Aging predisposes to increased morbidity and lethality to infectious diseases, which becomes apparent with the high mortality rates suffered by older people when infected with influenza virus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the root of this increased susceptibility to infections, is the widespread deterioration of the immune system.Early studies on immunological aging have mostly focused on adaptive immunity and found deficiencies in adaptive lymphocyte development and function. These include decreased T and B cell numbers and reduced effector and recall responses, leading to diminished anti-viral immunity . More rein vitro with West Nile virus had diminished nuclear translocation of STAT1, IRF7, and IRF1, resulting in decreased production of type one interferon (IFN-I) when compared to DCs from young controls [in vivo data, it is challenging to ascertain a causal relationship. However, the use of experimental mouse models provides a useful tool to investigate immunological aging.Because the innate immune system plays essential roles in the initial control of pathogens and also in the activation of adaptive immunity, age-associated changes in innate immune cells can potentially have a significant role in the increased susceptibility of the elderly to viral morbidity and lethality. Aging is known to affect human innate immune cells. For example, one study found that dendritic cells (DCs) from aged people infected controls . Similarcontrols . These eLike humans, aging increases the susceptibility of mice to morbidity and death after viral infections. For example, our laboratory has shown that while C57BL/6 (B6) mice survive footpad infection with ectromelia virus (ECTV) without significant disease signs when young, they gradually lose this resistance and all succumb to the infection once they reach 14 months of age. Years ago, we found that a reason for this increased susceptibility is that fewer natural killer (NK) cells get recruited to the popliteal draining lymph node (dLN) of aged mice. The result is a much more rapid systemic viral dissemination and uncontrolled viral replication in the liver and spleen. We also showed that NK cells in aged mice have an immature phenotype compared to young mice. Yet, the adoptive transfer of NK cells from young into older mice only partially restored their survival to ECTV infection . These rThe recruitment of NK cells to the dLN is one of many events that confer young B6 mice with resistance to footpad infection with ECTV. In a series of papers, we identified an intricate network of innate immune responses in the dLN that are indispensable for their resistance. We showed that following ECTV infection of young B6 mice in the footpad, infected and uninfected migratory DCs (mDCs) from the skin migrate to the dLN within the first 16 hours post-infection (hpi). At 24 hpi, infected mDCs in the dLN upregulate NKG2D ligands to activate group 1 innate lymphoid cells (G1-ILC) to produce interferon-gamma (IFN-γ). Simultaneously, infected and uninfected mDCs upregulate expression of mRNA for the chemokines CCL2 and CCL7, which recruit inflammatory MO (iMO) to the dLN. At the peak of iMOs recruitment (60 hpi), iMOs perform distinctive roles depending on their infection status. Uninfected iMOs produce CXCL9 in response to the IFN-γ secreted by the G1-ILCs to recruit NK cells to the dLN. Infected iMOs produce significantly less CXCL9, but are potent producers of IFN-I. Disruption of these events results in increased viral replication and death of infected mice [Because of their importance of mDCs and iMOs in recruiting NK cells and producing IFNs, we recently investigated whether aging may alter their function . We founIn summary, the lack of mDC accumulation in the dLN during the early stages of ECTV infection in aged B6 mice results in a highly dysregulate innate immune cascade. Consequences are reduced NK cell recruitment, inability to control early virus loads, rapid viral spread, and death from mousepox. Our data highlight previously unknown defects in innate immunity that contribute to increased susceptibility of the aged to viral infection. Future work could determine whether restoring mDC accumulation in the dLN can increase NK cell recruitment and reinstate ECTV resistance to aged mice. The results should open avenues to therapeutic intervention to reinvigorate the immune response in older people and promote their ability to control pathogens more effectively.


High-grade serous epithelial ovarian cancer (HGSC) is the most aggressive subtype of epithelial ovarian cancer. The identification of germline and somatic mutations along with genomic information unveiled by The Cancer Genome Atlas (TCGA) and other studies has laid the foundation for establishing preclinical models with high fidelity to the molecular features of HGSC. Notwithstanding such progress, the field of HGSC research still lacks a model that is both robust and widely accessible. In this review, we discuss the recent advancements and utility of HGSC genetically engineered mouse models (GEMMs) to date. Further analysis and critique on alternative approaches to modelling HGSC considers technological advancements in somatic gene editing and modelling prototypic organs, capable of tumorigenesis, on a chip. Epithelial ovarian cancer (OC) is the third most common gynaecologic cancer , with paPreclinical mouse models are essential for investigating carcinogenesis and testing new therapies, however, have proven somewhat difficult to achieve in OC. This is partly due to OC encompassing several distinct disease subtypes, each with a unique histological and molecular pathology. The four OC subtypes which have been classified histologically are mucinous, clear cell, endometrioid, and serous. In 2004, a broad classification separating ovarian cancers to type I and type II tumours was introduced , promptiHGSC accounts for the largest proportion of deaths from epithelial ovarian cancer, with over 70% of deaths being attributed to the disease ,37. HGSCThrough prognostic genomics it was shown that the immunoreactive subtype had the longest survival, the proliferative subtype had significantly worse overall survival than the immunoreactive subtype but obtained the greatest benefit from bevacizumab treatment, and that mesenchymal subtype patients had significantly worse overall survival than patients of the immunoreactive subtype . A studyInteractions between tumour cells and non-malignant stromal cells – such as endothelial cells, fibroblasts, adipocytes, mature and circulating progenitor immune cells – have been shown to promote tumour initiation, growth, and metastasis using both in vitro and in vivo models . The estThe path to developing a successful HGSC GEMM has been confounded by an initial lack of understanding of the cell-of-origin for HGSC. The primary consensus was that epithelial OCs, true to their name, originated in the ovarian surface epithelia (OSE) , howeverAmhr2/MISIIR). As such, these models did not specifically target the OSE or oviduct, leading to the development of uterine leiomyosarcomas, granulosa cell tumours, low-grade serous ovarian tumours or HGSC depending on the genes targeted [Early promoter-driven GEMMs relied on promoters that target the developing reproductive system such as anti-Müllerian hormone receptor 2  and oviductal glycoprotein 1 (Ovgp1). An inducible Pax8 promoter  was used by Perets et al. to target knockout of Brca1/2, Trp53 and Pten in the secretory cells of the mouse oviduct [Brca1 or heterozygous Brca2 mutations. Mutations to Brca1/2 resulted in significantly lower latency (13 weeks) than Tp53-/-/Pten-/- only mutants, with the models developing features consistent with serous tubular intra-epithelial carcinoma (STIC) lesions  and metastases patterns characteristic of HGSC. The success of this model is underpinned by the fact that PAX8 is a marker of secretory cells in the FT, but not the ovaries [Pax8 as a promotor for the development of oviduct derived HGSC GEMMs is limited by its lack of specificity as Pax8 is also highly expressed in the thyroid and kidney. Models using Pax8 driven perturbations in Tp53 and T121 have resulted in enlarged thymi resulting in respiratory distress and subsequent early lethality [More recently, promoters have been developed to target mutations to the oviductal epithelial cells. These include paired box 8  and mice with aberrant Brca1, Trp53 and Nf1 (BPN) mice had the highest penetrance characteristic of HGSC, with the former demonstrating shortest latency. Further characterisation of BPRN showed these mice develop tumours highly correlating in transcriptional signatures with human HGSCs of the immunoreactive and mesenchymal TCGA subtypes [Brca1 and Trp53 and the long latency to tumour development. Finally, tumour development in mice harbouring Brca1, Trp53 and Pten aberrations (BPP mice) was the most rapid and tumours displayed phenotypes not seen in the other genotypes such as mucinous metaplasia. Interestingly, these are the same genetic aberrations Perets et al. used in their Pax8-driven HGSC model. However, they did not observe these features, raising the possibility that Pax8 and Ovgp1 are initiating carcinogenic changes in different cell populations.  tissues  and repr tissues  crossed nic line  with varsubtypes . HoweverTrp53 mutation and Rb inactivation) to either the oviduct or the OSE to directly compare tumours arising from different cell lineages. This study was instrumental in not only showing that HGSC can arise in both the oviduct and the OSE, but that the cell of origin impacts tumour growth, metastasis and response to therapy. Importantly, as part of this study, they managed to develop a new promoter-driven model of OSE-derived HGSC using Lgr5; a gene involved with OSE homeostasis and repair, with concentrated expression in the ovarian hilum in adult mice [ERT2Lgr5Cre mice, Zhang et al. provide further evidence for the dual origin of HGSC tumours.Although later GEMMs have focused on FT/oviduct-derived HGSC, emerging molecular profiling suggests that the OSE may still represent the cell of origin for a significant proportion of HGSC ,60. Thesult mice . By succTP53, BRCA1/2, and RB, as well as alterations in genes not commonly associated with HGSC such as PTEN. It becomes clear from these GEMMs that to achieve the required genetic changes to induce HGSC in mice extensive breeding programs are required, which may act as an impediment for many researchers to use as they prove to be challenging, time-consuming, and expensive. To overcome this, SV40Tag has been widely used in the development of many GEMMs of cancer, including OC [Amhr2 promoter can induce ovarian tumours in a relatively efficient manner [Amhr2 promoter may provide limitations to its use as a model to recapitulate human HGSC. As demonstrated by the above models, GEMMs attempting to recapitulate serous epithelial OC have focused on known molecular pathognomonic markers, such as mutations or loss of uding OC ,65,66. It manner ,67,68. HDespite recent improvements to GEMMs of HGSC their widespread use has not eventuated, we surmise that this is due to several factors – extensive breeding requirements to introduce up to 4 or more genetic alterations to the OSE or FTE, long latency to tumorigenesis (in excess of 12 months in some instances), and instances of low penetrance and metastatic spread.GEMMs offer the ideal system to recapitulate spontaneous tumour development through a process of transformation of normal cells to development of precursor lesions before formation of invasive and metastatic tumours, which is why GEMMs remain the platform of choice for investigations into HGSC prevention strategies and early carcinogenesis. However, GEMMs