Tumor cells harbor genetic alterations that promote a continuous and elevated production of reactive oxygen species. Whereas such oxidative stress conditions would be harmful to normal cells, they facilitate tumor growth in multiple ways by causing DNA damage and genomic instability, and ultimately, by reprogramming cancer cell metabolism. This review outlines the metabolic-dependent mechanisms that tumors engage in when faced with oxidative stress conditions that are critical for cancer progression by producing redox cofactors. In particular, we describe how the mitochondria has a key role in regulating the interplay between redox homeostasis and metabolism within tumor cells. Last, we will discuss the potential therapeutic use of agents that directly or indirectly block metabolism. Deregulated redox homeostasis is a hallmark of cancer cellsIncreased ROS levels are able to promote tumor growth and malignant progressionIncrease antioxidant ability in malignant cells is a common featureAlteration of specific metabolic pathways in tumors is frequently foundTumors can be sensitized to chemotherapy and other antitumor treatment by disabling antioxidant defenses (NADPH and GSH) through metabolic inhibitionWhat are the redox-sensitive transducers that specifically promote signaling events in cancer cells?As metabolism can support the intracellular redox homeostasis by NADPH and GSH synthesis, what are the cancer-specific pathways/alterations that can be selectively targeted for therapeutic purposes?To what extent can the inhibition of antioxidant mechanisms be used to potentially enhance chemo/radiotherapy without inducing side toxicity on normal cells?Would it be possible to generate animal models that allow real-time detection of metabolic/redox intermediates with high spatial and temporal resolution during cancer progression?1 Although seemingly paradoxical, this pro-oxidant shift can promote tumor growth by inducing DNA damage and genomic instability,2 which then activate an inflammatory response,3 stabilizing the hypoxia inducible factor-14 and thus reprogramming metabolism.6 Due to the selective pressure induced by sustained ROS production, cancer cells have developed an efficient mechanism of ROS detoxification that presents a selective advantage over and upholds its survival under pro-oxidizing conditions. Therefore, the dependency of cancer cells from their antioxidant systems represents a specific vulnerability that must be exploited to induce targeted cell death. This can be achieved by increasing oxidative stress above the toxicity threshold, sparing normal cells, which are characterized by having lower intracellular ROS levels  levels as a consequence of genetic, metabolic and microenvironment-associated alterations. These are then compensated by an increased antioxidant ability from these cancer cells.S levels .7 Due toThis review focuses on the adaptive mechanisms that tumors use to face oxidative stress conditions. We will discuss the role of ROS in regulating metabolism and progression in cancer cells. Last, we cover potential therapeutic usage of agents that directly or indirectly alter the tumor redox balance.2−·) or the hydroxyl radical (OH•) as well as non-radical molecules including hypochlorous acid and hydrogen peroxide (H2O2).14 Both exogenous and endogenous sources of ROS production have been extensively described over the past decade.15 The most biologically relevant are represented by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, professional enzymes that catalyze the production of O2−· or H2O2 using NADPH as a reductant16 and the mitochondrial electron transport chain (mETC), wherein mainly complexes I and II generate O2− through univalent reduction of molecular oxygen as a consequence of electron leakage during mitochondrial respiration using nicotinamide adenine dinucleotide (reduced form) (NADH) and FADH.18Redox homeostasis is an essential requisite for aerobic organisms. They are dependent on the balance between the rate and the magnitude of oxidant production and their elimination over time. ROS are short-lived molecules with unpaired electrons deriving from partially reduced molecular oxygen that are perpetually generated, transformed and eliminated in a variety of cellular processes including metabolism, proliferation, differentiation, immune system regulation and vascular remodeling. These oxygen-containing derivatives are comprised of free radicals such as the superoxide anion (O2.−) to less reactive H2O2, (ii) catalase that reduces H2O2 to water and molecular oxygen and (iii) glutathione peroxidases that eliminate H2O2 using reducing power derived from glutathione. Other important defensive mechanisms and mediators of redox signaling are represented by the peroxiredoxin, the thioredoxin (TRX) and the glutathione/glutaredoxin systems.30 Due to intrinsic differences in the half-life, stability, chemical reactivity, cellular context, site and source of their generation (exogenous or endogenous), ROS can interact and modify different classes of biological macromolecules including DNA, lipids and proteins.37 The tight regulation of ROS production and detoxification over time and space represents the basis for the maintenance of an appropriate redox homeostasis and redox signaling events. Disruption of redox circuitries that control the turnover of ROS and the related redox signaling events has a profound impact on cellular physiology and in turn may lead to aberrant signaling, unrestrained accumulation of toxic byproducts, oxidative damage and cytotoxicity.38 Although low levels of ROS are believed to regulate redox signaling events, high doses are regarded as being responsible for cell toxicity.49 It is also well accepted that the efficacy of many anticancer therapies, including chemotherapeutics and radiotherapy, largely depends on their ability to induce ROS accumulation and evoke cell toxicity and death.56 The high levels of oxidative stress normally associated with malignant progression represent tumor-specific alteration that makes cancer cells vulnerable to further elevation of ROS and strongly dependent on their antioxidant defenses. Both extrinsic and intrinsic factors contribute to generate a persistent amount of high ROS levels in tumors. To prevent excessive oxidative stress and promote redox signaling, tumor cells strategically adjust multiple antioxidant enzymes and make extensive use of their metabolic pathways to provide an adequate supply of antioxidant molecules (such as reduced glutathione (GSH) and NADPH).1 On the basis of these observations, disabling the intrinsic antioxidant mechanisms by promoting ROS production has been conducted in several studies.63 Still, these studies highlight the growing interest in the scientific community towards therapeutic strategies that are aimed at disrupting the redox homeostasis of malignant cells. To identify new strategies and define redox regulation and ROS levels in the context of tumor progression, several laboratories have found success with new approaches on the basis of the metabolic blockade as anticancer treatment  superoxide dismutases (SODs) that convert superoxide , one-carbon metabolism and the pentose phosphate pathway (PPP).70 For this reason, it is important to analyze more in detail the major metabolic pathways that mainly control the redox homeostasis of cancer cells  fostering their uncontrolled growth. However, it is well recognized that part of the energetic substrates involved in these pathways can be also redirected into specific metabolic routes to generate not only antioxidant molecules (NADPH and GSH) but also redox cofactors  that can be readily used to maintain or restore an adequate redox homeostasis.er cells .71 As already observed by Otto Warburg in the 1924, tumor cells exhibit a prevalent use of the glycolytic pathway regardless the presence of sufficient oxygen tension, a phenomenon known as Warburg effect.64 Several studies indicate that the pro-glycolytic shift caused by oncogene activation and loss of tumor suppressors represents a selective advantage for tumors by providing essential precursors for building the macromolecules required to sustain growth and proliferation.73 As a matter of fact, therapeutic modulation of glucose metabolism and transport has been widely utilized as an effective anticancer strategy.80 It is now understood that glucose metabolism has an essential role in the control of redox homeostasis in tumors, as glycolytic intermediates can be shuttled into the metabolic pathways that directly or indirectly contribute to generate reducing equivalents, mainly PPP-derived NADPH or glutaminolysis-derived GSH. In this regard, a recent study showed that cancer cells exposed to glucose deprivation increase glucose metabolism to restrict the burden of ROS and prevent hydroperoxide-induced cell death.81 Also, inhibition of lactate dehydrogenase-A through the small-molecule FX11 impaired the malignant progression of lymphoma and pancreatic cancer xenografts by decreasing the intracellular ATP levels and inducing oxidative stress.82 Late, the inhibition of glycolysis and the PPP combined with the disruption of TRX system has proven to represent a successful strategy in selectively increase cytotoxicity in pancreatic and breast cancer cells but not in normal counterparts.83 These results suggest that a combined approach might be a better strategy in targeting malignant tumors when limited efficacy is observed with single agents.Glycolysis is an essential pathway occurring in the cytosol of mammalian cells through which glucose is transformed to pyruvate. Glucose is taken from the extracellular space by specific transporters . Glucose is then converted to glucose-6-phosphate by hexokinase enzymes and enters into a series of ten enzyme-catalyzed reactions culminating in the generation of pyruvate, adenosine tris-phosphate (ATP) and reduced cofactors in the form of NADH.84 The importance of FAO for NADPH homeostasis and redox balance of cancer cells prevent cell death during loss of matrix adhesion9 and metabolic stress conditions through the modulation of the liver kinase B1 (LKB1)/AMP kinase axis.85 Overexpression of the key FAO regulators, such as the carnitine palmitoyltransferase-1,86 occurs in both solid tumors and leukemia cells,87 whereas its pharmacological inhibition by etomoxir was found to impair NADPH production and promote oxidative stress-induced cell death in human glioblastoma cells associated with profound ATP depletion88 and to strengthen the pro-apoptotic effect of cytotoxic agents in human leukemia cells.89 Given its importance in many types of tumor, targeting the FAO represent a promising novel strategy to disrupt the redox homeostasis of malignant cells and interfere with biosynthetic or bioenergetics processes that regulate cancer cell survival