The Central Siberian Plateau is undergoing rapid climate change that has resulted in increased frequency of forest fires and subsequent alteration of watershed carbon and nutrient dynamics. Across a watershed chronosequence (3 to >100 years since wildfire) we quantified the effects of fire on quantity and composition of dissolved organic matter (DOM), stream water nutrient concentrations, as well as in-stream nutrient uptake. Wildfires increased concentrations of nitrate for a decade, while decreasing concentrations of dissolved organic carbon and nitrogen (DOC and DON) and aliphatic DOM contribution for five decades. These post-wildfire changes in stream DOM result in lower uptake efficiency of in-stream nitrate in recently burned watersheds. Nitrate uptake (as uptake velocity) is strongly dependent on DOM composition (e.g. polyphenolics), ambient dissolved inorganic nitrogen (DIN), and DOC to DIN ratios. Our observations and experiments suggest that a decade-long pulse of inorganic nitrogen and a reduction of DOC export occur following wildfires in streams draining the Central Siberian Plateau. Increased fire frequency in the region is thus likely to both decrease DOM and increase nitrate delivery to the main stem Yenisei River, and ultimately the Arctic Ocean, in the coming decades. When permafrost is degraded, interactions between nutrient availability and OM composition will play an important role in determining the net effects on delivery of dissolved organic carbon (DOC) and inorganic nutrients from upland landscapes to major arctic rivers and ultimately the Arctic Ocean3. Predicted and recently observed changes in the concentration and export of OM and nutrients vary geographically across the Arctic and as a function of permafrost structure, underlying parent material, vegetation, and disturbance4. Boreal forests have been burning with greater frequency due to longer growing seasons, warmer temperatures, and changing weather patterns7, thereby adding additional uncertainty to projections of how these ecosystems will respond to climate change. The response of arctic watersheds varies dramatically among regions, with both increases and decreases in DOC concentrations and inorganic nutrients following wildfire, and differing return intervals to baseline conditions13. Although various studies have documented the effects of fire on stream chemistry, few have evaluated how these changes will impact the processing and export of nutrients from Arctic fluvial systems. Arctic rivers mobilize large quantities of organic matter and nutrients to the Arctic Ocean14 which may be altered due to increasing permafrost thaw and wildfire frequency. Watersheds in the Central Siberian Plateau, a major component (2.5 million km2) of the Yenisei River Basin, provide a platform for understanding the long-term resilience and recovery of stream chemistry following fire in taiga landscapes. Streams of the Central Siberian Plateau drain watersheds with extensive larch forest (Larix gmelinii) growing on continuous permafrost (300–500 m deep) underlain by unglaciated basalt deposits16.Arctic soils store large amounts of organic matter (OM) and nutrients in permafrost3-) to downstream ecosystems20. Uptake rates  are commonly used to quantify nutrient demand and the efficiency with which in-stream biota remove and process nutrients at a given concentration22. Uptake velocity is typically determined via in-situ nutrient manipulations since uptake is difficult to infer from downstream changes in the ambient pool of nutrients. Generally, as nutrient concentrations increase, uptake efficiency of the microbial community declines, resulting in a larger fraction of nutrients exported downstream23. Uptake dynamics have not been evaluated extensively in arctic fluvial systems affected by wildfires10.Uptake and transformation of nutrients and OM by stream channel processes can result in losses of C and N through mineralization and denitrification, as well as alteration of the timing of delivery of DOC and nitrate  and ammonium (NH4+) to determine how fire influences the processing and export of nutrients in arctic fluvial systems. We assess changes to the composition of DOM via optical properties and Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR MS) to provide an ultra-high resolution assessment of molecular formulae for thousands of individual DOM molecules 25 in multiple water samples from each stream. Samples were taken in multiple years during June (freshet) and July (summer low flow).Here we examine how years since the last fire affect nutrient concentrations and the quantity and composition of dissolved organic matter (DOM) entering fluvial networks using a space-for-time approach spanning a 100-year well-constrained wildfire chronosequence across 17 watersheds  and DON  both increased with longer time since the last fire, with patterns consistent in June and July. Similar responses between June and July reflect consistency across changing flow regimes shifting from run-off dominated spring freshet in early June, and flows in July that are more representative of low-flow conditions typical of summer. In recently burned sites DOM inputs to the stream were likely reduced due to the combustion of the surface litter and organic layer, which reduces soil standing stocks of C and production of DOM from soil OM7. Post-fire declines in stream water DOC and DON concentrations can also be attributed to deepening of the active layer, which exposes mineral soil3 leading to increased adsorption of DOM26. Changes to concentrations of DOC and DON across the chronosequence indicate an ecosystem recovery time of approximately 50 years post wildfire disturbance, during which time the vegetation  undergoes regrowth re-establishing OM inputs into the soil. DOC concentrations in central Siberia exhibit a positive relationship with forest productivity and can serve as a proxy for forest C stocks16. The stabilization of DOC and DON concentrations after 50 years since the last fire suggests the reestablishment of an equilibrium as described by resilience theory and the ecosystem succession and nutrient retention hypothesis28.Fires altered concentrations of DOC, dissolved organic nitrogen (DON), and nutrients in streams of the Central Siberian Plateau. Concentrations of DOC  as described by molar DOC:DIN ratios . In the study streams, DIN is mostly composed of NO3− where ammonium (NH4+) is often at or below detection limit (~5 µg/L as N). During the post-fire interval when DIN is elevated and DOC is reduced, this balance between energy and nutrient demand (as measured by stoichiometry) is re-established after approximately ten years reflecting a system that returns to a state of inorganic-N limitation and high DOM availability. During this initial recovery period when DON concentrations are relatively low, the predominant in-stream nutrient source may shift from organic to inorganic forms of N. There was no statistically significant difference in ammonium and phosphate concentrations across the burn gradient (Supplementary Table Stream dissolved inorganic nitrogen (DIN) responded differently to forest fires than DOM. NOime Fig. , p«0.05;34. This is reflected in the spectral slope ratios (SR) shifting from low molecular weight to higher molecular weight values with increasing time since fire . Across the different models, years since the last fire and season consistently were the most significant predictors of DOC, DON, and DIN concentrations, DOC:DIN ratios, and DOM composition (Supplementary Table f) correlated negatively with DIN concentrations . This pattern has been observed in numerous studies