Ageing is the result of changes in biochemical and biophysical processes at the cellular level that lead to progressive organ decline. Here we focus on the biophysical changes that impair cellular function of human dermal fibroblasts using donors of increasing age. We find that cell motility is impaired in cells from older donors, which is associated with increased Young’s modulus, viscosity, and adhesion. Cellular morphology also displays parallel increases in spread area and cytoskeletal assembly, with a threefold increase in vimentin filaments alongside a decrease in its remodelling rate. Treatments with withaferin A or acrylamide show that cell motility can be modulated by regulating vimentin assembly. Crucially, decreasing vimentin amount in cells from older individuals to levels displayed by the neonatal donor rescues their motility. Our results suggest that increased vimentin assembly may underlay the aberrant biophysical properties progressively observed at the cellular level in the course of human ageing and propose vimentin as a potential therapeutic target for ageing-related diseases. Ageing is a complex process characterised by temporal changes in biological, biophysical and biochemical function that lead to a progressive whole-body decline throughout the lifespan of an individual. While age-related deterioration is most conspicuous at the organ level, it has been hypothesised that the underlying causes are likely to be dysfunctions at the cellular and tissue level . Age is The majority of cellular studies on ageing have focused on recursive passaging in vitro as a surrogate of ageing in vivo ,8,9,10. It is often assumed that actin is the main cytoskeletal network involved in the regulation of cell motility ,17, the In this study, we used a combination of biophysical approaches to assess how cell morphology and biophysical behaviour are altered due to ageing. Human dermal fibroblast from donors of different ages were used as a model to characterise how changes in cell motility and biophysical properties are associated to changes in cytoskeleton organisation. Fibroblast from older donors had reduced cell motility and increased cell stiffness, which was associated to changes in cytoskeletal assembly. In particular the age-associated aberrations in cell motility and biophysical properties appeared alongside vimentin accumulation and could be rescued using drugs believed to primarily target vimentin. Our findings suggest the importance of vimentin in donor ageing and point towards this cytoskeletal protein and associated signalling pathways as potential biomarkers for the diagnosis, prognosis, and treatment of a wide variety of different diseases associated with ageing.Human dermal fibroblasts were obtained from commercial sources. In brief, cells were derived from temple or labia tissue from ”apparently healthy” Caucasian female donors. Neonatal (N) and adult age 62 (A62) cells were purchased from  while adult age 21 (A21) and age 47 (A47) cells were purchased from . Vials of cells were shipped at passage 2, and all experiments were carried out in cells up to passage 7. NIH 3T3 cells were a gift from A.Mata group (Queen Mary University of London). All cell work was conducted in identical conditions among all donors, and culturing of cells was carried out in parallel. Cells were cultured on plastic plates in high glucose (4.5 g/L) DMEM medium  supplemented with 10% foetal bovine serum (FBS)  and 1% penicillin/streptomycin.2) onto 6-well tissue culture treated plates in antibiotic free medium and allowed to adhere overnight. After this, cells were transfected with plasmids using a specific dermal fibroblast transfection reagent . The concentrations of plasmids and reagent were scaled down according to the number of cells per well. The transfection was allowed for 6 hours and after the fresh antibiotic free medium was replaced. All live experiments with transfected cells were performed 48 h after transfection.Cells were transfected with actin  or vimentin ,  plasmids. Cells were seeded at low density  to reduce background fluorescence and photobleaching. Time-lapse recordings of single cell dynamics were acquired with a 20× objective by a Lumascope LS720 microscope  at a rate of 1 image every 10 min for at least 6 h. The miniaturised microscope was placed inside the incubator, so temperature and CO2 concentration were maintained throughout the time-lapse experiment.2 on 12-well plates with attached PDMS stencils and incubated for 2 days to confluence. The “wound” was initiated by removing the PDMS stencil, and the medium was aspirated and changed with fresh one. Cell migration was monitored by taking images every 30 min for 100 h.For wound healing assays, cells were seeded at 25,000 cells/cmThe algorithm to analyse time-lapse fluorescence videos is based on grey-scale images of the fluorescent channels, and there are two steps: (1) determination of the cell outlines for every frame and (2) calculation of the positions of cell centroids. Once the position of the cell’s centroid was determined for each frame, we computed the cell’s instantaneous migration speed and the persistence of the overall recorded migration path as previously described elsewhere . In brie2. If the cell exceeded that range, half or a quarter of cell was chosen including always a portion of the cell nucleus as well as the cell edge (Atomic force microscopy (AFM) was employed to investigate the biophysical properties of human dermal fibroblasts. The AFM system  was mounted on an epifluorescence microscope . Images of live, healthy fibroblasts were scanned under liquid conditions (DMEM medium with 25 mM Hepes supplemented with 10% FBS and 1% penicillin/streptomycin) at 37 °C with the V-shaped gold-coated silicon nitride cantilevers  with four-sided pyramidal tips in contact mode. The cantilever had a spring constant of 0.06 N/m, length of 200 µm, and width of 30 µm. The spring constant of the cantilever was calibrated using the thermal fluctuations method based on sensitivity calculation on the bare region of the substrate. Force maps of the cells were taken in quantitative imaging (QI) mode at a resolution of 32 × 32 pixels, using 4000 nm ramp length, 250 µ/s ramp speed and a force setpoint of 2 nN. Using these conditions, maximum indentation levels reached were ~2 µm, typically on the vicinity of the nucleus of the softest cells probed. The scan area depended on the cell size, with the maximum attainable range being 100 × 100 µmell edge .CSK) and the cell cortex (Ecort) was based on the approach proposed in Pogoda et al. [cort is obtained by fitting the force–indentation curve for data points corresponding to indentations <400 nm, whereas Ecsk is obtained by fitting the force–indentation curve for data points corresponding to indentations >750 nm. Cellular viscosity was determined from force–distance curves using the method described by Rebelo et al. [Biophysical properties such as Young’s modulus (E), viscosity (η), and non-specific adhesion work were determined from the force–distance curves. The force–distance curves were analysed using the BECC model for thin adherent cells on a stiff substrate , using aa et al. . In parto et al. .2) on 13 mm glass coverslips. The coverslips were coated with type I collagen at 10 µg/mL concentration for 1 h at 37 °C. After being rinsed with PBS, cells were seeded and allowed to adhere for 24 h. For drug treatment experiments, cells were seeded at the low density 24 h prior drug treatment. Cells were treated with 1 µM, 2.5 µM, and 5 µM concentration of withaferin A and with 2 mM, 4 mM and 6 mM concentrations of acrylamide for 3 h. Then, cells were fixed with 4% paraformaldehyde  for 20 min, washed with PBS, permeabilised with 0.25% Triton X-100  for 10 min, washed with PBS, and blocked with PBS supplemented with 3% bovine serum albumin (Sigma) for 1 h at room temperature.Dermal fibroblast samples were prepared by seeding cells at low density  and co-stained with vimentin monoclonal mouse , tubulin monoclonal rabbit , or yes-associated protein (YAP) monoclonal mouse , p21 monoclonal rabbit  or alpha-smooth muscle actin monoclonal mouse . Subsequently the secondary antibodies were used—anti-mouse Alexa 488  and anti-rabbit Alexa 488 . After staining, coverslips were mounted on glass slides with Prolong Gold antifade mounting medium with DAPI  to protect samples from drying out. Fluorescence images of the fixed cells were obtained using an inverted epifluorescence microscope  with ×20/0.5 NA objective lens and a charged coupled device (CCD) camera . Images were taken only on well-attached and not damaged cells using DAPI, FITC, and TRITC channels.Our pipeline for single-cell quantification of cytoskeleton and nuclear structures has been described in detail elsewhere . BrieflyThe reattachment experiments were carried out using the Lumascope LS720  microscope as above, using only healthy and well-attached transfected cells. To initiate the reattachment event, cells were treated with trypsin until they displayed a rounded up shape but before they were completely detached. Subsequently, fresh imaging medium was added to the wells and the process of cell reattachment was imaged. Fluorescence images were recorded every 10 min for 6 h using a 20× objective.Healthy vimentin-transfected and well attached cells were chosen and imaged for 1 h prior drug treatment. Subsequently, Withaferin A  with concentrations of 1 µM, 2.5 µM, and 5 µM, or acrylamide  with concentrations of 2 mM, 4 mM, and 6 mM was added and cells were imaged for 6 additional hours. Images were captured every 10 min. Cell velocity was calculated as described above on the same cells before and after drug treatment.t-test was used for the normally distributed data sets, otherwise, the non-parametric Mann–Whitney U test was adopted. Statistical significance was reported at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) unless otherwise stated. All experiments were performed using at least three replicates unless otherwise mentioned in the figure legend.Statistical analysis was performed using GraphPad Prism 5 software . The The purpose of this study was to evaluate the biophysical properties of human dermal fibroblast cells