triggering either apoptosis-dependent or -independent cell death.The FAO is composed of a cyclical series of controlled oxidations that occur in the mitochondria of mammalian cells, through which long- and short-chain fatty acids are shortened, generating NADH, FADH2 (flavin adenine dinucleotide (reduced form) and acetyl-CoA to support biosynthetic pathways and produce ATP. However, in cancer cells, a consistent fraction of the acetyl-CoA enters into the tricarboxylic acid cycle (TCA) cycle and generates citrate, which is therefore exported into the cytosol and funneled into metabolic reactions catalyzed by the malic enzyme (ME) and the isocitrate dehydrogenase 1 (IDH1), that ultimately produce large amounts of NADPH.90 Activation of the PPP represents a key hallmark of many tumors where this metabolic pathway is found at the crossroad between glycolytic activity, unrestricted proliferation and scavenging of excessive ROS.91 The transcriptional regulation of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, by TAp73 and TAp63α was recently described in U2OS osteosarcoma cells, wherein its overexpression enhanced the PPP-dependent production of NADPH.93 Additional mechanisms of G6PD regulation might directly depend on the availability of glucose: glucose funneling into the oxidative branch of the PPP directly controls the redox homeostasis of human clear cell carcinoma cells.94 This latest evidence underlines the importance of the PPP in the regulation of tumor cell survival and therapeutic resistance. In this respect, overexpression of G6PD promote doxorubicin resistance through increased GSH content and multidrug resistance-mediated efflux in HT29 colon carcinoma cells.95 In another study, the inactivation of the oncoprotein mucin1 C-terminal subunit restored the sensitivity of multiple myeloma cells to bortezomib, preventing the TIGAR-dependent glucose entry into the PPP and inducing massive ROS accumulation due to GSH depletion.96 Simultaneous inhibition of glycolysis and PPP through 2-deoxy-d-glucose and 6-aminonicotinamide, respectively, induced oxidative stress and sensitized malignant human cancer cell lines to radiotherapy presumably through the induction of multiple cell death modalities including apoptosis, necrosis and mitotic catastrophe.97 The functional inactivation of rate-limiting enzymes of the PPP or the hindrance of glucose funneling into the G6PD-dependent reactions could represent a promising strategy in overcoming intrinsic or acquired resistance to conventional chemo/radiotherapy in both solid and hematologic tumors.The PPP is a major catabolic pathway of glucose through which cancer cells produce large amounts of ribose-5 phosphate, a precursor of nucleotide synthesis and NADPH, a key molecule that is used to drive anabolic processes and to detoxify harmful ROS.98 Increased glutamine catabolism is a common hallmark of tumor metabolism reprogramming through which cancer cells support cell proliferation, signal transduction and redox homeostasis.99 It is generally assumed that the expression levels of certain oncogenes  or tumor suppressors  can strongly influence the extent of glutamine utilization and the metabolic profile of different tumors.102 However, emerging aspects of glutamine metabolism concern the potential mechanisms through which glutamine utilization can regulate the redox balance of malignant cells.103 Indeed, it is increasingly recognized that glutaminase enzymes directly contribute to glutathione synthesis converting glutamine into glutamate and promoting the uptake of cysteine through the Slc7a11 exchanger.104 Similarly, metabolic intermediates such as citrate can be diverted from the TCA cycle and exported into the cytosol, where ME or IDH1 use them to generate reducing power in the form of NADPH.84 This strategy helps tumors to keep the glutathione pool in a reduced state and support the TRX system.106 Additional mechanisms of redox modulation have been reported, wherein the mitochondrial enzyme glutamate dehydrogenase 1, by controlling the intracellular fumarate levels, positively regulates the enzymatic activity of the antioxidant enzyme glutathione peroxidase (Gpx).11 Given the paramount importance of glutamine metabolism in tumor progression and redox control, interfering with its function might represent an attractive anticancer strategy.107 With this respect, the glutaminase enzymes (GLS) as master regulators of glutamine metabolism have been the focus of recent anticancer research. Pharmacological inhibition of GLS1 with bis-2-ethyl sulfide (BPTES) impaired the proliferation of P493 B-cell lymphoma (BCL) cells inducing DNA fragmentation and apoptotic cell death, whereas the genetic silencing of GLS1 prolonged the survival of mice with Myc-induced hepatocellular carcinoma and markedly impaired the growth of P493BCL xenograft.108 Similarly, BPTES-induced GLS1 inhibition selectively suppressed the growth of glioma cells with the R132D mutation in the IDH1 isoform109 and blocked the proliferation of primary acute myeloid leukemia (AML) cells with mutations in the IDH1/2 enzymes.110 By comparing different AML cell lines, Goto et al.111 have reported that glutaminolysis inhibition was associated to the depletion of the intracellular GSH content and subsequent ROS generation, particularly in HL-60 cells characterized by glutamine addiction. In a former study, glutamine deprivation also decrease the GSH levels of neuroblastoma cells, leading to altered redox balance, impaired cell proliferation and increased chemosensitivity to the alkylating agent L-PAM.112 Finally, the combined inhibition of GLS1 and heat-shock protein 90 induced synthetic lethality in cancer cells and mouse embryonic fibroblasts with activating alterations of the mTORC1 pathway through increased ER stress and disruption of the redox balance caused by GSH depletion.83 Taken together, these studies underline the importance of glutamine in the maintenance of redox balance, cell growth and cell survival of both solid and hematologic tumors, setting the rational for therapeutic approaches aimed at the manipulation of glutamine metabolism to block malignancy.Glutamine is a non-essential amino acid that has a key role in tumor metabolism, serving as a source of carbon and nitrogen for biosynthetic processes, an intermediate for energy production and a precursor for glutathione synthesis.trans-sulfuration pathway.113 Folate, a vitamin B derivative, is reduced to THF by a series of metabolic reactions and converted into methylenetetrahydrofolate by serine hydroxymethyl transferase (SHMT). This product is either converted to F-THF or reduced by methylenetetrahydrofolate reductase to methylenetetrahydrofolate, whose demethylation completes the folate cycle. The carbon units therefore enter into the methionine cycle with the generation of S-adenosylmethionine by the methionine adenyltransferase, with further conversion by S-adenosyl homocysteine hydrolase into homocysteine.114 The last modular component of the one-carbon metabolism, the trans-sulfuration pathway, is functionally connected to the methionine cycle through the homocysteine, whose condensation with serine by cystathionine synthase generates cystathione, further metabolized to alpha-ketobutyrate and cysteine by cystathione lyase. The cysteine can therefore be diverted into GSH synthesis.113 For long time, SGOC has been associated with cancer cell due to its importance for the regulation of nucleic acid, lipids and protein synthesis of proliferating cells. More recent evidence indicates that this pathway is also crucial for redox balance.116 With this respect, the mitochondria have been shown to have a prominent role.117 Indeed, despite THF-derived carbon units are primarily used for nucleotide synthesis in the cytosol, new methods for tracing NAPDH compartmentalization indicate that serine is predominantly utilized in the mitochondria of mammalian cells to generate NADPH.118 An observation that was further substantiated by a recent study showing that both glycine and serine catabolism were responsible of NADPH production in the mitochondria of HEK-293 and MDA-MB468 cell lines.67 Interestingly, Myc-transformed cells subdued to hypoxia strongly upregulated the expression of the mitochondrial SHMT2, responsible for an abundant NADPH production. Conversely, knockdown of SHMT2 impaired antioxidant ability and increased cell death under hypoxia, but by a not yet known mechanism.119 Also, the antioxidant transcription factor Nrf2 can regulate the expression of key one-carbon metabolism enzymes, including 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase 1 and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in human non-small cell lung cancer (NSCLC) cells, supporting nucleotide and glutathione synthesis.120 Despite the fact that further studies will be necessary to assess potential therapeutic benefit of this approach, novel and selective inhibitors of SHMT2 and MTHFD2 enzymes might represent a promising anticancer strategy against hypoxic tumors characterized by otherwise limited tractability.117 Remarkably, the use of antifolate such as methotrexate and pemetrexed, which are known inhibitors of active SHMTD, still represents a cornerstone of antineoplastic therapy against solid and hematologic tumors including breast cancer, bladder cancer, acute lymphoblastic leukemia and lymphomas.121 Also, in therapy-resistant tumors, downstream pathways of one-carbon metabolism have been successfully targeted with agents that interfere with the nucleotide synthesis such as 5-FU for advanced colorectal cancer or gemcitabine for pancreatic cancer.113 Given the increased interest in metabolic alterations of cancer cells and the intimate connection between SGOC pathways and redox homeostasis of human tumors, key nodes in the one-carbon metabolism might represent a valid therapeutic target at the crossroad between the regulation of cancer growth and antioxidant capacity.The serine–glycine one-carbon metabolism (SGOC) is a complex network of biochemical reactions that integrate inputs from amino acids and glucose derivatives (mainly serine and glycine) and generates multiple outputs as carbon units (tetrahydrofolate (THF) and its derivate) that serve different cellular functions. The redistribution of these carbon units from serine and glycine rely on three pathways: the folate cycle, the methionine cycle and the 122 By hosting multiple redox-active complexes and metabolic enzymes that generate superoxide anion, the mitochondria represent a major source of endogenous ROS production. The most well-characterized site is represented by the mETC, through which the electrons from reduced metabolic intermediates  are transferred to the molecular oxygen. During the electrons flow, depending on the mitochondrial membrane potential status and the oxygen