in a variety of biomes, suggesting that the efficiency with which the microbial community removes DIN decreases with increasing DIN concentrations39. The changes in concentrations of DOC due to fires also influence DIN processing where streams with greater DOC:DIN molar ratios supported the greatest DIN removal . Relationships between uptake and molar ratios20 demonstrate the important role of DOM in DIN processing and indicate that the balance and interactions between DOM and DIN can influence the resilience of heterotrophic processes. Moreover, DOM compound groups such as polyphenolics, can enhance DIN removal ; increasing relative abundance of polyphenolics leads to higher DIN uptake velocities. The positive relationships between DIN uptake and polyphenolics (i.e. vascular plant derived DOM40) suggests that uptake will be greatest in watersheds that have longer recovery times since the last fires, where inputs of DOM sources from terrestrial plants can be elevated. In addition, polyphenolics which have typically been thought to be aromatic and less bioavailable may be important energy sources in potentially energy limited streams (low primary productivity) despite high ambient DOM41. Alternatively, greater availability of DIN could have enhanced DOM decomposition by relieving any DIN limitations that inhibit the breakdown of aromatic molecules like polyphenolics. Although nutrient pulse additions do not allow us to determine what specific processes are responsible for DIN removal from the water column, the data demonstrate a close relationship between DOM and DIN uptake, supporting the idea that these patterns are due to heterotrophic or assimilative processes that require an energy source such as DOM. With shorter fire return intervals, elevated DIN concentrations, and declines in both DOM quantity and DOC:DIN ratios we can expect greater export of DIN as the strength of watersheds as sinks is greatly reduced leading to larger amounts of nutrients reaching larger rivers and ultimately the Arctic Ocean. DOC:DIN ratios provide a valuable integrator of energy versus nutrient limitation in stream ecosystems, in which energy limitation (low DOC:DIN ratios) or nutrient limitation (high DOC:DIN ratios) can be important in determining the delivery of nitrogen and OM to downstream ecosystems20. Downstream exports of inorganic N will likely persist for several years to a decade post fire, until DOM inputs from the watershed are re-established.The response in stream chemistry due to fires influences the processing and export of DIN. Across all watersheds, DIN uptake velocities  and its continuity, hydrology, and vegetation communities (i.e. tundra vs. taiga) play in regulating elemental fluxes and the balance of energy and nutrient demand in arctic fluvial networks. A better understanding of the effects of large-scale perturbations like fires is important to develop a greater mechanistic understanding of how site conditions drive variability in responses, exports, and resiliency to increasing alteration of arctic landscapes.Across arctic watersheds, the response of stream chemistry to biomass burning is not consistent. Watershed recovery following wildfire across arctic watersheds appears to be a function of many variables, including composition of vegetation and rates of regrowth, nature and extent of OM consumed by fire, and the effects of both of these on active layer depth11 and 201310 are included in the main body of text. Samples were filtered through pre-combusted Whatman GF/F glass fiber filters and collected in acid washed high density poly-ethylene bottles , pre-combusted amber glass vials , and polycarbonate bottles (FT-ICR MS) and rinsed 3 times in the field. Samples were kept frozen except for DOM optical samples which were kept at 4 °C; all samples were transported and analyzed at the Water Quality Analysis Laboratory at the University of New Hampshire, except samples collected for FT-ICR MS which were analyzed at the National High Magnetic Field Laboratory at Florida State University.We collected stream water samples in June and July from 2016 to 2018, but data from 20113− as NaNO3, and NH4+ as (NH4)2SO4, with and without PO43+ as KH2PO4 but we report NH4+ uptake from both as PO43+ had no influence on NH4+ uptake metrics. Amendments were added along with NaCl as a conservative tracer mixed with stream water where we increased background nutrient and Cl concentrations 2–3X above background. Experimental reach lengths ranged between 15 m to 200 m  was determined as the negative inverse of the ratio between the natural log of the fraction of background corrected nutrient and Cl retrieved and the reach length distance. Uptake velocity (Vf) was determined as:w stream width, and Sw the uptake length. Uptake velocity is the best metric to compare across sites and dates as it is normalized for stream depth and velocity24. Uptake velocity is reported in terms of DIN (combining NO3− and NH4+ Vf) due to low statistical power if reported individually. Discharge was determined using the dilution gaging method42 using NaCl dissolved in stream water and added to the stream as an instantaneous slug addition and conductivity was logged every 5 seconds using a HOBO conductivity data logger .Calculations of uptake metrics follow the break through curve integration method3−) was analyzed with ion chromatography using an Anion/Cations Dionex ICS-1000 with AS40 autosampler. Ammonium (NH4+) was measured using a SmartChem 200 discrete automated colorimetric analyzer using the alkaline phenate standard method. Phosphate (PO43−) was analyzed as soluble reactive phosphorus by discrete colorimetric analysis on a Seal AQ2  by the ascorbic molybdenum blue method (EPA 365.1). DON was determined arithmetically by subtracting DIN (NO3− + NH4+) concentrations from TDN. Samples below detection limit were substituted with half the detection limit; method detection limit for NO3− (4 µg/L as N), NH4+ (5 µg/L as N), PO43− (1 µg/L as P), DOC (0.05 mg/L), TDN (0.035 mg/L). Stream chemistry data was also obtained from the same streams as this study during June 201111 and 201310. Those samples were also collected and analyzed as described here.All samples were analyzed for DOC and total dissolved nitrogen (TDN) using high temperature catalytic oxidation with a Shimadzu TOC-V CPH/TNM. Nitrate (NO254) was determined by dividing the UV absorbance at 254 nm by DOC concentration, where SUVA is an index of DOM aromaticity43. Fluorescence index (FI) was determined as the ratio of 470 and 520 nm fluorescence intensities at 370 nm excitation and FI used to identify sources of DOM 44. Humification index (HIX) was determined as the ratio between area under the emission spectra 435–480 nm and the sum of the peak area 330–345 nm and 435–490 nm and describes the degree of humification of DOM45. Spectral slope (S) was determined using a non-linear fit of an exponential function to the log transformed absorption spectrum in the ranges of 275–295 nm and 350–400 nm46. Slope ratio (SR) was determined as the ratio of the slope (275–295 nm) and slope (350–400 nm) which have been negatively correlated to DOM molecular weight and aromaticity46.All grab samples across chronosequence from July 2013, June 2016, and July 2018 and background nutrient pulse samples from June 2016 and July 2018 were analyzed for DOM optical properties. UV absorbance was measured with a Shimadzu photo diode array detector with HPLC at 1 nm intervals between 200 to 700 nm. Specific UV absorbance at 254 nm (SUVAExcitation emission matrices (EEM) were conducted on room temperature water at excitations ranging from 240 to 450 nm at 