obtained from donors of different ages, obtained at ages: Neonatal, 21, 47, and 62 years. To measure the cell velocity of single cells, a miniaturised live imaging system placed inside an incubator was used to perform long-term cell migration experiments in 2D at physiological conditions. Cells were seeded at low density onto six-well plates and transfected separately with a fluorescently-tagged vimentin plasmid. Transfected cells were allowed to recover for 48 h prior to migration experiments. Images were taken only of single cells that were clearly transfected, healthy, and well attached. Time-lapse fluorescence images were taken every 10 min for 6 h. The videos of cell migration were then analysed to measure migration velocity and directionality, by tracking the non-fluorescent circular area corresponding to the cell nucleus. The results show that human dermal fibroblast cells from the neonatal donor have a significantly higher velocity compared to all adult donors. The largest difference (twofold) was observed when comparing them to cells from the oldest donor A. IntereCell motility is associated with changes in biophysical properties, which are regulated by the cytoskeleton. We therefore examined whether donor age has an effect on cell biophysical properties using atomic force microscopy to measure viscoelastic properties. Individual cells from all groups were probed in QI mode, and our customised data-analysis pipeline was used to calculate cells’ Young’s modulus (E), viscosity, and adhesion work. When determining E, we found that cells from the oldest donor displayed a twofold increase compared to cells from the neonatal donor C. SimilaTogether, our results show that donor age significantly affects biophysical properties, and in particular induces a reduction in cell motility alongside increased cell elastic modulus, viscosity, and adhesion force.2  for cells for the neonatal donor, while for cells from adult donors, the surface area was larger and ranging from 3000–7000 µm2 (CoV = 60%), reaching a larger than twofold increase when comparing cells from neonatal donor to cells from oldest donor . Cells at low density were transfected with vimentin-GFP for 72 h prior to drug treatment, and time lapse fluorescence images were taken only on transfected and well-attached cells. Considering the large variability of single-cell motility, we decided to image the same individual cells before and after drug treatment. Therefore, cells were imaged for 1 h before treatment and 3 h after drug treatment. Cell velocity was measured as previously described. In parallel, a different set of cells treated with the same drugs were immunostained with phalloidin for F-actin and primary antibody against vimentin to quantify their assembly.First, we investigated the potential effect of withaferin A treatment on cell migration and vimentin assembly. Withaferin A treatment caused a reduction in cell motility and increased the amount of vimentin assembled in fibres for cells from the neonatal donor . SimilarNext, we investigated the effect of acrylamide treatment using the same approach as before. Surprisingly, acrylamide treatment had no effect on cell migration of cells from neonatal donor and showed a minor effect on vimentin and F-actin fibres assembly . HoweverCSK and Ecort of cells from the neonatal donor  and whether those effects depended on the mechanical structure being probed. Atomic force microscopy was employed to measure the viscoelastic properties of cells treated with withaferin A or acrylamide. Force indentation–curves were taken by probing cells treated with withaferin A or acrylamide after 3 h. We found that withaferin A treatment increased Eal donor , even thlt donor , suggestnor  has been shown to be regulated by cell senescence . Here, wVimentin has been known to play a key role in cell migration. In migrating fibroblasts, the nucleus is surrounded by an abundance of vimentin filaments, which extend into the tail of the cell. On the contrary, vimentin monomers and short filaments are localised at the leading edge. These intracellular regional changes in vimentin structure and organization are responsible for regulating protrusion activity. In addition, serum starvation in fibroblasts caused reduced motility and the local breakdown of the vimentin network . SimilarThe contribution of vimentin organization to cell motility and mechanical properties can be also assessed using drugs against vimentin. Of note, the use of drugs targeting the polymerization of vimentin monomers into filaments rather than the use of siRNA against vimentin protein expression is an approach that parallels the use of cytochalasin D or latrunculin A against the assembly of G-actin monomers into F-actin fibres to understand the structural role of stress fibres on cell mechanics. That being said, the existing biochemical toolkit to target vimentin is still very limited and not fully characterised. Accordingly, the two gold-standard drugs used in the literature, withaferin A and acrylamide, may also affect other cytoskeletal structures or signalling pathways in addition to modulating vimentin filament assembly. In our experiments, withaferin A treatment induced disruption of vimentin organization and led to the formation of aggregates, which are believed to be associated with changes in cell shape, reduction in cell motility , and celIn summary, and to highlight the relevance of our results, we show that vimentin dominates the changes in cytoskeleton organization and assembly in human dermal fibroblast cells and may thus play a key role in the aberrant behaviour and impaired function displayed by this cell type in the course of human ageing. Accordingly, we propose that vimentin might serve as a suitable therapeutic target especially for aging-related diseases. We further propose that biophysical properties such cell motility and mechanical properties are strongly correlated to vimentin amount and can thus be readily used as high-throughput biomarkers on drug screening assays in the search for new anti-aging therapies.


Adamantinomatous craniopharyngioma (ACP) makes up between 6 and 8% of pediatric brain tumors and is the most common pediatric tumor arising in the sellar/suprasellar region of the brain. The 10-year survival for patients diagnosed with craniopharyngioma ranges between 64 and 92%, but complicating factors such as location, common cyst formation, and potential hypothalamic infiltration cause significant morbidity in this population. There are a number of therapeutic options for children with ACP, including surgery, radiation, and cyst directed therapies such as interferon and bleomycin. Research has raised concerns regarding the efficacy and side effects associated with these conventional therapies, as well as with the difficulty in treating recurrent cystic ACP. Evidence from our group and others has shown that the cystic and solid tumor components of craniopharyngioma have high levels of IL-6R and IL-6, providing a potential target for therapy. Tocilizumab, a humanized monoclonal antibody, acts against soluble and membrane bound IL-6R, and has been widely utilized in pediatric patients. Two patients with recurrent cystic ACP were offered systemically administered tocilizumab or a combination of tocilizumab and bevacizumab on a compassionate use basis. Both patients' tumors had a significant response, with decreased cyst burden, supporting the assertion that tocilizumab with or without bevacizumab may be an option for patients suffering from cystic ACP. Adamantinomatous craniopharyngioma (ACP) is a neurologically devastating chronic disease with morbidity that far outweighs the mortality risk. It comprises 6–8% of pediatric brain tumors , 2 and iTherapeutic modalities for children with ACP include maximally-safe surgery, radiation, and cyst-directed therapies, such as interferon and bleomycin. None of these approaches, however, are directed against unique biological characteristics of ACP. Furthermore, after many years of use, each has been shown to be associated with shortcomings regarding clinical efficacy and/or side effects –11 especCTNNB1 gene  T-cell therapy for acute lymphoblastic leukemia. There is substantial experience with this well-tolerated medication in the pediatric oncology community. Adverse events associated with tocilizumab are infection, neutropenia, thrombocytopenia, and elevated liver enzymes. This report discusses the management of two patients with cystic ACP who failed first line cystic-directed therapies and were eventually offered systemic administration of the IL-6R antibody, tocilizumab, on a compassionate use basis.Primary patient samples were obtained from Children's Hospital Colorado and collected in accordance with local and Federal human research protection guidelines and institutional review board regulations. The protocol was approved by the Colorado Multiple Institutional Review Board (COMIRB 95–500). Written informed consent was obtained for all specimens and clinical information collected.A 3-year-old male presented to the Emergency Department after hitting his head during a fall from a crib. CT scan revealed a suprasellar mass with extensive cysts extending throughout the right middle and bilateral posterior cranial fossae, as well as to the atrium of the right lateral ventricle. MRI  confirmeA cyst catheter was placed into the dominant right temporal cyst. Three days after discharge, he developed significant facial and scalp swelling accompanied by vomiting. This swelling was ultimately attributed to cyst fluid tracking along the outside of the catheter and under the Ommaya reservoir into the subcutaneous soft tissues. He was successfully treated with a course of oral dexamethasone.Approximately 6 weeks following placement, he was treated with intracystic IFN-α according to previously utilized protocols , 18. OveFollowing 5 weeks of intracystic bleomycin, he presented emergently with new onset left VI and VII cranial nerve palsies. MRI demonstrated new edema involving the left pons, middle cerebellar peduncle, and anterior right cerebellar hemisphere, in continuity with one wall of the cyst receiving bleomycin . BleomycThe patient remained off of therapy with routine MRI