Altogether, we reveal a self-organising feature of the lateral line system that ensures a proper distribution of sensory organs along the body axis.Animal organs are typically formed during embryogenesis by following one specific developmental programme. Here, we report that neuromast organs are generated by two distinct and sequential programmes that result in parallel sensory lines in medaka embryos. A ventral posterior lateral line (pLL) is composed of neuromasts deposited by collectively migrating cells whereas a midline pLL is formed by individually migrating cells. Despite the variable number of neuromasts among embryos, the sequential programmes that we describe here fix an invariable ratio between ventral and midline neuromasts. Mechanistically, we show that the formation of both types of neuromasts depends on the chemokine receptor genes  Summary: Two parallel sensory lines in medaka share a common origin and are composed of identical organs that are, nevertheless, generated through different morphogenetic programmes. Oryzias latipes).The diversity of body forms across the animal kingdom is astonishing. Even within taxa, species show major differences in body size and relative shape. Teleost fish are arguably the most extreme example of such diversity among vertebrates, considering the range from a seahorse to an eel, a pufferfish, a tuna and a zebrafish. When reviewing all these diverse forms, we wondered how peripheral sensory systems could adapt to such dissimilar body shapes. Here, we tackle this question by focusing on the embryonic formation of the posterior lateral line (pLL) system in medaka  promoter to follow the dynamic formation of the pLL in vivo. We observed that neuromasts of two parallel pLLs in medaka are formed in a sequential manner by cells coming from a common primordium. The first set of organs are deposited from the rear of a migrating primordium and shortly after start to migrate ventrally forming the ventral pLL. A second set of neuromasts is formed by ICs and cells that escape from previously formed organs, organise into a cluster and migrate dorsally to form the midline pLL. We demonstrate that Cxcr4b and Cxcr7 are used iteratively: initially to drive the migration of the primordium and afterwards in individual cell migration during secondary organ formation. Finally, we show that this mode of organogenesis, whereby already generated neuromasts can produce second generation organs, is controlled within the lateral line and propose that this could facilitate the adaptation of the sensory system to the variety of body shapes found among teleost fish.We generated a transgenic line in medaka that specifically labels neuromasts using the Astyanax, in which most neuromasts are found along one line   B,C. The 22 pLLs) C. The alin vivo, we created transgenic lines in which the promoter of the medaka eyes-absent 1 gene (eya1) drives the expression of different fluorescent proteins. Previous studies indicated that eya1 is a bona fide marker for the lateral line in medaka, as eya1 mRNA in situ hybridisation labels neuromasts during embryonic and early post-embryonic stages . The alternated distribution of neuromasts in Tg(Eya1:EGFP) embryos was identical to non-transgenic controls , with around ten neuromasts in the ventral pLL  and between six and seven in the midline pLL . All mature neuromasts from the different post-embryonic lateral lines were labelled by Tg(Eya1:EGFP) and by Tg(Eya1:H2B-EGFP) as indicated by co-staining with specific vital dyes  for the differentiated neurons of the organ . In addition, our transgenic lines allow the visualisation of immature neuromasts that have not yet generated differentiated cells . Therefore, Tg(Eya1:EGFP) recapitulates the endogenous eya1 expression and allows neuromast formation to be visualised in vivo.To follow lateral line formation in medaka c stages C  to follow embryonic pLL morphogenesis dynamically, and observed a primordium that delaminates near the otic vesicle from the lateral line placode at stage 26 and migrates posteriorly (Eya1:EGFP) embryos indicated that the neuromasts produced by the primordium migrate ventrally and form the ventral pLL (Movie 1). Intriguingly, we could never detect a second primordium as reported in zebrafish and, yet, we could observe the appearance of about seven midline pLL neuromasts distributed along the myoseptum on the following days  embryos were developed until juvenile stages. We observed that although the control side displayed complete ventral and midline pLLs, early primordium ablation on the experimental side resulted in the absence of all neuromasts both on the ventral and the midline pLLs  (n=4)  A,B. Lates) (n=4) C, schemes) (n=4) C. These Eya1:EGFP), we observed that ventral migration of the neuromasts occurs soon after the primordium deposits each organ (Movie 1). Therefore, the most anterior neuromasts start their ventral migration while the primordium is still on its way towards the tail (Movie 1). After the initiation of ventral migration, we detected the appearance of new neuromasts (hereafter termed secondary neuromasts) between the ones that were originally deposited by the primordium (hereafter termed primary neuromasts) (Movie 2), and reach their final position just dorsal to the myoseptum  A,B. The romasts) A,C, exclyoseptum C. Overalyoseptum C.Fig. 4Eya1:EGFP) ranges from five to eight  and so do larvae displaying eight secondary neuromasts . This observation suggests that the number of secondary neuromasts is not decided by the empty spaces in the surrounding tissues (the occupancy theory), but rather is an intrinsic property of the lateral line systems.The formation of secondary neuromasts could be driven by signals outside or within the pLL system. Empty inter-somitic spaces could function as a call for the generation of secondary organs , but ourto eight D. Larvaen=14 secondary neuromast formation in 11 embryos). Intriguingly, instead we observed that all six to eight ICs group together in the middle of two consecutive primary neuromasts (Movie 3), start dorsal migration (Movies 2 and 4) and later form a secondary neuromast. In addition, +eya1 cells escape from primary neuromasts and get incorporated in the forming secondary organ (Movies 2 and 4). We observed migration of cells from primary neuromasts in all cases of secondary neuromast formation  and using different transgenic lines . Our movies also indicate that primary neuromasts contribute cells that join secondary-forming organs from both sides. Altogether, our data shows that secondary neuromasts in medaka are formed by two types of individually migrating cells, i.e. +eya1 ICs and +eya1 cells from primary neuromasts.Our previous observations demonstrated that the mpLL neuromasts are formed after vpLL neuromasts, which temporally resembles the formation of new neuromasts from ICs in zebrafish . There, cxcr4b and cxcl12a in medaka . At later stages, however, we noticed cxcr4b expression in the ICs , in the migrating cells that form the secondary neuromasts and in the secondary organs (cxcr4b:EGFP) embryos  to show EGFP+ cells that migrate from primary to secondary organs (Movie 5). By the time secondary neuromasts reached their final position, cxcr4b expression was restricted to secondary neuromasts and absent in primary ones . The dynamic expression of cxcr4b suggests that the same chemokine receptor involved in collective migration of the primordium might play a later role during secondary organ formation.Secondary neuromasts are generated by individual migration of ICs and cells from primary neuromasts. The Cxcl12a/Cxcr4b/Cxcr7b pathway is crucial for the collective migration of the primordium  and for n medaka  largely y organs A,B. We cary ones B. The licxcr4b mutant kazura, which exhibits severe phenotypes in the pLL due to the impaired migration of the primordium , a proportion manages to generate up to five neuromasts  , all were primary organs  . In the y organs D. We onlin situ hybridisations for cxcr7, a second receptor for Cxcl12a that in zebrafish is necessary for generating a signalling gradient along the primordium by acting as a sink for Cxcl12a at the rear . At later stages, however, cxcr7 is not observed either in the ICs or in forming secondary organs  although a fifth of the mutant larvae still display eight or more primary neuromasts . Among these, however, most larvae showed no secondary neuromasts  (n=12 pLLs). Our results indicate that primary neuromasts in yanagi mutants fail to produce secondary organs, and therefore indicate a role for Cxcr7 in secondary neuromast formation.Next, we carried out the rear . We detey organs E,F. The   G and thekazura and yanagi mutants, we followed the dynamics of ICs in both genotypes. Live imaging of Tg(Eya1:EGFP) kazura mutants revealed that most ICs, instead of grouping together in between organs, migrate back to the closest primary organ (Movie 6) (n=8). Secondary organs therefore fail to form in kazura mutants because there are insufficient ICs in place (Movie 7). yanagi mutant ICs, however, are seen dynamically shuttling between primary neuromasts, but are capable of neither coalescing nor being picked up by those organs (Movies 8 and 9) (n=5). Our 4D data on the mutants strongly implies that Cxcr4b and Cxcr7 have distinct roles during secondary organ formation.To understand how secondary organs fail to form in /cxcr4b chimeric primordia, wild-type cells are maintained at the front of the primordium whereas mutant cells located in the middle and rear part eventually form mutant organs  directed against cxcr4b. Both gRNAs worked in vitro and in vivo on the cxcr4b locus  and similar approaches have been successfully used in medaka to generate F0 chimeras (n=198) (cxcr4b in 100% of the cases (7/7 embryos), therefore linking the aberrant phenotype to the mosaic genotype.In zebrafish wild-typet organs . In ordechimeras . We obse (n=198) A,B, with (n=198) B. We gen+ cells were transplanted into either kazura or yanagi mutants. We observed that the transplanted wild-type cells rescue the full migration of the primordium in both mutants (n=5 larvae each). Furthermore, wild-type cells also rescued the generation of mature secondary organs in kazura and yanagi backgrounds  and all but one secondary organ generated in yanagi mutants  contained wild-type EGFP+ cells. Altogether, our results show that Cxcr4b and Cxcr7 are used iteratively during posterior lateral line formation in medaka, initially driving the collective migration of the primordium and later in secondary neuromast formation.To explore secondary organ formation further, we generated chimeric embryos in which wild-type EGFPkgrounds C-F. RescRemarkable plasticity in body shape has made teleost fish a very diverse animal class, containing roughly 80% of all vertebrate species. This plasticity comes with inherent problems, i.e. the changing ratio among organ sizes, different proportions of cell types within and expanded regions that need to be innervated and vascularised. We dynamically followed the posterior lateral line formation in an uncharacterised species to reveal a counter-intuitive morphogenetic event, in which a ventral pLL is generated along the myoseptum and migrates ventrally, whereas the midline pLL is