availability, semiquinone radicals can be generated at the level of complexes I, II and III, promoting the univalent reduction of oxygen into superoxide.123 Other sources of mitochondrial-dependent superoxide production include the 2-oxoglutarate dehydrogenase, the pyruvate dehydrogenase in the mitochondrial matrix, the mitochondrial glycerol-3 phosphate dehydrogenase and the electron transfer flavoprotein-ubiquinone oxidoreductase mitochondrial system (flavoprotein-ubiquinone oxidoreductase) located in the inner membrane.38 The generated superoxide can then leave the mitochondrial district through different ways. One well-established route is through its conversion into H2O2 by SOD2, whereas the second mechanism, still a matter of intense debate, postulates its direct diffusion into the cytosol through the voltage-dependent anion channel (VDAC),125 wherein is spontaneously or enzymatically transformed into H2O2 by SOD1. In this way, the mitochondria produce picomolar to nanomolar amounts of peroxide that then leave the site of generation and promote retrograde signaling to the nucleus or regulate activity, localization and stability of redox-sensitive target proteins that are located in the cytosol.126 On the basis of their central role in redox control and metabolism, the mitochondria represent attractive targets for anticancer therapy. Several studies have investigated the effect of mitochondrial-targeted antioxidants and their impact in tumor biology.127 MitoQ is an orally active antioxidant that not only mimics the role of the endogenous mitochondrial antioxidant coenzyme Q10 (CoQ10) but also substantially augments the antioxidant capacity of CoQ10 in a mitochondrial membrane potential-dependent manner.128 MitoQ has been found to kill breast cancer cells and unhealthy mammary cells, supporting a role for MitoQ and similar compounds to be further evaluated for novel anticancer activity.129 Among other aspects, the relative contribution of different metabolic pathways involved in NADPH generation  has received significant attention102 with particular emphasis on the role of one-carbon metabolism.113 With this respect, mitochondrial NADPH mainly derives from serine catabolism regulated by SHMT2 and MTHFD2, two mitochondrial-specific enzymes that are frequently overexpressed in cancer cells acting as important regulators of tumor redox homeostasis, but absent or underrepresented in normal tissues.130 Genetic depletion of SHMT2 altered the redox balance of Myc-transformed tumors during hypoxia and induced significant cytotoxicity,119 whereas knockdown of MTHFD2 in overexpressing breast cancer tumors has been shown to impair cell migration and invasion, and to sensitize malignant cells to methotrexate by inducing caspase 3/7-independent cell death.131 Taken together, these observations imply that targeting multiple mitochondrial functions with single agents or in combination with inhibitors of different metabolic pathways might represent a promising approach to improve the efficacy of conventional chemotherapeutics, in particular in those tumors wherein the apoptotic machinery is not functional.One emerging aspect in the study of molecular mechanisms controlling redox balance and metabolism in mammalian cells regards the existence of a clear compartmentalization of specific biochemical reactions in different cell organelles. It is increasingly known that mitochondria are key organelles for the regulation of redox signaling and redox homeostasis of normal and cancer cells . By inte127 By regulating the generation of ROS, ATP and other metabolites driving bioenergetic and biosynthetic processes, mitochondria have a key role in cancer progression.133 Oncogenic activation of MYC and KRAS promote increased glucose utilization and addiction in different types of tumors,134 while also enhancing the mitochondrial-dependent biosynthesis of macromolecules through increased ATP levels and TCA cycle intermediates.135 To replenish the TCA cycle substrates and fuel their uncontrolled growth, cancer cells utilize specific mechanisms of anaplerosis on the basis of the oxidation of glutamine to α-ketoglutarate and its conversion to oxaloacetate.136 As a consequence of oxidative metabolism, physiological amounts of ROS are produced at the level of the mETC, inducing the pro-tumoral activation of redox-sensitive pathways.31 To prevent ROS-induced toxicity, cancer cells redirect the metabolic intermediates coming from glutamine and one-carbon metabolism into alternative pathways that generate NADPH and GSH, antioxidants molecules readily used by several ROS-scavenging enzymes.118 Interestingly, tumors with impaired TCA cycle activity (or mutations in the mETC complexes) and that rely on glycolysis for ATP synthesis, shift to reductive glutaminolysis to mediate biosynthetic processes and cell survival.138 Such tumor cells depend on mitochondrial-derived ROS to promote cell proliferation and metastasis formation.141 In contrast to what is generally assumed, many tumors still use mitochondria for ATP production, despite the Warburg effect should provide sufficient amounts of ATP for their biological needs.142 Indeed, poorly vascularized and other subsets of tumors growing under limited glucose conditions heavily rely on oxidative phosphorylation for ATP synthesis,144 a weakness that might be targeted with drugs that limit glucose utilization and block mitochondrial bioenergetics.146 With this respect, two anti-diabetic biguanides, such as metformin and phenformin, show promising antitumoral effects.147 Epidemiological studies have shown that metformin decreases the incidence of cancer and prolongs the survival rate of patients with solid tumors.149 More studies have reported also that the in vivo antitumorigenic effects of metformin depended on the inhibition of the mETC complex-I activity and the decrease of circulating glucose and insulin levels.152 Metformin was also able to impair the growth of NSCLCs by blocking the activation of the Akt/mammalian target of rapamycin (mTOR) pathway and by potentiating the pro-apoptotic efficacy of ionizing radiation.153 Also, metformin selectively killed breast cancer stem cells through profound depletion of triphosphate nucleotides presumably reflecting a major impairment of the energetic metabolism.154 Therefore, another biguanide, called phenformin, has emerged as a potential anticancer agent due to its liposolubility, higher affinity for mETC complex-I and stronger antineoplastic activity.155 Phenformin enhanced the efficacy of the BRAF inhibitor PLX4270 both in vitro and in genetic models of melanoma driven by BRAFV600E mutations triggering apoptotic cell death upon inhibition of the mTOR pathway.156 Phenformin also induced selective apoptosis in a subset of NSCLCs with loss of the tumor suppressor LKB1 and oncogenic mutations in K-ras lowering the ATP levels and eliciting the activation of caspase 3.157 Despite the encouraging results, it should also be mentioned that the use of phenformin has been associated with lactic acidosis, an important pitfall that might limit its clinical applicability.158 For this reason, novel compounds that target the mETC complex-I  or alternative strategies based on the translation of specific mitochondrial proteins  have been designed and currently successfully employed in preclinical studies.159 Although the potential utility of these compounds still needs to be validated in long-term clinical trials, encouraging evidences suggest that targeting the bioenergetics function of the mitochondria might represent a valid therapeutic option for cancer treatment.Despite the fact that mitochondrial dysfunction has long been considered a metabolic hallmark of cancer cells, studies have also indicated that tumor cells not only have functional mitochondria but also that their activity is essential for tumorigenesis.14 In many studies, either metabolic inhibition or the blockade of certain antioxidant systems was shown to strongly sensitize cancer cells to apoptotic cell death induced by further elevation of ROS levels.164 However, due to the undesired side effects including cardio-toxicity and nephrotoxicity, extensive research has been pursued to identify novel ROS modulators with a safer therapeutic profile. With this respect, another class of quinone-based compounds, including menadione and other vitamin K3 derivatives, are emerging as promising anticancer agents.165 Remarkably, this combinatorial approach has the ability to engage multiple cell death modalities, not limited by the activation of the intrinsic and extrinsic apoptotic pathways, and therefore might be useful to overcome the mechanisms of therapy resistance due to the overexpression of anti-apoptotic proteins or compromised activation of the caspase cascades. With this respect, the induction of ferroptosis, an iron- and ROS-dependent form of non-apoptotic cell death characterized by altered mitochondrial morphology, is receiving increasing attention for its intimate connection with cellular metabolism and redox balance.167 Indeed, large B lymphomas and renal cell carcinomas with Ras mutations were found to be particularly susceptible to ferroptosis upon BSO-mediated GSH depletion, decreased Gpx4 activity and accumulation of lipid peroxidation.169 In another study erastin, a potent inhibitor of Xc− cystine importer was shown to induce ER stress and trigger ferroptosis in different cancer cell lines.170In light of recent research, the inhibition of metabolic pathways or ROS-scavenging mechanisms, followed by the administration of pro-oxidizing agents (i.e. chemo-radiotherapy), represents a promising therapeutic option for tumors characterized by resistance to treatment . With thIn conclusion, it is becoming clear that redox signaling events require the simultaneous regulation of sources, transducers and scavengers in a precise spatial and temporal framework, whose alteration may disrupt key redox nodes and promote aberrant signaling. The loss of control over specific redox circuitries is likely to represent a tumor-specific alteration that may trigger unrestricted proliferation and malignant progression. Moreover, metabolomics studies associated to computational models on the basis of the integrative bioinformatics are becoming increasingly accessible and will surely guarantee a rapid progress in the field of redox biology, in particular addressing how metabolism and its subcellular compartmentalization can influence ROS signaling and redox cofactors. We are now approaching a new era wherein ROS biology and their effects in the physiopathology of cancer may be dissected with unprecedented detail, bringing potential therapeutic benefits derived from selective manipulations of cancer redox balance to be uncovered, paving the way to novel and exciting investigations in the fight against cancer.