may not be essential to researchers wanting to test new therapies and further investigate the biology of established tumours, metastases, and tumour microenvironment. To this end, there are other approaches in use and emerging that may appeal to researchers as an alternative to GEMMs due to the models’ higher efficiency and reduced cost  injection in order to mimic the widespread metastatic disease seen in HGSC patients. The other major methods of engraftment include sub-cutaneous (s.c.) and sub-bursal injection. S.c. injection allows for rapid tumour development and ease in measuring tumour volume with a caliper, whilst i.p. and intrabursal injection allow for a physiologically relevant microenvironment . HoweverTrp53, Brca1, Brca2, Nf1, and Rb1 [Trp53 knockout (Trp53-/-) and another double knockout model with the addition of Brca2-/-. It was shown that the Trp53-/- and Trp53-/-;Brca2-/- models had significantly shorter median survival than the native ID8 orthotopic model when grown i.p. in mice, with the single knockout having the shortest median survival of the two , Nf1 , Pten  and a triple knockout with Pten . When comparing survival in mice with Trp53-/- tumours, it was shown that there was no significant difference in survival of mice with Trp53-/-;Brca1-/- tumours (46 days), but Trp53-/-;Nf1-/- and Trp53-/-;Pten-/- tumours displayed significantly shorter survival  had significantly shorter survival (40 days) than Trp53-/- and Trp53-/-;Brca2-/- tumours (57 days), but longer survival than Trp53-/-;Pten-/-. Mutants containing edits to Brca1/2 were significantly more sensitive to PARP inhibition with rucaparib treatment in vitro than those with single mutations in Trp53 only. There was no significant difference in rucaparib sensitivity between Trp53-/-;Brca2-/- and Trp53-/-;Brca2-/-;Pten-/- cells in vitro and tumours in vivo. Whilst results for cisplatin therapy mirrored those of rucaparib in vitro, in vivo cisplatin administration lead to significant differences in survival times with mice harbouring the Trp53-/-;Brca2-/- tumours exhibiting the longest survival and Trp53-/-;Nf1-/- and Trp53-/-;Pten-/- tumours having the worst survival. Thereby demonstrating that tumours derived from differing mutations, will respond differently to treatment and highlighting the importance of choosing multiple syngeneic cell lines for preclinical testing of therapies. Further characterisation of these cell lines is required to determine which human HGSC subtype they most closely resemble.A follow up study , again usurvival . TumoursPax8-rtTA; TetO-Cre; loxP/loxPBrca1; mutTrp53; loxP/loxPPten GEMM [+ T cells in all four polyclonal cell-derived mouse tumours compared to human HGSC. Upon assessing the stroma, it was shown that the models demonstrated enhanced expression of collagens, matrisome glycoproteins, proteoglycans, ECM regulators, and ECM-secreted factors compared to omental controls. As with human HGSC tumours, those developed in the syngeneic mouse models had an increased expression of matrisome genes, comparable ECM stiffness, and a correlating increase in the Matrix Index [Four polyclonal cell lines derived from the oxP GEMM  backcrosoxP GEMM . The stuix Index . The uprix Index  in humanThe examples listed above demonstrate how a syngeneic mouse model can be used to circumvent the extensive and time-consuming breeding programs required to establish a GEMM, by using a more cost-effective transplantable model of HGSC derived from either the OSE or FT.TP53, and loss of RB1 and PTEN and a maintained methylation profile after passaging. It was further demonstrated that HGSC-modelling organoids can be genetically modified in vitro in a stable manner. Organoid lines can be retransplanted into an immunocompromised mouse as an orthotopic xenograft and have been shown to develop tumours that maintain high fidelity with the original tumour and organoid in bladder [Organoids are a way of modelling 3D multicellular interactions in vitro and have recently been gaining more attention for cancer modelling. Briefly, organoids are grown in culture using pluripotent stem cells or organ progenitors that differentiate and self-organise to form an organ-like structure that behaves much like the archetypical organ in vivo. Such cellular organisation is feasible due to the use of a 3D cell culture matrix comprised of Matrigel, small molecules and growth factors that mimics the basement membrane. Organoid lines derived from surgically resected primary human HGSC tumours and metastatic lesions have been developed from both FT and OSE origin . It shou bladder  and live bladder  cancer. Pax8 and Lgr5+ respectively, and shown to develop tumours comparable to human HGSC tumours. The utility of this model is compounded by being able to transform organoids in vitro to direct mutations to the right cell of origin and circumvent off-target issues associated with the use of the Pax8 and Lgr5+ promoters. Another promising feature of organoids includes the ability to form human microtissues from primary cancer cells [Another efficient manner of using organoids for HGSC modelling in vivo is similar to the syngeneic cell lines discussed above, wherein the organoid is developed directly from a GEMM, transformed in vitro and then transplanted back into recipient mice. This method has been carried out effectively by Zhang et al.  to develer cells .A common criticism of GEMMs in cancer is that many of them are developed by using promoters that are expressed embryonically, resulting in loss of tumour suppressor genes and/or expression of oncogenes early on in development rather than in the adult organ. This can be circumvented by using inducible CRE systems such as the TetOCre or tamoxifen-inducible CRE. However, as seen in the recent paper by Zhang et al. , at timeThe CRISPR/Cas9 system is a powerful tool capable of recognising specific gene sequences and inducing a double-stranded break through non-homologous end joining (NHEJ) to induce somatic gene edits without having to undertake the onerous, time-consuming and expensive crossbreeding involved with developing a GEMM. Further, by inducing such edits somatically in adult mice, CRISPR/Cas9 allows for a more accurate representation of the sporadic mutations typical of tumorigenesis. Modifications to the Cas9 nuclease has allowed for a wide scope of applications that have been comprehensively reviewed by Anzalone et al. . A signiTP53 knockouts in dog oviductal epithelia, wherein cells lost normal morphology, exhibited increased proliferation and DNA double strand breaks [TP53 knockouts had a decreased expression of PTEN and RB1 but no changes on BRCA1/2. This study is a promising first step in modelling HGSC on a chip, however further studies investigating interactions between the tumour and stroma are necessary to further validate this method.The tumour-on-a-chip systems are microfluidic devices that aim to recapitulate features of tumour physiology. The system substrate is commonly glass or optically clear polymers with perfused, hollow microchannels. Typically, two or more cell types are set up within these channels and the system can be set up to have the cell-to-cell interactions occurring directly or indirectly. The intricacy of this system and the addition of microfluidics adds a dimension of physiological mimicry and gives the researcher precise control over variables . The simd breaks . Upon geCompared to other tumour types, GEMMs of HGSC have been notoriously difficult to develop. Contention surrounding the cell of origin, the lack of robust and specific promoters, extensive breeding requirements, and long latency to tumour development have all contributed to these difficulties. Despite the development of several models that closely resemble HGSC, the aforementioned challenges have meant that these GEMMs have not been widely adopted for preclinical research. New technologies are rapidly emerging that will lead to the development of better GEMMs or provide strong alternatives for their use in preclinical research.


Aplastic anemia is rare disorder presenting with bone marrow failure syndrome due to autoimmune destruction of early hematopoietic stem cells (HSCs) and stem cell progenitors. Recent advances in newer genomic sequencing and other molecular techniques have contributed to a better understanding of the pathogenesis of aplastic anemia with respect to the inflammaging, somatic mutations, cytogenetic abnormalities and defective telomerase functions of HSCs. These have been summarized in this review and may be helpful in differentiating aplastic anemia from hypocellular myelodysplastic syndrome. Furthermore, responses to immunosuppressive therapy and outcomes may be determined by molecular pathogenesis of HSCs autoimmune destruction, as well as treatment personalization in the future. Acquired aplastic anemia is a rare disorder presenting with bone marrow failure syndrome due to autoimmune destruction of early hematopoietic stem cells and stem cell progenitors , 2. It hIt was originally thought that AA was associated with damage to the haematopoiesis leading to the loss/destruction of hematopoietic stem cells (HSCs) by the following identifiable processes such as autoimmune mechanisms, direct damage to HSCs by drugs, chemicals or irradiation; virus infection and some inherited or acquired clonal and genetic disorders resulting in peripheral pancytopenia and a profound hypocellular bone marrow composed of fat cells and stroma . HoweverBesides the primary role of cytotoxic T lymphocytes with the impact of interferon gamma and TNF on inhibition of hematopoietic stem cell colony production and enhance expression of FAS  receptor resulting in HSC immune-mediated destruction –19. Ther+ cells of patients with AA were observed [There are data on the intrinsic abnormalities of hematopoietic stem cells and progenitors in patients with AA. A decreased number of hematopoietic stem cells and their progenitors, measured as myeloid colony-forming cells, with defective stem cell function with regard to repopulating ability and increased apoptosis , 25, 26,observed . An undeobserved . Geneticobserved .Some recurrent somatic alterations have been demonstrated in patients with acquired AA. One of these alterations is copy number – the neutral loss of heterozygosity of chromosome arm 6 p (6p CN-LOH), which is the major histocompatibility complex (MHC) region reach in the multiple human leucocyte antigen (HLA) loci. The 6p CN-LOH is more frequent in AA compared to other bone marrow failure syndrome . The 6p RTEL1 (regulator of telomere elongation helicase 1) were identified in bone marrow failure, which included AA (1–2%), they were likely benign or of uncertain significance [Telomeres are protective chromosome caps that shorten during cell replication and following exposure to stressors. One biomarker that has been used to assess the cumulative effects of these conditions experienced in early and late life is the length of telomeres . Becauseificance .TERT or TERC mutations [FANCG), c-myb, c-myc, cyclin-dependent kinase 6 (CDK6), CDK2 and cyclins E and A which would also be consistent with the ultimate development of premalignant or aneuploid cells and conversion to MDS/AML [It has been suggested that telomere length at diagnosis is correlated with outcome –62. Moreutations , 65 and utations , 66, 67. MDS/AML . It has  MDS/AML . The ide MDS/AML , 70.In has been shown that sex hormones or other pharmacologic agents might reduce the risk of clonal evolution inducing therapeutic upregulation of telomerase .Although, a number of studies have reported that cytogenetic abnormalities occur from 4 to 13% of AA –76. It mIt has been reported that female patients with AA exhibited a clonal