generated at ventral positions and migrates dorsally.n=90 pairs of pLLs). The same genetic information can therefore result in different morphological outputs on each side of the same organism. How is the relative number of midline and ventral pLL neuromasts defined? If the two lines compete for empty spaces along the medaka trunk, we would expect that a fish with a high number of primaries would generate more secondaries, and one with fewer primaries would need several secondaries to occupy all available positions. However, a pair of primary organs generates just one secondary neuromast, regardless of the total number deposited by the primordium. Taken together, our data indicate that the decision to generate a secondary organ resides within the lateral system and is not triggered by external cues. The autonomous control of the lateral line organs was highlighted by Ghysen and Dambly-Chaudière in the context of neuromast size . Interestingly, this process does not involve cell proliferation, which resumes only when secondary organs reach their final destination . We report a novel way to make embryonic neuromasts that results in a new, separate lateral line. Our data using kazura and yanagi mutants indicate that these receptors are being re-utilised by the system to generate more neuromasts, although mediating different processes, i.e. individual cell migration. Overall, eya1-expressing cells use the same chemokine receptors to generate identical neuromasts in two different manners.There are three known ways of generating a fish neuromast during homeostatic conditions: (1) direct deposition by the primordium during early embryogenesis , (2) pro/kazura and wild type/yanagi revealed that wild-type cells can rescue both primordium migration and secondary organ formation. The distribution of wild-type EGFP+ cells along the pLL of each mutant suggests similar roles of Cxcr4b and Cxcr7 during primordium migration to those reported in zebrafish. In wild-type/kazura chimeras, H2B-EGFP+ cells accumulate towards the most posterior neuromasts (as expected if Cxcr4b is necessary at the leading part of the primordium) whereas in wild-type/yanagi chimeras EGFP+ cells accumulate towards the most anterior neuromasts (as expected if Cxcr7 is necessary at the rear of the primordium). The proportion of EGFP+ cells that rescue the mutant phenotype was different in each case. To rescue primordium migration fully in yanagi mutants, the lateral line should contain a majority of wild-type EGFP+ cells; if there are just a few wild-type cells in the initial primordium, these will be deposited on the first neuromasts and the primordium will stop caudal migration (observed in two larvae). On the other hand, in kazura mutants, a few EGFP+ cells were enough to rescue full primordium migration.The generation of chimeras wild typekazura and yanagi mutants, our results suggest that both Cxcr4b and Cxcr7 are used during secondary organ formation. The high ratio of transplanted cells prevents us from making statements about the specific role of Cxcr7 during secondary organ formation. Integrating the expression data, it might well be that Cxcr7 is operating from the primary organs in an indirect manner. On the other hand, the fact that every secondary organ contains EGFP+ cells indicates that cxcr4b is necessary in the migrating cells initiating the formation of new neuromasts, in accordance with the expression of cxcr4b in ICs. cxcr4b+ ICs could be responding to a Cxcl12a ligand. Based on our data, we cannot state whether +cxcr4b ICs are migrating in a Cxcl12a-dependent or -independent manner. Interestingly, it has been shown that fibroblast growth factor (FGF) works as an attractant cue for isolated cells towards the primordium during early migration in zebrafish  constitutes an ideal tool. Second, how can a species lose an entire developmental module, i.e. a primordium? Recent work has demonstrated that small changes, either in non-coding sequences or in temporal expression of key regulators, can have drastic effects on the unfolding of morphogenetic processes  stocks were maintained as previously described (Medaka (escribed . Fish wekazura (cxcr4b mutant) and yanagi (cxcr7 mutant) (GaudiLoxPOUT (ubiquitous H2B-EGFP expression) , Tg(Eya1:mECFP), Tg(Eya1:H2B-EGFP) and Tg(cxcr4b:EGFP). For generation of Tg(Eya1:EGFP), a 3.2 kb fragment of the medaka eya1 promoter sequence was retrieved from the fosmid GOLWFno481_e16 (NBRP Medaka) by PCR with a specific primer set . The PCR fragment was A-tailed and cloned into pGEM T-easy (Promega), and subcloned using ApaI/SalI sites into I-SceI vectors (cxcr4b:EGFP), a 1.9 kb fragment upstream of the presumptive first exon of the endogenous cxcr4b locus was amplified from Cab genomic DNA using a specific primer set . The PCR fragment was cloned into an I-SceI vector (The strains used in this study are: Cab (wild-type population),  mutant) , GaudiLoression) . The folescribed : Tg linearised DR274 plasmid (Addgene clone number: #42250). This was followed by in vitro RNA transcription using T7 MEGAshortscript Kit (Ambion) and a clean-up step using RNAeasy kit (Qiagen). The gRNAs were co-injected at 10 ng/μl each along with 150 ng/μl of CAS9 mRNA into Tg(Eya1:EGFP) one-cell-stage medaka embryos. The sequences of cxcr4b gRNAs are GUGAAAACCUGGUACUUCGGAGG and CAAGUGGAUUUCUAUCACCGAGG.Two gRNAs targeting the coding sequence of the endogenous ng CCTop  and gRNAng CCTop . Briefly2O together with 5 g of NaH2P4.2H2O  and used as a 20× stock solution. Embryos were mounted in 0.6% low melting agarose in ERM (embryo rearing medium) . Tricaine (2 g) was dissolved in 500 ml of ddH medium)  on glassin situ hybridisations were performed as previously reported , and used in reactions with T7, SP6 (antisense) or T3 (sense) RNA polymerases and Dig-UTP, to obtain labelled probes that were stored at −20°C in hybridisation solution. Hybridisations were performed overnight at 65°C and samples were incubated afterwards with an antibody against anti-digoxigenin conjugated with AP Fab fragments . Staining was performed using NBT/BCIP (Roche) and followed under a binocular microscope.Probe generation and reported . BrieflyHair cells in living embryos were detected by using the vital dye 4-Di-2-ASP (Sigma-Aldrich) as previously described . BrieflyIn situ hybridisation pictures were taken using an Axio microscope connected to an HRC camera (Zeiss).Embryos were screened and imaged using an Olympus MVX10 binocular coupled to a Leica DFC500 camera, a Macro-scope Nikon AZ100 coupled to a Nikon C1 confocal, and the confocal laser-scanning microscopes Leica TCS SPE (40× oil objective) and Leica TCS SP5 II . For long-term time-lapse imaging, we used a Microscope Slide Temperature Controller (Biotronix). eya1+ spinal cord neurons was achieved using Fiji software.All image analysis was carried out using standard Fiji software. Image stitching was performed automatically using 2D and 3D stitching plug-ins on ImageJ or using Photoshop to align images manually. Pseudocolouring of pigments and/or Eya1:GFPs) medaka embryos was ablated either at 2 (early) or at 3 (middle-way) days post-fertilisation (dpf). A TriM Scope 2-photon microscope  mounted on a Nikon FN-1 upright stand, equipped with a Chameleon Ultra II femtosecond Ti:Sa laser  and a water-dipping 16× NA 0.8 LWD objective (Nikon) was used. Ablations were performed with 740 nm wavelength and laser power between 150 and 700 mW at the objective output, depending on the depth of the ablation.The lateral line primordium of Tg(


Chronic venous insufficiency and varicose veins occur commonly in affluent countries and are a socioeconomic burden. However, there remains a relative lack of knowledge about venous pathophysiology. Various theories have been suggested, yet the molecular sequence of events is poorly understood. Transforming growth factor-beta one (TGF-β1) is a highly complex polypeptide with multifunctional properties that has an active role during embryonic development, in adult organ physiology and in the pathophysiology of major diseases, including cancer and various autoimmune, fibrotic and cardiovascular diseases. Therefore, an emphasis on understanding its signaling pathways (and possible disruptions) will be an essential requirement for a better comprehension and management of specific diseases. This review aims at shedding more light on venous pathophysiology by describing the TGF-β1 structure, function, activation and signaling, and providing an overview of how this growth factor and disturbances in its signaling pathway may contribute to specific pathological processes concerning the vessel wall which, in turn, may have a role in chronic venous insufficiency. The term chronic venous disorder (CVeD) includes the full spectrum of morphological and functional abnormalities of the venous system, from telangiectasia to venous ulcers. Functional abnormalities of the veins of the lower extremities producing edema, skin changes, or venous ulcers are clinically known as chronic venous insufficiency (CVI)—a term reserved for advanced CVeD .Although it is extremely common, the exact prevalence of CVeD remains elusive. Reports of prevalence of CVI vary from <1% to 40% in females and from <1% to 17% in males . PrevaleThe recurrent nature of the disease, the socioeconomic burden and the ineffectiveness of current treatment modalities emphasize the need for more CVI-related research. Several theories about venous pathophysiology and varicosities genesis in the lower limbs  are outdated or have been refuted .More recent hypotheses proposed that a dysfunctional venous system follows venous wall and valvular damage, which are triggered by venous hypertension and are the result of sterile inflammatory reactions ,11,12,13TGF-β1 is a highly complex polypeptide with a key role in the regulation of cell function  and physiological processes  ,21. Not Aimed at shedding more light on the processes involved in venous pathophysiology, this review will focus on describing TGF-β1 structure, function, activation and signaling, and on providing an overview of how TGF-β1 and disturbances in its signaling pathways may contribute to specific pathological processes in the vessel wall which, in turn, may have a role in CVI.TGF-β1 is a highly complex polypeptide that belongs to the superfamily TGF-β, which contains more than 30 structurally related polypeptide growth factors in mammals. In general, the family members are subdivided into two functional groups: (I) the TGF-β like group that includes TGF-β (1 to 3), activins, inhibins and some growth differentiation factors (GDF); (II) the bone morphogenetic proteins (BMP) like group comprising BMPs, most GDFs and anti-müllerian hormone (AMH) . The TGFTGF-β1 is the most important isoform of the family in the cardiovascular system and is present in endothelial cells (EC), vascular smooth muscle cells (VSMC), myofibroblasts, macrophages and other hematopoietic cells .Knockout studies of TGF-β1 signaling components in mice offered the first indication of their critical role in vascular development and function . Both ECIn addition to the angiogenic effect, TGF-β1 can induce a process called endothelial-to-mesenchymal transition (EndMT), by which EC lose apical-basal polarity and acquire a