Amdoparvovirus is a newly defined parvoviral genus that contains four species (Carnivore amdoparvovirus 1–4), including the well-known Aleutian mink disease virus (AMDV). Amdoparvoviruses cause an immune-associated and often lethal wasting syndrome in Mustelidae and Caninae hosts. In this study, we molecularly investigated amdoparvoviruses detected in 44 striped skunks (Mephitis mephitis) found dead in and around Vancouver, British Columbia, Canada. Some of the animals exhibited pathological changes compatible with amdoparvovirus-associated disease. The nearly complete genomic sequence was obtained for seven different strains and our analyses show how this virus, which we named skunk amdoparvovirus (SKAV), should be classified as a separate species within the genus (proposed Carnivore amdoparvovirus 5). We detected co-infections, recombinant genomes, at least three separate viral lineages, and preliminary evidence for geographic segregation of lineages. Furthermore, we proved that similar viruses, only partially characterized in previous studies and labeled as AMDV, circulate in skunks from other distant areas of North America  and found evidence for spillover events in mink (Neovison vison). Although SKAVs are capable of causing disease in infected animals, a high proportion of sub-clinical infections has been observed, suggesting these animals might act as asymptomatic carriers and pose a threat to wild and captive carnivores. Finally, we highlight the need for more specific diagnostic tests and further molecular investigations to clarify the epidemiology and host- and geographical distributions of amdoparvoviruses in terrestrial carnivores, especially because the whole spectrum of viral diversity in this group is likely still unknown. Amdoparvovirus, within the family Parvoviridae and subfamily Parvovirinae, was recently defined by the International Committee on Taxonomy of Viruses (ICTV)1 after the discovery of several novel parvoviruses sharing genetic and virological characteristics with the Aleutian mink disease virus (AMDV) between 2011 and 2014.2 In addition to AMDV, classified as Carnivore amdoparvovirus 1, whose primary hosts are members of the Mustelidae family and possibly other furbearing animals, this genus includes a variety of viruses found in the Caninae. These are the gray fox amdoparvovirus (GFAV) or Carnivore amdoparvovirus 23 and the proposed Carnivore amdoparvovirus 3 or raccoon dog and fox amdoparvovirus (RFAV)4 and Carnivore amdoparvovirus 4 or red fox fecal amdovirus (RFFAV).5 These small single-stranded DNA viruses possess a genome of ~4.8 kb that contains two major open reading frames (ORFs).2 These ORFs, one encoding three non-structural proteins  and the other two structural proteins (VP1 and VP2), are characterized by different evolutionary dynamics.6The genus 6 Farm-derived strains also represent a threat for wild animal populations as escape of animals can result in the introduction of viruses to new geographic areas, where novel host species can get infected.8 Furthermore, infected animals do not always develop clinical signs and healthy carriers might have a significant role in the diffusion and distribution of viruses in the wild and to farms by acting as reservoir hosts.11 As disease severity seems to be influenced by both host and viral genetic factors,2 the study of amdoparvoviral infections in several different hosts is essential to achieve a full understanding of the transmission dynamics and pathogenicity potential of these viruses.Amdoparvoviruses are known to cause an immune complex-mediated wasting syndrome that is often fatal, especially in highly susceptible hosts, and are responsible for the occurrence of vast epidemics in farms, frequently associated with great economic losses.14 the proportion of sub-clinical infections seems to be high, suggesting that infections in these animals are primarily asymptomatic.15 Despite their potentially fundamental role in amdoparvoviral epidemiology, only a few studies have looked at the genetic makeup of AMDV-like viruses in these animals 13 and, therefore, their genetic diversity and molecular features are largely unknown. In this study we fully molecularly characterized amdoparvoviruses detected in mainly asymptomatic striped skunks (Mephitis mephitis) from in and around Vancouver, BC, Canada, and we studied their molecular epidemiology and distribution in North America. Furthermore, our sequence and phylogenetic analyses showed that this virus, which we have named skunk amdoparvovirus (SKAV), is divergent from all known members of the genus Amdoparvovirus and we evaluated its potential classification as a novel amdoparvoviral species (proposed Carnivore amdoparvovirus 5).Amdoparvoviruses have been found to be highly prevalent in skunks and, despite pathological examinations confirming lesions compatible with amdoparvoviral infection in several cases,10 The causes of death of these animals were various (mainly traumas and not related to infectious diseases) and only 4.6% showed pathological evidence of Aleutian disease. A complete description of the post-mortem examinations for this population is available in Britton et al.10 The nearly complete genomic sequences of seven viruses and the complete NS1 ORF of one additional virus were obtained as previously described.6 The primers AMDO_3F (5′-GGA TGG TTA CTW TGC TGC TG-3′) and AMDO_2R (5′-ACA TKC CTG GTG TTA YTT TRG-3′) were used to amplify and sequence a 998-nt long fragment of the NS1 region  for the molecular epidemiological investigation. For viruses showing evidence of multiple infections or intra-host polymorphisms, amplified fragments were cloned into a plasmid vector before sequencing.6We analyzed viral DNA isolated from 44 striped skunks found dead or injured severely in and around the Vancouver area , BC, Can6Ten spleen samples were also obtained from AMDV-positive mink from five different farms throughout British Columbia for further comparisons. DNA was extracted from mink spleen tissues using the DNeasy Blood and Tissue kit  and the complete NS1 ORF was sequenced as previously described.16 donor and acceptor sites were confirmed using NNSPLICE,17 and splicing events were reproduced in silico to determine the complete coding sequences for all viral proteins, which were then translated into amino acid sequences.Sequences obtained in this study were compared to several AMDV, RFAV, GFAV and RFFAV sequences downloaded from GeneBank  for sequence pairs within and between groups were calculated with MEGA .Nucleotide and protein sequences were aligned with ClustalX 2.122 and only events supported by at least three methods (P<0.05) were accepted for confirmation. All amdoparvoviruses for which a complete genomic sequence is known were included in the analysis but viruses other than SKAV were used only for tree topology estimates. Potential recombination events were evaluated with SimPlot software 3.5.123  and novel phylogenetic trees were built with sub-portions of the alignment included between putative breakpoints.Alignments of complete genomes were analyzed for the presence of recombinant strains using the RDP 4.55 software packageAll sequences obtained in this study have been deposited in GenBank under accession numbers KX981972–KX981981 for partial AMDVs, KX981927–KX981971 for partial SKAVs and KX981920–KX981926 for the nearly complete SKAV genomes.We obtained the nearly complete genomic sequences of seven SKAVs that were identified in mainly asymptomatic striped skunks from the Vancouver area , including those originating from the farm where one of the skunks under investigation was found, clustered within the AMDV clade (see in extenso).Two distinct phylogenetic trees were built using the complete NS1 and VP2 protein sequences  that incParvoviridae state that the NS1 sequences of viruses from one species must ‘show >85% amino acid sequence identity, while diverging by >15% from viruses in other species’,27 and we therefore concluded that SKAVs could be classified as a separate viral species, tentatively named Carnivore amdoparvovirus 5.Finally, pairwise sequence identities within and between each group were calculated for both proteins . Overall28 including AMDV,6 and we therefore investigated whether chimeric genomes were present among the fully sequenced SKAVs. We found evidence for at least two recombination events and identified two sequences with the same chimeric pattern  and two putative breakpoints, located at approximately nt 1000 and 3200 of the SK-1 genomic sequence. The second event involved one chimeric sequence (SK-16) and the two putative breakpoints were located at approximately nt 2300 and 3000 of the SK-1 genomic sequence. Both events could be clearly visualized by the BootScan analysis performed with Simplot , where tFinally, phylogenetic trees were built with sub-portions of the alignment that included the regions between putative breakpoints . As expe10 from which we also obtained AMDV sequences from mink (BCM-1 and BCM-3). The chromatograms obtained from several samples showed the presence of double peaks at >1 site, potentially indicative of multiple infections, and such amplicons were subjected to cloning before sequencing. Overall, this resulted in 52 SKAV nucleotide sequences considered in our molecular epidemiological investigation. In most cases, clones obtained from the same animal differed from each other at only a few positions and belonged to the same phylogenetic group, making the distinction between intra-host mutation and co-infection impossible. The presence of multiple distinct viruses simultaneously infecting one animal could be confirmed in only one case (SK-7) as viruses from two different lineages (see below) could be identified in this animal  that was found dead on a mink farm (cause of death was cellulitis)s animal .n=22), and SKAV-2B, which contained three viral sequences sharing lower sequence identities with each other  and that could potentially represent three separate sub-lineages. The complete set of sequence identities within and between clades and sub-clades is available in the The phylogenetic tree built with an ~950-nt region of the NS1 ORF  revealedNo clear geographical distribution of strains was observed. However, the SKAV-2 lineage was more diverse in terms of geographic origin as it included viruses obtained from each sampled location, including the only two viruses sampled south of the Fraser River. Interestingly, sub-clade 2B contained viruses sampled in the most isolated areas: Lulu Island (where Richmond is located), Mission (~40 km from the next nearest sampling location) and the mink farm . Finally, no temporal pattern was observed in the data, although no viruses from 2011 and 2012 were observed within the SKAV-2 lineage.13 Interestingly, evidence of SKAV infections was also found in one likely escaped domestic mink32 and another mink of unknown origin6 in Ontario. However, all of these viruses were not completely characterized in the previous studies and were therefore not recognized as a separate species and were labeled as AMDV.A thorough search of previous literature and in the NCBI database, which considered every amdoparvoviral sequence ever deposited in GenBank or reported in publications about AMDV-like viruses in skunks, revealed that similar viruses are also circulating in other locations in North America but distant from British Columbia (Western Canada). Specifically, these were identified in Ontario (Eastern Canada) and California (Southwestern USA) and corresponded to one Californian and 15 Canadian striped skunks.We studied the relationships among SKAVs identified in different locations and in different hosts by performing additional phylogenetic analyses. Unfortunately, only partial sequences of different portions of the genome were available for the viruses from California and Ontario. Therefore, we were not able to compare all viruses together and three separate phylogenetic trees were required to properly compare the various sequences . Sequenc2 The virus we describe in this study is likely responsible for a similar disease in striped skunks as a proportion of the studied infected animals showed pathological evidence of Aleutian disease.13 This virus, which we named Skunk amdoparvovirus (SKAV) as this is the only amdoparvovirus identified so far capable of establishing a clinically evident disease in skunks, is divergent enough from all the other members of the genus Amdoparvovirus to be classified as a separate species. According to the most