X-chromosome inactivation, not only a polyclonal immunoreceptor rearrangement pattern of T cell receptor gamma and an immunoglobulin heavy chain gene but also clonal somatic mutations in up to 32% of cases can be found –85.Although AA was regarded as a non-clonal disorder of hematopoietic stem cells and stem cell progenitors, recently several studies reported the high frequency of clonal abnormalities and haematopoiesis , 85–87. In most cases, the clinical relevance of somatic mutations in AA has been studied in the context of prognosis, response to the immunosuppressive therapy and differences between severe and non-severe AA and the risk of progression to MDS/AML. Kulasekararaj et al. postulated that somatic mutations are related to the transformation to MDS/AML which has been found in 40% of cases and also the longer duration of the disease in comparison to the group without somatic mutations . YoshizaOverall, clonal haematopoiesis of stem cell in the course of AA can be divided into two groups , 89, 91.The second group of patients is younger and it is characterised by the most commonly mutated genes in AA include PIGA, BCOR, BCORL1, which are known to be relatively benign/indolent mutations . The PIGIn general, in other clonal haematopoiesis these mutations are not found and are known to be unique to AA. Probably, they may promote the autoimmune response of the T cells. The second group of mutations is of great importance for clinicians because PIGA and BCOR may predict a favourable response to immunosuppressive therapy with improved progression-free survival and overall survival. Other mutations associated with a poor response to immunosuppressive therapy include: DNMT3A, ASXL1, JAK3, RUNX1, TP53, CSMD1 .Although some mutations may drive clonal expression in other disorders such as TET and JAK2, the impact on clonal evolution in patients with AA is less prominent.Due to analyses of the somatic mutations primarily involved in the pathways of immunity and transcriptional regulations, Babushok et al. reported somatic mutations in 72% of all analyzed AA patients including 66% of pediatric AA, while MDS-associated genes were only in 9% . One of The differences between patients with severe and non-severe AA in genomic features were recently reported by Patel J et al . AlthougImi et al. reported that in AA patients, hematopoietic stem progenitor cells (HSPCs) clones lacking an HLA class I allele which escape the cytotoxic T lymphocyte (CTL) attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations .The process of “inflammaging” refers to chronic, low-grade, sterile inflammation that develops in hematopoietic tissues with advanced age . This leThe increased incidence of AA in the elderly may be associated with age-related alterations in hematopoietic stem cells and progenitor cells (HSPCS)  includinInflammaging is also characterized by release of microvesicles and exosomes from stressed or damaged cells . These sAnother study reports a significant negative correlation between the expression of miR-144-3p and ten-eleven translocation 2 (TET2) which cause reduced osteogenic capacity of bone marrow mesenchymal stem cells taken from patients with AA, this may be a therapeutic strategy in future .There are several factors contributing to the destruction of hematopoietic stem cells in the pathogenesis of AA. Recent advances in understanding the molecular pathogenesis of AA, which tends to be a clonal disorder of hematopoietic stem cells and their progenitors with somatic cell mutations, including HLA-mediated immunity mutations, defective telomerase function, inherited and acquired cytogenetic aberrations, may allow, in some situations, for the prediction of the results of immunosuppressive therapy and a forecasting of an unfavourable outcome. Identification of somatic mutation predisposing to MDS/AML or accelerated telomere attrition preceded aneuploidy and malignant transformation in the course of AA may allow for the appropriate and well-timed management of patients at high risk of clonal evolution including stem cell transplantation. Figure


This study investigated the effect of non-periodized training performed at 80, 100 and 120% of the anaerobic threshold intensity (AnT) and a linear periodized training model adapted for swimming rats on the gene expression of monocarboxylate transporters 1 and 4 , protein contents, blood biomarkers, tissue glycogen, body mass, and aerobic and anaerobic capacities. Sixty Wistar rats were randomly divided into 6 groups (n = 10 per group): a baseline , a control group , three groups exercised at intensities equivalent to 80, 100 and 120% of the AnT  at the equal workload and a linear periodized training group (GPE). Each training program lasted 12 weeks subdivided into three periods: basic mesocycle (6 weeks), specific mesocycle (5 weeks) and taper (1 week). Although G80, G100 and G120 groups were submitted to monotony workload (i.e. non-modulation at intensity or volume throughout the training program), rodents were evaluated during the same experimental timepoints as GPE to be able comparisons. Our main results showed that all training programs were capable to minimize the aerobic capacity decrease promoted by age, which were compared to control group. Rats trained in periodization model had reduced levels of lipid blood biomarkers and increased hepatic glycogen stores compared to all other trained groups. At the molecular level, only expressions of MCT1 in the muscle were modified by different training regimens, with MCT1 mRNA increasing in rats trained at lower intensities (G80), and MCT1 protein content showed higher values in non-periodized groups compared to pre-training and GPE. Here, training at different intensities but at same total workload promoted similar adaptations in rats. Nevertheless, our results suggested that periodized training seems to be optimize the physiological responses of rats. Chronic exercise results in positive adaptations that occur throughout the body. This involves molecular adaptations including changes in gene and protein expression levels, which are regulated by metabolic processes . HoweverDespite the fact that use of an animal model provides a robust way to control independent variables, experiments with exercise commonly neglect to control for, and characterize, exercise volume and intensity. Thus, the “dose-response” to training intensity in this scenario remains unanswered. For example, some studies have compared the responses of animals to differing intensities of physical activities , 5, howeThe literature also lacks work in animals investigating the effect of modulating exercise intensity and volume (periodization) over a specific training period. In humans, periodization models are used to optimize gains associated with physical training . In animTraining adaptations are preceded by the molecular modulation of mRNA and protein levels, which directly affect physiological machinery during exercise. For instance, monocarboxylate transporters (MCTs) have an intrinsic relationship with physical performance and adaptation to physical training. Among the 14 isoforms previously sequenced , MCT1 anThus, the present study investigated the effect of non-periodized training performed at different intensities  and a linear periodized training model adapted for swimming rats by assessing morphological, physiological, molecular and performance adaptations. To investigate the effects of different training programs, we evaluated the protein contents, blood biomarkers, tissue glycogen stores, body mass, and aerobic and anaerobic capacities of these rodents. At the molecular level, we measured the gene expression and protein contains of monocarboxylate transporters 1 and 4 (MCT1 and 4) in different tissues (soleus and gastrocnemius muscles), due these proteins are known to be involved in the adaptation to both, aerobic and anaerobic metabolic changes. In our hypothesis, despite being trained at different intensities, rats will adapt similarly to training program if the same total workload is maintained. On the other hand, the periodized training will significantly enhance the effectiveness of adaptation as a result of their exposure to varied stimuli.ad libitum. Since rats are nocturnal, all handling procedures and exercise sessions were conducted during the dark period (~1 h after the lights had been turned off), and due to their high degree of light sensitivity, a red light  was used to reduce their exposure while allowing researchers to visualize and manipulate the animals . Insoluble materials were removed by centrifugation (10 000 × g) for 25 min at 4°C. Protein concentrations within supernatants were determined using the Biuret dye binding method. Supernatants were resuspended in Laemmli sample buffer and boiled for 5 min before separation via SDS-PAGE using a miniature slab gel apparatus . Electrotransfer of proteins from the gel to a nitrocellulose membrane was performed for 90 min at 120 V (constant). The membrane blots were probed with specific antibodies (anti-MCT1/bs-10249R and anti-MCT4/bs-2698R). Subsequently, blots were incubated with HRP-conjugated secondary antibodies . Results were visualized via chemiluminescence imaging  and band intensities were quantified using optical densitometry with ImageJ (1.50i) software. Band intensities were normalized to the loading control, α-tubulin.The body mass of each rat was measured weekly on the night in which rats were not exercised. Food and water intake were measured when water bottles and chow containers were refilled, which occurred every two to three days, before the supply of food and water of each cage ran out. Because rats were housed collectively, data regarding food and water intake was calculated as an average value per kilogram  per day.Data are presented as mean ± standard error of mean (SEM). Normality and homogeneity were verified using Shapiro-Wilk and Levene tests, respectively. One-way ANOVA was used for the identification of different training programs on dependent variables obtained post-euthanasia . Two-way ANOVA was applied to identify the effects of groups (intensities) and training periods on anaerobic threshold (AnT). When necessary, the Newman−Keuls post hoc test was used to detect the differences amongst groups. For all analysis, a 5% significance level was adopted.Aerobic capacity (AnT intensity) data were expressed relative to body mass overload at pre-training timepoints and after each mesocycle . RegardiWhen the exercise intensity of each day of training was fixed, the workload of each animal varied according to the total volume (duration) of exercise performed. Expected (blue) and performed (red) exercise volumes of each group during the 12-week training period are illustrated in th week (end of basic mesocycle). Food and water intake of all groups were reduced during training period, and no differences were observed between trained and control groups . All trained groups presented statistically reduced uric acid and creatine kinase values when compared to the GC group. Despite the fact that LDL levels were decreased in all trained groups, only mean LDL values of the GPE group were significantly lower than BL values and those of the GC group. Levels of FFA was also reduced in GPE and G100 groups vs BL (P < 0.05).Concentrations of biomarkers within blood samples are presented in Glycogen stores were not affected by training in predominantly oxidative  and glycExpression levels of MCT genes were not influenced by exercise. Some differences in levels were likely age-related, such as increased MCT4 mRNA levels observed in the gastrocnemius that occurred in trained and non-trained animals . Only MCOur results revealed that subjecting rats