scanning to monitor for disease progression. During the initial 7 months, there was radiographic improvement of the cystic portions of his lesion however, imaging at 14 months off therapy demonstrated progression of his cystic disease , graph.Given his young age and the large potential radiation field, the risks of radiation therapy were thought to outweigh the benefit, and he was offered treatment with tociliziumab (12 mg/kg IV q2 weeks) on a compassionate use basis, which he received for 6 months and tolerated well. Imaging 6 months following the start of therapy demonstrated a decrease in volume of some cysts and an apparent slowing of expansion of others . FollowiSystemic IV tocilizumab in combination with IV bevacizumab every 2 weeks was generally well tolerated. There were three delays in therapy of 2 weeks for the first two delays and 4 weeks for the third delay due to neutropenia . There were no fevers or signs of systemic illness associated with these episodes. In total, he received 14 months of combination therapy over 28 months with MRIs showing a significant decrease in cystic disease . FollowiThree months off of therapy, an MRI showed a minimal increase in some cystic components while the remainder of the solid and cystic components remained unchanged. Six months off therapy the patient was seen in the Emergency Department with seizure activity. At this time, an MRI demonstrated an overall increase in his cystic mass . BevacizA 7-year-old male presented with a 1 month-long history of nausea and vomiting. A head CT was performed for concern for increased intracranial pressure and demonstrated a large cystic frontal mass that crossed the midline, with perilesional edema. His past medical history was significant for a diagnosis of “lazy eye” 1 year previously and short stature (later determined to be due to growth hormone deficiency). Family history was unremarkable.MRI brain demonstrated a large cystic sellar/suprasellar mass, most consistent with craniopharyngioma . MRI volThe patient returned to the outpatient clinic for weekly cyst aspiration. Three months after catheter placement, MRI demonstrated an increase in cyst size , and repThe patient was offered systemic tocilizumab (12 mg/kg IV q2 weeks) on a compassionate use basis. After 6 months of therapy, the cyst exhibited a partial response with measurements improved from 26 × 28 × 27 mm (AP × TRV × CC) to 18 × 24 × 18 mm (AP × TRV × CC) . He has Here we report the first use of tocilizumab alone or in combination with bevacizumab for the treatment of cystic ACP in pediatric patients. The biological basis for this was based on recent studies that demonstrated elevated IL-6R levels and IL-6 transcript levels in ACP cyst fluid and solid tissue, respectively. The patient in case 1 had known elevated IL-6 in cyst fluid prior to therapy with tocilizumab and level were in line with other samples tested . Case 2 Current therapies for pediatric ACP can cause significant morbidity, and safer, more effective treatment is needed . ResearcThese two case studies suggest that the combination use of systemically administered tocilizumab and bevacizumab may be effective in pediatric patients with primarily cystic craniopharyngioma. Future evaluation of this combination of this drug is needed to further evaluate the potential of these drugs in the management of ACP.SG did primary case reviews and writing of original draft of manuscript. DM provided MRI analysis and figures. AD and NF provided laboratory data. ND and LH provided patient care and manuscript preparation. TH and JM provided primary patient care, conceptualization, manuscript preparation, and project management.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Enormous variability in skin colour and patterning is a characteristic of teleost fish, including Salmonidae fishes, which present themselves as a suitable model for studying mechanisms of pigment patterning. In order to screen for candidate genes potentially involved in the specific skin pigment pattern in marble trout (labyrinthine skin pattern) and brown trout (spotted skin pattern), we conducted comparative transcriptome analysis between differently pigmented dermis sections of the adult skin of the two species.gja5, clcn2, cdkn1a and tjp1) of the ten candidate genes tested. The potential role of these genes in skin pigment pattern maintenance is discussed.Differentially expressed genes (DEGs) possibly associated with skin pigment pattern were identified. The expression profile of 27 DEGs was further tested with quantitative real-time PCR on a larger number of samples. Expression of a subset of ten of these genes was analysed in hybrid (marble x brown) trout individuals and compared with the complexity of their skin pigment pattern. A correlation between the phenotype and the expression profile assessed for hybrid individuals was detected for four  contains supplementary material, which is available to authorized users. Skin pigmentation and pigment patterning play an important role in the survival of animals. Pigments can influence thermoregulation, social communication, mate choice and predator avoidance, among others, and provide protection against the harmful effect of solar radiation , 2. EvenThe genetic basis of pigment production and subsequent skin pigmentation is well understood in model species such as zebrafish –12, and Much research on zebrafish has been undertaken on artificially produced mutants. However, much less is known about the genetics of skin pigment patterns in natural populations. Salmonidae fishes are characterised by a huge variety of naturally present pigment patterns and thus present themselves as a suitable model for studying the mechanism of pigment pattern formation and maintenance.One factor that may cause great variety in salmonids could be the relatively recent whole genome duplication (WGD) event  that tSalmo: marble trout (S. marmoratus), with labyrinthine skin pattern, and brown trout (S. trutta), with spotted skin pattern , some zebrafish mutants , partiax41.8 M7 , and alsx41.8 M7 , as wellntinalis ).Fig. 1MMorphological differences in chromatophore ultrastructure, and position or arrangement, or both, in the dermis of marble and brown trout have been revealed using transmission electron microscopy . In zebrhdac1, vps18, dct and scg2a—and it has been proposed that the formation of the labyrinthine pattern at least partially depends upon the Wnt signalling pathway and is based upon a reaction–diffusion mechanism. Only genes involved in the biological process ‘pigmentation during development’ were considered as informative [Salmo, we employed high-throughput mRNA sequencing technology on differentially pigmented dermis sections of marble trout and brown trout. These first RNA-seq data on differently pigmented dermis regions for these two species enabled (i) an overview of the transcriptome in dark, white and red coloured skin regions, and (ii) identification of differentially expressed genes (DEGs) possibly associated with skin pigment pattern. The expression profile of 27 DEGs was tested further with quantitative real-time PCR (qPCR) on a larger number of samples, while expression of a subset of ten of these genes was analysed in hybrid trout individuals (marble x brown trout) and compared with the complexity of their skin pigment pattern.Recently, microarray analysis has been undertaken to compare gene expression profiles of whole skin samples of marble and brown trout, with dermis and epidermis, and all differently pigmented regions, considered as a single sample . Four caormative . In the S. salar) genome (INSDC: AGKD00000000.4) [Sequencing of RNA isolated from trout dermis resulted in a total of 812,144,586 pair-end reads of 100 bp each  that were mapped to the gene model of Atlantic salmon (00000.4) . All traThe top 50 genes most highly expressed in trout skin included genes of the keratin family and collagen proteins, with keratin, type II cytoskeletal cochlear-like the most highly expressed. The other genes highly expressed in marble and brown trout skin are reported in Additional files dct, tyr, tyrp1, pmel, ednrb) showed differences in expression profiles between the two species, as did some genes involved in cell–cell junctions  and the establishment of membrane and action potentials . Among these groups of differentially expressed genes, 27 were selected for additional testing by qPCR.Using NOISeq numerous DEGs between marble and brown trout pigment cell genes were identified. A total of 52,012 predicted genes were identified as expressed differentially between marble and brown trout skin. Of these, 3715 genes were up-regulated (≥ 2 fold) and 4327 down-regulated (≤ 2 fold) in the skin of marble trout compared to that of brown trout. Among the up- and down-regulated genes several are known to encode membrane proteins involved in, for example, signalling, cell junctions, or ion permeability. The 100 most up- and down-regulated genes that differed between marble and brown trout samples are reported in Additional files K-mean clustering was used to group genes with a similar pattern of expression among samples. Many genes were overexpressed specifically in the red spot of brown trout compared to other skin regions of both species, indicating that there are specific pathways involved in either carotenoid metabolism or communication between these specific pigment cells and their surrounding cells, or both, resulting in the formation of a red spot.http://www.geneontology.org.DEGs were annotated through GO classification analysis and grouped into three categories  based on their putative functions. Salmon genes were annotated with BLAST , 38 agaiFor the GO analysis, 18,087, 61,751 and 87,737 DEGs between marble and brown trout were grouped by molecular function, cellular component and biological process categories, respectively. Altogether, DEGs were mapped to 61 categories, as presented in Fig. KEGG pathway analysis was carried out to categorise and annotate the DEGs, which were mapped and visualised in a way similar to the KEGG Mapper tool. The pathways with the largest number of genes expressed differentially between marble and brown trout were Neuroactive ligand-receptor