recent ICTV classification rules,27 and also taking into account the latest published discoveries,5 we propose the taxonomic species designation of Carnivore amdoparvovirus 5 for SKAV.Amdoparvoviruses cause persistent infections in their hosts that can lead to a serious wasting syndrome, also known as Aleutian disease, associated with hypergammaglobulinemia, kidney failure and often death. AMDV, the first-identified and most-studied member of the group, is also known to cause an acute respiratory infection in mink kits, which can progress into fulminant pneumonia, and has been associated with complications during pregnancy.33 which are strikingly difficult to overcome and prevent as there are no available cures or treatments and because the great environmental stability of the viral particles makes them difficult to eradicate.2 However, because of the wide range of potentially susceptible hosts, these viruses have also raised major concerns outside of the fur industry as they represent a serious risk for wild animals and pose a threat to endangered species.8 The presence of asymptomatic individuals, whose existence has already been proven among mink and ferrets,35 certainly facilitates viral dispersion, making new hosts more accessible. The vast majority of the skunks that were sources or viruses for this study presented with an apparently sub-clinical infection,10 consistent with a presumed role of these animals as amdoparvovirus carriers and natural reservoir hosts, as already postulated by others.11 However, it is also possible that in some cases the infection was detected before signs of disease became evident, as the most common cause of death/killing in these animals was trauma, and further studies are required to clarify the full spectrum of clinical signs associated with SKAV infection. We have also identified two cases of SKAV-infected mink, demonstrating the capability for SKAVs to cross species barriers and infect other hosts. Furthermore, one of the skunks under investigation was found dead within the borders of a mink farm, illustrating how close contacts between skunks and mink are possible and that skunks can carry these viruses into farms, where close contacts between animals facilitate the establishment of co-infections and lead to recombination.6 Interspecies recombination has been already documented among members of the family Parvoviridae36 and farmed mink might represent a potential mixing vessel for AMDV and SKAV. It is currently unknown whether SKAVs can infect other carnivore species other than mink and future molecular investigations will be required to answer this question.Amdoparvoviruses are principally studied because they cause vast epidemics in farms, often associated with great economic losses,37 a high diversity was observed among SKAVs, with recombinant genomes and at least three separate viral lineages detected. Nevertheless, we acknowledge that the whole spectrum of genetic variability of SKAVs is likely still largely unknown and hypothesize that additional diversity and lineages remain to be discovered. This is primarily based on the observation that highly divergent viruses forming single-strain clades were identified, and because there have been only a few studies investigating genetic characteristics of amdoparvoviruses in skunks. Furthermore, as we already postulated elsewhere,6 it is reasonable to believe that the whole spectrum of amdoparvoviruses infecting terrestrial carnivores is currently unknown and we reemphasize here the necessity of performing more molecular epidemiological investigations in these animals to expand our current knowledge of diversity within this viral group.As already observed for AMDV,11 Although the genetic similarity of viruses detected in these three areas is high, it appears that distinct lineages might be circulating in different locations. However, the available sequence data from Ontario and California were insufficient to perform a detailed comparison and, therefore, further sequencing efforts should be carried out to complete this investigation. At a more local level, when examining viruses obtained from Vancouver and surrounding areas, we have also found evidence for geographic effects on the relationships among strains. Viruses obtained from Lulu Island, Mission and the mink farm  were genetically different from what was identified in Vancouver-proper and the other surrounding districts. Furthermore, our results suggest that water could act as physical barrier for viral spread as a different distribution of strains was observed in areas north and south of the Fraser River and within Lulu island. However, the number of analyzed samples was very small and a more intense and prolonged sampling within these areas is needed to better resolve the epidemiological dynamics, transmission behavior, and phylogeographic pattern of SKAVs.On the basis of our epidemiological investigation, these viruses are circulating in at least three distinct geographical regions covering distal regions of continental Canada (British Columbia and Ontario) and in the southern USA  and, according to available epidemiological data, these viruses are highly prevalent .6 Because of this, primers normally utilized for the molecular diagnosis of AMDV could be used to amplify SKAVs11 and the two viruses could be confused without a more thorough genetic characterization. In addition, this high genetic similarity most likely translates into an elevated antigenic similarity, which might result in cross-reactivity between antibodies and antigens of different viruses and result in misdiagnosis when immunological assays are employed. It is therefore possible that previous studies that reported evidence for AMDV in skunks actually detected other amdoparvoviral species or antibodies against them and, therefore, viral sequencing has to be included in future investigations for correct data interpretation. Two previous studies have analyzed the genetic characteristics of amdoparvoviruses in skunks,13 and the detected viruses can be classified as SKAVs in both cases. Indeed, to our knowledge, SKAV is the only amdoparvovirus ever identified in skunks and more studies are necessary to evaluate whether AMDV is actually capable of infecting striped skunks and if other amdoparvoviral species can be found in these animals. Obtaining sequence data from suspected cases of Aleutian disease in skunks, including those already published if possible,15 therefore has to be considered a priority, together with the development of more specific diagnostic tests.Even though SKAV and AMDV are sufficiently genetically distant to be considered separate species, they still share a high genetic similarity and this is especially pronounced within the gene encoding the capsid protein, which is known to be more conserved among amdoparvoviruses.In summary, we describe here a novel amdoparvovirus that infects striped skunks (skunk amdoparvovirus or SKAV), which is highly prevalent in Canada and appears to be widespread in North America. Infected animals show signs of disease typically associated with other amdoparvoviruses but a high proportion of sub-clinical infections has been observed, suggesting asymptomatic carriers could be common. These animals might be responsible for an efficient distribution of viruses in the wild and transmit the infection to other animal species, and this hypothesis is supported by the observed spillover events in mink. As this virus might pose a threat to wild and captive carnivore populations and the entire genetic spectrum of circulating lineages is unknown, we strongly highlight the need to perform more molecular epidemiological investigations to study the distribution and diversity of amdoparvoviruses in skunks.


Supplemental Digital Content is available in the text. Tenets of chest wall reconstruction for malignancy are well known; however, the insertion of synthetic prosthetic material to achieve rigid and nonrigid skeletal reconstruction could be associated with more complications including infection and extrusion especially in high risk patients. This includes fungating, infected tumors, previous radiotherapy, scars, smoking, diabetes, and morbid obesity. Bioprosthesis Acelluar Dermal Matrix Strattice would be a substitute to resist infection and provide stable coverage.A retrospective data analysis study on 8 high-risk patients who underwent chest wall resection and reconstruction with bioprosthesis (Strattice) with/without titanium plates in the period between 2012 and 2017 was performed. Patient’s demographics, risk factors, clinico-pathological, radiological, operative details, adjuvant therapy, postoperative morbidity, and follow-up data were recorded.2 . Reconstruction was performed using Strattice only in 4 patients, whereas in 4 it was combined with titanium plates. All patients required flap reconstruction (3 pedicled and 5 free). The follow-up ranged from 9 to 52 months . Minor complications occurred in 2 patients; however, good functional outcome was achieved in all.Sarcoma was the pathology in 7 and recurrent breast cancer in 1 with age range from 21 to 71 years  and preponderance of female patients (n = 5). Defects were located anterior/anterolateral with size ranging from 270 to 1,050 cmStrattice would act as a safe alternative modality for chest wall reconstruction to resist infection in high-risk patients with extensive defects. It should be considered as a valuable tool in the armamentarium of chest wall reconstruction. The indication to consider bioprosthetic material Strattice included high-risk patients having contamination of the operative field , previous local radiotherapy and surgery and/or associated general risk factors as diabetes, active smoking, chemotherapy, and morbid obesity. All patients were managed through a multidisciplinary approach including thoracic surgeon (M.K.) and reconstructive surgeon (H.K.) to optimize their perioperative treatment plan. This included preoperative preparation, pulmonary function test to assess fitness, radiological investigations including computed tomographic (CT) scan and/or angiography and magnetic resonance imaging to plan the surgical resection, possible skeletal and soft-tissue reconstructive options and oncological adjuvant therapy.A prospectively maintained database was used to identify the patients, and their records were assessed retrospectively. The demographic information and potential identifiable risks and comorbidities, previous history of malignancy, and treatment were collected. Tumor characteristics and details of adjuvant therapy including either chemotherapy or radiotherapy were also documented. The results of the radiological diagnostic workup included standard chest x-ray, CT scan, and magnetic resonance imaging to delineate the extent of the tumor in the chest wall, and involvement of adjacent surrounding structures was noted. The operative details identified the numbers of ribs and costochondral junctions along with other structures that were resected as part of the tumor extipiration and included intercostal muscles, chest wall muscles, lungs, diaphragm, and overlying soft tissue. The sizes of the skeletal and soft-tissue defect were recorded in all patients. Documentation of whether Strattice was used solely or in adjunct with titanium plates and rib clips  to restore chest wall integrity was noted. In addition, the methods of soft-tissue coverage were also documented. The postoperative outcomes in terms of length of stay, complications particularly SSI, wound dehiscence, chest wall stability, and respiratory function were identified from the patient records.18 and continued routinely for 48 hours postoperatively followed by oral therapy for the duration of time the drains remained in situ. All patients received structured physiotherapy and rehabilitation program for both chest and donor site depending on flap selection.All patients included were considered for surgery after been reviewed by the multidisciplinary thoracic oncoplastic team in the sarcoma and breast multidisciplinary team meeting (MDT). The resection and reconstruction were performed as a 1-stage procedure in all patients with a 2 team approach. In the majority of cases (5/8), the 2 teams worked simultaneously when free flap reconstruction was performed, whereas in 2 (2 of 8), the thoracic surgeons initially performed the resection and restored the skeletal wall integrity; this was followed by harvesting and insetting of the selected regional flap. On the other hand, in 1 patient, the