to the training models (periodized or non-periodized) resulted in significant adaptation to exercise at molecular, morphological and physiological levels. No differences were observed among animals that were subjected to non-periodized training regimes. The response of the GPE group to training was optimized. After training, the group had increased hepatic glycogen stores and reduced blood lipid levels, which confirms our initial hypothesis.The aerobic capacity (AnT) of animals of the GC group after the basic mesocycle were significantly decreased relative to the pre-training period. At the end of specific mesocycle and taper, LMT could not be used to determine AnT in animals of the GC group. It is possible that the AnT of the GC group decreased to below 4% bm , which resulted in the continuous accumulation of blood lactate and the absence of the U-shaped curve required for the determination of AnT . In two One of the main goals of the present study was to investigate the isolated effects of exposure to different training intensities in animals subjected to equal external workload levels. Animals of the G80, G100 and GPE groups completed 97.5, 92.8 and 96.6% of the total volume expected, respectively. However, rats of the G120 completed only 72.6% of the total volume expected. This resulted in a significantly reduced total workload of training when compared to other groups. This suggests that the internal workload increases proportionally to the external workload until the AnT is reached. While above AnT, the external workload has a significantly enhanced impact on internal workload. This hypothesis was supported by data showing that above AnT, blood lactate levels increase and intracellular pH decreases disproportionally to exercise intensity, which leads to hyperventilation and subsequent exercise interruption . This fith week of training, which persisted until the end of the experimental period. Thus, all different training models prevented the exacerbated gains in body mass that was observed in sedentary animals (GC). Exercise prevents obesity by enhancing the resting metabolism of animals and reducing food intake. Nevertheless, neither food nor water intake were significantly affected by exposure to training. Thus, we believe that the energy expenditure associated with physical training and alterations in the resting metabolisms of animals were the main factors that prevented increase in body mass.The natural growth of animals can be illustrated by the body mass values throughout the progression of experimental procedures . From thIn accordance with body mass results, subjecting animals to all training models positively influenced adipose tissue profiles. Trained animals had significantly reduced levels of retroperitoneal adipose tissue and increased BAT levels in comparison to animals of the GC group. These results are of great interest to health and/or performance-related experimental researchers. Since we were able to demonstrate that regardless of intensity or workload variability throughout training, chronic exercise reduces weight gain and levels of adipose tissue, which indicates that the body compositions of trained animals have improved ratios of lean and fat tissue. It has also been showed that BAT levels play a significant role in thermogenesis, metabolic regulation and body mass control. As a result, researchers have assessed whether altering diet  or takinIncreased albumin levels were found in both G120 and GPE groups. Albumin represents 50% of the total protein in the bloodstream, and plays an important role in the control of osmotic pressure, hormone levels and free fat acid (FFA) transport . TherefoDespite the fact that altered levels of LDH were not observed, CK levels of all trained groups were significantly less than those of the GC group. In both humans and animals, blood CK activity can be most prominent 24–72h after muscle damage , 29. Thu+ and the adenosine molecule in ATP. During exhaustive exercises, the inadequate ADP phosphorylation leads to increased levels of inositol monophosphate, which is then further converted to UA [Uric acid (UA) levels were also reduced in all trained animals when compared to animals of the GC group. UA is a metabolite of purine, which is a nucleotide that comprises DNA, coenzymes like NADed to UA . TherefoWe investigated the effect of exercise intensity and periodization on glycogen repletion. Hepatic glycogen stores of exclusively the GPE group were significantly increased after training in comparison to levels observed in other groups. Similar results were determined in another study that assessed swimming periodization in rats . IncreasMonocarboxylate transporters 1 and 4 were chosen as variables of interest due to their close relationship with pH regulation during exercise and metabolic adaptation in response to training , which cSeveral studies have reported that gene expression and protein levels of MCTs increased as a result of exercise , 33. TraMCT mRNA levels were altered in both trained and control animals, which may suggest that expression of the gene is age-dependent. MCT1 mRNA increased in a tissue- and intensity-dependent manner due to increased levels were observed in the gastrocnemius of animals of the G80 group. However, protein content in gastrocnemius was not altered. In contrast, MCT1 protein content was significantly increased in animals not subjected to periodized training in soleus muscle, even when MCT1 mRNA levels remained unaltered in the tissue. These results are in accordance with literature reports that have shown that regulation of MCTs may occur post-transcriptionally, and thus, protein content is not completely dependent in increased levels of gene expression , 39. AddTherefore, we tested whether reduced workloads that occur throughout the tapering period reduced MCT1 content and observed that levels post-taper were decreased. This suggests that the regulation of MCT1 content is transitory and dependent on the last training session experienced by the animal. Nevertheless, the reduction did not affect the aerobic performance (AnT) or physiological parameters of the GPE group.Finally, our results corroborate the literature regarding three conjectures: i) the expression of the MCT1 gene and protein are more sensitive to training than MCT4; ii) the protein content did not follow the corresponding gene expression, although our experimental design does not included a time course response; and iii) different intensities and training models affect the expression of the MCT1 gene and its protein content.According to our results, all training models successfully prevented typical decreases in aerobic capacity that are commonly observed in animals with sedentary lifestyles or age. At the molecular level, MCT1 isoform was more sensitive to physical training than MCT4, with mRNA increasing in G80 and protein content in all non-periodized trained groups. Further, all training models prevented body mass and fat gain, and reduced the appearance of muscle damage markers and purine metabolites. Apparently, training at different intensities but at same total workload promotes similar adaptations. Nevertheless, despite similar total workload, animals of the GPE group had significantly reduced FFA and LDL levels, and increased hepatic glycogen stores. Although observed differences were modest, these results suggest that periodization models may be used to optimize the physiological responses of rats to swimming training.S1 File(PDF)Click here for additional data file.


Despite the fact that different genetic programmes drive metastasis of solid tumours, the ultimate outcome is the same: tumour cells are empowered to pass a series of physical hurdles to escape the primary tumour and disseminate to other organs. Epithelial-to-mesenchymal transition (EMT) has been proposed to drive the detachment of individual cells from primary tumour masses and facilitate the subsequent establishment of metastases in distant organs. However, this concept has been challenged by observations from pathologists and from studies in animal models, in which partial and transient acquisition of mesenchymal traits is seen but tumour cells travel collectively rather than as individuals. In this review, we discuss how crosstalk between a hybrid E/M state and variations in the mechanical aspects of the tumour microenvironment can provide tumour cells with the plasticity required for strategies to navigate surrounding tissues en route to dissemination. Targeting such plasticity provides therapeutic opportunities to combat metastasis. Metastasis is the major cause of mortality associated with solid tumours. Tumour cells escape from the primary tumour mass, move through surrounding tissues, enter the circulation, and colonise distant organs to form secondary tumours. During this process, tumour cells have to navigate mechanical hurdles consisting of various extracellular matrix (ECM) structures and layers of cells. Cross talk between intrinsic properties of the tumour cells and mechanical aspects of their surroundings drives cellular plasticity that enables tumour cells to make this journey.2 The TME comprises cancer-associated fibroblasts (CAFs), blood vessels and lymphatic vessels, immune-inflammatory cells, and neuroendocrine and adipose cells, all of which are embedded in an ECM, a structural network that sustains and shapes the three-dimensional architecture of tissues and organs. Within the TME, tumour cells are subjected to chemical  and physical cues that originate from the cellular elements as well as from the ECM. Together, these cues impinge on cellular signalling cascades in tumour cells thereby promoting tumour development and metastasis.The cells of solid tumours are typically surrounded by a dense fibrotic tissue composed of cellular and acellular elements—the tumour microenvironment (TME)—which plays an active role in the aggressive metastatic behaviour of cancer.3 This process is orchestrated by signalling molecules such as transforming growth factor (TGF)-β and Wnt, which induce downstream pathways that regulate a network of transcription factors to control the balance between key epithelial proteins  and mesenchymal proteins (such as vimentin).5 Transcription factors such as TWIST, SNAIL and ZEB induce EMT whereas GRHL2 and OVOL2 suppress EMT.7 EMT is important in embryonic development for cell migration and regulation of tissue differentiation and homoeostasis,9 but has also been associated with cancer initiation, development, and progression.11 However, the idea that a full transition from an epithelial to a mesenchymal state is required for metastasis has been challenged by observations from pathologists and studies using genetically modified mouse models.14What triggers a cluster of tumour cells to transit to a motile state, crawl through surrounding tissues, and start the metastatic process? One concept is that this involves an epithelial-to-mesenchymal transition  cells may initiate movement is derived from active matter physics. It describes how changes in mechanical and geometric parameters such as extracellular pressure, cell density, and cortical tension, can trigger a shift from solid to fluid-like behaviour in cell clusters, without the need for transcriptional alterations such as those underlying EMT15 Fig. . This shHere, we focus on the early stage of the metastatic cascade where tumour cells leave the primary tumour, invade surrounding tissues, and enter the circulation. We present an overview of mechanical properties of the TME and discuss roles for  EMT and unjamming in tumour cell migration strategies. We then explore bidirectional cross talk between the TME and partial EMT and discuss how this may contribute to