1 nm intervals and emission ranging from 350 to 550 nm at 2.5 nm intervals using a Jobin Yvon Horiba Fluoromax-3 fluorometer. For 2016 samples EEM emission range was extended from 300 to 600 nm, but for purposes of Parallel Factor Analysis (PARAFAC) modeling these were clipped to match samples from previous years.47. Modeling of PARAFAC components followed established procedures and was normalized to reduce collinearity with reversal of the normalization prior to exporting the model48. Prior to PARAFAC modeling, data in the region of first-order and second-order scatter was removed.Parallel Factor Analysis (PARAFAC) was performed on 132 samples of stream water and soil pore water collected in 2011, 2013, and 2016. PARAFAC is a multivariate technique that decomposes fluorescence spectra into individual fluorescence components49. Briefly, each sample was acidified to pH 2 and passed through a Bond Elute PPL (Agilent Technologies) cartridge. Residual salts were rinsed from the cartridge with acidified (pH 2) Milli-Q water. The stationary phase was then dried with a stream of N2 and the DOM was eluted with 100% MeOH (JT Baker) to a final concentration of 50 µg/mL C and stored at 4 °C prior to analysis. Negatively charged ions were produced via direct infusion electrospray ionization at a flow rate of 700 nL/min and analyzed with a custom-built FT-ICR MS equipped with a 21-tesla superconducting magnet50.Samples analyzed for FT-ICR MS were collected in June 2016 and July 2018 only. DOM was obtained for FT-ICR MS by a solid phase extraction (SPE) method51 developed at the NHMFL. The mass measurement accuracy did not exceed 200 ppb. Each molecular formula was classified based on stoichiometry; condensed aromatic (modified aromaticity index (AImod) ≥ 0.67), polyphenolic (0.67> AImod > 0.5), unsaturated, low oxygen , unsaturated, high oxygen , aliphatic , peptide-like 53. Although FT-ICR MS allows for the precise assignment of molecular formulae to peaks that may represent multiple isomers, they describe the underlying molecular compounds comprising DOM, therefore the term compound may be used when describing the peaks detected by FT-ICR MS. Signal magnitude was converted to relative abundances by dividing the signal magnitude of an individual peak by the sum of all assigned signals. Subsequently, the percent contribution of each compound category was calculated as the percent that the relative abundance in each compound category contributed to the summed abundance of all assigned formulae.Each signal>6σ RMS baseline noise was assigned a molecular formula with EnviroOrg©™ softwaref and DIN concentrations, stoichiometric ratios, and DOM compound groups. DIN concentrations and DOC:DIN ratios were log transformed to normalize data. To determine if watershed characteristics were potential predictors of patterns in stream chemistry across the fire chronosequence, we used stepwise multiple regression analysis with backward selection. Watershed characteristics , season (as freshet period or low discharge), and years since the last fire were loaded as independent variables and DOC, DON, DIN, DOC:DIN, relative abundance of combined aliphatics and N-containing aliphatics, condensed aromatics, and polyphenolics, and slope ratio as the dependent variables. Final models were used as those with lowest AIC scores. For all statistical analyses α=0.05 and were conducted in RStudio  using the stat, rcompanion, and MASS packages. The PARAFAC model was validated using split-half analysis using the drEEM toolbox48 in MATLAB .Normality was tested for all parameters using the Shapiro-Wilk normality test (α ≤ 0.05). Differences across parameters that follow the assumptions of normality were determined using a one-way ANOVA and nonparametric Kruskal-Wallis rank sum test for parameters that violate the assumptions of normality. We used simple linear regressions to determine relationships between DIN VSupplementary Dataset 1.


Inflammatory processes are implicated in the aetiology of Major Depressive Disorder (MDD); however, the relationship between peripheral inflammation, brain structure and depression remains unclear, partly due to complexities around the use of acute/phasic inflammatory biomarkers.MDD cases = 271, Ncontrols = 609).Here, we report the first large-scale study of both serological and methylomic signatures of CRP (considered to represent acute and chronic measures of inflammation respectively) and their associations with depression status/symptoms, and structural neuroimaging phenotypes (T1 and diffusion MRI) in a large community-based sample  and energy levels , along with reduced entorhinal cortex thickness . DNAm CRP was significantly associated with reduced global grey matter/cortical volume and widespread reductions in integrity of 16/24 white matter tracts . In general, the methylation-based measures showed stronger associations with imaging metrics than serum-based CRP measures (βaverage = −0.15 versus βaverage = 0.01 respectively).Serum CRP was associated with overall MDD severity, and specifically with current somatic symptoms- general interest (β = 0.145, PThese findings provide evidence for central effects of peripheral inflammation from both serological and epigenetic markers of inflammation, including in brain regions previously implicated in depression. This suggests that these imaging measures may be involved in the relationship between peripheral inflammation and somatic/depressive symptoms. Notably, greater effects on brain morphology were seen for methylation-based rather than serum-based measures of inflammation, indicating the importance of such measures for future studies. It is aOne of the most common approaches to assess peripheral inflammation is by measuring serum levels of C-reactive protein (CRP). CRP plays a key role in human inflammation and can provide a proxy estimate for inflammatory activity . It is wOne hypothesis is that peripheral inflammation contributes to depressive symptoms through effects on the brain. There is increasing evidence that peripheral inflammation has effects on the brain via humoral and neural routes subsequently impacting upon neural cell function, changes in functional/structural connectivity and behavioural changes including depressive symptoms . HoweverWe therefore investigated a methylation marker of chronic inflammation using a recent large epigenome wide association study (EWAS) of CRP . From thHere we present findings from both serological measures of CRP and a novel methylomic approach to investigate the role of inflammation in MDD with a comprehensive range of structural brain phenotypes, including white matter microstructure (n = 189 phenotypes). The current study examines a large community-based sample (n = 880) to investigate (i) associations between serum CRP and DNAm CRP and MDD symptoms, (ii) associations between both CRP (serum CRP and DNAm CRP) measures and structural imaging phenotypes (TI and diffusion MRI) and (iii) interaction effects between both measures of CRP and MDD diagnosis to determine the differential relationship of these inflammatory markers and imaging associations in depression.22.1The participants in this study were recruited as part of the ‘STratifying Resilience and Depression Longitudinally’ (STRADL) study (2015–2019) which re-contacted participants from the Generation Scotland: Scottish Family Health Study (GS) via post for further assessment of mental health, specifically depression. Full details of the STRADL cohort and GS protocol are published elsewhere . A totalEthical approval for STRADL was formally obtained from the NHS Tayside committee on research (reference 14/SS/0039), and