mesenchymal migratory phenotype . EndMT iTGF-β1 has also been shown to be a key regulator of ECM synthesis and remodeling. Specifically, it has the ability to induce the expression and deposition of ECM proteins, as well as to stimulate the production of protease inhibitors that prevent their enzymatic breakdown ,31,32,33The widespread expression profile of TGF-β receptors in all immune cell types suggests TGF-β1 participation in broad activities of the immune system . AccordiIt is worth mentioning that this multifunctional growth factor is also known by its dual action. Indeed, the TGF-β1-elicited response is highly context-dependent throughout development and across different tissues. For instance, some authors reported that TGF-β1 protects bovine aortic EC from apoptosis , in contTGF-β1  is synthLTBP is required for secretion and correct folding of TGF-β1. The association with LTBP results in the storage of latent TGF-β1 in ECM structures rapidly after secretion ,46,47,49In summary, latent complexes act to control TGF-β1 activity by sequestering it in the ECM or by mediating interactions with integrin receptors to release the mature peptide . Thus, aTGF-β1, as any other TGF-β family member, elicits its cellular effects by binding to different receptors. These are heteromeric complexes comprised of type I  and type II (TGF-βR2) transmembrane serine/threonine kinase receptors. Type II receptors are constitutively active kinases capable of binding TGF-β1 alone, while type I receptors can only bind the ligand in cooperation with type II receptors. There are also type III receptors or coreceptors , that bind to TGF-β1 and regulate this growth factor binding to its corresponding receptors, though they do not signal directly . The typThrough intracellular mediators, known as Smads, the TGF-β1 pathway can directly transduce extracellular cues from the cell-surface transmembrane receptors to the nucleus. This well conserved family  can be divided into three functional classes: receptor-regulated Smads , common-mediator Smads (Co-Smad: Smad4) and inhibitory Smads (I-Smad: Smad6 and Smad7) ,22. All Consistent with other TGF-β family members, TGF-β1 uses several intracellular signaling pathways to regulate a wide array of cellular functions. In addition to the canonical pathway  there are noncanonical pathways  .Mechanisms of Smad-mediated TGF-β1 signaling are shown in A number of noncanonical signaling cascades that operate in a context-dependent manner and contribute to cell-specific biological responses have also been elucidated . Among tTGF-β1 signaling is tightly regulated at different levels, from the availability of the ligand, to the amount of receptors on the cell surface and the downstream transcription factors that will ultimately dictate the outcome of TGF-β1 signaling . The regTGF-β1 signaling is also regulated at the level of its receptors, with FK506-binding protein 12 (FKBP12) that binds type I receptors (thus competing with type II receptors) to maintain them in an inactive conformation . Once acAt the Smad level, Smad2/3 binding to FYVE domain proteins  retains them in the cytoplasm awaiting activation by their respective type I receptor . InteracTGF-β1 signaling regulation proceeds into the nucleus, where Smad complexes target specific promoters to regulate gene expression patterns. Given the low-affinity Smad/DNA binding, Smads are generally dependent on direct interaction with specific high-affinity DNA binding proteins (FOXH1 protein is just one of a growing list ) for recOnce the pathway is activated, a number of feedback mechanisms are turned on to regulate the duration and strength of the signal. For example, I-Smads, whose production is directly increased by TGF-β signaling, act as negative regulators of the pathway by forming stable complexes with activated type I receptors and thereby blocking the phosphorylation of R-Smads . They alGiven the complexity of TGF-β1 signaling and its regulation, it is not a surprise that aberrant expressions of the signaling components and signaling pathway dysregulation often result in human disease, as explained below.Mechanical forces imposed by pulsatile flow of blood, which include frictional wall shear stress, circumferential distention and blood pressure, play an important role in maintaining vessel structure and function ,73. EC lSeveral studies were able to link TGF-β1 production and vascular remodeling induced by shear stress. It was demonstrated that human arterial and venous VSMC exposed to chronic cyclical mechanical strain responded, in a “dose-dependent” fashion, with an increase of TGF-β1 mRNA expression and matrix accumulation . AccordiOther authors could confirm that low shear stress  was a pathological inducer for vascular remodeling by upregulated migration and proliferation of EC and VSMC . An incrFurthermore, observations on vein graft remodeling have identified hemodynamic forces  as the primary stimuli that induce active reorganization of the graft wall, and a role for TGF-β1 in this event. Evidence supporting the concept that increased wall stress after vein graft implantation induces the recruitment of adventitial fibroblasts, mediated by a connective tissue growth factor  and TGF-β1, and the conversion to a myofibroblast phenotype has been provided ,79. ThisThe potential role of TGF-β signaling in mediating the protective effects of physiological shear stress on EC was also studied. Interestingly, the results revealed that shear stress induced TGF-β3 signaling and a subsequent activation of Kruppel-like factor 2 and nitric oxide (NO), indicating that TGF-β3 (but not TGF-β1) has a critical role in the maintenance of endothelial homeostasis in a hemodynamic environment .In physiological conditions, fibrosis is a process of normal wound healing and repair, activated in response to injury, to maintain the original tissue architecture and its functional integrity. It involves a complex multistage process with recruitment of inflammatory cells, release of fibrogenic cytokines and growth factors (such as TGF-β1) and activation of collagen-producing cells . HoweverAs mentioned before, TGF-β is a key regulator of ECM, thus its excessive signaling has long been implicated in the pathogenesis of vascular fibrosis and other fibrosis-related diseases. Moreover, it acts as a mediator of vascular fibrosis induced by several agents involved in vascular diseases  ,72,82,83Several genes encoding ECM proteins that are known to be important in driving fibrosis are directly regulated by TGF-β signaling, through Smads ,86,87 asA member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like four (LOX-like 4), was also identified as a direct target of TGF-β1 in EC and evidence that LOX-like 4 was extracellularly secreted and significantly contributed to ECM deposition was provided . Such reIt has been suggested that TGF-β1 and CTGF synergize to promote chronic fibrosis  Figure . CTGF isSimilarly, it seems that osteoprotegerin promotes fibrogenesis in VSMC via a TGF-β1 autocrine loop  Figure . This meThe inhibition of ECM degradation (leading to excessive ECM accumulation) can also be reinforced by TGF-β1 action on TIMP expression, which once stimulated helps to decrease collagenase production and activity ,88 Figu.An additional contributor to fibrosis is the previously mentioned EndMT process , which iIntriguingly, these well-documented effects of TGF-β1 activity on ECM remodeling  seem to be part of a vicious cycle that drive cell fibrogenesis, since ECM straining and stiffening have an important impact on the activity and availability of this pro-fibrotic growth factor . AccordiHealthy veins of the lower extremities are equipped with efficient walls, contractile VSMC and competent valves in order to withstand the high hydrostatic venous pressure in the lower limbs and allow unidirectional movement of deoxygenated blood toward the heart ,18. In cThe primary reason for this extensive ECM remodeling and structural weakness of the vein wall has still not been clearly explained, but numerous factors, including TGF-β1, seem to be implicated not only in the pathogenesis of varicose veins, but also in numerous complications associated with varicose veins  ,113,114.Data regarding TGF-β1 expression/activity in patients with CVI remains inconclusive . Some stFurthermore, as tissue responsiveness to TGF-β1 depends on several factors  rather than on its activation/availability alone, a few studies  evaluateThe conflicting results regarding TGF-β1  expression/activity may be partially explained by important methodological differences: for example (no) use of effective control samples , when some argue that CVeD is a generalized disorder in the venous system ,112;  and inflammation are among the potential factors that could modulate the expression/activity of TGF-β1 in the vascular wall. An increase in venous hydrostatic pressure in the lower extremities  could lead to EC injury, increased permeability, activation of adhesion molecules and leukocyte infiltration—collectively these factors could contribute to vein wall inflammation ,123,124 Due to TGF-β1 broad influence on ECM remodeling, vein wall imbalance of elastin and collagen content and the growth factor expression/activity (mostly in association with MMP/TIMP ratio changes) have also been an object of study. Depletion of vein elastic components is a common observation in the venous wall of elderly and CVI patients  ,127 thatChanges in TGF-β1 expression/activity have also been associated with CVeD progression and with numerous CVI-related complications. Several studies concerning advanced forms of dermal pathology in CVI patients  have identified CVI dermal fibroblasts as an important target for leukocyte-derived TGF-β1, and established a link between CVI progression and increased tissue levels of TGF-β1, MMP2 activity and decreased TGF-β1 induced mitogenic responses of fibroblasts ,129,130.TGF-β1 plays a key role in many biological and pathological processes, some of which have an impact on cardiovascular homeostasis. Considerable progress has been made over the past several years in the understanding of biomechanical and structural aspects of this growth factor, including its activation and intracellular signaling  pathways.Despite its high prevalence and detrimental socioeconomic impact, CVeD is far from being completely understood due to major reasons: a multifactorial aetiology and a great difficulty to identify the primary stimulus and map the sequence of pathological events . It has become widely accepted that vein wall changes and valvular dysfunction (appointed as primary events) are triggered by blood stasis and venous hypertension and are the result of sterile inflammatory reactions. All the main mechanisms postulated to contribute to the disease onset and development  seem to affect or be affected by TGF-β1 expression and activity, which can be dysregulated.While valuable research efforts have been made to understand the bimolecular mechanisms of severe complication of CVI, such as chronic venous leg ulcers, there is still much to understand about TGF-β1 role in the pathophysiology of both early and late stages of CVeD. To date, there are only a few studies regarding TGF-β1 expression/activity in CVeD patients, with a significant number of them reaching opposite results. The field requires not only more studies, but mostly methodological uniformity . Given the great number of components and regulation factors involved in TGF-β1 signaling, future studies should focus on the expression/activity of molecules beyond TGF-β1 itself . They should also take into account CVeD progression, as these molecules expression/activity may evolve throughout the disease stages. An example would be studying a few major TGF-β1 signaling components in segments of healthy and varicose veins, grouped by different disease stages and specific anatomical regions, to better understand how differences, if any, in these molecules expression/activity may explain vein wall structural evolution throughout CVeD progression.Among CVeD treatment modalities, the outlook for pharmacotherapy targeting the TGF-β1 signaling pathway seems promising, yet challenging—current drugs targeting TGF-β1 and its signaling components are not selective for pathological signaling pathways and require careful side effects monitoring . It is p