plasticity and unjamming. While a detailed description of underlying molecular pathways is beyond the scope of this review, we discuss candidate therapeutic opportunities for targeting the TME and the hybrid E/M state to break crosstalk and plasticity in order to interfere with metastatic strategies.2 Increased stiffness and pressure, both solid and fluid, are the main macroscopic mechanical alterations that can be observed in the tumour bulk.Tumour cells are subjected to multifaceted physical cues within the TME.2 This process triggers a series of intercellular feedback loops: tumour cells recruit and activate stromal cells; these stromal cells contribute to the increased production and secretion of ECM, which, in turn, stimulates tumour progression. Ultimately, these events result in a stiffer TME, which confers increased resistance to physical deformation. This alteration in tissue tensional homoeostasis has been reported to enhance cancerous transformation.17 The dysregulation of ECM deposition, named desmoplasia, involves not only changes in terms of ECM quantity, but also its architecture and organisation.18 In particular, the main components of ECM that are dysregulated and associated with cancer progression are fibrillar collagens, fibronectin and hyaluronic acid (HA).19 These alterations in ECM contribute to the increased stiffness of the TME, which has been associated with increased malignancy and invasiveness in pancreatic ductal adenocarcinoma, breast cancer, colorectal cancer and prostate cancer.24The components of the TME are not malignant per se—in fact, they are an important source of support for tissues in physiological conditions. However, as cancer progresses, many of these components are exploited by the tumour cells, causing a change in the mechanical properties of the TME. For example, CAFs can arise from resident fibroblasts and become activated in response to the release of growth factors such as TGF-β to acquire a tumour-promoting function.25 Expansion of the tumour bulk compresses tumour-associated blood and lymphatic vasculature, which, in turn, can affect the vascular integrity, ultimately leading to leaks and impaired drainage of lymphatic vessels. This impairment of the normal function of vessels leads to an increase in interstitial fluid pressure, which contributes to therapy resistance by inhibiting drug delivery to the tumour.26 In addition, impaired vascular integrity creates hypoxic regions, which induce activation of the transcription factor hypoxia-inducible factor (HIF)-1α, leading to tumour invasion and promotion of angiogenesis.27Besides alterations in stiffness, the mechanical TME is affected by increased solid and interstitial pressure as the tumour increases in size. ECM components such as HA and proteoglycans absorb water, which leads to an increase in solid pressure due to the resistance conferred by the surrounding tissue. In addition, proliferation of tumour cells generates solid pressure, as an increased uptake of soluble factors results in enhanced conversion into insoluble biomass.25 In a positive-feedback loop, the stiffer environment triggers an increase in actomyosin contractility and force application by tumour cells, causing further ECM stiffening.28 The tensile forces on the ECM also lead to the unmasking of new binding sites for integrins, further promoting cell–ECM interactions.29 In addition, tumour cells and CAFs remodel the ECM by enhancing collagen alignment through a process that requires contractility mediated by the GTPase Rho and its downstream effector Rho-associated kinase (ROCK), which has been associated with tumour invasion and attraction of vascular endothelial cells.32 Moreover, tumour cells can enhance crosslinking of collagen fibres in the ECM, which further augments stiffness of the tumour stroma. The main enzymes responsible for this crosslinking are tissue transglutaminase 2 and lysyl oxidases (LOXs), the expression of which is upregulated in several solid tumours. LOX enzymes, in particular LOX2, are upregulated in response to hypoxia and high levels of TGF-β, both of which are characteristic of the TME and associated with tumour progression and metastasis.34The physical alterations that occur within the tumour stroma are not just passive consequences of tumour growth. Tumour cells and CAFs actively change the mechanical properties of the TME through their interaction with the ECM. They adhere to ECM components through integrin receptors and use contractility mediated by the actin cytoskeleton and myosin motors to apply force onto these adhesions, causing cell-mediated deformation of the ECM proteins (termed strain stiffening), which contributes to the stiffening of tumour stroma.35 A force-transmitting cytoskeleton is essential for cells to sense the mechanical properties of the environment and several signal transducers have been implicated in this process, including ion channels, cell matrix adhesion complexes and membrane-associated phospholipases. Within cell matrix adhesion complexes, mechanoresponsive elements including integrin receptors and associated cytoplasmic proteins such as focal adhesion kinase (FAK)36 couple the ECM to the cytoskeleton across the plasma membrane, providing mechanical homoeostasis between cells and the ECM.37 In conjunction with chemosensory signalling pathways (such as those activated by TGF-β and hypoxia mentioned earlier), this bidirectional signalling controls cell shape and migratory and invasive behaviour, as well as cell survival and proliferation.39The altered mechanical cues in the TME help to create a niche that supports tumour growth, invasion of surrounding tissues, and therapy evasion. Tumour cells sense the above-mentioned mechanical changes and transduce the mechanical input into intracellular biochemical signalling.41 This ‘intra-tumour heterogeneity’ provides plasticity and confers a survival advantage on tumour cells to migrate, invade and reach distant organs.43 The conversion from a localised tumour into a full blown, disseminated cancer requires that tumour cells activate migration. EMT and unjamming represent two concepts explaining the acquisition of migratory capacity in tumours.Changes in the TME induce adaptive mechanisms, such as metabolic reprogramming in tumour cells, that, in addition to the intrinsic lack of homogeneity within tumours, contribute to the generation of tumour cell populations with diverse gene expression patterns and phenotypic features within a tumour mass.45 For example, ErbB2 is a metastasis-promoting oncogene that is frequently overexpressed in non-invasive ductal carcinoma in situ. However, only a subset of ErbB2-overexpressing cells progressed to invasive breast cancer in animal models and patient tumours and in this subpopulation ErbB2 was accompanied by overexpression of 14-3-3ζ, which led to EMT.46 The notion that EMT represents a critical step for the initiation of metastasis is challenged by the lack of evidence for EMT in the histopathology of metastatic tumour tissues as well as in several studies using animal models.48 For example, depletion of the key EMT-promoting factors SNAIL or TWIST in a mouse model for pancreatic cancer or lineage-tracing using Fsp1 as an EMT marker in a mouse model for breast cancer failed to support a role for EMT in metastasis.47 On the other hand, a study using loss of E-cadherin as an EMT marker in a mouse model for breast cancer, associated the occurrence of spontaneous EMT in a small subpopulation of tumour cells with increased migration capacity.48 The interpretation of studies in favour of- and arguing against a critical role for EMT remains an ongoing debate.50 Importantly, defining EMT based on the expression of a single marker underestimates the dynamic nature of EMT as this process is likely to be a transient event in cancer.51 Moreover, EMT is a non-linear programme that can be defined and controlled by distinct gene networks in a cancer-type specific manner.53 It has been shown that a pro-metastatic effect of EMT depends not only on the final state but on the molecular route that leads tumour cells to that state.54 The reverse process, mesenchymal-to-epithelial transition (MET), occurs as tumour cells arrive at distant organs, and might be important for the formation of metastatic lesions, as disseminated tumour cells locked in a mesenchymal state fail to effectively colonise these organs.57EMT can contribute significantly to tumour heterogeneity and plasticity and has been proposed to drive the initiation of metastasis.58 or mesenchymal stem cells.59 In addition, CAFs can arise from epithelial cells through EMT or from endothelial cells through endothelial-to-mesenchymal transition (EndMT) and both conversions are induced by TGF-β.61 It is largely unknown how these CAF populations differ in functionality, but they are all characterised by a myofibroblast phenotype that drives stiffening of the TME as described above.Notably, EMT also plays a role in other cell types in the TME including the generation of CAFs. CAFs can originate from normal resident tissue fibroblasts65 One advantage of maintaining an epithelial phenotype, such as expression of E-cadherin in a hybrid E/M state is an increased survival fitness through cell–cell contacts in tumour clusters in the circulation.66 Hybrid E/M is also associated with increased stemness, which, in turn, is linked to elevated plasticity and self-renewal capacities as compared with completely E or M states in breast cancer.68 Additionally, a tumour that harbours subpopulations of cells residing at different stages of a fluid, cancer-associated hybrid E/M state might have an optimal capacity to cope with variations in the TME and progress towards metastasis. A hybrid E/M state confers phenotypic and molecular diversity, which provides cellular plasticity, empowering tumour cells to navigate various physical hurdles during their journey to metastatic sites while maintaining expression of epithelial markers and intercellular adhesion.72 Indeed, in a mouse model for breast cancer, a hybrid E/M state induced the formation of tumour cell subpopulations with varying degrees of invasiveness and metastatic potential.63 The existence of hybrid E/M cell populations and their association with enhanced metastatic features including migration and intravasation, were corroborated by studies on ovarian and pancreatic cancers.74 A biophysical model also showed that hybrid E/M states give rise to heterogeneous clusters migrating collectively and leading to the circulating tumour cell clusters as observed in animal models and patients.75Rather than a complete EMT, transient subtle changes in the balance between pro- and anti-EMT transcription factors that result in a partial EMT or ‘hybrid E/M’ state might be more relevant in the context of cancer Fig. . Indeed,15 In this case, changes in mechanical and geometric parameters in the tissue trigger fluidisation in absence of EMT15  and moving states (“unjammed”) in cell aggregates.T15 Fig. . In epit83 Likewise, circulating tumour cell clusters can arise from collective cell migration and intravasation in renal cell carcinoma, lung cancer and invasive melanoma.86 Whether cluster invasion in the complete absence of a partial EMT fully explains these findings is unresolved. EMT-like changes have been detected in circulating tumour cells.87 Yet, clusters of circulating tumour cells are largely epithelial and evidence in favour of E/M hybrid clusters is still scarce, suggesting that unjamming of fully epithelial tumour tissues may occur.If and how the early steps of metastasis follow similar principles, represents an urgent, unresolved issue. In breast cancer, clusters of invading tumour cells are more prone than