus are similarly equidimensional  are nearly equidimensional in shape and lack the transversely narrow profile of modern cetaceans with a caudal peduncle Figs  and 16, nsional : Fig 7C)Albertocensional , well within the range of extant delphinoids, and chronicling a drastic jump in EQ across the Eocene-Oligocene boundary. Further study of appropriate body size estimation is needed to investigate the proposed Eocene-Oligocene explosion in odontocete encephalization.3. The endocast of Albertocetus meffordorum permits the first basic examination of ontogenetic trends in stem Odontoceti. Ontogenetic study of Albertocetus meffordorum identifies several sutures of the dorsal braincase and facial region of interest for assessing ontogenetic status in stem Odontoceti , to be confirmed with larger samples of undescribed xenorophids . Postcranial epiphyseal fusion is achieved earlier in ontogeny than cranial suture closure in A. meffordorum.4. The sample size of Albertocetus meffordorum, like basilosaurids, Mysticeti, and sperm whales, is a "pattern 1" species with no anteroposterior specialization of the vertebral column. This indicates that dorsoventral undulation occurred through the entire flexible lumbocaudal series; this appears to characterize stem odontocetes. Rectangular caudal vertebrae indicate the presence of caudal flukes. Surprisingly, no caudal vertebrae are transversely narrower than tall, suggesting the absence of a transversely narrowed peduncle as in all extant Mysticeti and Odontoceti. Such a feature would imply that the narrow peduncle evolved independently. However, skeletons of stem odontocetes and mysticetes with a more complete caudal series are required to further evaluate this hypothesis.5. Vertebral proportions indicate that S1 Table(XLS)Click here for additional data file.


Arabidopsis and its relative the extremophile Eutrema salsugineum. This data enables a better understanding of the contribution of the lipidome in determining the ability to tolerate suboptimal temperature conditions. Collectively this information will allow us to identify the key lipids and pathways responsible for resilience, enabling the development of new approaches for crop tolerance to stress.Environmental constraints limit the geographic distribution of many economically important crops. Cold stress is an important abiotic stress that affects plant growth and development, resulting in loss of vigour and surface lesions. These symptoms are caused by, among other metabolic processes, the altered physical and chemical composition of cell membranes. As a major component of cell membranes lipids have been recognized as having a significant role in cold stress, both as a mechanical defence through leaf surface protection and plasma membrane remodelling, and as signal transduction molecules. We present an overview integrating gene expression and lipidomic data published so far in  Climate change is driving greater temperature fluctuations, resulting in frequent periods of cold temperatures. Susceptible crops exposed to cold temperatures have impaired growth and development, this limits the use of valuable varieties and lowers yields. Therefore, it is important to understand the mechanisms underlying cold stress responses in the search for more tolerant crops and those that maintain yield under temperature stress. Sensing and reacting to cold stress is a complex process that appears to require a multitude of mechanisms and pathways. The strategies that plants utilise to endure stressful conditions are varied and involve a multitude of molecular, metabolic and physiological adaptations. These strategies make changes that occur in the first instance to protect the plant, followed by cold acclimation enhancing the plant survival under the low temperature stress Eutrema salsugineum, an extremophile belonging to the Brassiceae family, which is highly resistant to salinity stress, . In addition to its halophytic properties it also shows a high tolerance to drought and cold stress. Furthermore, it has a close evolutionary relationship with the model plant Arabidopsis thaliana (Arabidopsis), with high genome homology and similar plant morphology Eutrema salsugineum (Eutrema) is now utilised as a model for abiotic stress studies Extremophile plants, that can endure extremes of abiotic stress, are often proposed as models to study resilience to severe environmental conditions. One such example is Arabidopsis and its relative the extremophile Eutrema, to better understand the contribution of the lipidome in determining the ability to tolerate suboptimal temperature conditions. Collectively this information will allow us to identify the key lipids and pathways responsible for resilience, enabling the development of new approaches for crop tolerance to stress.This review presents an overview integrating gene expression and lipidomic data published so far in 22+ channels, kinases and G-protein associated receptors. Cold temperatures typically trigger a signalling cascade which includes Ca2± flux and −dependent protein kinases (CDPKs), mitogen-activated protein (MAP) −kinase mediated cascades, and the consequent generation of lipid signalling molecules e.g. phosphatidic acid, diacylglycerol and inositol phosphate, which collectively contribute to responsive gene expression Plants are poikilothermic and sessile, as such they are highly influenced by the surrounding environment. Of the many abiotic challenges faced by plants, low temperature often defines the natural distribution of plant species, and limits the survival range of many agricultural crops. Cold stress reduces plant growth and development, altering the physical and chemical composition of cell membranes, causes electrolyte leakage, decreased protoplasmic streaming and changes in cell metabolism The accumulation of abscisic acid (ABA) in response to abiotic stress mediates signal transduction and gene expression. The role of ABA in the cold stress response results from its function as a plant hormone, regulating growth, sugar content, leaf senescence, seed dormancy and germination, cell division and elongation Arabidopsis and other economically significant species, such as rice and wheat, have yielded a multitude of potential candidate genes participating in the low temperature stress response; however, some of their functions are still unknown Arabidopsis Functional Genomic Network Project, AtGenExpress, has allowed the detection of 24000 protein-encoding genes via the Affymetrix ATH1 gene chip, enabling the identification of more than 2000 cold response genes (1335 up-regulated and 1061 down-regulated genes), of which more than 170 genes encode cold-induced TFs ArabidopsisLow temperature-induced gene expression can be described as transient or long term. Transient expression is generally considered up to five hours of cold exposure, whereas long term expression genes are transcribed and remain activated during the entire cold stress period. Transcriptome analyses in 3Arabidopsis has tolerance mechanisms restricted to mild, but not extreme conditions even amongst the most resistant ecotypes ArabidopsisArabidopsis thaliana and Arabidopsis lyrata occurred approximately 43.2 million years ago (MYA), and from Schrenkiella parvula (or Thellungiella parvula), a close relative of Brassica rapaKnowledge about how plants can cope with extreme environments is economically valuable as these useful traits could developed in crop species. Extremophile plants operate in the most challenging environments, such as extreme cold, drought and salinity, in combination with a broad range of other stresses Thellungiella salsugineaArabidopsis, although its leaf shape may change with the growth conditions, appearing waxier and serrated. Despite similarities in flower morphology between Arabidopsis and Eutrema these two species cannot be crossed successfully Arabidopsis with 95% homology Arabidopsis allow a direct comparison between genes and their function, rendering most of the experimental procedures developed for Arabidopsis highly adaptable. This constitutes a major advantage in stress related studies, opening the possibility of expressing genes of interest from the extremophile into Arabidopsis facilitating their characterization, and more importantly, their potential role in stress resilience.Eutrema, formerly known as Arabidopsis may be the key to the robust resilience mechanisms found in the extremophile, gene duplication mechanisms and transposable elements which increase genome size might be involved in evolutionary stress coping mechanisms 2 ratios of salt vs. control were ≥1.5, whereas the same experiment in Arabidopsis showed 40 such salt stress-inducible genes. These differences are attributed to a constitutive high level of expression of stress-responsive genes in Eutrema in comparison with Arabidopsis. This constitutively high level of some transcripts is thought to contribute to its extreme salt tolerance. Another proposed hypothesis is that Eutrema has a photosystem that rapidly and efficiently responds to abiotic stress. Wong et al. Arabidopsis, than either salt- or drought-induced sequences. Of the down regulated transcripts under cold stress 15% of them code for genes involved in photosynthesis, indicating that Eutrema has a significant capacity for mitigating the effects of photoinhibition The difference in genome size between Eutrema and 4Lipid