The classification of human papillomavirus (HPV) intratypic lineages by complete genome sequencing is a determinant in understanding biological differences in association with this disease. In this work, we have characterised complete HPV genomes from southern Brazil. Fifteen cervicovaginal Pap smear negative samples previously categorised as HPV-positive were sequenced using ultradeep sequencing, and 18 complete genomes from 13 different HPV types were assembled. Phylogenetic and genetic distance analyses were performed to classify the HPV genomes into lineages and sublineages. This is the first report describing the distribution of HPV intratype lineages of high and low oncogenic risk in asymptomatic women from southern Brazil. Human papillomavirus (HPV) is the most prevalent sexually transmitted infection worldwide and is a major factor in the development of cervical cancer zur . With ovDistinct HPV types differ by more than 10% in their L1 sequences, whereas the classification of intratype lineages and sublineages requires complete genome information. Nucleotide distances between 1 and 10% distinguish intratype lineages, whereas differences between 0.5 and 1% define distinct sublineages .Biological differences have been proposed for HPV lineages and sublineages . StudiesFifteen samples of cervicovaginal smears originated from HPV-positive women previously typed by ® XT DNA Sample Preparation kit  and tagmentation and polymerase chain reaction (PCR) according to the protocol established by the manufacturer. Libraries were purified according to the manufacturer’s protocol and quantified by real-time PCR in an ECO System (Illumina Inc.) using the Kapa Library Quantification kit . Pooled libraries were subjected to clustering in a cluster C-bot station and sequenced in an Illumina® HiSeq 2500 system. After sequencing, reads were filtered in the Sickle-Master programme (available from: https://github.com/najoshi/sickle) to select reads with a quality ≥ 28 in the Phred scale and with a length ≥ 20 bp. Reads were evaluated with FastQC software  for their amount and mean quality.HPV DNA was first enriched by rolling circle amplification (RCA) using the Illustra TempliPhi DNA Amplification kit . Libraries were prepared for each sample using a NexteraBWA  was usedp-distance method in MEGA5  in 12 out of the 15 samples analysed. The remaining three samples did not provide enough information that enabled HPV complete genome assembly.The 18 complete genomes, spanning 13 different HPV types of the alpha genus (HPV6 (n = 3), HPV18 (n = 1), HPV31 (n = 2), HPV35 (n = 1), HPV39 (n = 1), HPV51 (n = 1), HPV56 (n = 1), HPV58 (n = 1), HPV61 (n = 1), HPV67 (n = 1), HPV68 (n = 2), HPV82 (n = 2) and HPV85 (n = 1)), were aligned with reference sequences from different lineages and sublineages of each HPV type . Newly aThis report is the first describing the distribution of HPV lineages/sublineages of high and low oncogenic risk in asymptomatic (Pap smear-negative) women from southern Brazil. We showed herein that the use of NGS enabled the detection of co-infections with multiple HPV genotypes due to its greater sensitivity when compared to Sanger sequencing, as shown previously by our group and others da .Interestingly, in one of the samples (07) the HPV type identified by Sanger sequencing was not detected by NGS, even though the latter is more sensitive. One possible explanation for this observation may be related to the use of the rolling circle amplification (RCA) enrichment during the preparation of samples for NGS, a technique that enriches episomal (unintegrated) HPV genomes. Thus, we hypothesize that HPV33, which was previously detected by Sanger sequencing in sample 07, is likely integrated, and its detection was disfavoured by the RCA protocol, while HPV68 may be episomal, and may have been enriched during RCA and hence detected. This may be considered an important limitation to our study and to the use of NGS for HPV typing. While amplicon-based NGS approaches could circumvent this bias , they doIn this study, the lineages identified that HPV6, the aetiological agent of anogenital warts and laryngeal papillomas, belonged to the A lineage and B1 and B3 sublineages. These results are in agreement with the multicentric study by With respect to HPVs with high oncogenic potential, HPV18 is the second most common type associated with cervical cancer. Our study has identified an European sublineage of HPV-18 (A3) in a sample of a woman with no cervical intraepithelial lesions. Previous work has also identified European HPV-18 lineages as predominant among asymptomatic women in southeastern Brazil . A high HPV58 is the second highest oncogenic risk type in prevalence in the city of Rio Grande, Brazil . In thisHPV is the most prevalent sexually transmitted infection worldwide and a major factor for the development of cervical cancer. Infection by multiple HPV types or lineages can be pivotal to the virus’s oncogenic potential in infected women. Next-generation sequencing becomes a powerful research tool to identify co-infections by different HPV types and/or lineages worldwide.


In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein’s activity. We find that the redesigned LacI retains a robust activity  Escherichia coli represents one of the structurally  is the total concentration of LacI, [RT] is the total concentration of the Cy5-labeled dsDNA oligonucleotide and Kd is the dissociation constant. This equation is a full quadratic expansion of the binding polynomial obtained when solving for the concentration of the complex RL in a reversible binding reaction of the type R + L ↔ RL under conditions where the concentration of one of the binding partners is near the Kd of the complex /[LacI dimer] = 57%), was calculated by separately determining the LacI and Cy3 fluorophore molar concentrations based on absorbance measurements at 280 nm and 550 nm, respectively.Labeling of LacI with Cy3-maleimide was performed in a buffer containing 100 mM potassium phosphate pH 7.0, 150 mM NaCl, and 5 mM EDTA. The labeling buffer was degassed by sonication for 15 min (VWR ultrasonic cleaner) and kept on ice for 5 min. To 300 μL of the labeling buffer TCEP and LacI were added to final concentrations of 100 μM and 10 μM, respectively, followed by a 30 min incubation of the mixture at room temperature in the dark. The 300 μL LacI sample was subsequently mixed with 30 μL of Cy3-maleimide dissolved in DMF  and incubated at room temperature for 2 h, followed by an overnight incubation at 4Δ(araD-araB) 567 rph-1 Δ(rhaD-rhaB) 568 hsdR514] containing the pKD46 plasmid  -coated quartz microscope slides through biotin-streptavidin linkage. Binding of LacI was observed at room temperature upon infusing the sample chamber with the imaging buffer supplemented with 500 pM Cy3-LacI using a syringe pump (Harvard Apparatus). DNA-bound and therefore immobilized Cy3-LacI was excited with a 532 nm Nd:YAG laser (Cobolt) and fluorescence emission from Cy3 was detected using a prism-type TIRF microscope, filtered with a 585 nm bandpass filter (Chroma Technology), and imaged onto a CCD camera  is replaced with a 6xHis affinity tag to facilitate the purification of the labeled protein from excess fluorophore. This initial LacI variant harbouring the 6xHis tag but carrying an otherwise wild-type amino acid sequence will be referred to as “LacIΔCterm” . Due to etramers –53. Howein vivo [Cys-Lite”  as sites of fluorophore attachment. Among these, cysteines Cys107 and Cys140 appear buried within the “core” of LacI , suggestin vivo . The remin vivo , 62, we in vivo , 58, 69.ys-Lite”  and thatCys-Lite” lacks the C-terminal tetramerization helix. Since the effects of mutations affecting LacI dimerization are more pronounced in the absence of tetramerization [Cys-Lite” variant prior to the site-specific insertion of non-native cysteine residues , consistent with its being a dimer  used  used . Taken tCys-Lite might be responsible for its loss of dimer formation and DNA binding ability. Given its central location in the monomer-monomer interface of the LacI dimer, the cysteine at position 281 represents one plausible candidate  similar to that of LacIΔCterm  site using total internal reflection fluorescence (TIRF) microscopy  .1 operator sequence on a surface immobilized DNA, indicating that our labeling scheme is compatible with the functional activity of LacI. The fluorescently labeled LacI represents a useful tool for mechanistic in vitro studies of LacI target search at the single molecule level. In conjunction with an electroporation-based delivery into live cells [in vivo as the higher photostability of synthetic dyes compared to autofluorescent fusions allows the recording of longer single-particle trajectories, thus potentially revealing multiple diffusional regimes/binding modes of LacI during its target search.In this proof-of-principle study, we have generated a functionally active variant of LacI for a site-specific fluorescence labeling with maleimide-based fluorophores. Labeling this variant with Cy3 in the DNA-binding “headpiece” resulted in a fluorescent LacI that was able to bind to its native Ove cells , the fluS1 FigThe calibration of the HiLoad 16/600 Superdex 200 column was carried out with a Gel Filtration Markers Kit (Sigma-Aldrich) according to manufacturer’s instructions.(PDF)Click here for additional data file.S2 Fig1 operator DNA to LacI calculated from a quantification of the integrated band intensities. Data points were measured in triplicates, with error bars representing the standard deviation. Data points were fitted to a quadratic binding isotherm as described in the Methods section. Deviations of the fitted curve from the measured data points, in particular at low LacI concentrations, are likely due to the cooperativity observed for dimeric LacI binding to DNA that arises from the LacI monomer-dimer equilibrium [Data points represent the fractional binding of the 5’-Cy5-labeled Oilibrium .(PDF)Click here for additional data file.S3 FigPhotobleaching steps were counted in three independent replicate experiments, each with >140 binding events.(PDF)Click here for additional data file.