all participants provided their written informed consent.2.2A full medical history was obtained and updated from previous GS baseline assessment  and any Participants in STRADL completed a broad range of tests designed to assess depression incidence/severity. All participants were screened for a lifetime history of MDD. A research version of the Structured Clinical Interview for DSM disorders (SCID)  was used2.3To obtain serum CRP levels, venepuncture was employed using a butterfly needle kit. Blood was extracted into clot activator gel for serum separation. CRP samples were taken and sent to NHS laboratories  for analysis. The low-sensitivity assay utilized in CRP analyses possessed a detection threshold of 4 mg/L and CRP levels below the 4 mg/L detection threshold were recorded as 0. For analyses purposes, CRP levels were stratified based on this threshold limit into clinically relevant groups: <4 mg/L  and ≥ 4 mg/L  . Blood s2.4Blood samples used to generate the DNAm CRP score were collected at the Generation Scotland baseline appointment (between 2006 and 2011) and DNA methylation was profiled using the Illumina Human-MethylationEPIC BeadChip in two different sets. Pre-processing and quality control steps for both sets of methylation data have previously been fully reported .Full details of the calculation of the DNAm CRP score in GS have been reported previously . Briefly2.5STRADL participants were scanned at two centres: the Ninewells Hospital in Dundee and at the Aberdeen Royal Infirmary in Aberdeen. Participants in Dundee were scanned using a Siemens 3T Prisma-FIT  with a 20-channel head and neck coil and a back-facing mirror . In Aberdeen, participants were imaged on a 3 T Philips Achieva TX series MRI system  with a 32-channel phased-array head coil with a back-facing mirror . Tract Based Spatial Statistics (TBSS) was carried out according to the ‘The Enhancing NeuroImaging Genetics through Meta-Analysis’ (ENIGMA) Consortium DTI protocol (http://enigma.ini.usc.edu/protocols/dti-protocols/). Region of interest (ROI) extraction analyses were then performed also using ENIGMA protocols to extract fractional anisotropy (FA) and mean diffusivity (MD) measures (http://enigma.ini.usc.edu/protocols/dti-protocols/). White matter tracts were categorised using the Johns-Hopkins University DTI-based white matter atlas  were set as covariates in mixed-effect linear models for both serum CRP and DNAm CRP analyses. Additionally, smoking status and pack years smoked were included in all DNAm CRP analyses given the effects of smoking on a range of methylation-based measures . For uni2.6.2For DTI measures, age, sex, BMI and assessment centre were included as covariates for all analyses and methylation set/smoking variables were additionally included for DNAm CRP analyses. Global integrity was determined by applying principal component analysis (PCA) on the 24 tracts to extract a latent measure. Scores of the first unrotated principal component of FA/MD were extracted and set as the dependent variable . We then separately examined four subsets of white matter tract for which scores of the first unrotated principal component were also extracted: (a) association fibres, (b) commissural fibres, (c) projection fibres and (d) thalamic radiations. The tracts included in the four subsets are provided in supplementary materials (Table S1). Finally, we examined each white matter tract individually. Mixed-effect linear models were used for the measures of bilateral white matter tracts correcting for hemisphere as a within-subject variable, consistent with above, while general linear models were used for the unilateral midline tracts.2.6.3FDR in this report, using the ‘p.adjust’ function in R and all betas were standardised. FDR correction was also applied over each sub-analysis. We investigated: (i) associations between serum CRP and DNAm CRP and depression symptoms, (ii) associations between both CRP measures and structural imaging phenotypes (T1 and DTI) and (iii) CRP × MDD interaction effects using both measures of CRP (serum and DNAm), and case/control MDD status. Further analyses controlling for concurrent smoking in serum CRP-MDD associations (Table S22) and controlling for days between methylation and serological/MRI appointments in DNAm CRP associations are also provided in supplementary materials (Supplementary Tables S24-28).All analyses were conducted using R (version 3.2.3) in a Linux environment. As GS is a family-based study we randomly included one participant per family in order to have an unrelated sample. Randomisation was conducted in R using the ‘rnorm’ function to create a random seed variable for each participant and one individual per family with the highest random number was included in subsequent analyses, excluding all other family members. Linear mixed-effects models (function ‘lme’ in R package ‘nlme’) and general linear models (function ‘glm’ in R package ‘stats’) were used to investigate structural brain metrics . False D33.1Descriptive statistics of the key variables as well as sample numbers for each analysis in this study are presented in 3.23.2.1FDR = 0.033; FDR = 0.027) and decreased general interest .There were no significant associations between serum CRP and case/control MDD status. However, increased serum CRP levels were significantly associated with increased depressive symptoms as measured by the total QIDS score , with general interest remaining FDR significant after additional correction for smoking status .A further supplementary analysis controlling for smoking status in serum CRP associations with depression is presented in supplementary materials (Table S22). This sample had reduced power of 101 participants, however, effect sizes for the main findings remained similar, total QIDS  and associations with serum CRP and DNAm CRP were also null (Table S23).3.33.3.1FDR = 0.037; There were no significant associations between serum CRP status and any global or lobar brain summary measures. For individual regions however, we found elevated CRP status was significantly associated with reduced thickness of the entorhinal cortex , we report significant associations between an increased DNAm CRP score and smaller global grey matter  and smaller global cortical volume  and the anterior limb of the internal capsule .With regard to DTI measures (n = 565), increased DNAm CRP scores were significantly associated with decreases in white matter microstructure, including negative associations with gFA , FA in the external capsule  and thalamic radiations . We also tested the effects of the DNAm CRP score for MD of individual tracts and found FDR significant effects for 16/24 white matter tracts (FDR = 0.042).There were also significant associations with MD values for projection fibres .Results for an additional analysis controlling for the time interval between methylation data collection and imaging/serological collection (Tables S24–28) were largely similar to the above findings. Results remained the same for all global and FA findings, and 14/16 of the previous MD tract findings remained FDR significant, with the largest effect sizes in the same tracts .4Using data from a large community-based sample, we report associations between serological and methylomic markers of CRP with 189 structural neuroimaging phenotypes and their interaction effects with MDD diagnosis. We show that serological CRP exhibited significant associations with overall current MDD symptoms, in particular somatic symptoms, as measured by QIDS scores. In terms of brain morphology, serum CRP was associated with decreased thickness of the entorhinal cortex, whereas