interaction, NOD-like receptor signalling pathway, PI3K-Akt signalling pathway, Pathways in cancer, Focal adhesion, and MAPK signalling pathway , mitf, pmel, mc1r, ednrb, sox10, aox1, cdkn1a, wnt10a and wnt9b—, and (2) genes with a molecular function in cell binding known to be involved in ion exchange, chromatophore interactions and pigment pattern: gja1, gja4, gja5, gja9, gjb1, gjb3, gjb4, gjd2, tjp1, tspan3, slc7a2, slc01c1, kcnc2, clcn2 and pcdhac2. qPCR  of the 27 selected genes had a significantly higher level of expression in brown trout skin than in marble trout skin, four others  were expressed significantly more, and one (tyr) significantly less, in the red spot of brown trout, while five genes  had significantly higher expression levels in the skin of marble trout than in brown trout. Gene gja5 exhibited an interesting pattern of expression, significantly higher in (black and red) spots of brown trout than in other skin parts of either species. The qPCR results were mostly consistent with the data from transcriptome sequencing, with 23 out of 27 genes having the same expression profile.In order to validate the transcriptome sequencing results, we selected 27 DEGs for qPCR to determine their relative expression in marble and brown trout skin Table . These gdct, cdkn1a, gja5, tjp1, wnt10a, gjd2, slc7a2, kcnc2, clcn2 and pcdhac2) with the most differential expression between the two species were selected and their expression further tested in differently pigmented skin regions of hybrid samples.From analysis of the qPCR products, ten candidate genes 5). The cype Fig. . The oveout Fig. . FiguresVarious mechanisms are thought to be responsible for the great variety in skin pigment patterns observed in animals. Recently, the role of homotypic and heterotypic intercellular interactions has been demonstrated in pigment progenitor cell migration, and in the formation and maintenance of a specific pigment pattern in zebrafish , 17–19. KEGG pathway analysis showed that a substantial number of DEGs between the two species are associated with pigmentation-related pathways—e.g. MAPK signalling pathway, Wnt signalling pathway, Melanogenesis, Tyrosine metabolism—and cell–cell communication, such as Focal adhesion, Cell adhesion molecules (CAMs), Calcium signalling pathway, Tight junction, Adherens junction and Gap junctions.pmel, tyrp1, dct) were overexpressed in the black spot of brown trout when compared to other pigmented regions of brown trout skin.Differential expressions were also observed when differently coloured regions of skin from the same individual fish were compared, most probably associated with specific expression profiles of individual pigment cell types predominating in a particularly coloured skin region . When comc1r, tyrp1, dct, wnt6), and (2) genes with a molecular function of binding, including those coding for proteins involved in cell–cell communication and ion channels , known to act as factors that can affect the organization of pigment cells in the dermis [The results from the present study indicate that DEGs with a possible role in trout skin pigment pattern maintenance can be classified into two major groups: (1) genes involved in pathways that have a known role in pigmentation A positive correlation between phenotype and the expression profiles assessed in hybrid individuals with a variety of different skin pigment patterns was observed for four of the tested candidate genes: cdkn1a  encodes a cyclin dependent kinase inhibitor 1a that was up-regulated in skin with labyrinthine pattern (marble trout). This gene functions as a regulator of cell-cycle progression in the phases G1 and S. Furthermore, it has been shown that cdkn1a is involved in patterning of eye-spots in the pigmented epithelium of the retina [cdkn1a is known to be important in the development of melanocytes, promoting their differentiation and melanogenesis [Gene e retina . Anothere retina , which cogenesis .cdkn1a and the Rho-pathway and thus could affect the final skin pigment pattern. To substantiate our hypothesis KEGG analysis of the light region versus the black spot was conducted. Pathways with the largest numbers of DEGs included PI3K-Akt signalling pathway, Focal adhesion, ECM-receptor interaction, Cell adhesion molecules, Regulation of actin cytoskeleton and Axon guidance. These results show that different cell movement and attachments could indeed affect the perpetuation of spots.Šestáková et al.  reportedclcn2, encodes a plasma membrane voltage-gated chloride channel Clcn2 . As with other chloride channels, this is a transmembrane protein involved in the transport of chloride ions and, through that, the maintenance of chloride ion homeostasis and the transmission of electrical signals between cells. The functions of chloride channels include membrane potential stabilization, signal transduction, regulation of cell volume and of a tight junction, and transepithelial transport [clcn2 gene has not been described previously to be involved in pigment patterning. Meanwhile, another chloride channel—CIC-7—has been shown to have an important role in pigmentation in mice [A second candidate gene, ransport . To the  in mice , while i in mice .tjp1 and gja5, are involved in cell–cell junctions. The gene tjp1  encodes a protein involved in tight junctions and was up-regulated in spotted skin (red and black spots in brown trout). It has already been demonstrated that the expression of this gene can affect pigment patterning. In a previous study, a zebrafish with a mutation that disrupts the functioning of the tjp1a protein was examined [tjp1a was expressed in dense iridophores but its expression was down-regulated in loose-form iridophores and in melanophores and xanthophores. In the mutant, the dense iridophores interrupted the dark stripes, which reorganized to form spots. Iridophores in trout skin were not recognized as loose or dense forms, but rather were classified based upon the size of their inclusions, into large (L) and small (S) types [tjp1 might play a distinct role in the functioning of these iridophores. Namely, type L iridophores were present exclusively in the light area of brown trout skin [tjp1, these cells could prevent melanophores from forming or from maintaining the labyrinthine pattern, or both, but rather organizing into spots. Another, very likely, role of the tjp1 gene in skin pigment patterning is connected to the observation that Tjp1 protein is a scaffolding protein that links tight junction proteins (such as claudins and occludins) to the actin cytoskeleton and could, as a consequence, affect the rearrangement of the cytoskeleton and the movement of the cells [cdkn1a, which has a similar role, the expression profile of these two genes could explain the diverse movements of two types of melanophores and their communication with other pigment cells resulting in different pigment patterns [The other two candidate genes, examined . The mutS) types . Neverthout skin . With thhe cells . Togethepatterns , 48.gja5  was overexpressed in dark and red spots in brown trout. This gene codes for a protein that is a member of the connexin family, which forms another type of cell junction: gap junctions. These are connections between cells, have a role in cell–cell communication and are involved in the maintenance of ionic and metabolic homeostasis and transmission of various ions and electrical signals [gja5 might play an important role in communication (heterotypic interaction) between these two cell types or between erythrophores (homotypic interaction), resulting in a spotted pattern. The role of gap junction proteins in pigment pattern formation and maintenance has been confirmed several times in zebrafish, mainly through studying a range of mutants with various pigment patterns as a consequence of the modified cx41.8  gene  [gja5. Nevertheless, to the best of our knowledge, this is the first study demonstrating a connection between gja5 expression level and skin pigment pattern. Differential expression of another connexin—gja1—between light and dark pigmented regions has been shown previously in sticklebacks [gja5 in red spots of hybrids showed a direct correlation with phenotype, with an overexpression in red spots present in skin with a general marble trout phenotype, or labyrinthine pattern. We additionally examined the correlation between gja5 expression in red spots and overall colour tone that is directly connected to the labyrinthine pattern. The hybrids formed two groups, where higher gja5 expression was connected to a darker overall colour tone  , 23, 50,klebacks , where anophores . The connophores . The invIn summary, we sequenced transcripts from the skin of marble trout and brown trout for the first time and identified more than 8000 DEGs (with a fold change ≥2) in the skin of the two species. These DEG data provide a reference for screening genes for their effect on pigment patterning and other processes taking place in trout skin. The results significantly enhance our understanding of the composition of the trout skin transcriptome and the potential differences in gene expression associated with skin pigment pattern maintenance, and provide a foundation for future studies. We proved the validity of the mRNA-seq results using qPCR, where 27 candidate genes were tested on a large number of samples. Our results suggest an important role of cell–cell interactions, junctions and cytoskeleton rearrangements in trout skin pigment pattern maintenance as a consequence of differential gene expression.Adult marble trout individuals (of age > 2 years) were collected from Tolminka fish farm in Tolmin, Slovenia, and adult brown trout (of age > 2 years) from Bled fish farm, Bled, Slovenia. With an age of at least two years these trout had reached sexual maturity and already developed their adult skin pigment pattern. Their body length was approximately 30 cm. All fish were first-generation offspring reared in their respective fish farm from wild-caught parents originating from Zadlaščica stream (marble trout) and Malešnica stream (brown trout), Slovenia. Individuals had been fed with Biomar INICIO Plus (Denmark) as a starter feed and Biomar EFICO Enviro 920 when adult. Prior to skin sample collection