biosynthesis is composed of a multitude of processes that span different cellular organelles, such as plastids, the endoplasmic reticulum (ER), and cytosol, employing complex pathways and trafficking mechanisms. Fatty acid synthesis utilizes the carbon flux derived from photosynthesis in the form of pyruvate and it is localized in the plastid, after which the fatty acyl chains are then channelled to produce more complex lipid molecules in the plastid, or transported in the cytosol and endoplasmic reticulum (ER). This is regulated according to the supply and demand for acyl chains, and its production is balanced according to requirements. The regulation of fatty acid synthesis and further modifications is complex and has been extensively reviewed Arabidopsis sfr3 mutants which are sensitive to freezing, present an alteration of cuticle permeability due to the variation of wax composition and morphology, in the cold and in flower development. This phenotype arises from a missense mutation of the ACC1 gene, that encodes a cytoplasmic acetyl-CoA carboxylase (ACCase) involved in de novo fatty acid biosynthesis The first line of physical defence against abiotic stresses is the leaf cuticle which incorporate intra- and epi-cuticular waxes. Cutin and suberin, present in plant cell walls, are complex fatty acid derived polymers that confer chemical and physical barriers that protect cells from external pathogens and control the movement of gases, water and solutes Plasma membranes are considered as the primary barrier between the organism and external environment, being the first to experience the injurious effects of stress. Alterations in lipid composition/structure in the plasma membrane under environmental stresses are crucial to maintaining membrane stability and functions. In general, biological membranes have a crucial role not only in the protection, homeostasis and metabolism of the cell, but also in signal recognition and signalling cascades which are proposed as key regulatory processes both under optimum and stress conditions mediated by protein-lipid interactions. 4.1Cell membranes are composed of three main classes of lipids, glycerolipids  sphingolipids and sterols. Whereas glycerolipids are synthesized on the cytosolic leaflet of the endoplasmic reticulum (ER), sphingolipid production is completed in the ER/Golgi, and later presented to the outer monolayer. With regard to sterols, they have a higher affinity for sphingolipids and are thought to fill the voids between the sphingolipids, increasing the packing of lipids trans-16:1-phosphatidylglycerol species Arabidopsis, pldδ mutants showed impaired freezing tolerance and reduced PA levels, whereas overexpression of this gene has the opposite effect Low temperature damage, also associated with freezing injury, can arise from changes in the intercellular water temperature which affects water potential through the plasma membrane, affecting also its own conformation causing structural damage and hence increasing ion leakage. Formation of non-bilayer lipid structures in both plasma and chloroplast membranes, such as the inverted hexagonal (HII)-type structures, can occur under cold temperatures causing lesions in the membrane. Under such conditions changes in the lipid composition, and membrane repair systems, mediated by many lipid pathway enzymes, are activated in order to maintain its structure, keeping membrane integrity and avoiding damage Arabidopsis (fad mutants) it was possible assign a role in cold tolerance to some of this genes Arabidopsis fad2 plants become chilling sensitive at 12 °C, and die when exposed to 6 °C Arabidopsis, transgenic plants were enriched in the linoleic acid content of the mitochondrial membranes, showing higher viscosity, and, hence, reduced rigidity with cold stress Arabidopsis. Although the specific mechanisms are yet to be understood, the role of this enzyme could be crucial in cold tolerance response and is a candidate worthy of further research Arabidopsis sfr3 mutant is freezing sensitive due to a missense mutation in ACC1 (Acetyl-CoA carboxylase1). This enzyme is responsible for the cytosolic pool of malonyl-CoA to be used for the production of Very-Long-Chain-Fatty-Acid (VLCFA), essential for the assembly of complex lipids such as sphingolipids The ability to adjust membrane fluidity according to temperature changes is often attributed to the regulation of membrane fatty acid desaturation  and chaiArabidopsis COR15 proteins are encoded by two nuclear homologous genes COR15A and COR15B (82% sequence identity) and targeted to the chloroplast stroma Arabidopsis transgenic plants, overexpression of COR15 genes further stabilizes chloroplast membranes and increases freezing tolerance arabidopsis mutants show extensive intracellular damage after freezing recovery, with rupture in both chloroplasts and tonoplasts likely through the fusion of destabilized membranes in these organelles 1 and 2 genes (ADS1 and ADS2) genes are paralogous genes, sharing a 75% of similarity at the amino acid level, that encode cold inducible desaturases affecting cold acclimatization response and chilling/freezing tolerance, respectively ADS1 encodes a soluble Δ9-desaturase that is localized exclusively in the chloroplast and catalyses the desaturation of stearic acid (18:1) of MDGD ADS2 encodes a 16:0 desaturase for MGDG and PG Arabidopsis, affecting chloroplast membrane desaturation.The specific composition of the inner and outer chloroplast membranes also influences the cellular chilling response. The 4.2The mechanical protection of biological membranes also has an important role in the production of stress responsive signalling lipids, which can be derived from membrane lipids, such as phospholipids and sphingolipids, through enzymatic reactions Arabidopsis knockout mutants (acd5) Sphingolipids play an active role in cold transduction pathways. The fast and transient production of phyto-S1P, by a specific LCB kinase isoform (LCBK2), has been reported to have an effect in the regulation of the expression of cold-responsive gene expression 4.3Arabidopsis seeds, and positively effects environmental stress tolerance during seed germination and plantlet establishment Modulated responses to stress are regulated by stress-induced gene expression in a complex regulatory gene network mediated by TFs A. thaliana Col-0 and Eutrema salsuginea where subject to 4 °C for 3 days (manuscript in preparation/unpublished data). We could identify reads for 17 of the 27 TFs, of which eight were differentially expressed in Arabidopsis, Eutrema or both. Those showing differences were predominantly associated with the fatty acid elongation and wax biosynthesis pathway . To date 27 TFs related to lipid metabolism has been identified (Supplementary Table S1)  pathway . Interes5Cold stress initiates a plethora of responses from the initial sensing and TF cascade to the cellular remodelling of membrane structures. Collectively these responses define the resilience of the plant to extremes of cold temperature. This review has sought to demonstrate the intimate role lipids play in defining the cells response to cold stress from protection to signalling. Indeed, lipids and their role in cold response mechanisms allow the plant to not only survive such stress, but to thrive under those conditions (e.g. Eutrema). Plant lipids are extensively studied mainly due to their economic significance as energy-storage compounds. More recently their active role on plant stress metabolism has been explored, although there remains still much to understand. The study of lipid biosynthetic pathways has benefitted greatly from improvements in mass spectrometry allowing the science of lipidomics to develop Arabidopsis have underpinned research into plant responses to perturbation, however, now much can be learned from the application of these approaches to extremophiles. However, to further establish Eutrema salsugineum as an extremophile plant model is still a challenge, and for the sake of reproducibility of experimental conditions and experiments, the improvement and adaption of existing protocols is essential. Techniques such as transformation Beyond the direct measurement of lipid species, Next-Generation Sequencing (NGS) has already revolutionized the study of transcriptomes Research into the response of the plant lipidome during periods of cold stress is a relatively recent area of work incorporating metabolite and transcriptomic approaches. The principle obstacle to overcome is the low level and high turnover of some  lipid species. Recent advances in the sensitivity and the accuracy of mass spectrometry and its linked chromatography are offering new insight into some of these hard to quantify lipids which when coupled with the power of RNA-Seq can identify the complex network of proteins that protect the plant from damage at low temperatures. The mechanism of action both in sensing low temperature and altering transcription and lipid turnover is unclear and further research must address the specific role of TFs in lipid homeostasis during cold stress. Addressing the question of whether the regulation of lipid species is predominantly transcriptional, translational or post-translational or a combination of these mechanisms will target the knowledge gap that presently exists and will enable the development of breeding strategies that deliver climate resilient crops.