DNAm CRP was more robustly associated with widespread imaging features including decreased global cortical volume, grey matter and decreased white matter integrity, with the greatest loss of integrity in the external capsule and the ALIC (as indexed by decreased FA and increased MD). Neither serum CRP or DNAm CRP had significant interaction effects with MDD case/control status with brain structure, indicating these relationships are not specific to the presence of a formal lifetime clinical diagnosis.We found that increased serum CRP levels were associated with increased depressive symptoms, particularly two somatic symptoms, general interest and energy levels, which is consistent with the existing literature. Previous studies of inflammation and depressive symptoms have also found that inflammatory markers are associated with somatic/neurovegetative symptoms of depression including fatigue, impaired sleep and activity, rather than psychological or cognitive symptoms . These fAcross the whole sample, we also report that higher serum CRP was associated with a thinner entorhinal cortex. This region has also been associated with CRP in previous research. A study by Bettcher and colleagues found CRP was associated with smaller left medial temporal lobe volumes, which included the entorhinal cortex, and found that those with detectable levels of CRP demonstrated poorer performance in cognitive tasks . This arThe most prominent findings in this study are the global and regional imaging associations with DNAm CRP. To our knowledge, there are no previous studies on the relationship between peripheral blood DNA methylation CRP scores and brain structure in the context of depression. We found that DNAm CRP scores had a greater number of associations with imaging traits, and with larger effect sizes compared to serum CRP. DNAm CRP scores were associated with several structural neuroimaging measures including reductions in global grey matter and global cortical volume. We also found that increased DNAm CRP scores were associated with widespread white matter changes including reductions in FA in the external capsule and ALIC and increases in MD for projection fibres and thalamic radiations. This is highly consistent with regions implicated in previous imaging studies of MDD . The ALIThese findings could also be considered in the context of the ‘inflammaging’ theory of ageing and accelerated ageing models of MDD . ChronicOur findings indicate utility in combining serological and methylomic markers of inflammation in the investigation of MDD. Methylation markers of other traits have proven useful in the prediction of MDD in previous studies,  and futuThis study has a number of strengths, in particular, the STRADL study is a large community-based sample with in-depth phenotypic assessment and neuroimaging data (n = 880 in the current study). Our study also benefits from the inclusion of methylation data which provided a long-term signature of chronic/low-level inflammation, overcoming issues with single timepoint CRP measurement which fluctuates in response to numerous factors and is prone to measurement error. We have also utilized a data-driven approach by not selecting neuroimaging regions of interest prior to analyses. This allowed an assessment of the effects of inflammation and depression on a wide range of structural neuroimaging measures.Limitations of this study included a small number of current MDD cases in comparison to the number of controls. As this study is a community-based sample, this indicates that the participants with MDD are relatively well in comparison to MDD cases who are hospitalised. The mean depression symptom score  in the lifetime MDD group was 7.1, indicating mild depression levels in this group. Therefore, we are potentially not capturing the effects of moderate to severe forms of MDD. However, community-based sampling results may be more generalizable to the population than those obtained in a clinic. Furthermore, although CRP is widely used clinically to determine peripheral inflammation, it is ultimately a proxy for inflammatory activity  and the In conclusion, we found that serum CRP was associated with depressive symptoms, in particular somatic symptoms, and with a reduction of entorhinal cortex thickness. This study also utilized a methylomic signature of C-reactive protein to capture chronic signatures of low-level inflammation. Here we found significant widespread associations with several structural neuroimaging measures, in particular, differences in white matter integrity, including regions previously implicated in MDD such as the external capsule and ALIC. This study highlights the utility of using both serological and methylation markers in a multi-level approach to study brain imaging and psychiatric phenotypes. Furthermore, as peripheral inflammation was associated with both changes in brain morphology and depression symptoms, inflammation may represent an important and clinically relevant therapeutic target for depressive symptoms.5www.generationscotland.org.Access to and use of GS and STRADL data must be approved by the GS Access Committee under the terms of consent. Full details of the application process can be found at Generation Scotland received core support from the Chief Scientist Office of the Scottish Government Health Directorates [CZD/16/6] and the Scottish Funding Council [HR03006] and is currently supported by the Wellcome Trust [216767/Z/19/Z]. Genotyping of the GS:SFHS samples was carried out by the Genetics Core Laboratory at the Edinburgh Clinical Research Facility, University of Edinburgh, Scotland and was funded by the Medical Research Council UK and the Wellcome Trust  Reference 104036/Z/14/Z). CG is supported by The Medical Research Council and The University of Edinburgh through the Precision Medicine Doctoral Training program. SRC is supported by the UK Medical Research Council [MR/R024065/1] and a National Institutes of Health (NIH) research grant R01AG054628.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Nicotiana tabacum plants.Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in We expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive β-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility.Expression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants. The genome of all living organisms is continuously exposed to exogenous genotoxic agents and endogenous factors that can result in critical DNA lesions such as DNA double-strand breaks (DSBs). To maintain genome integrity, eukaryotic cells have evolved powerful and complex DNA damage repair mechanisms –3. DSBs − 6, is extremely low in somatic tissues that are frequently used in plant transformations and further stimulation of the HR machinery will be needed to make HR-mediated GT routinely achievable. Early work on HR in plants suggests that such stimulation might be achieved by expression of heterologous recombinases (i.e. genes involved in DNA repair and recombination) in plants. When expressed in Nicotiana tabacum plants, E. coli RecA and RuvC recombinases have been shown to increase intrachromosomal HR by 10- to 11-fold -methionine labelled products from a wheat germ TNT® system showed all the expected individual protein products were present, along with some high molecular weight uncleaved polyprotein. For example, translation of the RecA:RecG:RuvC polyprotein construct  are powerful tools that will be very useful in precise gene manipulation in gene therapy and crop improvement. The development of CRISPR/Cas9 endonuclease systems has made genome editing routinely feasible in higher eukaryotes including plants . CRISPR Rad52 is essential in the choice of DSB repair pathway by HR in yeast and animals , 38 but The translocase RecG is crucial in DNA repair of stalled replication forks , 42 and E. coli RuvC, is necessary for the resolution of Holliday junctions -Methionine . Radiolabelled protein products were separated in 10% SDS–PAGE [Polyprotein constructs were used with wheat germ transcription–translation system  according to the manufacturer’s instructions in the presence of [SDS–PAGE  and deteThe EcoRV fragment containing sulphonamide resistant gene under nopaline synthase transcriptional signals (nos-Sul) was inserted into HpaI close to the left border of pGreenII 0000  to make Agrobacterium tumefaciens strain LBA4404 containing pSoup plasmid by electroporation method. Agrobacterium clones were used to transform tobacco seedlings of N1DC4 line as described by Abbott et al. [The constructs in pGSC binary vector were transferred into t et al. . TobaccoT1 seeds were plated on MS medium in the presence of 100 mg/l of sulphonamide and scored for resistance after 2 weeks growth under 16 H light regime. The lines showing 3:1 segregation ratio were selected to make homozygotes. Six resistant seedlings per line were grown in the glasshouse and T2 seeds collected. These seeds were screened in the presence of sulphonamide to detect homozygous lines (100% resistance). Three plants per homozygous lines and N1DC4 control were grown in the glasshouse and their pollen and seeds were collected. The seeds of ten dry pods per plant were pooled and their number estimated by weight method.N1DC4 is a homozygous line containing β-glucuronidase (GUS) based transgene as a substrate for Intrachromosomal recombination (ICR) , 25. TheP-value less than 0.05.A separate linear regression model was fitted for each of the responses  with the genotypes included in the model as an explanatory factor. Using corner point parameterisation of the genotype factor, each of the test genotypes was compared to the control genotype N1DC4 and was taken to be significantly different from the control if the t statistic had an associated


Rhizoclosmatium globosum, that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by β-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor.Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid,  The phylum Chytridiomycota (chytrids) diverged from other fungal lineages approximately 750 Mya and, with the Blastocladiomycota, formed a critical evolutionary transition in the Kingdom Fungi dedicated to osmotrophy and the establishment of the chitin-containing cell wall . W. WRhizocycle (d) , easy la2.Detailed materials and methods are provided as electronic supplementary material.(a)Chytrid plasma membranes were labelled with 8.18 µM FM 1–43 and imaged using a Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss). Z-stacks of rhizoids were imported into the neuron reconstruction software NeuronStudio ,17 and s(b)4) for 30 min, then with 5 µg ml−1 Texas Red-conjugated wheat germ agglutinin (WGA) in PEM for 30 min.To label F-actin and the cell wall throughout the rhizoid system, cells were fixed for 1 h in 4% formaldehyde in 1×PBS  and stained with 1 : 50 rhodamine phalloidin in PEM ) buffer at pH 6.9, 1 mM EGTA (ethylene glycol tetraacetic acid), and 0.1 mM MgSO(c)Caspofungin diacetate (working concentration 1–50 µM) was used to inhibit cell wall β-glucan synthesis and cytochalasin B (working concentration 0.1–10 µM) was used to inhibit actin filament formation. Cells were incubated for 6 h, which was found to be sufficient to observe phenotypic variation.(d)R. globosum was processed for β-glucans using a commercial β-glucan assay (Yeast & Mushroom)  following the manufacturer's protocol. Briefly, samples were processed by acid hydrolysis then enzymatic break-down and β-glucans were quantified spectrophotometrically with a CLARIOstar Plus microplate reader (BMG Labtech), relative to a negative control and positive β-glucan standard. A sample of shop-bought baker's yeast was used as an additional positive control.(e)R. globosum genome were identified using the JGI MycoCosm online portal. GT2 functional domains were identified using DELTA-BLAST . T. Ta) [22o et al.  when scaSuch similarities also extend to rhizoid branching patterns, where lateral branching dominates over apical branching. This branching pattern is also the predominant mode of hyphal branching, where apical branching is suppressed by a phenomenon termed ‘apical dominance’ . These fR. globosum. Normal rhizoid branching was disrupted by inhibition of cell wall synthesis and actin polymerization, both of which are known to control branching and growth in hyphal biology [R. globosum are similar to disruption of normal hyphal branching reported in Aspergillus fumigatus (Ascomycota) in the presence of caspofungin [Neurospora crassa (Ascomycota) in the presence of cytochalasins [Chytriomyces hyalinus similarly disrupts normal rhizoid branching [Given the apparent hyphal-like properties of rhizoid development, we sought a greater understanding of the potential subcellular machinery underpinning rhizoid morphogenesis in  biology –33. Thespofungin , and in halasins . Recent halasins ,37 and iranching . In thisR. globosum exhibiting feeding and searching phenotypes, respectively. In addition, exogenous carbon starvation has also been shown to be associated with a decrease in branching in the multicellular dikaryan fungus Aspergillus oryzae (Ascomycota) [We also show that rhizoid development is plastic to resource availability, with chytrid cells displaying an adaptive searching phenotype under carbon starvation. Adaptive foraging strategies are well described in multicellular hyphae ,39, and omycota) . OverallFinally, we report the spatial and functional differentiation of feeding and searching sections of anucleate rhizoid systems from individual cells. The simultaneous display of both rhizoid types in the same cell indicates a controlled spatial regulation of branching and differentiation of labour within single chytrid rhizoid networks. Functional division of labour is prevalently seen in multicellular mycelial fungi ,39 inclu5.The improved understanding of chytrid rhizoid biology related to substrate attachment and feeding we present here opens the door to a greater insight into the functional ecology of chytrids and their environmental potency. Our approach of combining live cell confocal microscopy with three-dimensional rhizoid reconstruction provides a powerful toolkit for morphometric quantification of chytrid cell development and could shed light on the biology underpinning chytrid ecological prevalence. In the future, the application of this approach to different systems could provide a detailed understanding of chytrid parasitism and host interaction, development under different nutrient regimes and degradation of diverse carbon sources.From an evolutionary perspective, the early-diverging fungi are a critical component of the eukaryotic tree of life ,43, inclR. globosum is able to control rhizoid morphogenesis to produce a searching form in response to carbon starvation and, from an individual cell, is capable of spatial differentiation in adaptation to patchy substrate availability indicating functional division of labour. The potential for convergent evolution aside, we propose by parsimony from the presence of analogous complex cell developmental features in an extant representative chytrid and dikaryan fungi that adaptive rhizoids are a shared feature of their most recent common ancestor.Perhaps our key discovery is that the anucleate chytrid rhizoid shows considerable developmental plasticity.