fish were sedated in anaesthetic Tricaine-S  and killed by a blow to the neck.In order to verify any correlation between the expression of ten candidate genes and the skin pigment pattern, hybrids between the two species with a variety of pigment patterns were sampled from Volarja stream  where stocking with brown trout has been intensive for several decades and where ‘pure’ marble trout could not be found due to hybridisation. Hybrids with various levels of brown trout introgression are a result of hybridisation between marble and introduced brown trout, and backcrosses. Level of introgression was assessed by genotyping a panel of 36 diagnostic nuclear SNPs as reported previously . The intAll methods described were carried out in accordance with relevant guidelines and regulations. Fish skin sampling was approved by the Ministry of Agriculture and Environment, Slovenia, under decision letter number U34401–60/2013/4.Small pieces of skin from the lateral part of the trunk of the body Fig.  were obtTotal RNA was isolated from cryosections of skin using a PicoPure RNA Isolation Kit  according to manufacturer’s instructions. All samples were treated with DNA-free DNA Removal Kit (Thermo Fisher Scientific) to remove traces of genomic DNA. Concentration of total RNA and RNA integrity value (RIN) were checked using an RNA 6000 Pico LabChip of Agilent 2100 Bioanalyzer . RNA extracts with the highest RNA integrity of identically pigmented skin samples from three individuals per species were pooled. All RIN values were > 8.5. In total, five libraries from 1 μg of total RNA isolated and pooled from six individual fish (three per species), labelled as MTD, MTL, BTB, BTL and BTR, were prepared using random hexamer primers. cDNA libraries were sequenced by Beijing Genomics Institute  using 2x100bp pair-end high-throughput mRNA sequencing (RNA-seq) on two lanes  of Illumina Hiseq 2000 .Paired end sequences from the mRNA-seq were mapped with Bowtie2  against P-value [www.geneontology.org. The output ECM gene list could be cross-checked for DEGs from NOISeq. KEGG pathway analysis [Differential expression analysis was performed using the R/Bioconductor package NOISeq . FoP-value , 63 was analysis  was perfSamples for RNA isolation were prepared using biopsy punches to selectively dissect differently pigmented parts of the skin. Skin pieces were then homogenised with Percellys24 homogenizer (Bertin corp.) with the use of zirconium oxide beads (Bertin corp). Total RNA was isolated using RNeasy Plus Universal Mini Kit according to manufacturer’s instructions and subsequently treated with RNase-free DNase I (Thermo Fischer Scientific). Only RNA with good purity, checked with a NanoVue spectrophotometer , with values A260/280 ~ 2.0 was used for cDNA synthesis, performed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific).rps20, hprt1, RNA polII, ppia1, pgk, sdha, b2m, ef1a, β-actin, 18 s—were analysed to identify two that could be used as reference genes: rps20 and pgk1 were chosen as having a stable expression, i.e. at the same level in all tested samples. The expression of candidate genes was analysed with ten biological replicates of marble  and ten of brown trout , each performed in triplicate. A no-template-control (NTC) was used to check for potential external contamination. Standard curve analysis was performed for reference and target genes to assess amplification efficiency, which was comparable between genes. The conditions for all reactions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. At the end of every run, melting curve analysis was performed in order to confirm a unique amplicon reaction.qPCR was performed using SYBR Green PCR Master Mix  and conducted on a Viia7 Real-Time PCR System . All primers for selected candidate genes Table  were desExpression of ten candidate genes with the highest differential expression between the two species was additionally tested in differently pigmented skin regions of hybrid samples using the protocol described above.p < 0.05 were considered statistically significant.Differential gene expression results were calculated using the Pfaffl method , normaliPSS) is defined as the area-weighted mean isoperimetric quotient of the contours extracted from each image:Pigment patterns of hybrid animals and genetically pure brown and marble trout were quantified and analysed as described previously . BrieflySi and Li are, respectively, the surface area and the perimeter of each contour. The overall colour tone of a pattern was defined and calculated as the proportion of non-pigmented area (white pixels in a binarized image). The proportion of red spot regions was calculated from the average proportion of reddish pixels in each image.where Additional file 1:Table S1. Summary results of transcriptome sequencing and mapping. (PDF 21 kb)Additional file 2:Table S2. 50 most expressed transcripts in marble trout. (PDF 51 kb)Additional file 3:Table S3. 50 most expressed transcripts in brown trout. (PDF 61 kb)Additional file 4:Table S4. 100 transcripts with the highest fold change between marble and brown trout samples. (PDF 108 kb)Additional file 5:Table S5. 100 transcripts with the highest fold change between brown and marble trout samples. (PDF 111 kb)Additional file 6:Figure S6. Gene expression patterns of four candidate genes in differently pigmented regions in the skin of hybrids. Red, red spot; dark brown, dark region; light brown, light region. (PDF 345 kb)Additional file 7:Table S7.. Level of brown trout introgression in hybrid individuals. (PDF 417 kb)Additional file 8:Figure S8. Correlation between the expression of gja5 gene in the red spots of hybrids and overall colour tone of the skin in hybrids. (PDF 315 kb)


Given no consensus on optimal timeframe of periarticular multimodal drug injection (PMDI) in knee osteoarthritis patients undergoing total knee arthroplasty (TKA), this study was aimed to compare the postoperative pain and the functional recovery in patients who underwent simultaneous bilateral TKA (SBTKA) and received PMDI at the different intraoperative time points.This prospective, randomized, double-blinded controlled trial study included 48 patients who underwent SBTKA and received PMDI mixture, either before prosthetic implantation (late PMDI), or just after knee arthrotomy (early PMDI). Each subject’s knees were randomly selected to different PMDI administration time points. The outcome parameters were postoperative pain assessed by using a visual analog scale (VAS), the maximal angle of knee flexion, and quadriceps function from day 1 to 6 weeks after surgery.Late PMDI revealed slightly higher VAS at 6 and 12 h after the operation than early PMDI administration. Afterward, the VAS tended to be lower in the late than early PMDI administration until the end of the study, but without statistical significance. The time difference between early and late PMDI had no effect on postoperative VAS, while older age resulted in significantly less pain. No statistical differences between the two groups in all other outcome parameters were observed.Postoperative pain reduction and functional recovery of SBTKA with early and late PMDI administration were not significantly different. The time interval of PMDI between knees did not confound the comparison of postoperative pain and functional recovery in SBTKA.TCTR20170617001 on 16 June 2017.The protocol of this study was retrospectively registered in the Thai Clinical Trials Registry database No. Total knee arthroplasty (TKA) is one of the most frequently performed surgical procedures by orthopedic surgeons, with effective treatment and a high success rate in patients suffering from knee osteoarthritis , 2. HoweExaggerated pain response following TKA is caused by both peripheral and central sensitization, which may persist up to 3 months after surgery . PreventCurrently, periarticular multimodal drug injection (PMDI) is trendiness because of its simplicity, effectiveness, and limited adverse effects , 15. TheThis study was a prospective randomized controlled, double-blinded trial of patients with the diagnosis of bilateral primary knee osteoarthritis and scheduled for SBTKA. The study protocol was approved by the Institutional Review Board of Naresuan University. The written informed consents were obtained before the enrollment. Patients who had any previous knee surgery, knee infection, inflammatory joint disease, history of the thromboembolic event, or a neuromuscular disorder, were excluded from the study.Fifty-five patients who were scheduled for SBTKA were assessed for the study. Of 55 patients, seven patients were excluded . The remaining 48 patients had enrolled and completed the study. All surgical procedures were performed by a single surgeon under spinal anesthesia with 2.8–3.6 mL of bupivacaine . The cemented fixed-bearing, posterior stabilized, knee prostheses were implanted via a standard medial parapatellar approach in all cases. The PMDI mixture consisting of 100 mg bupivacaine, 30 mg Ketorolac , 5 mg morphine sulfate, and 300 μg adrenaline  were diluted with a sterile normal saline solution to a total volume of 150 mL. The surgery was randomized using a sealed envelope to start on one side and the second randomization determined two different timings of PMDI that would be administered in the first operated knee. A group of “late PMDI” was the knees which received 75 mL of PMDI mixture prior to the prosthetic implantation as common practice of surgeons, whereas a group of “early PMDI” received 75 mL of the mixture immediately after knee arthrotomy . Therefore, the contralateral knee would receive the PMDI in the opposite timing. The time lag between PMDI administration was recorded and defined as the interval between PMDI. The flow diagram of patients in the study was shown in Fig. All patients were assessed and evaluated by two independent investigators who were blinded to the randomized process and the surgical procedures. Postoperative pain assessment using the 10-cm visual analog scale (VAS) was recorded during rest at 6, 12, 24, 48, 72, 96 h, and at 2 and 6 weeks after surgery. The maximal angle of knee flexion reached by the CPM device was noted every day during hospitalization and the active