The increased stiffness of a tumor triggers a multitude of responses that aid cancer cell dissemination. Stiffness-induced expression of CCN1 mediates autocrine signaling in the endothelium to upregulate N-Cadherin levels. This permits more stable interactions with cancer cells and increases their ability to spread into the circulation. Metastasis is the leading cause for cancer related deaths. The vascular endothelial cells (ECs) are key players in this process by providing a major route for cancer cells to escape the primary tumor and metastasize to distant sites. Cancer cell intravasation is facilitated by leaky tumor vessels and by the expression of cellular adhesion proteins on the cancer and endothelial cells. However, the effect of the tumor microenvironment on the ECs and the impact this has on metastasis is not well defined.3,4. In ECs the dominant receptor is αvβ3, mediating migration, survival and tubule formation.The extracellular matrix (ECM) is heavily remodeled during tumor development and is dynamic both in its composition and structural properties. Alterations in the ECM during tumor progression often lead to increased tumor stiffness, promoting primary tumor growth and metastasis by directly affecting cancer cells through the activation of integrin signaling.To assess how tumor matrix stiffness affects ECs, we cultured primary human umbilical vein endothelial cells (HUVECs referred to as ECs) on polyacrylamide gels representing physiological (400 Pa) or tumor stiffness (22000 Pa).CCN1 was significantly upregulated at high stiffness in cultured ECs and in an orthotopically transplanted breast cancer model. CCN1 was also found to be upstream of stiffness-induced N-Cadherin expression, which is partly dependent on β-catenin activity.loxP/loxPCyr61 mice induced a partial vascular recombination. Together with the B16F10 subcutaneous syngeneic tumor model, Cyr61 knockout resulted in fewer lung metastases and detectable circulating cancer cells, without affecting primary tumor growth, vascularity, hypoxia or pericyte coverage. Conversely, incidence of lung metastases when B16F10 cells were intravenously injected was not affected in an EC-specific Cyr61 knockout mouse model.We recapitulated the initial stage of metastasis in vitro and measured the ability of N-cadherin expressing cancer cells to stably adhere to the endothelium and to transmigrate through a microvascular endothelial monolayer. ECs expressing CCN1 at high stiffness increased the binding of cancer cells via N-Cadherin interactions, and CCN1 loss prevented cancer cell binding and TEM. Furthermore, intravital imaging analysis of the interaction between fluorescently labelled B16F10 cancer cells and the vasculature demonstrated that loss of endothelial CCN1 reduces cancer cell attachment to the endothelium, whilst increasing motility along the blood vessels. Additionally, injection of a soluble Cre protein intravenously into This data enabled us to draw a model through which endothelial CCN1 controls cancer cell metastasis. The desmoplastic reaction that accompanies tumor aggressiveness induces increased levels of endothelial CCN1, which, in turn, upregulates the N-Cadherin available at the surface of the blood vessels. Endothelial N-Cadherin facilitates homophilic heterotypic interactions with aggressive cancer cells that have undergone EMT thus facilitating their intravasation in the blood stream to form distant metastasis .Figure Other cell-cell adhesion proteins were also increased with matrix stiffness in the ECs, some of which have been implicated in EC-leukocyte interactions. For example CD166 antigen  and poliovirus receptor, which are both crucial in TEM of monocytes.This recent work


Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment.Reverse phase protein arrays were generated from laser capture micro-dissected carcinoma and stromal cells from fresh frozen colorectal cancer tissues. Phosphorylation and total protein levels were measured for 30 cancer-related signaling pathway endpoints. Strength and direction of associations between signaling endpoints were identified using Spearman’s rank-order correlation analysis and compared to TRIM28 levels. Expression status of TRIM28 in tumor epithelium and stromal fibroblasts was assessed using IHC in formalin fixed tissue and the epithelium to stroma protein expression ratio method.We found distinct proteomic networks in the epithelial and stromal compartments which were linked to expression levels of TRIM28. Low levels of TRIM28 in tumor stroma (high epithelium: stroma ratio) were found in 10 out of 19 cases. Upon proteomic network analyses, these stromal high ratio cases revealed moderate signaling pathway similarity exemplified by 76 significant Spearman correlations . Furthermore, low levels of stromal TRIM28 correlated with elevated MDM2 levels in tumor epithelium (p = 0.01) and COX-2 levels in tumor stroma (p = 0.002). Low TRIM28 epithelium to stroma ratios were associated with elevated levels of caspases 3 and 7 in stroma (p = 0.041 and p = 0.036) and an increased signaling pathway similarity in stromal cells with 81 significant Spearman correlations .By dissecting TRIM28-associated pathways in stromal fibroblasts and epithelial tumor cells, we performed comprehensive proteomic analyses of molecular networks within the tumor microenvironment. We found modulation of several signaling pathways associated with TRIM28, which may be attributed to the pleiotropic properties of TRIM28 through its translational suppression of the family of KRAB domain transcription factors in tumor stromal compartments.The online version of this article (10.1186/s12967-018-1465-z) contains supplementary material, which is available to authorized users. The crosstalk between epithelial cells and the non-epithelial cellular component of tumor stroma exerts substantial influence on the severity and aggressiveness of cancer . StromalTripartite motif-containing 28 (TRIM28) is a nuclear corepressor involved in transcriptional regulation of a large number of transcription factors belonging to the Krüppel-associated box (KRAB) repressor domain family of zinc finger proteins , 10. TRIWe have previously shown that TRIM28 is overexpressed in colorectal cancer  and thatThe study was approved by Ethics  Research Committee at Beaumont Hospital, Dublin, Ireland. Informed consent was obtained from all patients. Patients with a history of cancer and neo-adjuvant treatment were excluded. A total of 19 cases with late-stage (stages III and IV) colorectal cancer (CRC) were investigated. All patients were diagnosed with CRC between 2012 and 2013. The median age of the patients at the time of diagnosis was 67 (range 47–88 years) with 10 male and 9 female patients. In total, 15 patients had colonic carcinoma, whilst 4 had rectal carcinomas. Clinical and pathological parameters of the patient cohort are shown in Table Both, fresh-frozen and formalin-fixed and paraffin embedded (FFPE) tissue samples were obtained for each case. FFPE tissues were used for immunohistochemical analysis, while fresh-frozen tissues were used for laser capture microdissection (LCM) and reverse phase protein array (RPPA) analysis. A pathologist identified and collected areas of invasive carcinoma from the tumor mass for fresh-frozen preservation and FFPE. Local resection and standard fixation protocols were carried out in all cases. Each FFPE block was sectioned and stained with hematoxylin and eosin (H&E) and graded by a consultant pathologist to confirm pathological stage and grade of the tumors. All fresh tissue samples for RPPA analysis were snap-frozen in liquid nitrogen and processed uniformly and rapidly to ensure preservation of molecular endpoints. The time from removal of a colectomy specimen to snap-freezing of samples was < 20 mins. Fresh-frozen tissue samples were stored at − 80 °C.Immunohistochemistry (IHC) and scoring were carried out using an anti-TRIM28 rabbit monoclonal antibody (mAb) , as previously described . The degLaser capture microdissection (LCM) was performed to isolate separate populations of epithelial and stromal cells for cell signaling analysis as described previously –25. BrieReverse phase protein arrays (RPPA) were generated as previously described , 28, 29.RPPA slides were blocked  for 2 h prior to immunostaining. Immunostaining was conducted on a Dako Autostainer  kit, Dako). Each slide was incubated with a single primary antibody at room temperature for 30 min. The negative control slide was incubated with antibody diluent (Dako) instead of a primary antibody. For normalization purposes, a slide was incubated with anti-ssDNA antibody . The secondary antibodies were goat anti-rabbit , or rabbit anti-mouse IgG  depending on the host species of the primary antibody. Amplification was achieved using horseradish peroxidase-mediated biotinyl tyramide with chromogenic detection . In order to determine the total protein concentration in each sample, two RPPA slides were stained with Sypro Ruby Protein Blot Stain  and visualized with NovaRay Image Acquisition Software .In total, 30 primary antibodies specific to known signaling endpoints were used to measure phosphorylation and protein levels using RPPAs  and spot intensity was analyzed using the Image Quant v5.2 software package (Molecular Dynamics). To normalize data, the relative intensity for each protein spot was divided by the ssDNA relative intensity for the corresponding spot  and datap ≤ 0.01 was used to build proteomic network graphs . Proteomic networks were drafted based on 18 CRC cases (one excluded) and classified into two groups based on their epithelial to stromal TRIM28 expression ratio score; TRIM28 low ratio group (n = 9) representing good patient prognosis and TRIM28 high ratio group (n = 9) representing poor patient prognosis [Proteomic network analysis was performed as previously shown , 33. SpeNodes in the network represent proteins quantified by RPPA; the bigger the node, the more significant correlations relative to that protein. Each line connecting 2 nodes represents a significant correlation between the nodes; the thicker the line, the higher the Spearman ρ correlation between those two proteins. Proteins within the network are grouped into various subgroups on the basis of Spearman ρ values and the number of connections among a group of nodes. Strongly correlated nodes are represented close to each other and with the same color, with each color representing a different subgroup. The number of subgroups within each proteomic network is determined by the software.p ≤ 0.01 was considered significant. An independent-samples t test was used to compare the expression levels of each endpoint between groups, the findings were considered statistically significant at p < 0.05.All tests were analyzed using SPSS 21.0 software  and JMP 5.1.2 . The Spearman rank correlation coefficient, ρ, was calculated for each protein pair in the RPPA cohort; ρ ≥ 0.75 with We have previously shown that the assessment of TRIM28 expression levels, when it encompasses epithelial and stromal tissue compartments, can serve as an independent marker of relapse and patient survival in colorectal cancer . In ordeThe tissue-compartment protein expression ratio method developed for reciprocal tumor microenvironment assessment , allowedp ≤ 0.01) between stroma of TRIM28 high and low ratio cases  exclusively in the epithelium of TRIM28 high ratio cases and were entirely absent in TRIM28 low ratio cases   and autophagy  pathways. Activated AKT mediates downstream responses, including cell survival, growth, proliferation, cell migration and angiogenesis by phosphorylating a range of intracellular signaling proteins. Survivin, on the other hand, is a member of the apoptosis (IAP) inhibitor family that inhibits caspase activation, thereby leading to negative regulation of apoptosis or programmed cell death. Activated caspases 3 and 7 are noticeably diminished in this protein interaction network. In contrast, the TRIM28 low ratio epithelial proteomic network is associated with pro-apoptotic proteins including activated caspase-7 and the apoptotic regulator BCL-2.Our earlier studies propose that during the epithelial to mesenchymal transformation of tumor cells, some carcinoma cells can take on characteristics of stromal fibroblasts . EMT is We demonstrate that there is significantly more epithelial than stromal MDM2 in TRIM28 High Ratio patients and this is in line with our previous findings that TRIM28 is overexpressed in epithelial cancer tissue. MDM2 plays an important role in the regulation of p53 by inhibiting its transcriptional activity, controlling its subcellular localization, and by modulating its protein stability . A numbeLck is one of the dominant nodes in both of the TRIM28 low ratio networks, and Lck overexpression has previously been linked to improved survival . In receWe have previously shown that the expression levels of COX-2 is related to lymph node metastasis, advanced Dukes staging, and poorer long-term outcome for patients with colorectal cancer . In thisTaken together, our findings suggest that the proteomic architecture of the stromal tissue compartment is significantly different in TRIM28 high and low ratio patients, favoring apoptotic processes in the low ratio patients, which may be ultimately linked to better outcome seen in this patient cohort. The study highlights the importance of evaluating the expression levels of biomarkers directly in the tumor microenvironment as an alternative to other in vitro models. By dissecting the effects of TRIM28 in stromal fibroblasts and epithelial tumor cells, we were able to elucidate the complex relationship between stromal and epithelial compartments in CRC. Our approach offers insights into the tumor suppressive and tumor promoting effects of the highly pleiotropic protein TRIM28, thereby reinforcing its value as a prognostic biomarker and a potential therapeutic target.Additional file 1: Table S1.Additional file 2: Table S2.Additional file 3. Additional examples of TRIM28 expression ratios.Additional file 4: Table S3. Significant Spearman’s Rho correlations from RPPA measurements of stromal cells.Additional file 5: Table S4. Significant Spearman’s Rho correlations from RPPA measurements of epithelial cells.