individual cells to survive. These clusters promote metastasis formation in mouse models and give rise to oligoclonal clusters in the circulation that are associated with poor prognosis in patients.88 Such movement has been suggested to be critical for cell mixing inside the tumour, which allows the most aggressive clones to effectively replace all other cells.89 In the outer regions, tumour cells are prone to mechanical stress due to a high abundance of ECM, which results in further unjamming.15Tumour cells in the centre of a tumour mass are likely to be jammed but increased pressure might drive a switch from a solid to a fluid-like state. Indeed, multiphoton microscopy in a spontaneous mouse model for intestinal cancer has shown coordinated migratory patterns in the tumour core that are indicative of a fluid-like behaviour.91 Likewise, unjamming of breast cancer cells triggered by a cascade of growth factor receptor internalisation, activation of extracellular signal-regulated kinase/mitogen-activated protein kinase and cytoskeletal remodelling, induces collective migration.81 Glioma cells infiltrate the brain as multicellular networks and breaking cell–cell interactions by downregulating p120-catenin was found to decrease infiltration capacity, again indicating that the ability to maintain cell–cell contacts is important.92 It is likely that the interaction between molecular programmes induced by hybrid E/M and local, physical cues in the TME creates routes for subpopulations of tumour cells to unjam and start disseminating.93Unjamming, as well as a hybrid E/M state, leads to a fluid-like migration of clusters of tumour cells that maintain cell–cell contacts. It has been reported that high expression of EMT-promoting transcription factors such as Snail and Twist leads to the collective migration of tumour cells that exhibit epithelial and incomplete mesenchymal features.94 Single cells can move through ECM networks by adopting amoeboid or spindle-like mesenchymal shapes:95 amoeboid cells generate few ECM adhesions and stress fibres whereas mesenchymal migration is associated with strong ECM interaction and actomyosin contractility.93 Collectively migrating cells adopt different morphologies such as sheets, strands, multicellular tubes and masses with irregular forms  cooperates with ECM confinement to drive unjamming, fluidisation and, ultimately, cell individualisation.101 Thus, the interaction between molecular features of tumour cells and local properties of the TME can drive metastasis by mediating interconversions between collective and individual behaviour , which facilitates ECM remodelling and regulates canonical signal transduction pathways that control cell fate.102 Mechanical cues from the TME, such as increased ECM density and stiffness, can stimulate EMT106 and act in concert with soluble EMT-stimulating factors, such as TGF-β.108 Important mediators of mechanically-induced EMT are the transcription factors TWIST1 and YAP/TAZ,110 which, upon matrix stiffening and subsequent intracellular transduction of mechanical signals, are induced to translocate to the nucleus to influence the expression of several genes that promote EMT  leading to the up-regulation of hyaluronic acid synthase 2 (HAS2) and increased HA production.113 Thus, the chemical composition and stiffening of the TME can promote  EMT in tumour cells. Notably, cells appear to possess a “mechanical memory” i.e., prolonged exposure to a stiff ECM causes EMT-like behaviour with nuclear localisation of YAP, high actomyosin contractility, and large cell matrix adhesions and this phenotype is maintained when the cells move to a soft environment for as long as the factors mediating the mechanical memory suppress a transcriptional switch.115Tumour cells sense and respond to mechanical stimuli from the TME.EMT Fig. .103,110–116 tumour cells themselves have been observed to be “more deformable” or “softer”.117 EMT might play a role in such softening of tumour cells. Cells undergoing EMT change their morphology, lose adhesive properties and undergo actin cytoskeletal rearrangement, which all influence cell stiffness and tension with neighbouring cells and the ECM.118 Mesenchymal-like cells tend to reduce their stiffness and become softer in response to force application, while epithelial cells are more likely to stiffen in response to the same force application.119 Accordingly, EMT-promoting transcription factors such as SNAIL and TWIST1 promote increased cellular deformability,120 which facilitates migration through ECM networks and intravasation.119Whereas stiffening of the TME drives EMT and the aggressive behaviour of tumours,121 This interaction is important for tuning the mechanical properties of the nucleus during migration in confined spaces. Indeed, nuclear deformability is a rate-limiting step for cell migration and some level of nuclear rupture has been observed during the migration of tumour cells in a confined space.124 The nucleoskeletal lamins regulate stiffness of the nuclear envelope and thereby determine a cell’s migratory capacity in confinement.125 How  EMT affects nuclear mechanics remains to be elucidated but a hybrid E/M will increase cellular and, perhaps, nuclear deformability to increase plasticity, allowing tumour cells to adapt to confinement and enhance migratory potential.Actin fibres connect integrin-containing adhesions with the nuclear envelope through the linker of nucleoskeleton and cytoskeleton (LINC) complex, thereby creating a physical connection between the ECM and the nucleus.127 The number of invadopodia—specialised actin-based membrane protrusions in which localised proteolytic activity degrades ECM—is also increased in tumour cells that are subjected to a stiffer environment or dense fibrillar collagen structures.129 Likewise, EMT induced by transcription factors including TWIST1 and ZEB1, promotes the formation of invadopodia in tumour cells.131 Thus, the interconnection between stiffening of the TME and EMT discussed above might enhance the ability of tumour cells and tumour-cell clusters to proteolytically degrade the ECM and break through tissue barriers. The importance of proteolytic ECM degradation, however, depends on the migratory strategy. While enzymatic breakdown of ECM is necessary for collective migration, individually migrating cells can either proteolytically remodel their surrounding ECM or adapt their shape to the already existing gaps.93 EMT driven by ZEB1 also leads to increased expression of LOXL2,132 which not only causes enhanced collagen crosslinking and TME stiffening but has been found to stimulate an EMT-associated transcription network,133 providing yet another positive feedback loop between EMT and the TME.As tumour cells undergo EMT, they also increase the production of soluble proteases or membrane-anchored MMPs, which allows invading tumour cells or tumour cell clusters to remove barriers or create tracks.135 vimentin,136 Rho/ROCK and actomyosin contractility137 and FAK.138 Notably, however, interfering with tumour–TME interactions can also have unexpected and undesirable effects. For example, whereas inhibition of FAK in a mouse model for pancreatic cancer attenuated the cancer-promoting activity of the fibrotic stroma, limited tumour progression and enhanced survival,138 depletion of CAFs, which might be expected to have a similar effect, actually led to more aggressive tumours and reduced survival.139 One explanation is the heterogeneity of CAFs in pancreatic and other cancers that may have diverse impacts on tumour growth and progression within the TME, including immune-modulation.141Interfering with the metastatic process remains a major challenge. Crosstalk between tumour cells and the TME is complex and dynamic and provides plasticity that allows tumour cells to adapt to different environments and escape therapy. We have discussed the mechanical interplay between the TME and tumour cells and a role for partial EMT in this process. Several candidate targets exist, which, when inhibited, might block this mechanical interaction and prevent tumour cell plasticity, including integrins,142 A network topology-based modelling approach has been applied to identify approaches for interfering with feedback loops in EMT networks, which may point to new strategies to interfere with plasticity and, hence with metastasis.143 Signal transduction cascades and transcription factors promoting a stable hybrid E/M state might serve as promising therapeutic targets, including GRHL2, OVOL2, NUMB and NRF2.145 Such a strategy has been successfully explored in breast cancer cells, in which the expression of SNAIL is associated with the hybrid E/M state. Deletion of SNAIL or either deletion or overexpression of ZEB1 pushed cells either in a complete E or in an M state, in each case resulting in attenuated capacity to form tumours.146 Despite these promising results, strategies that drive hybrid E/M cells into MET pose the risk of driving metastatic outgrowth of already disseminated tumour cells.57 On the other hand, strategies that lock cells in the M state might attenuate the outgrowth of primary and secondary tumours but drive the dissemination of individual tumour cells.56 An alternative promising strategy that exploits the highly plastic hybrid E/M state has made use of a combination of peroxisome proliferator-activated receptor γ (PPARγ) activation and MEK inhibition to enforce transdifferentiation of the tumour cells into post-mitotic adipocytes.147 This points to an exciting possibility that while plasticity allows tumour cells to adapt to different environments during metastasis it also represents a state that is vulnerable to differentiation therapy.Strategies that simultaneously target different mechanisms of tumour cell plasticity, including the hybrid E/M state, might prevent tumour cells from adapting to changes in the TME.In this review, we have discussed the dynamic interactions of tumour cells with the TME. In particular, we highlighted the importance of tissue mechanics and the role of  EMT in the early steps of the metastatic cascade. The TME provides a pathological mechanical environment that tumour cells sense and respond to. The initiation of the metastatic cascade requires acquisition of a migratory phenotype that is influenced by this environment. The role of EMT in this process is likely different in different tumour types and in most cases involves a partial EMT or hybrid E/M state. EMT and unjamming provide distinct mechanisms to initiate movement and to what extent hybrid E/M sets the stage for unjamming of epithelial tumour cell clusters is poorly understood. The hybrid E/M state provides tumour cells with plasticity affecting stemness, tumour growth, and migration, allowing them to navigate variations in the mechanical TME as they use collective strategies to invade local surrounding tissues and enter the circulation. It is the bidirectional cross talk between partial EMT-driving molecular programmes in the tumour cells and the heterogeneous local mechanical properties of the environment that drive the early stages of the metastatic cascade. Further insight into the dynamic nature of this process at different stages of the metastatic cascade is required. This will depend on integration of multiscale theoretical models, in vitro models incorporating tumour heterogeneity and relevant mechanical variations in the TME, and in vivo models that capture the full complexity of the metastatic process. Disrupting mechanical tumour–TME interactions and/or tumour plasticity at the level of the hybrid E/M state offers promising avenues for therapeutic strategies. In this area, we have only just begun to scratch the surface of what might be possible.