They do this with 37 genes encoded in their own genome and more than 1,200 genes from the nuclear genome, whose gene products are targeted to mitochondria after being translated on cytosolic ribosomes. This complex intergenomic communication is a hallmark of eukaryotic life and has been under continuous refinement for more than 10al cells , the metal cells , the genal cells , 9, the Most questions about mitonuclear interactions focus on the oxidative phosphorylation pathway (OXPHOS), the ATP producing process that includes four multisubunit protein complexes of the electron transport chain (ETC) and a fifth complex that phosphorylates ADP to produce ATP. These OXPHOS complexes, embedded in the mitochondrial inner membrane, include all of the 13 proteins encoded in animal mitochondrial DNA (mtDNA) plus ~78 proteins encoded on nuclear chromosomes. Two other classes of mtDNA genes, transfer- and ribosomal- RNAs, are needed to express mtDNA encoded proteins, and their functions require nuclear encoded proteins for transfer RNA (tRNA) charging, ribosome function, and RNA processing. Because these sets of genes involve physical interactions between mtDNA- and nuclear-encoded gene products, they are logical targets for studies of mitonuclear communication, coordination, and coevolution. But mitochondria do much more than make ATP; for example, they maintain redox and calcium balance, mediate apoptosis, regulate amino acid and lipid metabolism via the Krebs cycle and beta-oxidation, and influence TOR signaling, among many other functions . So, theTesting hypotheses of molecular coadaptation in the age of genomics is potentially rather easy. One can sequence the genomes or transcriptomes of different organisms and ask if the interacting genes or proteins show parallel patterns of molecular evolution. Because mtDNA genes usually evolve faster than nuclear genes, those that interact with mtDNA-encoded partners should show elevated rates of divergence, implying coevolution. For functional tests, genetic replacement of a foreign mtDNA in to the nuclear background of a different species should disrupt mitonuclear coadaptation and cause fitness defects. There are some nice cases in support of these molecular  and genePLOS Genetics, Baris et al. [Fundulus heteroclitis to search for a signature of adaptive mitonuclear interactions, taking advantage of a natural experiment that is a geographic legacy of glacial retreat along the east coast of North America. They sampled a population of fish in Mantoloking, New Jersey, harboring two distinct mtDNA haplotypes that diverged during a period of geographic isolation imposed by the Hudson River basin that drained meltwater from the Wisconsin ice sheet. Following glacial retreat, “northern” and “southern” populations came in contact, and today, the New Jersey population has a ~50:50 ratio of two mtDNA haplotypes and a ~80:20 ratio of nuclear alleles typically found in Maine and Georgia. Baris et al. used genotyping by sequencing (GBS) to sample the nuclear genomes of fish from the New Jersey population and stratified the analysis of nuclear allelic variation by the mtDNA haplotype that each fish carried. The question they ask is whether there is evidence for population subdivision of nuclear alleles within a single population, associated with the divergent mtDNA haplotypes segregating in that population . Importantly, these SNPs show higher levels of allele frequency difference between mtDNA partitions within this population (measured as Fst “outliers”) than they do between the focal New Jersey population and geographically isolated populations in Maine and Georgia, a clear rejection of neutral expectations. Baris et al. quantified OXPHOS activity (ADP-dependent state-3 respiration) of mitochondria extracted from heart tissue from four mitonuclear genotypes that define pure parental and admixed hybrid individuals  and show that these mitonuclear genotypes explain a significant portion of the variation in OXPHOS function.Baris et al. found a few hundred single nucleotide polymorphisms (SNPs) spread across the nuclear chromosomes of Fst outlier status of SNPs partitioned by mtDNA, by testing 1,000 alternative data partitions of >9,000 nuclear SNPs. None of these “nuclear-nuclear” Fst outlier tests uncovered as many outlier SNPs as the original mitonuclear outlier test, suggesting that the main result is not littered with many false positives.It is tempting to invoke adaptive mitonuclear epistasis to explain these data, in which mtDNA haplotypes offer a sort of alternative metabolic niche for nuclear alleles within a population, akin to the classical Levene model of balancing selection via opposing selection in alternative habitats . Baris eFst outlier loci map to known nuclear genes encoding subunits of the OXPHOS complexes. If this study really has identified loci important in mitonuclear coadaptation, it suggests that long-range cis- or trans-regulatory factors, or downstream pathway effects, are mediating the epistatic interactions between mtDNA haplotypes and nuclear loci other than those encoding subunits of OXPHOS complexes. Understanding the genetic basis of fitness variation in the wild is a central goal of ecological and evolutionary genetics, and the genes of central metabolism have long fascinated biologists as a logical place to search for the source of this variation [One unexpected result from these analyses is that that none of the 349 ariation . Given t


On the basis of data published recently, we discuss the possibility that gingival CD8+ T cells contain a pool of cells with regulatory/suppressor properties involved in the maintenance of gingival tissue integrity by constitutively downregulating inflammation under homeostatic conditions and initiating repairing mechanisms in case of tissue injury. These basic physiological roles could be surpassed and hidden when a potent and/or chronic immune response against pathogenic bacterial colonization occurs, thus leading to bone loss. Elucidation of the basic physiological roles of particular CD8+ T cells present in periodontal tissue and the rules they follow in order to cope with minor versus major disruption of tissue homeostasis can improve our understanding of how they react to changes in their environment and ultimately allow the development of novel therapeutic approaches to favor anti-inflammatory responses and bone repair.Chronic periodontitis is a multifactorial disease characterized by the presence of dysbiotic microbial communities that, together with genetic and environmental factors, results in chronic inflammation of the periodontium, which ultimately may trigger alveolar bone resorption . The rep+ T cells is their heterogeneity, as they differ in terms of T cell receptor (TCR) diversity and antigen specificity, which allows them to monitor for shifts in peptide antigens presented in MHC class I molecules expressed on the plasma membrane of all nucleated cells. CD8+ T cells acquire functional properties after being activated, normally in secondary lymphoid organs, by antigen presenting cells (APC). As a result, some acquire innate receptors, including NK receptors, enlarging the kind of stimuli they can receive  since they have the ability to control other leukocytes to avoid excessive immune activation and its pathological consequences , suppressive cytokines or tissue-specific chemokine receptors, or adoptive transfer of CD8+ Treg should provide insights into the bone-protective role of gingival CD8+ T cells (+ T cells with the periodontium and enable the development of novel therapies to inhibit bone loss under pathological conditions.While scant, there is evidence that resident gingival CD8ry cells  will lik T cells , 49, 50.EC did bibliography search and wrote the manuscript. FA did bibliography search and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bone metastases are a common complication of epithelial cancers, of which breast, prostate and lung carcinomas are the most common. The establishment of cancer cells to distant sites such as the bone microenvironment requires multiple steps. Tumour cells can acquire properties to allow epithelial-to-mesenchymal transition, extravasation and migration. Within the bone metastatic niche, disseminated tumour cells may enter a dormancy stage or proliferate to adapt and survive, interacting with bone cells such as hematopoietic stem cells, osteoblasts and osteoclasts. Cross-talk with the bone may alter tumour cell properties and, conversely, tumour cells may also acquire characteristics of the surrounding microenvironment, in a process