knee flexion angle was measured with a goniometer at 2 and 6 weeks postoperatively. The postoperative quadriceps function was determined by the degree of active straight leg raising (SLR) and extension lag (EL). The operative time, drain output, and the patient’s preferred side at the last admission day before discharge from the hospital, and at 2 and 6 weeks after surgery were also recorded.p-value < 0.05. A sample size of 48 knees for each group was anticipated to detect a difference in VAS between early and late PMDI, with a standard deviation of 1.5 [All measured demographics and clinical outcomes were summarized with descriptive statistics including mean and standard deviation. Normality of data was checked with the Kolmogorov-Smirnov test. We compared the outcomes between groups by using the student’s t-test for normally distributed data, and using the Wilcoxon matched-pairs signed rank test if the data were not normally distributed. Generalized estimating equations (GEE), adjusted for age, body mass index (BMI), American Society of Anesthesiologists (ASA) physical status classification, and interval between PMDI, was used for comparing the change of pain scores, knee flexion angle, SLR, and EL between the early and late PMDI along the study period . Statistical significance was defined as n of 1.5 , 90% powp > 0.05 at any time point) as shown in Fig. p = 0.80) higher VAS than the late PMDI. Interval between PMDI, BMI, and ASA physical status classification had −0.003 , 0.00 , and −0.45  changing of VAS scores, respectively, while each additional year of patient age significantly reduced VAS score by 0.05 points . However, all these factors had no effect on the change of other outcomes throughout the study period.A total of 48 patients with bilateral knee osteoarthritis undergoing SBTKA were included in the study. Patients’ demographics and baseline characteristics were provided in Table On the last admission day, 21 patients (43.8%) preferred the knee with early PMDI administration, while 16 patients (33.3%) preferred the side with late PMDI, and 11 patients (22.9%) could not differentiate between both knees. At 2 weeks postoperatively, 14 (29.2%) and 18 (37.5%) patients preferred the knee injected immediately after the arthrotomy, and just prior to the implantation, respectively, and 16 patients (33.3%) felt similarly. At 6 weeks after surgery, most of the patients  could not feel a difference between both knees. Four patients experienced minor complications. In both groups, superficial wound infection was suspected and managed by oral antibiotics prophylaxis. One deep vein thrombosis (DVT) was questionable in each group which further study by venous ultrasonography confirmed no clotting. These clinical findings were improved later without specific treatment.Postoperative pain management is one of the essential elements in improving the patient’s satisfaction, rehabilitation, and functional recovery following the TKA, especially in SBTKA. The PMDI is commonly administered before implantation of the prosthesis or before wound closure by most surgeons , 20. HowAccording to our study, we injected a similar multidrug mixture into the identical location of both knees under direct vision, but at a different timeframe. Therefore, we could directly compare the efficacy between early and late administration of PMDI. We found that the knees receiving the PMDI immediately after arthrotomy had slightly lower drain output at the day after surgery, and lower pain scores at postoperative 6 and 12 h when compared to the other group without statistical significance. The PMDI that was delivered just before implantation seemed to reduce pain better than early administration of PMDI at 24 h and afterward. However, there was no statistical significance along the study period.In addition, the previous study showed that the first operated knee would have higher pain scores significantly with less patient satisfaction compared to the latter side in SBTKA . In thatCurrently, multimodal pain management is widely accepted as a principle of contemporary pain management , 13. SevThis study has some limitations. First, the participants were predominantly female gender, which might be a factor affecting the postoperative pain and function . In our The early administration of the PMDI is able to slightly reduce pain in the first 12 h after surgery without statistical significance. In the same way, late administration of the PMDI is not able to significantly extend the period of pain relief. Therefore, the results suggest that the timing of the PMDI has no effect on pain relief and functional recovery following TKA. Additionally, the interval of PMDI between knees is not a confounding factor for comparison of the postoperative pain score and functional recovery following SBTKA.


We then designed and tested an in vitro protocol of resistance exercise (RE) inducing fatigue in arm samples. We showed that, upon the establishment of fatigue, a transient increase in mTORC1 phosphorylation reaching a pick 30 min after exercise was induced. Our data indicate the activation of mTORC1 pathway in exercise paradigm and possibly in the regulation of energy homeostasis in octopus and suggest that mTORC1 activity can be used to monitor animal response to changes in physiological and ecological conditions and, more in general, the animal welfare.Cephalopods are highly evolved marine invertebrates that colonized almost all the oceans of the world at all depths. This imposed the occurrence of several modifications of their brain and body whose muscle component represents the major constituent. Hence, studying their muscle physiology may give important hints in the context of animal biology and environmental adaptability. One major pathway involved in muscle metabolism in vertebrates is the evolutionary conserved mTOR-signaling cascade; however, its role in cephalopods has never been elucidated. mTOR is regulating cell growth and homeostasis in response to a wide range of cues such as nutrient availability, body temperature and locomotion. It forms two functionally heteromeric complexes, mTORC1 and mTORC2. mTORC1 regulates protein synthesis and degradation and, in skeletal muscles, its activation upon exercise induces muscle growth. In this work, we characterized Octopus vulgaris mTOR full sequence and functional domains; we found a high level of homology with vertebrates’ mTOR and the conservation of Ser Cephalopods are an important component of marine ecosystems, they count around 800 living marine species, and reached over time unprecedented environmental adaptability. They are not only abundant but also ecologically and economically important . Their iCephalopods have large, well-developed brains, and their brain-to-body-mass ratio exceeds that of all the other invertebrates. Muscles represent their major body constituent and, octopuses in particular, have a rapid growth rate reaching up to 5% body weight per day and manifest a high feed conversion rate, with 30–60% of ingested food being incorporated in their own weight . Based oSeveral investigations have been carried out so far to elucidate the bases of their muscle metabolism and energy consumption and its relation to environmental condition . Yet, liSimilar to vertebrates, muscles of their body are typically striated and for several functional aspects, they can be assimilated to skeletal muscle fibers . In vertSeveral signaling pathways, including the evolutionary conserved mTOR signaling cascade, maintain this process. mTOR is serine/threonine protein kinase that exists in two functionally distinct protein complexes, the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTOR complex 2 (mTORC2), that are defined by the presence of Raptor (regulatory protein associated with mTOR) and Rictor (rapamycin-insensitive companion of mTOR) .mTORC1 plays a critical role in muscle homeostasis because it senses and integrates a broad range of cellular signals from growth factors, hormones, cytokines, amino-acid availability, cellular energy levels and muscle activity . Genetic2448) -mediated hypertrophy requires mTORC1 activation, mainly through the phosphorylation of mTOR at serine 2448 (Ser2448) . The resIn this work, for the first time in a cephalopod, we investigated the conservation of mTOR and its role during a particular class of training such as the RE inducing muscle fatigue. Muscle fatigue is defined as the inability of muscles to maintain the required power output. Upon establishment of fatigue, muscle contractile force shows a rapid decline reversible by rest. We tested samples of arm with a specifically designed RE and showed the induction of a fatigue state. We next assessed mTOR pathway following tetanization and show a sharp increase in its activation state and its time dependency.We believe that studying mTOR pathway may be important to understand the ability of muscles, over time, to adapt to various physiological and ecological conditions such as exercise response, metabolic stress and nutrient availability and may prompt to the development of advanced methods for monitoring cephalopod welfare.O. vulgaris were collected from local anglers of the Ligurian coast. Adult animals of both sexes (n = 9) ranging between 150 and 250 g and not showing signs of damage and/or regeneration of the arms employed (L2 or L3) were selected for this study.Specimens of Following captures, the animals were placed in 80 × 50 × 45 cm marine aquarium tanks filled with artificially prepared sea water  and kept at a temperature of ∼18°C at 12 h light/dark cycle. Octopus environment was enriched with sand substrate and clay pot dens. Water cleaning and oxygenation were assured by a pump-filter and aeration system which continuously circulated the water through biological filters; all relevant chemo/physical water parameters were checked daily to prevent unhealthy or stressful conditions for the animals. Animals were left to adapt to captivity for at least 10 days before experimentation. Octopuses were inspected daily and fed with shrimps 3 times per week. Particular attention was paid to housing, animal care, and health monitoring. All our research conformed to the ethical principles of the three