Of 54 SNPs differentiating East African strains, ten were under positive or episodic diversifying selection and 20 occurred in the clinical strain despite strong purifying selection at those loci. Candidate markers were in functional domains of the RNP complex (n = 19), the matrix protein (n = 4), on CDV glycoproteins (n = 5), and on the V protein (n = 1). We found mutations at two loci in common between sequences from three CDV outbreaks of clinical distemper in African lions; one in the signaling lymphocytic activation molecule receptor (SLAM)-binding region of the hemagglutinin protein and another in the catalytic center of phosphodiester bond formation on the large polymerase protein. These results suggest convergent evolution at these sites may have a functional role in clinical distemper outbreaks in African lions and uncover potential novel barriers to pathogenicity in this species.Canine distemper virus (CDV) is a multi-host pathogen with variable clinical outcomes of infection across and within species. We used whole-genome sequencing (WGS) to search for viral markers correlated with clinical distemper in African lions. To identify candidate markers, we first documented single-nucleotide polymorphisms (SNPs) differentiating CDV strains associated with different clinical outcomes in lions in East Africa. We then conducted evolutionary analyses on WGS from all global CDV lineages to identify loci subject to selection. SNPs that both differentiated East African strains and were under selection were mapped to a phylogenetic tree representing global CDV diversity to assess if candidate markers correlated with documented outbreaks of clinical distemper in lions ( Felis catus [Mustela putorius furo [Panthera leo [Lycaon pictus [Canine distemper virus (CDV) is a notoriously promiscuous multi-host pathogen that is typically associated with domestic dogs but capable of infecting taxa from at least twenty-two families across five orders . CDV is is catus ) to up tius furo ). Furthehera leo  African n pictus ,7). WhatCanine distemper in a susceptible host occurs as a result of two successive infection stages. The first stage involves virus invasion of host lymphatic cells which is achieved when the CDV glycoprotein hemagglutinin (HA) recognizes and binds the host signaling lymphocytic activation molecule receptor (SLAM). Subsequently, CDV is amplified and spreads systemically to secondary lymphoid organs and massively suppresses the immune system . Though As the initial site of host invasion, the biochemical compatibility between viruses and host cell receptors is often a major determinant of the susceptibility of a host to infection . HoweverThe role of viral evolution in driving CDV emergence in alternative hosts has been reported in observational studies and investigated experimentally. These studies find that specific amino acid changes at sites under selection in the SLAM-binding region of the viral HA are associated with distemper emergence in non-dog hosts ,20. WhilPanthera leo) are susceptible to CDV infection; however, the pathogenicity of CDV infection in lions is apparently variable. For example, serological evidence suggests that CDV infection in captive African lion populations without manifestation of distemper is not uncommon [African lions . uncommon , while sCo-infection with other pathogens can play a significant role in morbidity and mortality in CDV-infected hosts, given the massive toll that infection has on the immune system. Indeed, co-infection with Babesia was associated with higher mortality in lions during the 1993/1994 outbreak in the Serengeti lion population and in a second high-mortality CDV outbreak in the nearby Ngorongoro Crater in 2001 . SignifiIn the present work we identify viral genotypes correlated with clinical phenotypes of CDV in African lions, to explore the hypothesis that viral evolution facilitates cross-species pathogenicity in this species. Whole CDV genome sequences were generated representing clinical and apparently subclinical CDV strains in lions in East Africa. We report 28 candidate markers, single-nucleotide polymorphisms (SNPs) that differentiate clinical and subclinical strains in lions at loci experiencing selection pressure. Mutations at two candidate markers were found in common between three independent CDV outbreaks that were characterized by distemper clinical signs and distemper-associated mortality in African lions; one in the SLAM-binding region of the HA and another in a highly conserved region of the RNA-dependent RNA polymerase critical for polymerization during viral transcription and genome replication. Our results suggest a role for viral evolution in CDV pathogenicity in a novel host and reveal new insights into potential mechanisms for surmounting host barriers in this virus.n = 98) were sourced from public archives for subsequent analyses   and a sianalyses . The datTo identify loci that could be associated with CDV pathogenicity in lions we identified single-nucleotide polymorphisms (SNPs) differentiating strains in circulation in East Africa associated with distemper in lions  from those that were not associated with overt clinical signs . The clinical group comprised 17 sequences during a single outbreak during which lions exhibited distemper clinical signs and high mortality (1993–1994). The subclinical group comprised eight sequences collected over three periods of peak CDV infection in lions that occurred in Laikipia, Kenya in 2000 and in the Serengeti ecoregion 2006–2007 and 2011, during which no CDV clinical signs or increased mortality was observed in lions.n = 6), P (n = 6), M (n = 4), F (n = 7), H (n = 6), and L (n = 17), and in intergenic untranslated regions (UTRs) (n = 3). Three mutations occurred in the overlapping open reading frames (ORF) of P, V, and C. Thus, taking translation into account 54 total SNPs were observed. Nonsynonymous substitutions accounted for 22 (40.7%) of the SNPs, 28 (51.8%) were synonymous, and three occurred in UTRs (5.6%). The 54 SNPs observed between clinical and subclinical strains reveal loci that may have potential clinical relevance. If mutation at a locus improves CDV fitness in a given host, then we expect those sites to experience detectable selection pressures.In total, SNPs at 49 loci in the CDV genome differentiating clinical and subclinical strains were identified based on a multiple sequence alignment . SNPs beAll available CDV WGS spanning CDV geographic and host distribution, including those sequenced for this study and publicly available WGS, were analyzed to evaluate the potential role of recombination in shaping genetic diversity and to characterize selection pressures acting on individual loci across the CDV genome. Here we identify candidate markers of CDV pathogenicity in lions as loci that differentiated clinical and subclinical strains in East Africa and at which positive, negative, or episodic diversifying selection was detected.Nyctereutes procyonoides, Canidae) genome sampled in China in 2013 (GenBank Accession KJ994343). No putative recombinant CDV strains were associated with pathogenicity in lions. Putative recombinant strains were removed from selection analyses.Recombination analyses detected 16 unique putative recombination events in 11 sequences  from 119https://www.datamonkey.org/), identified 118 sites with pervasive positive or episodic diversifying selection (p ≤ 0.1) (p ≤ 0.1) with SLAC and FEL  representing all gene products using single-likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), and mixed effects model of evolution (MEME) methods on the Datamonkey Adaptive Evolution Server (p ≤ 0.1) , while 1 and FEL .Of the 54 loci differentiating clinical and subclinical strains in East Africa, 29 (53.7%) were subject to selection  accordinPhylogenetic analysis of partial H gene sequences (717 bp) revealed that two genetically distant CDV lineages were circulating in or near Dallas, Texas in 2013, when a clinical outbreak of distemper impacted African lions at a big cat rescue facility . A cladePhylogenetic analysis of 65 CDV WGS indicated that 10 previously recognized global clades were represented in our data A. SequenMapping SNPs at candidate markers to the tips of the CDV WGS phylogeny representing global diversity revealed that (1) nine of 28 loci segregating clinical and subclinical East African strains also segregated canids and non-canids during the clinical East African outbreak (including P/V280 which accounts for one site on two genes), (2) six candidate genetic markers  are shared between the 1994 Serengeti outbreak and a strain causing clinical distemper infection in lions and other big cats in North America in 2013, and (3) mutations at two candidate loci were found in all documented outbreaks of distemper in lions for which there is WGS data B.In East Africa, by definition, the consensus sequence of clinical strains differs from subclinical strains at all 28 candidate markers. However, nine of the 28 candidate markers differentiating clinical and subclinical East African strains also segregated the canid and non-canid sequences within the 1993–1994 outbreak, with three exceptions involving a single lion. Lion PLE658 shares the same SNP at 3 sites on the L gene as the sequences found in canids during the outbreak, shares SNPs at 23 candidate markers with other lions and non-canids during the outbreak, and shares one SNP with the subclinical genotype found in East African (no data is available for one candidate site from PLE-658) B.Mutations at six candidate genetic markers  are shared between the clinical CDV strain (of Africa-2) and a strain causing clinical distemper infection in lions and other big cats in North America in 2013. A third outbreak in African lions and other big cats in 1992–1993 shares two of these. CDV strains from both clinical outbreaks in lions outside of Africa belong to the America-2 global lineage. Thus, in the search for a genetic signature of pathogenicity, we found at least two and up to six loci associated with independent, clinical outbreaks in lions. Furthermore, candidate markers of pathogenicity in African lions identified in this study were also observed in other novel host species and vaccine strains across the phylogeny.In this study we sought to identify candidate viral markers associated with pathogenicity of CDV in African lions to gain a better understanding of potential barriers to clinical spillover in this species. Fifty-four SNPs differentiated genomes from CDV strains in circulation in East Africa during CDV infection peaks that were not apparently pathogenic in lions from genomes of CDV sequences which were pathogenic in lions. Of these, ten SNPs were at loci experiencing pervasive positive or episodic diversifying selection, and twenty occurred at loci despite the strong purifying selection acting on these sites. Most of these mutations mapped to functional domains of the RNP (polymerase) complex and matrix protein, implicating the processes of transcription and replication, and viral budding as potential barriers to clinical CDV spillover in lions. We investigated whether the mutations correlated with pathogenicity in lions in East Africa were involved in other CDV outbreaks affecting African lions in captive populations in North America. We found mutations at two loci in common between all available sequences from CDV outbreaks of clinical distemper in African lions—one in the SLAM-binding region of the hemagglutinin protein (Y549H) and a synonymous mutation in the catalytic center of phosphodiester bond formation on the large protein (710). These results suggest convergent evolution at these sites may have occurred allowing CDV to surmount species barriers, and potentially uncovered a novel mechanism of CDV pathogenicity in African lions.Our results support the hypothesis that viral genetic factors are associated with CDV pathogenicity in African lions. We found that 28 SNPs at loci under selection were associated with clinical strains of CDV in African lion populations in East Africa. During the catastrophic 1994 distemper outbreak in lions , 13 mutations separated sequences from canid and non-canid species . MutatioAlthough mutations at candidate markers identified here are correlated with clinical outcome of CDV in East Africa, it is not possible to determine whether these mutations have a functional role in clinical infection in lions with our observational data. One lion, PLE658, with an intermediate haplotype between canid and non-canid strains sampled during the 1993–1994 Serengeti outbreak, provides anecdotal evidence to this end. Although the sequence is ancestral to all other non-canid sequences, as determined by previous analyses of 21 time-stamped CDV sequences from non-canids and canids sampled between December 1993 and December 1994, this sequence was sampled from a lion found dead in the center of the study area during the middle of the outbreak . FurtherOur data identified candidate loci associated with clinical outcome in African lions within a single CDV lineage (Africa-2) and provided a basis for testing the hypothesis that a common mutational signature  explains the CDV pathogenicity in African lion populations  infected with other CDV lineages. Assessing the distribution of mutations at the 28 identified candidate loci over all available WGS revealed mutations at two sites associated with all outbreaks of clinical distemper