Mycobacterium tuberculosis is a facultative anaerobe and its characteristic pathological hallmark, the granuloma, exhibits hypoxia in humans and in most experimental models. Thus the host and bacillary adaptation to hypoxia is of central importance in understanding pathogenesis and thereby to derive new drug treatments and vaccines. Thus, TB now ranks alongside HIV as a leading cause of death worldwide, and the rate of HIV-TB co-infection worldwide in 2014 was 12% Since tuberculosis (TB) was declared a global health emergency in 1993 in vitro interferon gamma release assay (IGRA), in the absence of clinical signs and symptoms of active disease Mtb is transmitted by the cough of an infected person (aerosolized) and inhaled into the alveoli of a new host. This process can lead to three possible outcomes: i) a minority develop active primary progressive TB disease and develop a detectable but ineffective acquired immune response (immune sensitization), ii) the majority develop latent TB infection that is contained throughout their life by an effective acquired immune response, and iii) a small proportion of those latently infected develop post-primary TB as a result of reactivation of their latent infection, which can be triggered by immune suppression such as HIV-1 infection Conventionally, LTBI is conceived as Mtb remaining in an inactive, stationary phase in the granuloma as a stable latent population of bacilli capable of surviving under stressful conditions generated by the host in vivo are hypoxic in vitro and animal models are limited in the extent to which they recapitulate the multifactorial environment created by the host to arrest mycobacterial growth. Nonetheless, many conceptual advances have been achieved in recent years in our understanding of mycobacterial physiology under low oxygen conditions, particularly in the areas of gene regulation, metabolism, and energy homeostasis.Direct measurement of lesional oxygen tension in rabbits 2in vitro “Wayne” model of hypoxia-induced dormancy in vitro under hypoxic conditions and clinical cases of latent infection have made the Wayne model a key tool for investigating the molecular basis of mycobacterial dormancy. A key caveat is that many of these studies were performed using laboratory strains of Mtb that have been passaged aerobically over many years, these findings therefore need to be revisited using recent clinical isolates.The existence of a coordinated and inducible response of Mtb to low oxygen conditions was initially revealed by Wayne and colleagues, culminating in the now widely employed 2.1dosR results in a relatively mild loss of viability under hypoxia in vitro (2–3 logs decrease in CFUs after 30–50 days incubation) in vivo in multiple animal models Early work on gene expression analysis of Mtb undergoing hypoxic challenge identified a suite of almost 50 genes that were significantly and consistently upregulated relative to aerobic controls. Further work identified that this regulon was controlled by a transcription factor subsequently named DosR , the activation of which was mediated through two classic two-component system-type transmembrane sensor histidine kinases, DosS and DosT Mycobacterium smegmatis homologue of the Rv0079 - a gene within the same operon as Rv0081 - has also been shown to have functional importance in this bacterium during hypoxia in stabilizing ribosomes in the 70S form, in contrast to the higher order structures seen in many enteric bacteria grown under similar conditions Outside of the DosR response, other transcriptional regulators have been identified as playing significant roles under hypoxic conditions, although precise functions have not been determined The roles of gene regulation at the posttranslational level have also been assessed in hypoxic Mtb, and both proteases and Serine/Threonine Protein Kinases (STPKs) have been found to play functional and essential roles 2.2tgstgs mutants in the initial stages of hypoxia relative to wild-type bacteria  transcript, protein, and activity levels are also observed under hypoxia 2 releasing TCA cycle steps, or alternatively in the maintenance of the membrane potential and/or the proton motive force (PMF) via secretion of Icl-produced succinate through a succinate/H+ symport system fas, kasA, cholesterol catabolism regulation, gluconeogenic pckA) in vivo, in mouse lung infection, suggesting that a metabolic shift towards lipid metabolism is a general stress response rather than being specific to hypoxia or due to the nature of the provided/available carbon source A reduction in net carbon flux is a hallmark of hypoxia-induced dormancy in Mtb, suggesting a need to conserve carbon and energy sources for prolonged survival and later resumption of growth when conditions improve. Consistent with the dormancy/hibernation programs of other organisms, Mtb accumulates intracellular triacylglycerides (TAGs) under hypoxia, which correlates with increased expression of the DosR-regulated TAG biosynthetic gene bacteria  [34]. AlHypoxia-challenged Mtb also substantially down regulate many genes involved in the oxidative direction of the TCA cycle , changes in glutamine biosynthesis 2.3de novo ATP synthesis via the ETS (as opposed to via substrate level phosphorylation) is a strict requirement for bacterial survival under these conditions ndh; essential for survival under hypoxia cydAB) and down regulation of the proton-pumping type I NADH dehydrogenase  and consistent with depleted stores of terminal electron acceptors (TEAs) nase nuo. The sursdh-1; Rv0247c-0249c), the major aerobic SDH, abolishes the ability of bacteria to regulate oxygen consumption  when approaching hypoxia which subsequently led to increased bacterial death at later stages of anaerobiosis sdh-1; Rv3316-3319) may have a key role during hypoxia, either as a canonical succinate dehydrogenase/fumarate reductase sdh-2 null mutant under hypoxia is lethal Succinate dehydrogenase, which physically links the TCA cycle and ETS, has recently been shown to play a key but enigmatic role in mycobacterial adaptation to hypoxic conditions. Genetic deletion of succinate dehydrogenase (SDH) 1  biosynthesis is cidal to anaerobic bacteria In the absence of molecular oxygen many facultative anaerobes can switch to alternative external TEAs to sustain respiration. Mtb contains all the genetic elements necessary for reduction of nitrate and nitrite, and both of these activities have been detected in growing cells 33.1Macrophages undergo substantial phenotypic changes when exposed to reduced oxygen tension and several lines of evidence suggest that hypoxia modulates central effector functions of this key innate immune cell . The resHIF-1α plays a key role for macrophages to adapt to low oxygen tension. Cell-specific deletion of HIF-1α or transient gene silencing in macrophages reduces inflammatory responses with regard to macrophage motility and invasiveness, phagocytic capacity and most importantly bacterial killing This phenomenon of aerobic glycolysis in immune cells, resembling the Warburg effect in tumors 3.2Mtb infects macrophages, dendritic cells and neutrophils, with macrophages most extensively studied. Infection with Mtb leads to a wide array of cellular responses, most of which have been studied under normoxia. The evolutionary success of virulent mycobacteria likely depends on cross-species-conserved mechanisms operative in infected cells 2, which is closer to tissue conditions, did not affect the growth of free-living bacteria but strikingly reduced the growth of intracellular mycobacteria. The growth inhibitory effect was not due to a putative differential response to IFN-γ or TNF-α at low oxygen conditions, but was associated with a shift from oxidative toward glycolytic metabolism, consistent with earlier work in which macrophages cultured at low pO2 showed a metabolic shift toward glycolysis 2), when compared to human macrophages kept at 20% Human monocyte derived macrophages cultured in 5% oxygen, corresponding to the physiological tissue concentration, permitted significantly less growth than those cultured at the 20% oxygen levels of ambient air 2) accumulate triacylglycerols (TAG) in lipid droplets This hypoxia-mediated control of Mtb replication is at the same time associated with a significant metabolic reprogramming of its host cell. Human macrophages cultured for 24 h under hypoxia  was unimpaired in log phase growth and persisted in IFN-gamma-activated human macrophages, suggesting that Rv0251c is dispensable. The protein Acr2 is strongly recognized by cattle with early primary Mycobacterium bovis infection and also by healthy MTB-sensitized people (LTBI). Interestingly, within the latter group, those with recent exposure to infectious tuberculosis had higher frequencies of Acr2-specific IFN-gamma-secreting T cells than those with more remote exposure, suggesting infection stage-specific immunity to tuberculosis Amongst the first antigens to be investigated were those of the heat shock response: proteins induced under stress conditions, such as elevations of temperature causing denaturation of proteins during infection 4.3Several studies evaluated the above candidate genes, and many were found to encode MTB antigens that induce strong immune responses. One of the most abundant upregulated proteins during hypoxia is the 16 kDa  protein The finding that the IFN-gamma response to Rv2031c was higher in healthy BCG-vaccinated controls compared to those with extensive untreated tuberculosis led to the speculation that prolonged containment (LTBI) in humans may be contributed to by long-lived Rv2031c-specific cells, able to divide on re-challenge, and thus limit dissemination These findings prompted the investigation of the human immune response to other DosR regulon encoded genes, summarized in Ref. 4.4in vivo and thereby could be targets of the immune response A detailed analysis of MTB genes that are upregulated during the latent stage of infection was considered a priority to identify new antigenic targets for vaccination strategies in vivo or in vitro, to what is recognized by human T cells as antigens, a combined bioinformatic and empirical approach was employed as a novel genome based strategy, to guide the discovery of potential antigens. The fold induction of the top 100 highly induced genes at 7 days of hypoxia, their transcript abundance, population specific MHC class II-peptide binding prediction (ProPred), and a literature search was combined, leading to the selection of 26 candidate genes. Overlapping peptides were used in combination with two readout systems, ELISpot for IFN-γ as well as IL-2. Five novel immunodominant proteins: Rv1957, Rv1954c, Rv1955, Rv2022c and Rv1471, showed responses similar to the immunodominant antigens CFP-10 and ESAT-6 in both magnitude and frequency. These findings revealed that a number of hypoxia-induced genes are potent T-cell targets and therefore offers general support to the important role of hypoxia in the natural course of TB infection. Importantly however, only moderate evidence of infection stage specific recognition of antigens was observed In order to relate what is expressed by the bacilli M. bovis strains including BCG (Rv2658c and Rv2659c), respectively. When compared to the immunodominant molecules ESAT-6 and CFP-10, IFN-gamma responses to the RD11 proteins were inferior in both aTB and LTBI groups. A strong IL-2 recall response to Rv1986 was found in LTBI, targeted at two epitopic regions, containing residues 61–80 and 161–180 in vivo and are potent T-cell targets of the immune response. The results further our understanding of the biology of latent infection and offer general support to the hypoxia hypothesis and its relationship to the natural infection of MTB. While some of these findings did not provide support to the hypothesis of infection stage specific antigen recognition, they support an overlapping immunological spectrum between those with latent and active TB disease as suggested In light of the above findings, the hypoxia inducible MTB specific proteins absent from the BCG vaccine strains were also evaluated. One region of difference (RD) 2 and two RD11 encoded proteins were identified, that are absent from the commonly used BCG strains (Rv1986) and all