known as osteomimicry. Alternatively, these cells may also express osteomimetic genes that allow cell survival or favour seeding to the bone marrow. The seeding of tumour cells in the bone disrupts bone-forming and bone-resorbing activities, which can lead to macrometastasis in bone. At present, bone macrometastases are incurable with only palliative treatment available. A better understanding of how these processes influence the early onset of bone metastasis may give insight into potential therapies. This review will focus on the early steps of bone colonisation, once disseminated tumour cells enter the bone marrow. Bone metastases are a frequent complication of solid cancers . The estThe establishment of cancer cells in the bone marrow requires multiple steps, whereby cells need to leave the primary tumour and then adapt and survive in a physiologically different environment. The local microenvironment, or premetastatic niche, may be modified through the secretion of factors by cancer cells to establish favourable conditions for metastasis. For instance, cancer cell secretion of lysyl oxydase (LOX) is able to increase extracellular rigidity by reticulation of collagen and thus promote cancer cell anchorage . In orde2+) from resorbed bone promote tumour growth [Tumour cells that settle in the bone marrow enter a dormant state in specific niches and/or adapt to the bone microenvironment (osteomimicry). Disseminated tumour cells (DTCs) may become active years later as they proliferate and alter the functions of bone-resorbing (osteoclasts) and bone-forming (osteoblasts) cells, disrupting physiological bone remodelling and promoting skeletal destruction. In turn, the release of bone-derived growth factors (transforming growth factor β (TGFβ) or insulin-like growth factor 1 (IGF-1)) and calcium  receptor type 4 (CXCR4) and 6 (CXCR6) and their respective ligands, C–X–C motif chemokine ligand 12 (CXCL12) and 16 (CXCL16). In healthy breast and prostate epithelial cells, CXCR4 and CXCR6 are not expressed, but their expression levels are significantly increased in invasive cancer cells ,16. In t2+ concentration as a result of normal bone remodelling has been demonstrated to act as a chemoattractant for breast cancer cells in vitro [2+ acting on calcium sensing receptor (CaSR), in line with the role of CaSR in the localisation of HSCs to the bone marrow due to high local Ca2+ levels  is produced by osteoblast in bone and the RANK/RANKL pathway has a key role in osteoclast differentiation . RANK isAn important part in the mestastatic cascade, loss of cell-adhesion is firstly demonstrated to allow cells to dissociate from the surrounding matrix in order to extravasate into the circulation . After tIn a large-scale study of early-stage breast cancer patients, DTCs were identified in 30% of bone marrow aspirates at time of diagnosis . SimilarEndosteal and vascular niches are crucial regulators of normal and malignant stem-cell behaviour in the bone marrow. Quiescent HSCs are enriched in the endosteal niche lining the bone surface, whereas the vascular niche is oxygenated and stimulates proliferation and differentiation of HSCs . The adhBreast tumour cells were found to preferentially locate to osteoblast-rich regions following chemical alteration of the endosteal niche with the bisphosphonate zoledronic acid . SimilarSeveral clinical observations showed that DTCs in the bone marrow do not proliferate immediately but enter a state of dormancy, whereby a group of cancerous cells cannot grow beyond a certain size . It is pAs discussed, the annexin II and its receptor are expressed by osteoblasts and prostate cancer cells, respectively . The binTGFβ2 is a bone marrow derived factor which promotes DTC dormancy through TGFβ-R3 and MAPK p38α/β signalling . In the It is generally accepted that a majority of DTCs do not survive, due to the inefficient metastatic process. The bone marrow has been postulated to provide survival signals, whereby the bone marrow may be a protective environment for DTCs in patients undergoing chemotherapy ,76. For Bone turnover may have an important role on bone metastasis ,84. In oIn addition, the immune system is also involved in reducing tumour cell proliferation through a process known as “immune-surveillance”, which balances proliferating cells and dying cells . This is2+ can also activate dormant tumour cells [The latency period observed in metastatic relapse refers to the time for DTCs to adapt and escape the metastatic niche B. Intereur cells . Howeverur cells . For insur cells . Tumour ur cells . TherefoIn addition to the metastatic niche, a number of proteins have been reported to be able to reactivate dormant DTCs. For instance, microenvironments rich in type I collagen ,98 or fiMetastatic cancer cells are not solely responsible for the destruction of bone. This process mainly involves osteoblasts and osteoclasts and they are essential for metastatic tumour cell establishment in the bone C. OsteobIn addition, osteoclasts can secrete factors that promote tumour growth such as microRNAs (miRNAs), some of which have been shown to modulate bone function . A numbeIn addition to miRNAs, tumour cells can release factors that can interfere with normal bone resorption. This includes inhibitors of osteoblast differentiation such as dickkopf-1 (DKK-1) and sclerostin (SOST) . OsteoclWhile early detection and conventional treatments are effective for breast cancer management, the development of bone metastasis remains a major cause of death. Patients diagnosed with bone cancer are considered incurable. To date, no effective treatments for bone metastasis exist, only therapies that aim to limit the bone degradation such as denosumab  and bisphosphonates. In this review, we described the main mechanisms that allow cells to disseminate and colonise in the bone marrow by highlighting the importance of the bone microenvironment. It is clearly recognised that the tumour cells use the same processes as HSCs to migrate and anchor in the bone marrow. Furthermore, the dormancy mechanisms give rise to the formation of bone metastases, sometimes years after the detection of primary cancer. The study of DTCs in the bone marrow can be improved through the implementation of different in vitro and in vivo models of the bone metastatic niche . Through


Cutaneous squamous cell carcinoma (SCC) is among the most common cancers in humans, and many patients with SCC will develop multiple tumors within their lifetime. The field cancerization concept, originally proposed over 60 years ago, hypothesized that multiple primary cancers may arise simultaneously and coexist with subclinical precursor lesions within a defined field. Genetic sequencing of SCC and precursor lesions has identified what may be the earliest clonal proliferations in SCC development and confirmed that field cancerization in the skin is mediated by ultraviolet radiation. For patients with multiple SCCs and severe actinic damage, treatment of precursor lesions within a cancerized field can decrease the risk of subsequent cancer development. Sunblock is an effective intervention for field cancerization, even in patients with established disease. There is now direct evidence that field therapy with topical 5-fluorouracil is effective in reducing the incidence of subsequent SCC, and there is indirect evidence suggesting that topical imiquimod, topical ingenol mebutate, and photodynamic therapy are similarly effective. There is limited direct evidence to show that systemic acitretin or nicotinamide can decrease incident SCC in patients with field cancerization. In this review, an approach to the management of patients with multiple SCCs and field cancerization is presented along with the rationale to support field-directed therapy. Over half of patients with diagnosed SCC will develop multiple tumors within their lifetime. Although the majority of SCCs of the skin are localized tumors with an exceptionally good prognosis, approximately 5% of SCCs recur locally after definitive treatment, 2–3% exhibit nodal metastasis, and 1–2% are fatal3. In addition to causing between 4,000 and 9,000 deaths annually, SCC and its associated skin lesions have an enormous financial burden at over $4 billion per year in the United States4.Cutaneous squamous cell carcinoma (SCC) is the second most common malignancy in humans; up to 1 million SCC tumors are treated annually in the United Statesin situ is an intra-epidermal malignancy (not breaching the basement membrane) that often presents as a pink scaly plaque  or destruction with electrodesiccation and