reconstructive team harvested the selected regional flap first to facilitate tumor extipiration and also salvage the regional flap from being damaged through the conventional thoracic approach. Post tumor resection chest wall stability was achieved using firm Strattice ADM (30 × 30 or 15 × 15 cm), which was washed in vitro with 5 L of saline to remove any chemical preservatives, followed by fashioning of the sheet to the defect (onlay technique) and securing it to the surrounding rib edges with interrupted Ethibond 2/0 sutures. Titanium plates and rib clips (Stratos osteosynthesis MedExpert) were used in addition to the Strattice ADM in 4 patients due to the extent of the resection and the location of the tumor being anterolateral to maintain structural support and stability. The site, size of the defect, availability of tissues, and preference of the reconstructive surgeon determined the choice of the soft-tissue flap. Soft-tissue reconstruction was achieved utilizing loco-regional pedicled muscle flaps as latissimus dorsi with or without split-thickness graft. Free microvascular tissue was indicated in more extensive resection with large surface area or when loco-regional flaps deemed not feasible due to direct tumor involvement, vascular compromise, and previous radiotherapy or surgery. We were mindful of creating a suitable window in the Strattice sheet in the cases where the internal mammary pedicle was used as the recipient pedicle for the free flap to avoid any compromise of the vascular supply postmicrovascular anastomosis. As a routine, all surgical sites were copiously irrigated with pulse-jet lavage using 3 L of saline (0.9% Nacl) solution to mechanically remove any debris or loose diathermized tissue and to maintain moisture of the Strattice. A chest wall tube was placed in the thoracic cavity, whereas suction drains were inserted deep into the flap and subcutaneous tissue. The duration of the drainage depended on the clinical, chest x-ray, and biological follow-up. Intraoperative collagen implant impregnated with aminoglycoside antibiotic gentamicin  was inserted in the operative field before closure. All 8 patients were transferred to intensive therapy unit with 6 patients (6 of 8) extubated at the end of surgery and 2 within 12 hours. Standard flap observation chart protocol was followed, including Doppler signal, skin color, temperature, and capillary refill. At induction, all patients received antibiotics intravenous therapy, determined by appropriate tissue penetrability with special reference to bone and joint infections for prosthetic device2 .The average number of ribs resected with adjoining costochondral cartilage was between 3 and 10 . In addition, partial sternectomy was performed in 1 patient and complete sternal resection in another one. Adequate clear surgical resection margins were achieved in all the patients. The resultant chest wall defect was repaired using Strattice in all patients. In 4 patients, the defects were large, crossing midline and required additional rigid reconstruction using titanium plates and rib clips (Stratos). The repaired defects were covered in all patients with soft-tissue flaps successfully achieved with free microvascular tissue transfer in 5 patients [muscle sparing transverse abdominus myocutaneous (MSTRAM) type I (n = 4) and tensor fasciae latae ], whereas in 3 pedicled latissimus dorsi muscle with split-thickness graft and musculocutaneous flap were performed. The recipient vessels for the microvascular anastomosis were internal mammary pedicle (n = 4) and thoracodorsal pedicle .The patient, tumor, and chest wall defect characteristics including surgical management and outcomes for the cases are summarized in Table 2 mean, 51.The aveTwo patients experienced minor complications, 1 developed wound breakdown and superficial SSI 10 months postoperatively after receiving radiotherapy. This required minor surgical debridement with secondary closure. The same patient developed local recurrence and distant metastases (month 12 postoperatively) and was referred to the palliative team. The other patient developed superficial minor distal flap necrosis, which required debridement and flap readvancement day 14 postoperatively; in neither patients, the Strattice was exposed. During the follow-up period of 9–52 months , there was no local recurrence or distant metastases in any of the other patients with clinically good chest wall function. No readmissions were encountered within the 30 days from discharge. One patient died at 14 months due to systemic disease progression.7 Furthermore, if infection and/or exposure of mesh ensue in the event of being exposed due to dehiscence or breakdown of overlying skin, surgical removal is often required. Observation from previously published reports demonstrated the relatively high infection rate (6–22%) in chest wall reconstruction in noncontaminated defects with up to 42% requirement of removal of synthetic mesh.6 In this series, of extensive chest wall defects  in compromised surgical fields, Strattice ADM was used to substitute synthetic mesh to resist infection. The general consensus accepted for chest wall skeletal reconstruction that we also applied in our series is that any defect less than 5 cm in size in any location, and those up to 10 cm in size posteriorly do not need rigid reconstruction for functional reasons. On the other hand, posterior defects in proximity to the tip of the scapula and larger lesions mostly anterior and lateral are likely to produce paradoxical chest wall motion therefore requires rigid reconstruction.11 Four patients in our series required additional structural support with rigid skeletal reconstruction using titanium plates (Stratos) due to the extent and location of the defects in the anterolateral chest wall region to maintain chest wall integrity. To date, there are few case reports demonstrating the use of Strattice in chest wall reconstruction.17 Alternatively, other bioprosthetic meshes as Alloderm and Permacol in both human and animal models have shown to be efficacious in similar scenarios.13 The advantages of biological mesh materials over synthetic material have been reported, which includes superior handling properties, reduced visceral adhesions, resistance to infection, better integration into the autologous tissue due to host cell infiltration, and revascularization while maintaining structural integrity.13 In addition, bioprosthesis has better tolerance to exposure due to tissue breakdown, hence can be managed conservatively rather than surgical extipiration.19 Despite the ability of both Alloderm and Strattice to become revascularized and resist infection, previous case reports have highlighted the superiority of porcine-derived over human-derived biological mesh including less elasticity to avoid stretch, been harvested in larger and consistent sheets achieving coverage of larger defects with a single sheet.20 This was in concordance with our results, as 1 sheet was used per patient notwithstanding that the defect sizes reconstructed in this series were relatively larger from previously reported cases. The relatively longer follow-up  when compared with previous reports illustrated the durability of the functional properties of a remodeled mature biologic scaffold in terms of strength and stretch. During the follow-up period, all patients showed satisfactory functional outcome assessed clinically and with pulmonary function testes complimented with equally cosmetic satisfactory cosmetic outcomes. Hedgire et al.21 reported on significant enhancement of Alloderm Biologic Mesh Spacer in the abdomen and pelvis detected on follow-up multi detector CT scan through progressive rise in Hounsfield unit values. This has been attributed to the possible revascularization of the ADM as supported by other reports.22 In our study, this was also observed in the CT scan follow-up performed postoperatively in 4 patients, which revealed well-enhanced scar tissue denoting revascularization and maturation providing stable coverage. Arguably other synthetic mesh materials would have been used; however, there would be an increased risk of infection of these synthetic meshes within this compromised surgical field and their intolerance for contamination and /or cutaneous exposure, which has been highlighted in other studies.14 Eventually, this would lead to detrimental consequences as a result of the requirement of mesh removal, which has been also highlighted in previous studies.14 In his case report, Brunbjerg et al.14 reported the use of Strattice successfully in chest wall reconstruction in a patient with multiple recurrent breast cancer, chemotherapy, and radiotherapy requiring multiple chest wall resection and reconstruction with previous failure and extrusion of the polypropylene mesh. We previously reported the value of multidisciplinary thoracic oncoplastic approach in the reduction of infection (2.8%) in the presence of synthetic prosthetic material in noncontaminated chest wall defects.3 Adopting the same approach in this relatively small cohort of high-risk patients, the pertinence of bioprosthetic mesh is seen to be associated with decreased incidence of SSI, less extrusion rate without any compromise of the chest wall stability that has also been supported by other studies.14 Observation from previously published reports demonstrated the importance of the proximate collaboration between several disciplines in managing these patients; however, it underscores the occurrence of the wound morbidity even in the presence of soft-tissue coverage for rigid reconstruction ranging from 9% to 25%,8 whereas in other series, this relationship was not highlighted.23 The recruitment of well-vascularized soft tissue is paramount to provide coverage of the prosthesis, prevent infection, seal the pleural space, protect underlying viscera, obliterate dead space, contribute to structure integrity and healing, and enhance aesthetic outcome.25 A wide variety of flaps could be used including loco-regional or free flaps depending on the site, size, and availability of tissues from donor sites and the available expertise.26 Soft-tissue coverage was performed in all patients in this series due to the extent of the cutaneous defects and to provide protection for the underlying Strattice and titanium plates. The majority of the patients required free flap including MSTRAM and TFL  when loco-regional flaps deemed not feasible, extent of the resection and due to the logistics of positioning the patients to allow thoracic and reconstructive surgeons working simultaneously. The efficacy of the xenogeneic nature of biological mesh has been questionable in previous reports28; however, with the rapidly increasing use of porcine-derived ADM in several indications including breast, abdominal, head and neck, dural reconstruction and static reconstruction postfacial nerve paralysis over the last decade has demonstrated its advantages.33The synthetic prosthesis mesh is used in the majority of chest wall reconstruction following resection of tumors. However, there is an increased risk of SSI, particularly if used in compromised surgical fields including fungating infected necrotic tumors, previously local radiotherapy and surgical scarring and in patients with general risk factors as diabetes, active smoking, chemotherapy, and morbid obesity.The current literature is largely limited to case studies, and to the authors best knowledge, this would be the largest series from a single institute to be reported with long-term follow-up addressing the decreased incidence of SSI and durability of chest wall reconstruction with the use of Strattice ADM in high risk patients. However, the limitation for this study is the relatively low number of patients from a single institute mainly due to the rarity of these presentations, the lack of available control group for direct comparison, and the requirement of potential objective analysis comparing pre- and postsurgery chest wall biomechanics. This study is driven to evaluate the indications and functional outcomes in patients undergoing chest wall reconstruction with bioprosthesis Strattice ADM, which would act as a safe alternative modality for chest wall reconstruction to resist infection in high risk patients with extensive defects. It should be considered as a valuable tool in the armamentarium of chest wall reconstruction.The authors acknowledge the efforts and expert input of the Medical Illustration Team at Heart of England NHS Foundation Trust.