Defects in the glomerular filtration barrier (GFB) play a major role in the onset of human renal diseases. Highly ramified glomerular cells named podocytes are a critical component of the GFB. Injury to podocytes results in abnormal excretion of plasma proteins, which can lead to chronic kidney disease. The conserved paired nephron of larval zebrafish is an excellent model for assessing glomerular function and injury. The efficacy of two known podocyte toxins was tested to refine models of acute podocyte injury in larval zebrafish. The validated compound was then used to test a novel assay of the dynamics of abnormal protein excretion. Injected adriamycin was found to be unsuitable for induction of glomerular injury due to off-target cardiovascular toxicity. In contrast, puromycin treatment resulted in a loss of discriminative filtration, measured by excretion of 70 kDa dextran, and podocyte effacement confirmed by electron microscopy. The dynamics of dextran excretion during puromycin injury modeled the onset of glomerular damage within 24 hours postinjection. These data validate puromycin for induction of acute podocyte injury in zebrafish larvae and describe a semihigh-throughput assay for quantifying the dynamics of abnormal protein excretion. The GFB comprises two highly specialized cell types separated by a type IV collagen and laminin-rich glomerular basement membrane (GBM).2 The fenestrated endothelium forming the walls of glomerular capillaries is enwrapped by podocytes. Podocytes are highly ramified epithelial cells with primary and secondary foot processes, the structure of which depends on their actin cytoskeleton.3 The filtration slits between interdigitated podocyte foot processes comprise highly specialized gap junctions called slit diaphragms (SDs). The SD protein complex forms the barrier to protein filtration and is essential for maintained podocyte structure and function.4Anephrin (NPHS1),5transient receptor potential channel 6 (TRPC6),6Wilm's tumor suppressor gene 1 (WT1),7 and podocin (NPHS2).8 Most glomerular injuries are, however, not hereditary, but acquired in immune or nonimmune diseases, with 75% of cases having an unknown cause.9All identified monogenic mutations associated with nephrotic syndrome are in podocyte-specific genes, revealing the essential role of these specialized cells for glomerular function. Disease-causing recessive or dominant autosomal mutations have been identified, among others, in 10 The breakdown of the GFB in disease, or as a result of drug toxicity, causes proteinuria, the abnormal filtration of plasma proteins, including albumin. Proteinuria associated with nephrotic syndrome results in edema, likely due to increased sodium retention by the distal nephron.11Podocyte effacement, the gradual simplification of the interdigitating foot process pattern, is a key pathology common to most glomerular diseases.9 In rodents, acute nephrosis may be experimentally induced through insult to podocytes by nephrotoxic compounds. Injection of the antibiotic, puromycin aminonucleoside (PAN), causes either a mild or reversible glomerular damage resembling minimal change disease, or more severe lesions that mimic human focal segmental glomerulosclerosis (FSGS),13 the latter a hallmark of lasting and progressively damaging chronic kidney diseases.14 Similarly, a single injection of the chemotherapy drug, adriamycin, causes a severe form of FSGS in rats and mice.15Experimental animal models are required that recapitulate the molecular events that manifest during glomerular injury and the sequelae of pathological responses that lead to glomerulosclerosis and the loss of functioning glomeruli.in vivo experimentation, zebrafish have become a valuable animal model for renal research. The small size, rapid development, and translucency of larval zebrafish offer excellent opportunities for whole-animal chemical and genetic screening16 and, recently, for studies of renal cancer.17–21 Within the first 3 days of life, the pronephros of larval fish becomes functional and capable of discriminative filtration.23 Mature pronephric podocytes in larval zebrafish are anatomically similar to their mammalian counterparts and are nonmigratory, including during repair.24–26 With a developed glomerulus, segmented tubules, and renin-expressing cells, the single paired nephron of the zebrafish pronephros shares remarkable anatomical and functional similarity with the metanephric mammalian nephron.27–33With its unique set of experimental advantages, including genetic tractability and unparalleled opportunity for 34 A particular drawback to pharmacological models of acute renal failure in larval fish is that they are terminal, which limits the potential for follow-up studies.34 Genetic approaches for induction of glomerular injury have successfully utilized nitroreductase (NTR)35–37 to induce ablation of podocytes by cytotoxic metabolization of metronidazole (MTZ).38 This recapitulates proteinuria and has been successfully used to demonstrate the regenerative capacity of fish podocytes postinjury.37 However, the NTR/MTZ system is limited by side effects of MTZ and the prolonged time required for induction of apoptosis.Due to inherent limitations in current methods, the zebrafish model requires novel and refined renal injury techniqiues.39 A comparison of pharmacological agents for induction of podocyte injury in zebrafish is required to identify compounds exhibiting the greatest potency with the lowest off-target effects.While also often limited by off-target effects, pharmacological approaches are quick to implement and may be applied to a variety of wild-type or transgenic lines. Current methods for the pharmacological disruption of podocytes in zebrafish utilizing puromycin result in an increased glomerular permeability.in vivo dynamics of abnormal protein excretion.The aims of the present study were twofold, (1) to compare puromycin and adriamycin for pharmacological induction of glomerular injury in zebrafish larvae with an established GFB and (2) to develop a high-throughput assay to measure the 40 the effects of these compounds on heart rate and blood flow were also tested. After validating a puromycin model of glomerular injury, a novel dextran excretion assay was developed to assay the dynamics of glomerular permeability. Our study provides both a defined pharmacological model of acute podocyte injury and a novel assay enabling the measurement of protein excretion dynamics during glomerular injury and repair.Adriamycin and puromycin were tested for their ability to induce structural podocyte damage and increased glomerular permeability. Increased permeability was determined by the excretion of normally retained dextran, and changes in podocyte morphology were assessed by transmission electron microscopy (TEM). As glomerular function is tightly associated with cardiovascular function in larval fish,Danio rerio) were maintained at 28.5°C in 1 × conditioned water (CW)40 containing 0.1% (w/v) methylene blue. Where required, fish were anesthetized in CW containing 40 μg mL−1 MS-222 (tricaine methanesulfonate). Forty micrograms per milliliter MS-222 is sufficient for anesthesia without significant effects on the heart rate.41 Microinjections and microscopy were carried out at room temperature (26°C ± 0.5°C).All experiments utilizing fish >5 dpf (days postfertilization) were approved by the local ethics committee and conducted in accordance with the Animals (Scientific Procedures) Act 1986 in a UK Home Office-approved establishment. Unless stated otherwise, wild-type WIK zebrafish  70 kDa FITC-dextran was dissolved in phosphate-buffered saline (PBS). PAN  was administered at 5 or 25 mg mL−1 (17.0–85.0 mM) and adriamycin (doxorubicin hydrochloride)  at 1, 2, or 4 mg mL−1 . Shams were administered with vehicle (PBS) only.Dextran and pharmacological agents were administered under anesthesia by intravenous microinjection through the cardiac sinus venosus (SV) that drains the common cardinal vein (CCV).Three dpf fish were injected respective treatment compounds, or vehicle (PBS), and separated into Petri dishes in triplicate. Fish were examined immediately after injection and at 24 and 48 hpi (hours postinjection). At each time point, fish were scored for percentage survival and overt pericardial sac edema.The clearance of 70 kDa FITC-dextran was determined by measuring fluorescence intensity in the dorsal aorta.In summary, postinjection, fish recovered in CW (without methylene blue) before brief anesthesia and mounting in 3% methylcellulose for baseline imaging at 3 hpi. Sagittal images of FITC fluorescence in the dorsal aorta were taken using a stereomicroscope with fixed parameters. Embryos were returned to CW and standard conditions immediately after each imaging time point. For each fish, the mean intensity of three areas of the dorsal aorta was measured using Fiji—National Institutes of Health (NIH). Mean fluorescence intensity values were normalized against the baseline fluorescence and percentage intensity change was calculated.−1. Heart rate was assessed similarly by recording the video at a frame rate of 10 frames per second and heartbeats counted manually over a 10-s period. Both videos were taken of a lateral orientation.After respective treatments, 3 dpf embryos were left to recover in CW for three hours before mounting in 3% methylcellulose and immediate imaging under anesthesia. Bright-field videos over 120 s in length were acquired of the dorsal aorta at 50 frames per second. Single erythrocytes were then tracked through the cardinal vein anterior of the cloaca. The distance traveled by three erythrocytes per embryo was measured using Fiji and venous erythrocyte velocity calculated in μm s® plate (Nunc). Two hundred fifty-micrometer slits were used to create 48 paired wells  before distribution into a modified black, flat-bottomed, 96-well Microfluor 1ed wells . In eached wells . The two−1 puromycin or vehicle. At 8 and 24 hpi, fish were fixed for TEM as described by Lyons et al.43 Briefly, fish were fixed in modified Karnovsky's solution , postfixed in 2% osmium tetroxide in 0.1 M imidazole and 0.1 M sodium cacodylate, stained en bloc in saturated uranyl acetate, and dehydrated into 100% acetone. All of these steps were accelerated using microwave stimulation with a Panasonic microwave with inverter technology. Temperature throughout was maintained at 15°C in a cooled water bath. Following dehydration, embryos were embedded in Epon. Ultrathin sections (60 nm thick) were cut from selected areas and mounted on Formvar/carbon-coated copper slot grids. After drying, these were stained in 1% aqueous uranyl acetate and Reynold's lead citrate, and then viewed in a JEOL JEM-1400 Plus TEM.Three dpf fish were injected with 25 mg mLet al.44 The mean podocyte frequency per μm length (of basement membrane) and mean podocyte width were determined in all clearly identifiable sections of the GFB . All percentage data were arcsine transformed before analysis. Dextran clearance from the dorsal aorta, heart rate, and blood flow were subjected to two-way ANOVA. Foot process width and foot process number per μm GBM were analyzed by one-way ANOVA. Dextran excretion data were analyzed by linear regression and comparison of regression slopes. The first derivative of excretion data was determined for individual fish. Differences between the mean first derivative of control and treatment groups were determined for measures between 0–24 hpi and 24–48 hpi using 45 and, similarly in fish, affects formation of the subintestinal vessels and eye development.46 The measurement of FITC in peripheral vessels is therefore potentially problematic. Similarly, FITC measurements in the heart can be obscured by accumulation of dextran in the pericardial sac, particularly in cases of edema. To minimize any potential confounding effects on FITC measurements and to allow normalization to baseline values, measurements of FITC were determined in the dorsal aorta, as similarly performed by Rider et al.40The dose of a single intravenous injection of puromycin or adriamycin required to cause edema without effects on survival was tested. Achieving successful intravenous injections is considerably easier, and consequently more accurate, in 3 dpf fish due to the large CCV and SV. Due to a narrowing of the CCV by 4 dpf, injections in older larval fish are much more challenging and error prone. Adriamycin affects angiogenesis in mammals26 this age was selected for the injection of compounds to induce pharmacological injury. Edema is readily