curettage results in cure rates as high as 90–95% for typical SCCin situ lesions with either imiquimod or 5-fluorouracil (5FU) has been reported with cure rates of 73–86% and 48–69%, respectively17, but neither of these treatments is consistently effective for invasive SCC. Similarly, photodynamic therapy (PDT) with either aminolevulinic acid or methyl aminolevulinate may achieve clinical cure rates of 76–82% for SCCin situ18. Lesion-directed cryotherapy with liquid nitrogen  is commonly used for low-risk SCCin situ and precursor lesions of actinic keratosis (AK). Although cryotherapy is highly operator-dependent, one trial reported a complete response rate for SCCin situ of 67% at 1 year, which was comparable to 5FU and inferior to PDT17. Intra-lesional chemotherapy  with agents such as 5FU, methotrexate, bleomycin, or type I interferon has successfully treated SCC in several published case series, often specifically addressing the keratoacanthoma type of SCC that is characterized by rapid growth and occasional spontaneous involution19. Because intra-lesional chemotherapy has not been studied in controlled clinical trials, its use is often reserved for patients who are poor surgical candidates or who have such a high burden of individual lesions that surgery is not practical. Although medical treatment of SCC has decreased efficacy for individual lesions relative to surgery, several of these modalities have the benefit of potentially treating a broad field around the index lesion, which may inhibit the development of subsequent SCC. Patients with multiple SCCs often benefit from a combination of medical and surgical field-directed and lesion-directed treatment approaches as discussed below.For SCC not amenable to surgery, non-surgical treatments may be used, albeit with lower cure rates. Topical medical therapy of individual SCC20. Treatment of larger SCCs, poorly differentiated tumors, or tumors on the scalp, ears, or lips with superficial radiation may result in increased recurrence rates of up to 30%21. More intense radiation therapy with electron beam or megavoltage photons has been reported as monotherapy for advanced SCC, but high-quality outcome data are lacking22. In addition, because of the risk of secondary malignancy decades after radiation therapy, this treatment is relatively contraindicated in younger patients with a long life expectancy. Finally, advanced SCC with lymph node or visceral metastasis is best managed with multi-modal therapy, including surgery, radiation, and potentially systemic chemotherapy. More recently, immunotherapy with monoclonal antibodies to the immune checkpoint receptor programmed death 1 (PD-1) on T cells has been reported as a successful treatment to induce T cell-mediated rejection of advanced SCC that is unresponsive to standard therapy24. Whether this therapy can be adapted for patients with multiple primary SCCs remains to be determined.Radiation therapy has long been employed in the treatment of SCC and may be preferable to surgical treatment in specific circumstances. Low-risk SCC treated with superficial X-ray therapy has a reported 5-year recurrence rate of 5.8% in one series25. In that seminal report, the authors observed that all cases of oropharyngeal SCC had pathologic dysplasia and foci of SCCin situ in the adjacent mucosa that was grossly normal . Importantly, many of these changes were not contiguous with the primary tumor and represented separate islands of dysplastic epithelium. In addition, 11% of oropharyngeal SCCs presented with two separate primary lesions, a rate 10-fold greater than expected on the basis of disease prevalence. Together, these findings support the independent or co-incident development of multiple lesions rather than the linear progression of a single mutant clone. The paradigm of field cancerization was thus proposed on the basis of the hypothesis that an area, or field, of epithelium is altered by a regional carcinogenic activity. This regional injury, which may be acute or chronic, causes irreversible genetic changes in multiple clonal populations of cells, one or several of which eventually manifest in cancer.In 1953, Slaughter and colleagues described the concept of field cancerization on the basis of their study of pathologic changes in the epithelium surrounding oropharyngeal SCC26. In addition, both histopathologic biopsies and non-invasive imaging with optical coherence tomography of cutaneous regions of suspected field cancerization confirmed that 79% of grossly normal skin samples in these fields had evidence of dysplasia or occult carcinoma27. The field cancer paradigm has two important implications for the management of cutaneous SCC. First, because SCC arises from multi-focal areas of precancerous change, the presence of at least one SCC confers an increased risk of subsequent SCC. It has been shown that after a single SCC, there is a 42% risk of subsequent SCC within 5 years and this risk increases to 72% in patients with two or more SCCs28. Second, clinical recurrence of SCC after complete surgical excision may in fact represent the development of a new primary cancer, and recurrence risk may correlate with the degree of field cancerization. Indeed, it was found that patients with two to nine SCCs exhibit a twofold increased risk of recurrence compared with patients with a single SCC, and patients with a lifetime history of 10 or more SCCs exhibit a 12-fold increased risk of local recurrence as well as an 11-fold increased risk of nodal metastasis29. In these patients with a heavy burden of individual cancers, understanding the mechanism of field cancerization and SCC evolution from precursor lesions has spurred development of therapeutic strategies to prevent or suppress the development of subsequent SCC.Although this concept of field cancerization has long been observed in the skin, Slaughter’s original pathologic findings were recently recapitulated for cutaneous SCC. In excision specimens of cutaneous SCC, 57% had separate (discontiguous) foci of dysplasia that were not detected clinically but were pathologically diagnostic of AK30. From a mechanistic standpoint, UV radiation directly  and indirectly  damages epidermal DNA, leading to somatic mutations in genes that confer a growth advantage and facilitate malignant transformation31. Somatic mutations, both inactivating mutations in tumor suppressor genes and activating mutations in oncogenes, accumulate over time in a sequential fashion. According to the model of multi-step carcinogenesis, increasing somatic mutations induce clonal proliferations that progress from microscopic precursors to grossly visible lesions during the development of carcinoma32. A second mechanistic effect of UV radiation is local immunosuppression that facilitates epidermal carcinogenesis. Studies in human volunteers have defined both acute and chronic effects of UVB in inhibiting adaptive immune responses to contact allergens applied to the skin and shown that this UV-mediated immunosuppression correlates with SCC risk33. Given the established association of therapeutic immunosuppression  with dramatically increased SCC risk, it is clear that effective anti-tumor immunity plays an important role in limiting the progression of precursor lesions in the skin.The primary driver of epidermal carcinogenesis is solar ultraviolet (UV) radiation. Epidemiologic studies have demonstrated a 10-fold increase in SCC incidence with increasing ambient solar radiation35. AK is a clear clinical indicator of field cancer, and some patients may present with dozens of lesions in a single anatomic field, conferring a high risk of subsequent SCC  17 broad-spectrum sunblock (UVA and UVB protection) or placebo cream. Patients who received sunblock had a significant reduction in new AK, and this reduction was proportional to the total amount of sunblock used44. Of note, the use of a placebo sunblock would likely be considered unethical in 2018, but the benefits of sunblock were not yet established at the time of the trial. A second trial randomly assigned 1,621 unselected participants to one of four treatments: daily use of SPF 15 broad-spectrum sunblock, discretionary use of sunblock, daily sunblock plus oral beta-carotene, and oral beta-carotene alone with discretionary use of sunblock. Over a follow-up period of 4 years, daily sunblock use caused a significant 39% reduction in new SCC; the use of beta-carotene had no effect on skin cancer development45. Follow-up of these participants for an additional 8 years revealed a persistent 41% reduction in new SCC in the former daily sunblock