Rs  and of minimizing animal suffering, following the Directive 2010/63/EU  and the guidelines from For molecular biology experiments, 3 animals were anesthetized in ethanol 2% (v/v) in SW. Brain and arm samples (from L2 or L3), devoid of skin and suckers, were collected, frozen in liquid nitrogen and immediately stored at −80°C. Brain samples  were employed as an additional control in western blotting experiments.2 in SW, since ethanol exposure induced muscle stiffness. After anesthesia, a single segment (∼4–5 cm) per animal was cut from the middle-end of the L2 or L3 arm. Arm samples were moved to ∼18°C oxygenated artificial sea water (ASW) (pH 7.6) containing: NaCl, 460 mM; KCl, l10 mM; MgCl2, 55 mM; CaCl2, 11 mM; Hepes, 10 mM; glucose 10 mM. This temperature was the same as that of the aquarium where the animals were maintained and was within the temperature range of the Mediterranean sea.For biomechanical experiments a total of 6 animals were anesthetized in 3.5% MgClGiven the large portion of arm excised and/or the dissection of the brain, the animals underwent terminal anesthesia in order to prevent animal suffering or distress and following the Guidelines for the Care and Welfare of Cephalopods in Research published by Fiorito et al. , 2015.This study was carried out in accordance with the recommendations of Fiorito et al. , 2015. Tn = 3) using RNeasy Microarray Tissue Mini Kit (Qiagen) and contaminating DNA has been degraded by treating each sample with RNase-Free DNase Set (Qiagen). The purity of total RNA extracted has been estimated measuring 260/280 and 260/230 absorbance ratios. For each sample, 1 μg of total RNA extracted have been retrotranscribed with ImProm-II(TM) Reverse Transcription System (Promega) following the manufacturer’s instructions. mTOR gene has been divided into small fragments up to 1.5 kb and the correspondent primers were designed  using a Dual-Mode Lever Arm System (Aurora Scientific – 300C-LR) mounted on a muscle test chamber with integrated stimulating electrodes (Aurora Scientific – 801C). Field stimulation was delivered through a current-voltage bi-phase stimulator (Aurora Scientific – 701B). Data were acquired at a sampling frequency of 10 kHz, bandwidth filtered at 10 Hz - 3.3 kHz and further analyzed with a LabVIEW based Data Acquisition and Analysis System (Aurora Scientific – 604A and 605A). Test chamber was bath filled with temperature-regulated ASW continuously circulating at ∼18°C. Arm samples were placed in the chamber, tied tightly at the force transducer system and allowed to rest for about 5 min before starting the protocol. Recordings were made in isometric condition, thus enabling measurements of the force developed over time upon electrical stimulation of the sample. Muscle length was adjusted such that a transient passive force could be visualized , have been homogenized with TissueLyser (Qiagen) in 600 μL of extraction buffer  with protease inhibitor  and phosphatase inhibitor cocktails (Sigma-Aldrich). After incubation for 5 min at 100°C, samples have been sonicated and centrifuged at 4°C for 15 min at 13000 × rpm to remove cell debris. The soluble fraction was collected, and protein concentration was determined using the bicinchoninic acid (BCA) assay method (Thermo Scientific). For Western blotting, 80 μg of tissue lysates were boiled in 5X sample buffer , resolved by SDS-polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membranes . Mouse gastrocnemius muscle lysates were used as control samples  was used for statistical analysis. Normality of the dataset was first assessed with a normality test (Shapiro Wilk). All the dataset analyzed were not normally distributed hence non-parametric test were employed in further analysis. In particular, non-parametric Mann-Whitney Rank Sum Test was used to compare two data sets. Multiple comparisons were performed with Kruskal-Wallis One Way Analysis of Variance (Holm-Sidak correction). n = 3) and obtained a single O. vulgaris mTOR full-length cDNA sequence consisting of 7629 bp that includes 117 bp of the 5′UTR and an open reading frame (ORF) of 7512 bp encoding 2503 amino acid residues  were tested for their ability to develop peak tetanic tension under the designed RE protocol. As a control, arms that did not undergo RE were also tested for their ability to produce peak tetanic tension and no difference was observed over the experimental time scale. Typical traces of samples undergoing RE are shown in P < 0.05). In details, the force was reduced by ∼50% at the third repetition and by to ∼70% from the fourth repetition, maintaining a plateau until last stimulation session . For Western blotting analysis, we used a polyclonal antibody against residues surrounding Ser2448 of human mTOR protein. The antibody specificity for the phosphorylated form of mTOR has been largely confirmed in human, rat and mouse tissues. Based on the extent of protein sequence similarity , octopus arm and mouse muscle . These variations are the result of a cumulative effect of fatigue in the entire sample composed by muscle and nerve cord. A contribution of nerve cord on the phosphorylation state of mTOR cannot be ruled out; however, given the small volume occupied by the nerve cord , this is considered to be minor.Quantification analysis revealed a gradual increase of mTOR phosphorylation reaching a pick 30 minutes from the end of the RE and returning to pre-fatigued values within 60 min   and few of the genes involved in muscle formation such as NK4, a gene essential for cardiac muscle formation in a number of metazoans, were found to be expressed in cephalopod locomotory muscles  was found at a conserved location close to the catalytic domain PI3K and FAT C-terminal (FATC) domain, at the COOH-terminus thus suggesting a possible conservation of function of octopus mTORC1 in the control of muscle response to mechanical loading.In this study, we recognized  Ser1261 . Among te mTORC1  whose ca2386 mTOR. Octopus arms are mostly muscular and present a peripheral nervous system running in a central position controlling the simultaneous contraction of a relatively large group of muscles  muscle cells in cephalopod body mass are uninucleated, (2) cephalopods have a virtually indeterminate growth, and (3) they manifest high regeneration abilities . Any incIn conclusion, we believe that mTOR can become a versatile tool for measuring the animal health and predictive of diseases in both wild and captive animals. Its use as a marker of wellness may further provide hints for the improvement of animal maintenance procedures of both aquaculture and experimental animals.The datasets generated for this study can be found in the GenBank Accession No. KY774846.The animal study was reviewed and approved by the Local Ethical Committee  and by the Italian Ministry of Health (authorization no. 1111/2016-PR).LZ and AR conceived and designed the research, and interpreted the experimental results. SF performed the first experiments. FM and SG performed the experiments and analyzed the data. LZ carried out the statistical tests. LZ, AR, and FM drafted the manuscript. All authors revised and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to assess a novel ultrasound (US) scanning approach in evaluating knee femoral cartilaginous defects, compared with magnetic resonance imaging  and arthroscopy (gold standard).Sixty-four consecutive patients (65 knees) were prospectively evaluated between April 2010 and July 2011.κ = 0.76). Furthermore, agreement between grading by US and MRI was substantial (weighted κ = 0.61).The overall sensitivity (62.2 and 69.4%), specificity (92.9 and 90.5%), accuracy (75.4 and 78.5%), and adjusted positive (88.7 and 90.4%) and negative predictive (69.5 and 73.3%) were similar for both radiologists  contains supplementary material, which is available to authorized users. Progressive articular cartilage defects in the knee are a major cause of pain, disability, and medical expenses in the general population and particularly in the elderly . PrecisePrevious assessments were performed with a transducer capable of the highest frequency available for routine clinical use, already resulting in the highest sensitivity possible , 14. TheTherefore, this study aimed to assess a novel US scanning approach in evaluating knee femoral cartilaginous defects, compared with magnetic resonance imaging  and arthroscopy (gold standard).In this prospective study, 64 consecutive patients (65 knees) scheduled for knee arthroscopy between April 2010 and July 2011 were prospectively evaluated by US and MRI at our hospital. The study was approved by the research ethics committee of the Third Affiliated Hospital of Sun Yat-sen University. All patients signed an informed consent prior to participation in the study.Patients with a chief complaint of knee pain or disability and scheduled for arthroscopy were enrolled in this study. The patients underwent arthroscopy of the knee because of suspicion of internal derangement. Patients with prior knee surgery  and contraindications to MRI were excluded.US examination of the knee joint was conducted a week before arthroscopy using a LOGIQ 700  with a 7–9-MHz high-resolution linear transducer. A novel US scanning approach based on the functional anatomy of the knee Fig.  15] was was15] wA US grading system for knee femoral cartilage defects, based on the International Cartilage Repair Society (ICRS) classification , 19, 20,Two musculoskeletal radiologists  were trained for 2 months to perform the novel examination method before the present study. This training involved ten volunteers. The radiologists repeatedly scanned those volunteers, alone and together, and compared their results. In this study, the radiologists scanned each patient immediately one after another  and made assessments independently during real-time scanning, based on individual findings. A form with a schematic drawing of the articular surfaces of the femoral cartilage  was usedMRI examinations were performed within 1 week before arthroscopy on a Signa Excite 1.5 Tesla MR System , using a phased array knee coil. Patients were placed in the supine position with the knee fully extended. All MRI examinations consisted of sagittal T1- and