SLC4A1) gene could co-exist with red cell membrane abnormality, Southeast Asian ovalocytosis (SAO). Although this association is well described in Southeast Asian countries, it is less frequently found in Sri Lanka.Familial distal renal tubular acidosis (dRTA) associated with mutations of solute carrier family 4 membrane − 1 (We describe six patients who had dRTA co-existing with SAO. All of them initially presented with severe hypokalemia and paralysis. They presented within a period of six months to the Teaching Hospital Anuradhapura, Sri Lanka.All had metabolic acidosis indicated by low serum bicarbonate. Three of them were having underlying chronic kidney disease as well. Those three patients had mixed high and normal anion gap metabolic acidosis indicated by low delta ratio. In all dRTA was confirmed by presence of normal anion gap, hyperchloraemia, high urine pH and positive urine anion gap.Examination of blood films of all of them revealed presence of stomatocytes and macro-ovalocytosis compatible with SAO.In relation to complications of dRTA, two patients had medullary nephrocalcinosis. Three patients had biochemical evidence of osteomalacia, with two of them having radiological evidence of diffuse osteosclerosis. One patient had secondary hyperparathyroidism and a pathological fracture.Plasmodium vivax malaria and cerebral malaria cause by Plasmodium falciparum. Although two families of SAO was described earlier, SAO and dRTA combination was reported only once in a patient from Anuradhapura district.Erythrocyte in SAO is exceptionally rigid and this abnormality is said to be evolved as it protects against Distal renal tubular acidosis, SAO combination and its related complications including nephrocalcinosis, chronic kidney disease and metabolic bone disease was not described in Sri-Lankan literature. This case series emphasize the importance of investigating recurrent/ chronic hypokalemia to diagnose dRTA and its associations, as early correction of acidosis could prevent development of chronic kidney disease and metabolic bone disease. Distal renal tubular acidosis (dRTA) is characterized by impaired hydrogen ion secretion in distal nephron. This defect leads to an inability to excrete acid load causing hydrogen ion retention and hyperchloremic metabolic acidosis with an inappropriately alkaline urine. Distal RTA is commonly associated with hypokalemia causing muscle paralysis which could be the first manifestation of the underlying disease.AE1 (SLC4A1 gene product) or band 3 protein. In the kidney the protein is involved in acidification of the urine and in the red cell it functions both as an anion exchanger and as a membrane glycoprotein required for shape and stability. Autosomal recessive dRTA which may be associated with sensorineural deafness present mutations in the gene ATP6B1 encoding the B1-subunit of H + -ATPase. Autosomal recessive dRTA without sensorineural deafness may be caused by mutations in the gene ATP6N1B encoding the 116-kD subunit of H+ ATP-ase. Recent genetic studies has shown that autosomal recessive dRTA associated with AE1 gene mutation in many countries in Southeast Asia. Accordingly both dominant and recessive forms of dRTA can be associated with mutation of the SLC4A1 gene [AE1, SLC4A1) gene associated with dRTA are reported worldwide. Three mutations, (G701D), (A858D) and deletion of Val850 (DV850) described are restricted to South-East Asian populations particularly to Malaysia and Papua New Guinea [Mutations in genes encoding renal acid-base transporters have been identified as causes of inherited/primary dRTA . These s4A1 gene . SeveralSLC4A1) gene, may be co-inherited in trans resulting in dRTA [Familial dRTA and an inherited erythrocyte disorder ‘Southeast Asian Ovalocytosis’ (SAO), both caused by mutations in the solute carrier family 4 membrane − 1  and bilateral medullary nephrocalcinosis. Her blood gas analysis revealed a metabolic acidosis with alkaline urine. The presence of high anion gap with low delta ratio indicated mixed high and normal anion gap metabolic acidosis due to CKD and dRTA respectively Table . She hadA previously healthy 59-year-old female from Kahatagasdigiliya, Fig.  was admiA 62-year-old previously well male patient from Alayapaththuwa Fig.  presenteA 32-year-old male from Thanthrimale Fig. , was admA 55-year old male from Medawachchiya Fig.  was undeA 50-year-old female from Padaviya Fig.  with pasAll were advised on the need for long-term treatment to minimize nephrocalcinosis and MBD associated with dRTA. They were started on oral sodium bicarbonate1–2 meq/kg/day aiming at serum bicarbonate 22 to 24 meq/L. Patients who were intolerant to or showed inadequate response to sodium bicarbonate were given potassium citrate. All are being regularly followed up in outpatient medical clinic.All patients had hypokalemia and metabolic acidosis indicated by low serum bicarbonate (Table-1). Distal RTA (type-1) was confirmed by normal anion gap, hyperchloraemia, high urine pH and positive urine anion gap. Association with SAO could also help the diagnosis and suggest the hereditary nature of the dRTA. Patient 1 in 2016, and patient 3 and 4 had developed CKD and mixed metabolic acidosis (indicated by delta ratio) and all others had normal anion gap metabolic acidosis. Patient 1 & 5 had medullary nephrocalcinosis. All patients had SAO.Two families in Sri Lanka with SAO were reported in 2004 without referring to the association with dRTA . In the SAO has unique red cell morphology in the blood film with stomatocytes, ovalocytes, and macro-ovalocytes and found almost exclusively in South-East Asia . The eryDistal RTA is often associated with nephrocalcinosis, hypokalaemia and metabolic bone disease. Progression of nephrocalcinosis may lead to development of CKD . If deteThe usual type of metabolic bone disease (MBD) in dRTA is osteomalacia. There are rare case reports of diffuse osteosclerosis  and MBD Assessment of CKD in three out of six patients was challenging as recently there was an epidemic of CKD of toxic origin termed chronic interstitial nephritis in agricultural communities (CINAC) in the same area of Anuradhapura district . AlthougSix adult patients, 3 males and 3 females age ranging from 32 yrs. to 