group, even when returning to discretionary sunblock use after completion of the original trial46. Finally, there is an important role for sun protection in immunosuppressed organ transplant recipients as well. In a 24-month trial of organ transplant recipients at high risk of SCC, patients provided with SPF 50 broad-spectrum sunblock developed fewer SCCs (eight in the control group versus zero in the sunblock group) and exhibited a significant regression of existing AK47. Cumulatively, this suggests that rigorous sun protection will have significant and long-lasting benefits in both prevention and management of field cancerization.The first step in the management of the patient with multiple SCCs and field cancerization is rigorous sun protection. Because UV radiation is both a tumor initiator and a tumor promoter for skin cancer and because animal models demonstrate reduced precursor lesions when UV exposure is interrupted48. The hypothesis that treatment of precursor lesions within a cancerized field can reduce subsequent SCC development is supported by several lines of indirect evidence and, more recently, a clinical trial that documented clear efficacy of 5FU in SCC prevention. First, there is a proven benefit of field therapy over lesion-directed therapy for sustained clearance of AK within a defined field. One randomized trial found that although 5FU, imiquimod, or lesion-directed destructive therapy all had good immediate efficacy in AK clearance (68–96%), only field therapy with 5FU or imiquimod produced any significant clearance at 1 year 49. Second, long-term follow-up of patients randomly assigned to 5FU field therapy versus placebo demonstrated that the benefits of sustained lesion clearance are significant for over 3 years following a single treatment course, suggesting that topical field therapy produces a long-lasting remission in SCC precursors50. Third, a recent randomized, double-blind clinical trial in 932 patients with a history of skin cancer (39% of whom had prior SCC) identified a 75% reduction in incident SCC in the first year following a single course of topical 5FU treatment (five patients with SCC in the 5FU group versus 20 patients in the placebo group)51. This study is the first high-quality, prospective trial to demonstrate a significant reduction in SCC with field therapy and provides strong support for the concept of field-directed therapy in high-risk patients. Finally, preclinical and early clinical data suggest that topical 5FU can be combined with topical immunotherapy to enhance field-directed prevention of SCC. Topical calcipotriol, a vitamin D derivative shown to enhance cutaneous T-cell activation via the production of thymic stromal lymphopoietin (TSLP), can reduce the development of SCC in animal models and was shown to enhance the efficacy of AK clearance with 5FU in a preliminary human trial52.Apart from sun protection, the most widely used field-directed treatments are topical medications such as the pyrimidine analog chemotherapeutic 5FU. For many years, the observation that SCC precursors can be effectively treated with topical therapy provided the rationale for field treatment to decrease the incidence of SCC in high-risk patients. There is strong evidence to support the use of 5FU and other topical agents, such as the immune response modifier imiquimod and the non-steroidal anti-inflammatory drug diclofenac in the treatment of AK. Clinical evidence for these agents in AK treatment is extensive and beyond the scope of this reviewEuphorbia peplus plant, which itself is a known irritant and established botanical therapeutic53. Ingenol mebutate has direct cytotoxic effects on keratinocytes and induces a local inflammatory milieu via effects on the microvasculature and endothelial cells. Unlike other topical therapies, a short, 2- to 3-day treatment with ingenol mebutate has demonstrated efficacy in field treatment of AK, which may improve patient compliance55. Via elimination of SCC precursors such asTP53-mutated clones in the epidermis, ingenol mebutate can prevent or reduce the development of UV-induced lesions in an animal model of SCC56. Similarly, ingenol mebutate therapy in humans has been shown to decrease not only visible AK but also subclinical precursor lesions detected by optical coherence tomography and histopathologic analysis27. Although it appears likely that various topical agents such as ingenol mebutate and imiquimod can decrease the incidence of subsequent SCC in patients with field cancer, direct evidence of this effect is currently available only for 5FU51.Another topical field therapy has recently emerged in the form of ingenol mebutate, a cyclic diterpene ester derived from the sap of the57. Analogous to the topical therapies presented above, PDT is an effective field treatment for AK and is superior to lesion-directed destructive therapy58. Like other field treatments, PDT can significantly reduce the size and number ofTP53-mutated clonal precursors of SCC in human skin59 and has been demonstrated to decrease UV-induced tumor development in animal models60. Because PDT (and presumably other current field therapy options) does not completely eliminate mutated precursors from the skin, repeated treatments appear to be required to continually suppress the development of SCC. One observational study of 12 organ transplant recipients with high-risk field cancer treated with cyclic PDT every 4–8 weeks reported 79% and 95% reductions in incident SCC after 1 and 2 years, respectively, compared with the year prior to cyclic PDT61. In contrast, a prospective trial of 40 patients who received a single course of PDT found no significant decrease in SCC incidence after 2 years62. The optimal interval for cyclic therapy may be better defined by an ongoing trial investigating cyclic PDT every 6 months in organ transplant recipients at elevated risk for skin cancer; interim results of the trial have not yet detected any incident SCC63. If repeated treatments are required, one promising new alternative could be daylight PDT, in which photo-activation of protoporphyrin IX is achieved by natural daylight as opposed to more intense (and often more painful) artificial light sources. Although daylight PDT appears promising for the treatment of AK64, its effect on SCC prevention has not been investigated.An established option for field-directed treatment is office-based PDT. PDT is based on photo-activation of protoporphyrin IX in keratinocytes to generate reactive oxygen species and cytotoxicity. A prodrug (aminolevulinic acid or methyl aminolevulinate) is topically applied to the skin 1–18 hours prior to visible-spectrum light treatment, and because metabolic conversion of the prodrug to protoporphyrin IX is enhanced in malignant and pre-malignant cells, this treatment is relatively selective for SCC precursors in the skin65. Compelling evidence to support acitretin as field therapy comes from a prospective trial of 44 renal transplant recipients randomly assigned to either acitretin 30 mg daily or placebo for 6 months. Incident SCC was reduced by 88% in patients during acitretin treatment, although the rate of SCC development increased to the same as the control group in the 6 months following discontinuation of therapy66. Similar to PDT or topical therapy, field treatment with acitretin suppresses the development of SCC but does not completely eliminate clonal precursors of SCC and thus therapy must be continued for a prolonged period of time. Systemic side effects from acitretin can be significant but are predictable and often dose-dependent, including xerosis, mucositis, myalgias and arthralgias, hyperlipidemia, and potential hepatotoxicity. Acitretin, like all systemic retinoids, is an established teratogen and should be avoided in women of child-bearing potential because of its long half-life. Isotretinoin has a significantly shorter half-life and may be an alternative for younger women, although it is rare for this population to require systemic field therapy67. It should also be noted that the beneficial effects of systemic therapy are less pronounced in patients at lower risk for SCC and this therapy should be reserved for patients with few other options68.For patients with severe field damage at highest risk for subsequent SCC, systemic treatment may dramatically inhibit new tumor development. Acitretin is a systemic retinoid known to suppress proliferation and promote differentiation of keratinocytes and has been shown to induce regression of SCC-like tumors in animal models3) has recently been shown to have modest preventive