Trace fossils of sediment bulldozers are documented from terminal Ediacaran strata of the Nama Group in Namibia, where they occur in the Spitskop Member of the Urusis Formation (Schwarzrand Subgroup). They consist of unilobate to bilobate horizontal to subhorizontal trace fossils describing scribbles, circles and, more rarely, open spirals and meanders, and displaying an internal structure indicative of active fill. Their presence suggests that exploitation of the shallow infaunal ecospace by relatively large bilaterians was already well underway at the dawn of the Phanerozoic. Efficient burrowing suggests coelom development most likely linked to metazoan body-size increase. These trace fossils are the earliest clear representatives so far recorded of sediment bulldozing, an activity that may have had a negative impact on suspension-feeding and/or osmotroph communities, as well as on matgrounds, representing early examples of ecosystem engineering and trophic-group amensalism. The occurrence of sediment bulldozers may have promoted the establishment of gradients in horizontal and vertical distribution of organic material in connection with spatially heterogeneous environments on the sea floor at a critical time in Earth evolution. Ediacaran ichnofaunas are typically dominated by surficial to very shallow, non-specialized horizontal trails5. These trails are interpreted as produced by vagile bilaterian metazoans that are thought to exploit organic matter concentrated within microbial mats. Other less common, and more controversial, components of Ediacaran ichnofaunas are paired scratch marks associated with the enigmatic body fossil Kimberella, which has been traditionally assigned to a molluscan-grade producer9. However, it has been noted that no unequivocal derived molluscan features are present in Kimberella10, and even its assignment to bilaterians has been considered equivocal11. Dickinsoniid trace fossils, suggesting external digestion of microbial mats, are also known14; an alternative interpretation involving passive transport15 is less likely16.Ediacaran benthic marine ecosystems were dominated by microbial mats that sealed the sediment from the water column essentially in the absence of bioturbation, and which have been referred to as matgrounds1. In turn, the notion of a Cambrian substrate revolution highlights the evolutionary and ecological effects of substrate changes on benthic metazoans18. The shift from matgrounds to mixgrounds is thought to have been signalled by an increase in the spatial complexity and heterogeneity of marine environments20. However, the impact of this evolutionary event is still controversial, with some authors emphasizing its significance for sediment mixing and ecosystem engineering23 and others interpreting that bioturbation did not play a major role until later in the Palaeozoic25. Also, ongoing studies suggest that patchiness in the distribution of organisms may have been significant in the Ediacaran as well27.The demise of extensive microbial mats formed in normal marine settings and the concomitant disappearance of matground-related lifestyles as a consequence of increased sediment mixing by burrowing animals have been referred to as the Cambrian agronomic revolutioncirca 1 cm wide or larger) vagile, deposit-feeding animals that displace substantial volumes of sediment while burrowing within the sediment29 would have been particularly effective in promoting such major changes in benthic ecology. Sediment bulldozers backfill their own tunnels using the material dug out in front of the body and transported to the posterior area, typically (but not exclusively) producing wide and flat trace fossils30. It is generally believed that these bulldozers are absent in Ediacaran and lowermost Cambrian rocks, represented by the Treptichnus pedum Zone, and that they first appear in the overlying Rusophycus avalonensis Zone, where they are accompanied by bilobed arthropod trace fossils that may have had a significant effect on incipient sediment-mixing. The Ediacaran–Fortunian Nama Group of Namibia, with its thick, laterally continuous, virtually undeformed and relatively well-dated shallow-marine successions, is ideally suited for this search. In this paper we document the presence of moderately abundant, almost 1 cm wide, trace fossils produced by sediment bulldozers in shallow-marine deposits of terminal Ediacaran age in the Spitskop Member of the Urusis Formation   Fig. . This fi31. Historically the Ediacaran–Cambrian boundary in southern Namibia has been placed within an unconformity at the base of the Nomtsas Formation , with pronounced valley incision that locally has eroded deep into sediments of the underlying Urusis Formation34. This initial location of the boundary was consistent with ash beds, from the lower and upper part of the Spitskop Member , from which zircons had originally been dated to 545.1 ± 1 Ma and 543.3 ± 1 Ma respectively33. However, recalibration of the Spitskop radiometric data indicates revised dates of 542.68 ± 1.25 Ma  and 540.61 ± 0.67 Ma (within error of the Ediacaran–Cambrian boundary), respectively35. An ash bed from the Hoogland Member towards the base of the Nama Group  has yielded an age of 547.4 ± 0.3 Ma33,36, now slightly modified to 547.32 ± 0.31 Ma35. The lower part of the Nomtsas Formation has yielded an age of 539.4 ± 1 Ma, recently recalibrated to 538.18 ± 1.11 Ma35. A negative excursion in carbon isotopes within carbonates above the highest recorded Cloudina in Oman37 has been dated to around 541 Ma. This prominent negative excursion in carbon isotopes, recognized globally at a level close to the Ediacaran–Cambrian boundary, has not been detected in the Nama Group. However, carbon isotopes in carbonates of the Schwarzrand Group are close to +2 per mil PDB, similar to values found in latest Ediacaran successions in China and Oman35. The available radiometric and carbon isotope data therefore concur that the Ediacaran–Cambrian boundary in the Nama Group is situated below the Nomtsas incision surface, and probably within the uppermost part of the Spitskop Member.There are no radiometric dates for the level of the basal Cambrian GSSP on Newfoundland, but it is currently considered to be 541 ± 1 MaCloudina and Namacalathus  as well as Ediacara-type fossils, including Pteridinium and Swartpuntia in the upper part of the Spitskop Member in Swartpunt Farm38. The branching burrow Streptichnus, which occurs in the upper part of the Spitskop Member39 but above the Pteridinium beds also in Swartpunt Farm, is now within error of the Ediacaran-Cambrian transition, and may in fact be regarded as a representative of Cambrian-style bioturbation. The Nomtsas Formation contains unambiguous examples of the Cambrian-type trace fossil Treptichnus pedum in various sections, most notably in Sonntagsbrunn Farm40.Palaeontological evidence for this placement of the boundary is the presence in various outcrops of the Kuibis and Schwarzrand subgroups of the early skeletal fossils 42 allows for correlation of the Urusis Formation across the Witputs subbasin in southern Namibia , both sequences have been removed by erosion due to incision of the Nomtsas Formation  of the Nomtsas valley incision have been depicted42. However, detailed mapping in the area indicates that the Huns, Feldschuhhorn and Spitskop members are laterally persistent following a west-east trend43.A detailed stratigraphic framework for the Nama Group41. The Spitskop Member records deposition in a mixed carbonate-siliciclastic platform. Regionally, carbonate facies are dominated by stromatolitic limestone, cross-bedded calcarenite, domal bioherms and pinnacle reefs encompassing from the mid shelf to the carbonate slope41. Carbonate deposits interfinger landwards with offshore, offshore-transition and shoreface sandstone44. Shoreface intervals in the Urusis Formation commonly contain evidence of oscillatory flows, such as hummocky cross stratification and wave ripples, the latter showing crests oriented northwest-southeast, parallel to the palaeoshoreline42. However, evidence of tidal influence has been noted as well41 and, therefore, some of these sandstone units may be better referred to as tidal beaches or tidally modulated shorefaces46. This is consistent with our sedimentological analysis of ichnofossil-bearing beds in the Koelkrans area of the Fish River Canyon, which indicates a tidal origin for the sandstone deposits here (see SI Appendix). The absence of structures indicative of oscillatory flows and the dominance of tidal structures in these proximal deposits may suggest deposition in a more protected embayment, where tidal effects were amplified and open marine waves buffered.Integration of regional information and local stratigraphy indicates that the trace-fossil beds in the northern Fish River Canyon area occur in the clastic interval present in the lower half of the Spitskop Member within depositional sequence DVendotaenia antiqua  to the ichnofossil locality, providing further support for its stratigraphic position.Placement of the trace fossil-bearing beds within the Spitskop Member  is further supported on palaeontological grounds. The Spitskop and Feldschuhhorn member beds at Koelkrans campsite, just a few hundred meters west of the new trace-fossil locality, contain abundant vendotaenids, such as Even assuming strong diachronism within the Urusis Formation, the ichnofossil-bearing deposits clearly occur below the subaerial unconformity at the base of the Nomtsas Formation . This is attributed to significant erosion even in interfluve areas as a result of sediment bypass during the sea level fall that took place in the basin during the earliest Fortunian combined with general pinch out of the carbonate belt in a landward direction, a feature that has been long recognized in stratigraphic analysis of the Nama Group44.The trace fossil-bearing beds occur within a sandy clastic interval of the Spitskop Member that passes abruptly upward into thin-bedded Spitskop Member carbonates recording a major deepening episodeParapsammichnites pretzeliformis n. igen. and n. isp. (SI Appendix) consists of unilobate to bilobate, actively infilled, horizontal to subhorizontal trace fossils describing scribbles, simple circles and, more rarely, open spirals and meanders  heterogeneous (i.e. patchy) distribution on stratigraphic surfaces reflecting the ability to detect and imperfectly exploit an area enriched in organics; (2) active backfill and sediment displacement as illustrated by an envelope zone; (3) consistent width along the same toponomic level; (4) course characterised by scribbles, nested loops, pretzel-like forms and common self-overcrossing; (5) lack of preferential orientation ; and (6) absence of frayed or angular terminations51.In contrast to the usual debates surrounding Ediacaran trace fossils and problematic formsParapsammichnites pretzeliformis indicate it was produced by a bilaterian. Giant protists are known to produce locomotion trails52, but these structures are morphologically simple and typically short. Experiments with modern actinians and platyhelminths that creep by cilia on mucus ribbons indicate that they may produce trails having lateral raised margins53. Some actinians may even produce trails displaying diffuse crescentic ridges54. However, these simple structures lack the internal complexity of Parapsammichnites, in particular the meniscate backfill locally organized in sediment pads and the bilobate structure. Equally important in supporting the bilaterian origin, there is no evidence that actinians or platyhelminths may produce scribbles, spirals or pretzel-like pathway patterns.In short, all morphological features of Taphrhelminthopsis” circularis55 and Psammichnites gigas30. A characteristic feature of these Cambrian forms is that they are commonly preserved as full reliefs or positive epireliefs on sandstone tops and they reveal a complex internal structure involving a thin axial groove  and a laminated backfill structure. The axial groove, locally with a sinusoidal development, has been interpreted as reflecting the passing of a snorkel-like device and prompted attribution to molluscs56. In any case, disregarding detailed morphological differences, affinities with these ichnotaxa are based on a similar way of exploiting the sediment and exploring substrate heterogeneities, clearly supporting a bilaterian origin and continuity of the “Psammichnites evolutionary lineage” across the Ediacaran-Cambrian transition55.The general pattern of the excavations analysed is strongly reminiscent of a group of relatively large trace fossils with global distribution during the early Cambrian showing looping, circling, and self-overcrossing, including “57. Claims of an earlier appearance of bioturbators representing ecosystem engineers58 based on the presence of supposed meniscate backfill structures in Nenoxites, remain controversial59. The structures consist of serially repeated modules, identical to those in problematic body fossils such as Helanoichnus, Palaeopascichnus and Shaanxilithes, arguing