T2-weighted, sagittal fat-saturated (fs) proton density (PD)-weighted, coronal fs PD-weighted, and axial fs PD-weighted fast spin echo (FSE) sequences. The imaging parameters are presented in Additional file All MRI images were interpreted by one radiologist (XCM) with over 15 years of experience in musculoskeletal imaging. The radiologist was blinded to the clinical history and US findings. The cartilage was morphologically graded according to a modified ICRS classification system : grade 0Two orthopedic surgeons  were involved in this study. They were blinded to the US and MRI findings. Knee arthroscopy with fluid irrigation was performed using the standard anterolateral and anteromedial portals to assess the articular cartilage defects and potential comorbidities. The surgeons were free to flex and extend the patients’ knees. For cartilage evaluation by arthroscopy, the same schematic drawing of the femoral cartilage surfaces of the knee was used. The surgeons performed their assessment in consensus. The trochlear surface and lateral and medial femoral condyles were evaluated separately. The cartilage lesions were graded analogously to the US and MRI using the ICRS classification . In the With arthroscopic findings as a reference, sensitivity, specificity, and accuracy of US and MRI for femoral cartilage defects were assessed. In addition, positive predictive value (PPV) and NPV were calculated and adjusted for the prevalence of femoral cartilage lesions documented in arthroscopy at our institution (51.9%), based on previous recommendations . The thrκ statistics for the level of agreement among different methods and between radiologists (US) were used [t test was used to compare the κ values between grades assigned by arthroscopy and US or MRI. Error analysis was performed for the first blinded US evaluation by the same radiologists after the revelation of arthroscopic findings. All false-positive or false-negative results were analyzed, and the most likely reason for the diagnostic error was recorded.Weighted ere used . The StuP values < 0.05 were considered significantly significant.All statistical analyses were performed with SPSS 17.0  and STATA 11 . Two-sided Sixty-four consecutive patients (65 knees) with various abnormalities of the knee were assessed, including 21 men and 43 women, with a mean age of 42 years . Final diagnoses included OA (25 knees), meniscus injury (11 knees), traumatic arthritis (11 knees), chondromalacia patella (7 knees), anterior cruciate ligament tears (6 knees), dislocation of the patella (3 knees), and meniscus cyst (2 knees). Among the 65 knees, 90.8% (59/65) had a good flexion. Altogether, 195 knee femoral cartilage surfaces, including the trochlear surface (TS), lateral femoral condyles (LC), and medial femoral condyles (MC), were evaluated by US, MRI, and arthroscopy. Arthroscopy revealed that 84, 20, 30, 38, and 23 surfaces had grades 0, 1, 2, 3, and 4 defects, respectively , and adjusted NPV was 69.5–73.3% . Only a lower sensitivity of US was observed for radiologist 1  compared with MRI (P = 0.042). Other parameters showed non-significant differences between US and MRI (P = 0.060 to 1.000), as shown in Table Table For all cartilage defect grades on each articular surface, some differences were seen between US and MRI for specific surfaces Table , but theP = 0.083 to 0.317), but a lower detection rate with US for grade 1 defects was obtained for both radiologists (P = 0.025). Indeed, for radiologist 1, ten lesions were diagnosed as grade 0, eight as grade 2, and two as grade 3. For radiologist 2, eight lesions were diagnosed as grade 0, six as grade 2, and six as grade 3. The proportions of cartilage defects graded identically and matched within one grade of arthroscopic values using US by the two radiologists were 80.0 and 80.5%, respectively, with no significant difference between both radiologists . A moderate agreement between arthroscopy and US for grade assignment  was obtained, with no significant difference  in comparison with MRI data . In addition, substantial agreement was found between grades assigned using US and MRI .The respective detection rates and comparisons between US and MRI for cartilage defects of each grade are shown in Tables Error analysis was carried out to determine the reasons for false-positive and false-negative diagnoses by US, and the results are shown in Table In this study, we aimed to improve the diagnostic accuracy of US for the diagnosis of knee lesions with the intention of improving the accessibility and decreasing the costs of knee examination associated with MRI and arthroscopy, particularly for outpatients. Nevertheless, MRI and arthroscopy are still necessary. Therefore, this study assessed the diagnostic value of a novel US scanning approach in evaluating knee femoral cartilaginous defects and found that it allows a similar diagnostic performance as routine MRI, but with improved NPV compared with previous US scanning approach, which is of clinical significance. Different articular surfaces of the femur can be accessible with an external US probe by varying the angle range of knee flexion –25. InteIn the present study, a substantial interobserver agreement was observed, while similar PPVs and higher NPVs were obtained in comparison with recent reports , 14. TheIn full extension (0°), the patella rests over the supratrochlear fat pad and is almost completely proximal to the superior border of the femoral articular cartilage , 23. In MRI is considered a method of choice for thorough evaluation of cartilage morphology, but its routine use in all symptomatic patients with clinical suspicion of knee cartilage defects is limited due to unavailability in many district and community hospitals in China and high costs (in terms of money and time) . TherefoAs a non-invasive imaging modality, the novel approach needed further clinical validation. Therefore, the novel approach was compared with MRI, which is probably the most important method for cartilage imaging . PreviouThe first major problem is that although the novel approach allows a significant decrease of false-negative diagnoses, it should be highlighted that the risk of false negatives was still as high as 26.7–30.5%, representing the majority of erroneous diagnoses. This likely results from the inability to visualize the lateral and medial condyles near the intercondylar notch, even at the maximum angle  of the knee flexion, due to their continuous articulation with the lateral and odd facet of the patella . Thus, dThe second major problem is that only femoral surfaces can be seen by US, not the patellar and tibial surfaces, which precludes the technique from providing an overall assessment of the knee articular cartilage. Nevertheless, strong correlations (Pearson’s correlation coefficients = 0.75–0.77) between volume changes in femoral cartilage and that in tibial cartilage in OA patients have been reported , indicatThe US systems and technique are possible sources of error in US, as well as the operator. Although similar diagnostic accuracy between the novel approach and MRI was presented here, a substantial number of patients with small or superficial lesions (grade 1 defects) were misdiagnosed or missed by the US. Indeed, the US equipment available for routine clinical use can only assess conspicuous morphological changes of cartilage, not determining its internal characteristics, while MRI can. Therefore, subtle morphological changes in the early stage of cartilage defects might explain the misdiagnoses. More advanced equipment and techniques  may proThere were some limitations to this study. First, although routine 2D FSE MRI sequences were performed as previously described –34, it mAnother limitation is that the same cartilage lesion could be attributed to different sites between US and MRI or arthroscopy. To minimize such discrepancies, the same standard schematic drawing of the femoral cartilage surfaces was used for all techniques. Nevertheless, a lesion located on the very edge of three articular surfaces would be likely assigned to different surfaces in various examination methods. Therefore, an overall assessment of the femoral cartilage from all three sites is necessary before the diagnosis and subsequent treatment of cartilage lesion; this is not affected by the possible misplacement. Further studies regarding treatment evaluation are required to target the precise lesion localization of cartilage.In addition, the correlation between US findings and other assessment tools was not established. Indeed, in this initial study, we prioritized the associations of US with arthroscopy (gold standard) and MRI . In the future, the correlation between the novel US scanning approach and clinical assessment should be evaluated for its recommendation in routine clinical use, including evaluation of degenerative changes and therapeutic effects.The aim of the present study was to investigate the value of US as a screening tool for cartilage defects in patients with a chief complaint of knee pain (without any previous examination and diagnosis). It is indeed possible that some patients were not definitely diagnosed with OA. On the other hand, cartilage degeneration caused by OA may also present as cartilage defect. Therefore, it could be hypothesized that OA will not directly affect the capability of the US detection of cartilage defects, but this specific point will have to be examined in the future.The novel US scanning approach taking knee articular motion into consideration is more valid in a clinical setting to significantly decrease false-negative diagnoses compared with fixed-angle (120°) transverse scanning. It also has similar diagnostic performance, PPV, and agreement as routine MRI approaches for evaluating the knee cartilage defects in patients with a broad spectrum of knee diseases, but the NPV was higher than the previous US scanning approach. As a non-invasive, fast, inexpensive, and radiation-free imaging modality, US has a potential to be used for initial screening assessments of cartilage defects in first-visit patients with a chief complaint of knee pain and/or disability.Additional file 1:Table S1. Parameters for MRI sequences. (DOCX 15 kb)