effects on UV-induced skin lesions, presumably by promoting effective DNA repair and reducing the immunosuppressive effects of UV exposure69. In a prospective trial of 386 patients with prior skin cancer, those randomly assigned to oral nicotinamide 500 mg twice daily had a 23% reduction in new skin cancers (30% reduction in SCC) during 12 months of therapy. When patients were monitored for 6 months after cessation of nicotinamide therapy, there was a non-significant trend toward increased SCC incidence during this period70. Although nicotinamide produced only a modest reduction in SCC incidence, the lack of significant side effects associated with this agent makes it a potentially useful option, either alone or in combination with other field therapies. Second, capecitabine, an oral prodrug of 5FU that is US Food and Drug Administration-approved for the treatment of colorectal, gastric, and breast cancer, may be an effective prophylactic measure for patients with recalcitrant field cancerization. In one uncontrolled observational study, cyclic treatment with capecitabine produced a 68% reduction in incident SCC71. Because of the significant side effect profile of this systemic chemotherapy, treatment of cutaneous field cancerization with capecitabine is usually performed in conjunction with a medical oncologist.At least two other approaches for systemic field therapy may also be considered. First, nicotinamide (also known as niacinamide, the amide form of vitamin B72, those patients are not considered here. The type, frequency, and intensity of field treatment for an individual patient depend on the severity of field cancerization and the risk of subsequent SCC (73. Those not responding to PDT or topical therapy may benefit from systemic acitretin. Patients with more than three or four SCCs per year or more than 10 lifetime SCCs, as noted previously, are at high risk of both subsequent SCC and metastatic or fatal SCC and require aggressive field therapy. Initial therapy in these patients is often systemic acitretin, which may be combined with repeated topical therapy or PDT. Combination treatments may have synergistic efficacy, as reported in one recent series of combined lesion-directed destruction, topical 5FU, and PDT74. Combination systemic therapy with nicotinamide and acitretin is generally accepted as safe, but concurrent use of acitretin and capecitabine has not been studied and should be used with caution. Finally, organ transplant recipients require additional focus on management of field cancer, as the chronic use of immunosuppressant medications places these patients at exceptionally high risk of multiple and biologically aggressive SCCs75. Organ transplant recipients often require more frequent monitoring, earlier initiation of multi-modal field therapy, and potentially modification of their immunosuppression regimen in collaboration with their transplant team.In order to appropriately manage patients with cutaneous field cancer, it is necessary to define this term. A practical working definition of field cancerization requires three features: a defined region of skin, multiple AKs within that region, and at least one prior SCC. Although patients with severe actinic damage and multiple AKs without prior SCC may certainly require treatment for their AKsent SCC . All patSignificant progress has been made in understanding the pathogenesis and optimal management of field cancerization, yet several questions remain. It is clear that patients with a large burden of cutaneous SCC have a chronic disease with an increased risk of systemic involvement and early death; treating each lesion as an isolated neoplasm does not adequately address this disease. A systemic and systematic approach to field therapy is required for these patients. It is also clear that SCC evolves from microscopic precursors harboring carcinogenic mutations in grossly normal skin and that a significant fraction of epidermal cells may harbor these mutations in an actinically damaged field. It remains unclear, however, whether the number, size, total mutation burden, or specific mutation profile of these precursors is the critical determinant of progression to SCC. With additional data from genetic sequencing of normal but sun-exposed skin, it may be possible to more accurately determine the risk of SCC development, both for a single precursor lesion and for a cancerized field as a whole. Similarly, nearly all patients with more than 10 SCCs presented initially with a single lesion, yet current methods cannot prospectively determine subsequent field cancer risk. Additional study is required to identify those patients who would benefit most from field treatment at an early point in their field disease progression. Although there is now high-quality evidence to show that field treatment with topical 5FU can reduce the incidence of SCC for at least 1 year, additional clinical trials are needed to support the use of other therapies and the optimal interval of repeated field therapy for high-risk patients. Finally, anti-tumor immunity has been shown to be a critical factor in host defense against cancer, but the role of the immune response in the development and progression of precursor lesions has only been suggested. Future field treatments may more effectively harness the immune system to achieve a durable suppression of SCC development in a cancerized field.


Adipocytes are a significant component of the bone marrow microenvironment. Although bone marrow adipocytes were first identified more than 100 years ago, it is only in recent years that an understanding of their complex physiological role is emerging. Bone marrow adipocytes act as local regulators of skeletal biology and homeostasis, with recent studies suggesting that marrow adipose tissue is metabolically active, and can function as an endocrine organ. As such, bone marrow adipocytes have the potential to interact with tumour cells, influencing both tumour growth and bone disease. This review discusses the current evidence for the role of bone marrow adipocytes in tumour growth within the bone marrow microenvironment and the development of the associated bone disease. •Bone marrow adipocytes are a metabolically active source of lipids and adipokines.•Marrow adipocytes increase with age, but their role in bone metastasis is ill-defined.•Marrow adipocytes have tumour-promoting and -suppressive effects in bone metastasis. They constitute approximately 15% of the bone marrow volume in young adults, rising to 60% by the age of 65 years old 2The bone provides a unique and supportive microenvironment for a number of solid tumour metastases including breast, prostate and the haematological malignancy multiple myeloma Both breast cancer and MM cause osteolytic lesions, inhibiting osteoblast differentiation and thereby tipping the balance in favour of osteoclastic activity. In contrast, prostate cancer predominantly causes osteoblastic bone disease. Interestingly, increased marrow adiposity has been associated with both osteolytic and osteoblastic disease 3in vitro work suggests these changes are also important in the bone marrow Adipocytes located in close proximity to invasive cancer cells in the primary tumour exhibit profound phenotypic changes that include both morphological and functional alternations and are often referred to as cancer-associated adipocytes (CAAs). The morphological changes associated with these cells include loss of lipid content (delipidation) and acquisition of a fibroblast-like/preadipocyte phenotype (de-differentiation). Functionally they exhibit a decrease in expression of adipocyte-related genes such as adiponectin, FABP4 and resistin coupled with an increase in the production of pro-inflammatory cytokines IL-6, IL-1β 4Given the potential tumour-supporting role of adipocytes, targeting these cells either alone or in combination with common therapeutics may be a promising approach. Modulating levels of adipokines such as adiponectin has been shown to exert an anti-tumour effect. Pharmacological enhancement of circulating adiponectin by the apolipoprotein mimetic L-4F was shown to cause cancer cell death in mouse models of myeloma 5Over the last few decades the contribution of adipocytes to disease establishment and progression has become clearer. With aging and obesity resulting in increased numbers of bone marrow adipocytes, it is important to further understand the influence these cells are having on their environment. Targeting adipocytes and their products may open new therapeutic avenues in the fight against lethal metastatic cancers.