observed as a result of acute renal failure in larval fish.47 The concentration of puromycin was partially based on previous studies,39 and following preliminary studies, three concentrations of adriamycin were tested. One hundred percent survival was observed at both tested doses of puromycin (5 or 25 mg mL−1). Only the higher dose of puromycin resulted in edema  decreased survival to 90% after 24 h. Two milligrams per milliliter adriamycin resulted in transient edema in >10% of fish, conversely 4 mg mL−1 resulted in persistent edema in 75% of larvae, some of which had remaining yolk reserves suggestive of stunted development  significantly decreased the heart rate (−1) had no effect on heart rate, but reduced venous blood flow  impairs the establishment of discriminative filtration due to abnormal podocyte development.46 Waterborne delivery of adriamycin is also associated with off-target toxicity, particularly to the heart.50 We aimed to reduce off-target effects by using a more direct delivery of adriamycin by intravenous microinjection, as similarly used in the rodent adriamycin nephropathy model.15 In our model assessing the effects of injected adriamycin in older larval fish with a functional glomerulus, the clearance of coinjected 70 kDa dextran was likely confounded by off-target cardiovascular effects.Rodent models of adriamycin nephropathy are well established and characterized by increased protein excretion, tubulointerstitial inflammation, and fibrosis.40 In the present study, the adriamycin-induced reduction in heart rate and blood flow may have decreased glomerular flow rates and masked any increase in dextran clearance resulting from podocyte damage. Due to confounding off-target cardiovascular effects, adriamycin was considered unsuitable for modeling glomerular injury in zebrafish older than 3 dpf.The pronephric glomerular arterioles of zebrafish branch directly off the dorsal aorta, thus glomerular flow is tightly associated with flow in the central vasculature.51 To mitigate off-target effects and edema, a lower dose of puromycin (5 mg mL−1) was tested, but was found insufficient for the measurable induction of glomerular injury. By injecting 1 nL of 25 mg mL−1 puromycin, as opposed to the 2.3–4.6-nL volume used by used Hentschel et al.,39 we observed similar effects on dextran clearance and podocyte effacement, including the onset of terminal edema. Injection volumes were restricted to 1 nL for the avoidance of negative effects on cardiovascular function, which can arise when injecting larger volumes into the circulation of larval fish (unpublished).The injection of puromycin at 3 or 4 dpf in zebrafish larvae has been shown to result in podocyte effacement, loss of glomerular selectivity, and terminal edema.44 to quantitate podocyte foot process width and foot process frequency. The quantification of podocyte morphology established that measurable structural changes occur between 8 and 24 hpi.We employed image analysis39 The dose of puromycin established for acute podocyte injury was not associated with any measurable effects on tubular structure (data not shown), including the brush border. The cilia of tubular cells that form the zebrafish nephron are essential for fluid clearance.52 This suggests that in the pronephros, effects of puromycin are specific to the glomerulus, as similarly reported by Hentschel et al.39At a dose causing disruption of the GFB, puromycin also reduced blood flow by 24 hpi. Although decreased blood flow was observed, a higher dextran clearance was still detected, indicating a robust effect of puromycin on glomerular permeability. Previous studies have shown that FITC-dextran intensity in the vasculature is not significantly affected by edema-induced volume expansion.in vivo analysis of glomerular permeability in zebrafish, we developed a novel assay of dextran excretion allowing the continuous monitoring of glomerular permeability to high-molecular-weight (HMW) molecules. Repeated handling and anesthesia of fish induce stress responses and cause detrimental effects on cardiovascular function.54 This limits the frequency and precision at which FITC-dextran clearance may be measured from the vasculature of larval fish. Hence, an advantage of our semihigh-throughput plate assay is that after injection of FITC-dextran, it involves no further handling or anesthesia of fish. This dextran excretion assay complemented measurements of FITC-dextran clearance from the dorsal aorta and confirmed that increased excretion of HMW molecules occurred in the first 24 h after injection of puromycin. While our dextran assays suggest that in some individuals glomerular function may resolve by 48 hpi, systemic effects leading to terminal edema were not resolved, as reported in previous studies.39To advance methods for the 55 This study has identified the suitability of puromycin for induction of acute podocyte injury in zebrafish larvae. As observed in rodent models, the defined single dose of puromycin used in the present study results in podocyte effacement with a concurrent loss of glomerular selectivity. The onset of GFB changes within 24 h in fish is considerably faster than occurs in rodent models, which have a 4–5-day latency period after puromycin injection.56Puromycin- or adriamycin-induced nephrosis are among the oldest and most widely used models of human nephrotic syndrome, particularly using rats due to their susceptibility to both compounds.39 Further studies are required to assess the correlation between edema and GFB changes to establish if puromycin-induced injury is recoverable in larval fish, as observed in the genetic podocyte NTR-based podocyte injury model.37 A pharmacological glomerular injury model is yet to be established in adult zebrafish. Unlike larval fish, which can only repair their single paired nephron, glomerular injury in adult fish may induce neonephrogenesis, as documented with tubular injury.57In contrast to rodent models, puromycin administration in larval fish results in off-target effects on central blood flow, which may result in the terminal onset of edema.in vivo analysis of glomerular injury and podocyte repair in larval zebrafish.Our novel excretion assay for the monitoring of glomerular selectivity, a proxy of podocyte function, enables a semihigh-throughput and dynamic measurement of glomerular filtration without the confounding effects of repeated fish handling. This will facilitate the screening of small molecules that prevent glomerular injury or promote podocyte repair. The ability to measure the dynamics of GFB function provides an excellent tool for the


T2-weighted images using topological descriptors of image heterogeneity called Minkowski functionals (MFs).To develop an image analysis technique that distinguishes pseudoprogression from true progression by analyzing tumour heterogeneity in n = 50), and blinded to treatment response outcome, unsupervised feature estimation was performed to investigate MFs for the presence of outliers, potential confounders, and sensitivity to treatment response. The progression and pseudoprogression groups were then unblinded and supervised feature selection was performed using MFs, size and signal intensity features. A support vector machine model was obtained and evaluated using a prospective test cohort.Using a retrospective patient cohort  and classifier (Lasso) gave the same results. Although not apparent to the reporting radiologist, the T2-weighted MR images, which are acquired routinely in the clinic, has the potential to detect an earlier treatment response allowing an early change in treatment strategy. Prospective validation of this technique in larger datasets is required.Analysis of heterogeneity, in  T1-weighted MR images, representing non-specific blood-brain barrier disruption.Fluoroethyl)-l-tyrosine (FET) seems promising,[O-(2-[18F]Fluoroethyl)-l-tyrosine positron emission tomography to be used routinely, a separate radionuclide investigation in addition to MRI would be required. The advantage of the approach described here is that it relies only on T2-weighted MR images, which are acquired routinely worldwide, are high resolution and which require no specialized pre-processing prior to image analysis .Other imaging approaches for distinguishing progression from pseudoprogression are being investigated. DSC perfusion imaging has shown considerable promise  although the studies were also relatively small (14–25 patients for the training datasets).;31;56;57romising, probablyromising, FurthermT2-weighted images have been included in the new RANO criteria, although there is as yet no detail as to how these images should be assessed.. Sample sizes from these alternative test datasets were similarly small because many patients did not have a complete longitudinal dataset of T1-weighted post contrast imaging, which is required for determining the treatment response based on RANO criteria, and many had been entered into drug trials or had been treated with bevacuzimab. To demonstrate that discrimination of progressors from pseudoprogressors using the SVM classifier was meaningful we performed leave-one-out cross validation on the training dataset and then tested the classifier on a separate test set, which is a robust way of preventing overfitting using machine learning methods.[n = 7 to 17 [A strength of this study was that the treatment given to the retrospective training cohort was uniform and reflects routine management in the UK (and many other countries). This was important for allocation of treatment response status as well  methods. Such an  7 to 17 –75. More 7 to 17 ;44;76–78 7 to 17 –24 were T2-weighted MR images alone, which are acquired routinely in the clinic, that Minkowski functionals can differentiate pseudoprogression from progression. This was achieved by capturing image information on underlying tumour heterogeneity that is only rarely visually apparent to the reporting radiologist. The key conclusion is that if this technique is further validated prospectively in larger datasets, which will probably require multi-centre studies given the relative rarity of glioblastoma, then it may be used to detect treatment response at an earlier stage, allowing an early change in treatment strategy.In summary, several imaging techniques appear promising in being able to distinguish progression from pseudoprogression, however, none have been validated in multicentre prospective trials or are widely available to allow implementation in daily practice or in clinical trials, nor are S1 Figa) and prospective test data set (b). Note that no change of therapy was apparent in all included patients. (c) Kaplan-Meier survival curve for patients with progression (n = 11) and pseudoprogression (n = 6). The median survival was 234 days (range 39–370) for those with progression and 585 (329–1533) for those with pseudoprogression (with one censored patient surviving).Modified CONSORT flow diagrams of patient inclusion and exclusion criteria for the retrospective training data set ((PDF)Click here for additional data file.S2 FigPrincipal component score plot of normalized perimeter MFs from all four time points showing scans performed at 3 T (filled squares) and 1.5 T (empty squares). The modeled data were not scaled or centred. The displayed T2 Hotelling’s tolerance ellipse was set at the 0.05 significance level.(PDF)Click here for additional data file.S1 TableTreatment response status was significantly associated with location of the tumour and completion of adjuvant chemotherapy. As expected, the lack of completion of adjuvant chemotherapy occurred in both the progression and pseudoprogression groups predominantly because of perceived treatment failure.(DOC)Click here for additional data file.S2 TableThe results of the final linear mixed model that incorporated potential confounders. These are summarized by 2-way repeated measures ANOVA.(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 TableClassification of progression and pseudoprogression using a support vector machine with the training dataset.(DOC)Click here for additional data file.S5 TableCox proportional hazard regression analysis of the three training dataset SVM models that were the most accurate.(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 TableClassification using a Lasso model, trained on the retrospective training dataset after Rando.(DOC)Click here for additional data file.