against a trace-fossil origin60.Because of the controversial affinities of most Ediacara-type body fossils, ichnological information is of paramount importance to track the earliest evidence of bilaterian organisms in the Neoproterozoic23, it is becoming clear that more sophisticated behavioural strategies (i.e. branching burrow systems) had appeared by the terminal Ediacaran in shallow-water settings61. Parapsammichnites pretzeliformis is distinguished from these forms, namely Streptichnus narbonnei39 and the so-called treptichnids61, by the absence of branching. The latter also distinguishes the trace fossils documented in this study from Treptichnus pedum, extensively recorded in the same area in lower Cambrian rocks34. From a palaeoenvironmental standpoint, all these more complex trace fossils of the Ediacaran-Cambrian transition occur broadly in a similar depositional belt, situated around the fair-weather wave base, either right above, under shallow subtidal conditions, or right below, in offshore transition to upper offshore settings.Although global diversity and morphological complexity of Ediacaran ichnofaunas is low64. This new ichnotaxon records a significant innovation in burrowing style at the Ediacaran–Cambrian transition, namely sediment bulldozing by metazoans of a size comparable to that of Cambrian animals. Body size is significant in biomechanics and physiology, representing a constraint on morphological complexity64, as well as playing a major role in ecosystem functioning65. The exploratory pathway pattern of Parapsammichnites indicates sediment reworking and processing of sediment common in Phanerozoic pascichnial structures, but essentially absent in Ediacaran simple trails and burrows, which typically display very simple patterns and only very rarely may show a tendency to form crude spirals66. Other factors being equal, sediment disturbance is directly linked to the cross-sectional area of the bioturbator29. In addition, it has been indicated that mouth size is also important in increasing sediment reworking because small mouths tend to be associated to selective ingestion of small volumes or organic-rich detritus29. Although any discussion of mouth size of the producer of Parapsammichnites would be completely speculative, we note that the Ediacaran–Cambrian transition has been regarded as a time of significant evolution in metazoan mouthparts, albeit in connection with predation rather than deposit feeding67.The backfilled scribbling trace fossils documented here indicate the onset of a new style of burrowing exploration that indicates the presence of a coelom, whose origin was presumably associated with body-size increase68 and representing an example of ecosystem engineering69. The resultant physical instability directly inhibits newly settled suspension feeding larvae, preventing attachment of sessile epifauna and facilitating remobilization of sediment that may clog the filtering devices of suspension feeders68. In particular, sediment bulldozing involves the forcible pushing-aside of sediment with the animal’s body or the manipulation of packages of sediment with appendages30. Sediment bulldozers are of paramount importance in ecosystem function71. Bulldozing by deposit feeders, as well as by scavengers and predators, is commonly conducive to remobilization of fine-grained particles and faeces, fluidization of muddy substrates, disruption of matgrounds, clogging of suspension-feeding systems, deepening of the redox discontinuity surface, and creation of dynamic heterogeneous environments, among other aspects68. On the other hand, bioturbators, through promoting transformations of the habitat, represent a dynamic force in the self-structuring and function of marine ecosystems72.In modern environments, burrowing deposit feeders tend to impact negatively on immobile suspension feeders to the point of excluding the affected group, illustrating a phenomenon referred to as trophic-group amensalism75. Following these approaches, the Namibia trace fossils represent colonisation by a shallow-infaunal, highly mobile, deposit-feeder, moving through the sediment in a conveyor belt fashion, backfilling and transporting sediment. This style of sediment disturbance has not been previously documented convincingly from Ediacaran deposits. Although the density of trace fossils on the here described bedding plane is moderate by Phanerozoic standards, it is high if compared with other Ediacaran occurrences, which tend to consist of very low density suites of simple grazing trails16, but see76 for an exception. Earlier models imply a more or less gradual increase in the size of bioturbators since the beginning of the Phanerozoic29. However, the trace fossils documented herein demonstrate that an increase in size of bulldozing trace fossils to widths of 5–10 mm had already started by the terminal Ediacaran 86. In either case, sediment bulldozing may have negatively impacted on these communities. Although immobile suspension feeders were abundant and diverse in Cambrian soft substrates, this trophic group is remarkably absent in Fortunian strata. In fact, fabrics dominated by high densities of dwelling burrows of infaunal suspension feeders (e.g. Skolithos piperock) are known since Cambrian Stage 2 shallow-marine deposits, but are unknown in older strata23. Similar exclusion of immobile suspension feeders by bulldozers has been invoked as a macroevolutionary driver later in the Phanerozoic29. More specifically, the gradual demise of sessile suspension-feeding communities throughout the Phanerozoic has been attributed to the increased diversification of deposit feeders87. The Nama occurrence probably represents one of the earliest examples of ecosystem engineering and trophic-group amensalism88.The co-occurrence of trace and body fossils in the same Ediacaran facies belts suggests that bilaterians and Ediacarans probably co-existed for almost 20 My without the former impacting negatively on the latterWhile evidence of matgrounds is extensive through most of the Ediacaran strata included in the Nama Group, there is a notable absence of structures indicative of microbial mats in the Koelkrans ichnofossil-bearing siliciclastic deposits. Although it may be argued that different depositional environments may have been involved, matgrounds in the Nama occur in similar nearshore heterolithic and sandstone-dominated deposits, such as those exposed in the Koelkrans area, arguing against a facies control. This fact is consistent with the notion that increased substrate disruption by infaunal organisms was conducive to the demise of matground-dominated ecosystems. However, abundant comb-like vendobiont-type tool marks in these beds suggest that elements of the Ediacara biota may have been present in nearby settings.20. The overall rarity of trace fossils attributed to sediment bulldozers in terminal Ediacaran rocks suggests that their ecological impact may have been limited and raises the issue of potential diachronism in evolutionary innovations across the Ediacaran-Cambrian transition. This is in line with the persistence of matgrounds in the Fortunian78 and the observation that primary sedimentary fabrics show very little disturbance in Fortunian strata90. In any case, the well-documented presence of trace fossils produced by infaunal bilaterians in the Ediacaran–Cambrian transition of the Nama Group clearly shows that the evolutionary lineages and behaviours that led to the more dramatic shifts in substrate properties later during the Phanerozoic were already present by the end of the Ediacaran.In addition, the occurrence of sediment bulldozers in subtidal settings must have stimulated the establishment of new gradients in horizontal and vertical distribution of organic material, thereby promoting spatially heterogeneity on the sea floor. Increasingly heterogeneous environments may have been conducive to more sophisticated foraging patterns and development of macroscopic sense organs in mobile bilaterians, allowing exploitation of patchily distributed resources91. Measurements were taken of a number of parameters, such as trace fossil width and penetration depth. Density of trace fossils on bedding planes was assessed after estimating a percentage of covered surface using Adobe Photoshop and ImageJ softwares92.This study was based on an integration of sedimentological, stratigraphic and ichnological datasets. A bed-by-bed sedimentological log was measured in the succession containing the trace fossils. Conventional facies analysis was performed and beds were characterised in terms of lithology, physical sedimentary structures, bed contacts, and bed geometry. Bedform dimensions were measured at all scales. Interpretations in terms of depositional processes and sedimentary environments were based on parameters assessed during facies analysis. The succession was placed into a broader stratigraphic framework following regional mapping and correlation. The latter was based on the application of sequence stratigraphic tools, including recognition and delineation of depositional sequences, systems tracts, parasequences and surfaces of alostratigraphic importance. Trace fossils were described according to standard ichnological practice, which included the use of the concept of ichnotaxobases, distinctive morphological features of a trace fossil that display significant and readily detectable variabilitySupplementary Information


MC3T3-E1 cells were cultured with BDNF and were examined for cell proliferative activity, chemotaxis and mRNA expression levels of osteoblast differentiation markers. For in vivo study, inferior alveolar nerve (IAN) injury experiments and mandibular cortical osteotomy were performed using a rat model. In the osteotomy model, exogenous BDNF was applied to bone surfaces after corticotomy of the mandible, and we morphologically analyzed the new bone formation. As a result, mRNA expression of osteoblast differentiation marker, osteocalcin, was significantly increased by BDNF, although cell proliferation and migration were not affected. In the in vivo study, osteopontin-positive new bone formation was significantly accelerated in the BDNF-grafted groups, and active bone remodeling, involving trkB-positive osteoblasts and osteocytes, continued after 28 days. In conclusion, BDNF stimulated the differentiation of MC3T3-E1 cells and it promoted new bone formation and maturation. These results suggested that local BDNF produced by peripheral nerve injury contributes to accelerating sclerotic changes in the alveolar bone.Brain-derived neurotrophic factor (BDNF), which is released due to nerve injury, is known to promote the natural healing of injured nerves. It is often observed that damage of mandibular canal induces local sclerotic changes in alveolar bone. We reported that peripheral nerve injury promotes the local production of BDNF; therefore, it was possible to hypothesize that peripheral nerve injury affects sclerotic changes in the alveolar bone. This study aimed to evaluate the effect of BDNF on osteogenesis using  Dental treatments, such as third molar tooth extraction, oral surgeries, local anesthesia injection and implant placement, sometimes cause inferior alveolar nerve (IAN) injury. Clinically, we can observe a delay in natural healing of the tooth-extracted cavity after IAN injury and radiographic findings regarding some delays in bone regeneration with neurological symptoms such as sensory impairment and dysesthesia have sometimes associated with sclerotic changes in alveolar bone and/or deformity of the mandibular canal  1,2]. W,2. W1,2]in vivo experimental models of IAN injury and osteotomy.BDNF is a family of neurotrophic factors, and it serves a function in the survival and differentiation of central and peripheral neurons . BDNF isRegarding the relationship between the development of mandibular bone and IAN, the formation of a mandibular canal is known to follow IAN development ,13. The in vitro cell culture and in vivo cortical osteotomy experiments in the rat mandible, as well as to elucidate the causes of sclerotic changes in the surrounding bone after IAN injury.This study aimed to prove the biological effect of BDNF on bone remodeling using in vitro studies  was selected) were calculated separately. Osteopontin-positive bone areas in the central areas of each specimen (a grid [357 x 238 μm2] was selected) were calculated using graphics software (Adobe Photoshop CS4 for Mac). Data were obtained from 72 areas of 24 samples  in the osteotomy experiments. All of the data are presented as the means and standard deviations (SDs) of each group. A difference was considered as significant if the p value was less than 0.05 level.Mann-Whitney We performed IAN transection experiments to examine the expression of BDNF and involvement of BDNF during bone healing after IAN injury. At 14 days after surgery, the nerve tissue regenerated and new bone formation was observed in the vicinity of the injured nerve lesion . Althougin vitro. BDNF did not affect proliferation of MC3T3-E1 cells Click here for additional data file.