The aim of this review is to discuss recent advances in clinical aspects of stem cell therapy in chronic nonischemic heart failure (DCMP) with emphasis on patient selection, stem cell types, and delivery methods. + cells have been demonstrated to improve myocardial performance, functional capacity, and neurohumoral activation. Furthermore, allogeneic mesenchymal stem cells were also shown to be effective in improving heart function in this patient population; this may represent an important step towards the development of a standardized stem cell product for widespread clinical use in patients with DCMP.  Several stem cell types have been considered for the treatment of DCMP patients. Bone marrow-derived cells and CD34 The trials of stem cell therapy in DCMP patients have shown some promising results, thus making DCMP apparently more inviting target for stem cell therapy than chronic ischemic heart failure, where studies to date failed to demonstrate a consistent effect of stem cells on myocardial performance. Future stem cell strategies should aim for more personalized therapeutic approach by establishing the optimal stem cell type or their combination, dose, and delivery method for an individual patient adjusted for patient's age and stage of the disease. Despite significant advances in medical and device therapy over the past decades, chronic heart failure (HF) represents an increasingly common and debilitating disorder worldwide. Even in the 21st century, HF carries a dismal prognosis and is related to unacceptably high mortality rates , there hThus, the aim of this review is to discuss current status and recent advances of stem cell therapy in the treatment of DCMP, with emphasis on patient selection, stem cell types, and clinical efficiency of this treatment modality.DCMP is characterized by left or biventricular dilation with concomitant systolic dysfunction in the absence of extrinsic factors that may cause a similar phenotype such as coronary artery disease, arterial hypertension, valvular heart disease, or congenital heart disease. This disorder can develop at any age and regardless of gender or race . The eti+ cells and endothelial progenitor cells (EPC) in the earlier phases (NYHA I and II) of the disease and to the exhaustion of the pools of these progenitors in the later phases (NYHA III and IV) of the disease [Additionally, defective vascularization with impaired vasculogenesis and angiogenesis has also been documented in patients with DCMP  suggesti disease –10.Generally, stem cells are defined as a population of self-renewing cells with the potential to generate daughter cells with the capability to differentiate along specific cell lineages . In the Bone marrow is the source of mixed population of hematopoietic and nonhematopoietic stem cells. Both have been shown to possess the ability to transdifferentiate into different cell lineages if transferred to a tissue-specific cytokine milieu. Due to the easy access and straightforward procurement, this stem cell type has understandably gained the widest attention in preclinical and early clinical trials. Mostly, BMMC were studied in the setting of ischemic HF. However, several studies did analyse the safety and efficiency of this stem cell population also in patients with DCMP.In the TOPCARE-DCM study , intracoronary infusion of BMMC into the left anterior descending coronary artery was performed in 33 patients by an over-the-wire balloon catheter. This resulted in improved regional wall motion of the injected area and global left ventricular myocardial performance with an average increase in left ventricular ejection fraction (LVEF) by 3%. Furthermore at 12-month follow-up, NT-proBNP serum levels were persistently decreased suggesting a potential beneficial effect of BMMC infusion on LV remodeling process . RecentlTaken together, these data suggest that BMMC therapy may be of potential clinical benefit in patients with DCMP. However, the variations in BMMC populations, patient selection criteria, and delivery methods used in these studies make direct comparisons between them challenging and any extrapolation to a wider clinical utility very difficult.+ surface marker and have been shown to have a potential to differentiate into endothelial cells and thus promote target tissue neovascularization [HSC represent a part of the hematopoietic cell compartment of the bone marrow that differentiates along lymphoid and myeloid lineages to form mature white blood cells. HSC are positive for the CD34rization  directly+ stem cells obtained through G-CSF stimulation followed by peripheral blood apheresis and immunomagnetic selection. In comparison to the control group, a 5% increase in LVEF was observed in the stem cell group at 1-year follow-up. Additionally, improved functional capacity of the patients and reduced neurohumoral activation were observed [+ cell therapy [+ cell therapy in dilated cardiomyopathy; NCT 02248532) is evaluating the potential benefits of repetitive transendocardial CD34+ stem cell injections in DCMP patients, and the results are expected to be available by the end of 2017.In the first clinical trial assessing the effects of stem cell therapy, 28 DCMP patients received intracoronary application of CD34observed . Importa therapy . Recentl therapy . WhetherMSC represent a part of the nonhematopoietic cell compartment of the bone marrow and have been reported to differentiate into cardiomyocytes  and endo6/kg) in 22 DCMP patients. Although at 90 days no improvement of LVEF was observed, the data suggested better exercise capacity and quality of life after stem cell treatment [Two clinical trials explored MSC in DCMP. Butler et al. evaluated in a placebo-controlled crossover trial the safety and efficacy of intravenously applied allogeneic MSC  such as arterial hypertension, diabetes, and hyperlipidemia [The other, clinically more significant, reason is that sicker heart failure patients generate less stem cells that are also less potent –10. Althipidemia . Additioipidemia . It was ipidemia .It was also established that in comparison to patients with ischemic heart disease, patients with DCMP express significantly less myocardial homing factors, further reducing the efficiency of endogenous repair mechanisms of the myocardium in this patient population .To overcome these limitations of autologous stem cell therapy, allogeneic stem cells were recently explored , 29, 30.The ability of the damaged myocardium to attract circulating stem cells is fundamental to myocardial repair. It is currently suggested that acutely injured myocardium generates cytokine-mediated signals for the mobilization of stem cell pool from bone marrow to peripheral circulation. Afterwards, these circulating bone marrow-derived cells follow a SDF-1 cytokine gradient that enables them to home to the damaged regions of the myocardium . HoweverTo date, no consensus has been reached with regard to the optimal mode of stem cell delivery to the failing myocardium. Intramyocardial (IM) approach and intracoronary (IC) approach have mainly been used in the clinical settings . By far,Studies of stem cell therapy in patients with DCMP have demonstrated signs of improvement in LVEF  and exerP = 0.007).Our unpublished RECORD registry  data on 148 chronic HF patients suggest that stem cell therapy may lead to reduced hospital readmissions due to worsening heart failure. Within one year after stem cell therapy, we have observed a significant decrease in heart failure-related hospital readmissions when compared to the 1-year interval before cell therapy  and 35% in controls (55 patients) (P = 0.39) [P = 0.03).The longest follow-up of DCMP patients receiving stem cells published to date showed that after 5 years, patients who received CD34atients) . Additio = 0.39) , was fouAlthough these results are based on small single-center data and need to be confirmed in larger trials, they still offer an encouraging signal that stem cell therapy on top of optimal medical management further improves long-term prognosis of DCMP patients.In summary, the data of currently available clinical trials of stem cell therapy in DCMP have shown promising results regarding the improvement of LVEF, patients' functional capacity, and quality of life. This is in stark contrast to the clinical trials of stem cell therapy in ischemic heart disease that failed to consistently demonstrate the beneficial effect of this treatment modality, thus making DCMP an apparently more inviting target for stem cell therapy. Given the heterogeneity of clinical characteristics of patients with DCMP, it may be difficult to define a single “fit-all” stem cell therapeutic approach. Future stem cell strategies should aim for a more personalized therapeutic approach by establishing the optimal stem cell type or their combination, dose, and delivery method for an individual patient adjusted for underlying causes of heart failure and stage of the disease.