in African lions in our dataset. These are a tyrosine to a histidine at residue 549 (Y549H) on the hemagglutinin protein and a synonymous mutation at residue 710 on the large protein.Our finding of Y549H in African lions is consistent with that of McCarthy et al. 2007), who found positive selection and an association of histidine at this site in non-dog hosts . Multipl07, who fDespite the observed associations of the Y549H mutation with non-dog hosts, previous work showed no clear association of histidine at 549 with sequences obtained from Felid species . Our resThe mutation at residue 710 occurs in the 223-amino acid, conserved region (CR) of the L protein, CRIII. This functional domain is host to the catalytic center of phosphodiester bond formation, i.e., where polymerization occurs during transcription and replication . AlthougOur phylogenetic results indicate that the 1992 and 2013 North American outbreaks were associated with the same global lineage, America-2. Thus, mutations at common sites between these two outbreaks may be identical by descent, rather than by parallel evolution. Nevertheless, that the same global lineage was responsible for two of three well-documented outbreaks in lions supports the hypothesis that viral genetic factors are associated with lethal CDV spillover in lions. Furthermore, this also suggests that America-2 could have an intrinsic ability to cause lethal infection in big cats.The observation of two mutations in common across all lion outbreaks may be conservative. The 1994 East Africa and 2013 North America outbreaks shared mutations at six loci, while the 1992 North America strain shared two of these. Sequences from 1994 and 2013 were sequenced from clinical samples, whereas the sequences from 1992 were isolated and passaged in various types of canine cells and canine cell lines. Thus, any mutations that conferred an advantage in lion cells may have reverted or been lost in adaptation to cell culture on canine cells.In addition to America-2, adaptive mutations found in the clinical East African group were shared with CDV sequences in other lineages, the majority of mutations at shared sites (78.8%) were found in non-dog host species and/or vaccines. CDV vaccines were historically made by repeat passaging of a clinical isolate in alternative host species in vivo and in alternative host cells in culture until the isolate acquired so many mutations that it was attenuated in domestic dogs. Thus, the distribution of mutations in common with the clinical strain in a diversity of species (including vaccines) suggests that some of the mutations at candidate loci in our analysis may not be specific to lion infections, but rather related to the adaptation to alternative host species more generally. This is consistent with the thesis of Nikolin et al.  that CDVWhile mutations at the candidate loci identified were not found to be in common across all outbreaks in lions, these loci may have functional relevance influencing infection outcomes. Experimentally testing the relevance of candidate loci identified here was outside of the scope of this study. However, the functional domains of CDV and other related Paramyxoviruses are well characterized through previous studies that manipulate viral sequence and observe the resulting change in phenotypes.n = 19), and on the matrix protein critical for viral spread (n = 4). Mutations in these functional domains suggest that these processes may present barriers to clinical spillover in African lions. All mutations identified in this study are mapped in relation to functional domains on a model of the CDV genome in The majority of the 28 candidate loci correlated with pathogenicity in African lions were located in functional domains of the RNP complex essential for efficient viral transcription and replication  that research suggests both binds L and is pivotal in its correct positioning for polymerization initiation ,54. ThusOn the L protein, six conserved domains  are associated with enzymatic activity of mRNA transcription and virus replication, e.g., catalyzing polymerization and mRNA capping, methylation, and polyadenylation  . In addiInterestingly, the gene with the second most mutations associated with the clinical strain was the matrix protein (M). The M protein is essential for viral assembly and egress from the infected host cell, i.e., onward transmission. M interacts with the cytoplasmic tails of the virus glycoproteins, F and H, the N-tail of N, and host cellular actin filaments to assemble virus particles at the cell surface . The rolPrevious work showed that, in vaccine strains of CDV, six mutations occur on the M gene as compared to the virulent wildtype, interrupting its function . RecombiAdaptive mutations on the two glycoproteins of CDV (F and H) associated with clinical spillover of CDV in African lions in East Africa were fewer than expected, with two mutations on the fusion gene (F) and three on the hemagglutinin (H). The H protein is responsible for recognizing and binding the host cell, and works in concert with proximal F proteins to fuse the cell and viral membranes. Because this process is required to initiate infection, and the SLAM binding region has variable affinity to SLAM receptors from different host species, genetic diversity in the SLAM binding region has been considered the primary determinant of CDV host tropism. Indeed, SLAM binding defines susceptibility and host tropism; however, it does not determine host pathogenicity. For example, CDV recognizes and binds domestic pig and cat SLAM, and replicates in SLAM-positive cells, but does not cause clinical disease ,63. ConsIdentifying genetic markers of clinical outcome in African lions is a useful predictive tool and can improve surveillance when (1) the mutations are necessary and sufficient to cause disease, and (2) are present in the susceptible host community before spillover occurs, i.e., “off-the-shelf” . SpecifiThe presence of a common genetic signature requisite for clinical spillover in African lions might be precluded if there exist multiple ways, i.e., mutations, to achieve the same phenotypic effect. This may be that different mutations or combinations of mutations in a certain functional domain will have the same effect on the process, or that mutations affecting different processes are sufficient to produce the same phenotype in vivo, i.e., a clinical outcome in lions. Identifying sites in the CDV genome under selective pressure in clinical outbreaks of CDV has an intrinsic value in highlighting viral processes that may pose a barrier to CDV spillover in lions and other species, even if a diagnostic signature is elusive.The search for genetic signatures predicting clinical outcomes of disease exposure is the holy grail of those interested in predicting and preventing poor individual and population level outcomes of infection. To adequately test whether a pathogen genotype predicts phenotype in a given host necessitates experimental validation through mutagenesis and challenge experiments . In thisTo assess whether adaptive changes in the CDV genome are associated with pathogenicity in lions, samples were acquired from animals involved in three independent CDV outbreaks causing observable symptoms of distemper in African lions. These included two outbreaks in captive populations of exotic big cats in North America in 1992  and 2013Panthera pardus), and one free-ranging raccoon (Procyon lotor) were stored at −80 °C and acquired for this study. Virus isolation methods are described in [In 1992, 35 of 74 big cats at the Wildlife Waystation  showed clinical signs of distemper including respiratory, gastrointestinal, and neurological symptoms. Of these, 17 died , including seven African lions. Virus isolates on canine blood lymphocytes from one African lion, one common leopard (ribed in .n = 4), and seizures and death (n = 1). Samples from lions at the facility were not available, though a CDV-positive urine sample (by RT-qPCR) from a recovered tiger (Panthera tigris) from the same facility was obtained. Wild and domestic carnivores were sampled from the area contemporaneously. Tissue samples from wild rabies-suspect, rabies-negative mesocarnivores were acquired from the Texas Department of State Health Services including raccoons (n = 4) and a gray fox  collected between April and May 2013. CDV-positive cDNA was obtained from IDEXX Laboratories, Inc. from domestic dogs  that were sampled between November 2012 and December 2013 by Dallas Animal Services.In May and June of 2013, 21 captive big cats at a facility in Texas developed distemper disease, including five of eight resident lions . In totan = 1) and African wild dog  were collected in the Serengeti ecoregion in 2006 and 2007, respectively. Black-backed jackals  and domestic dogs (n = 2) were sampled at a ranch in Laikipia, Kenya in 2000 [n = 21) were published previously and include domestic dogs, bat-eared foxes , spotted hyenas (Crocuta crocuta), and African lions.Serological studies in the Serengeti ecoregion and in nearby Laikipia, Kenya, indicate CDV infection peaks in lions and other carnivores with no observed distemper disease or distemper-specific mortality in African lions ,26. SampAmplicon-based deep sequencing was used to generate near whole-genome sequences following previously described methods . All seqA partial H gene sequence (717 bp) was generated from a tiger that initially suffered loss of appetite, loose stools, and seizures, apparently recovered over 10 months, then suddenly and rapidly declined with undiagnosed neurologic symptoms and died. Viral RNA was extracted from a urine specimen from this tiger using the QIAamp Viral RNA Mini Kit  according to manufacturer’s instructions. A one-step RT-PCR reaction using previously published primers was performed using SSIII One-Step RT-PCR with Platinum Taq . The RT-PCR reaction conditions were as follows: 10 min at 55 °C, followed by 2 min at 94 °C, then 40 cycles of 30 s at 94 °C, and 30 s at 48 °C, and 2 min at 68 °C. Each reaction used 2-μL total RNA extract in a 50-μL volume reaction. Oligonucleotide concentrations were used according to the manufacturer’s instructions. Sequences were generated using the Applied Biosystems Automated 3730xl DNA Analyzer with BigDyeTerminator chemistry.n = 21). During this outbreak, CDV sequences collected from canid hosts (bat-eared fox and domestic dog) differed from sequences from non-canid hosts (African lion and spotted hyena) by 13 SNPs [To identify loci that may be associated with the clinical outcome of CDV in African lions, we first identified mutations differentiating the circulating CDV strains in East Africa with apparently different clinical outcomes in lions between 1992 and 2011. If CDV evolution has a functional role in cross-species pathogenicity, then sequences collected during an outbreak with observable distemper symptoms in lions  will differ from sequences collected during infection peaks with no observed distemper symptoms in lions  at functional domains. The East African sequence data were divided into two groups: clinical and subclinical. The clinical group comprised all CDV WGS collected during the outbreak in the Serengeti Ecological Region between December 1993 and December 1994, including domestic dogs and wild carnivores  software , includip-value cutoff of p ≤ 0.10 was used to determine significance in all analyses. This cutoff was used because the null model used for comparison in these methods assumes neutral evolution, which is likely to be violated by RNA virus evolution, which is dominated by purifying selection. Selection acting on mutations at intergenic sites was not investigated because the selection detection methods used only evaluate protein-coding sequence.Pervasive positive and negative selection, and episodic diversifying selection were assessed on a site-by-site basis, i.e., at each codon, using a counting method, SLAC, a fixed effects likelihood method, FEL, , and a mHigh-quality DNA for whole-genome sequencing was not available from any big cats infected during the outbreak at the North American facility in 2013; however, a partial H gene sequence was obtained from a recovered tiger. In addition, several WGS were generated from CDV strains in circulation in wild and domestic carnivores in the vicinity in the same year. To determine if these contemporaneous WGS sequences could be used as a proxy for the CDV strain causing the outbreak in the big cat population, a phylogenetic analysis of partial H gene sequences, including the contemporaneously collected wild and domestic carnivores, the recovered tiger, and publicly available partial H gene sequences from across the global distribution of CDV, was performed. Tests for the best-fit model of nucleotide substitution were implemented in jModelTest  and the To assess which candidate genetic markers were associated with discrete outbreaks of clinical distemper in lions and whether the strains causing these outbreaks were genetically independent, we performed phylogenetic analyses of CDV WGS using the methods above and mapped the mutations to the tips of the branches. The complete CDV WGS dataset was pruned to improve visualization by including only one sequence per species, per country of origin, per year. The frequency and distribution of mutations at candidate loci in host species and in space were visualized by mapping them to the tips of the phylogeny.