62 yrs. with dRTA in association with SAO, suggesting hereditary dRTA, were evaluated here. Recurrent admissions with hypokalemia with paralysis presenting during adult life, presence of several types of MBD were few distinctive feature in this cohort. Inability to perform genetic testing due to unavailability of resources in Sri Lanka was a limitation in this study.SLC4A1 gene may have evolved as SAO has protection against malaria and north central part of the country was repeatedly affected by malaria infection starting from 1029 AD. Six patients presenting to Anuradhapura hospital within 6 months indicate that the number would be more and probably under diagnosed. This case series highlight the importance of investigating patients presenting with hypokalemic paralysis and periodic paralysis as one cause would be dRTA. Early diagnosis and correction of acidosis are important in preventing complications. Delay in treatments lead to development of nephrocalcinosis, chronic kidney disease and metabolic bone disease.In conclusion, SAO and dRTA combination is less well described in Sri Lanka. A single case described earlier is also from Anuradhapura district of Sri Lanka. Mutations of


Severe peripheral neuropathy is a common dose-limiting toxicity of taxane chemotherapy, with no effective treatment. Frozen gloves have shown to reduce the severity of neuropathy in several studies but comes with the incidence of undesired side effects such as cold intolerance and frostbite in extreme cases. A device with thermoregulatory features which can safely deliver tolerable amounts of cooling while ensuring efficacy is required to overcome the deficiencies of frozen gloves. The role of continuous-flow cooling in prevention of neurotoxicity caused by paclitaxel has been previously described. This study hypothesized that cryocompression (addition of dynamic pressure to cooling) may allow for delivery of lower temperatures with similar tolerance and potentially improve efficacy.pre), after completion (NCSpost) and 3 months post-chemotherapy (NCS3m). Results were retrospectively compared with patients who underwent paclitaxel chemotherapy along with continuous-flow cooling and controls with no hypothermia.A proof-of-concept study was conducted in cancer patients receiving taxane chemotherapy. Each subject underwent four-limb cryocompression with each chemotherapy infusion (three hours) for a maximum of 12 cycles. Cryocompression was administered at 16 °C and cyclic pressure (5–15 mmHg). Skin surface temperature and tolerance scores were recorded. Neuropathy was assessed using clinician-graded peripheral sensory neuropathy scores, total neuropathy score (TNS) and nerve conduction studies (NCS) conducted before (NCSp < 0.0001). None of the patients experienced severe neuropathy (clinician-assessed neuropathy scores of grade 2 or higher). NCS analysis showed preservation of motor amplitudes at NCS3m in subjects who underwent cryocompression, compared to the controls who showed significant deterioration . Cryocompression did not significantly affect taxane-induced changes in sensory nerve amplitudes.In total, 13 patients underwent 142 cycles of cryocompression concomitant with chemotherapy. Limb hypothermia was well tolerated, and only 1 out of 13 patients required an intra-cycle temperature increase, with no early termination of cryocompression in any subject. Mean skin temperature reduction of 3.8 ± 1.7 °C was achieved. Cryocompression demonstrated significantly greater skin temperature reductions compared to continuous-flow cooling and control  contains supplementary material, which is available to authorized users. New research shows a growing interest towards cryotherapy or regional hypothermia for alleviating chemotherapy-induced toxicities such as oral mucositis, ocular toxicity, onycholysis and peripheral neuropathy . Of thesIn a recent trial reported by Hanai et al. , cryotheThe role of continuous-flow limb cooling in decreasing the incidence of CIPN has been previously described in studies by our group , 25. Coo3 or 3-weekly docetaxel, 75 mg/m3) chemotherapy were invited to participate in the study. All subjects were informed orally and in writing about the aims of the study and written consent for participation was sought and documented.The study was conducted at the National University Hospital, Singapore from November 2015 to February 2017 in accordance with the protocol approved by the Institutional Review Board of the National Health Group, Singapore. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and the 1964 Helsinki declaration and its later amendments. Cancer patients scheduled to receive taxane-based  aged 21–80 years; (ii) signed informed consent from patient or legal representative; (iii) no history of neuropathy; (iv) Eastern Cooperative Oncology Group (ECOG) performance status 0; and (v) no history of hospitalization in the past 6 months. Patients were ineligible for the study if they had: (i) open skin wounds or ulcers of the limbs; or (ii) history of Raynaud’s phenomenon, peripheral vascular disease, or poorly-controlled diabetes (HbA1c > 10%).During every cycle of chemotherapy, premedication drugs  were administered 30 min prior to the taxane infusion. The taxane chemotherapy was administered as a 1-h infusion , which simultaneously applied active cyclical pneumatic compression (cycling from 5–15 mmHg every 5 min) and cooling to the limbs through limb wraps attached to a thermoregulator. The system comprised of a portable thermoregulator control unit together with arm and leg cooling wraps, geometrically designed to comfortably fit around the subject’s limbs and could be easily applied and taken off. The control unit circulated the coolant (water) and air through the cooling wraps to cool and compress the wrapped limb. Skin surface temperature was measured using wireless temperature sensors  placed at evenly spaced locations on all four limbs . Temperapre), after chemotherapy (NCSpost) and 3 months after chemotherapy (NCS3m) , including evaluation of sensory and motor nerve functions, was conducted at three time points—before the start of chemotherapy (NCS3m) Fig. . Tests wWe also compared the efficacy of cryocompression and continuous-flow cooling by comparing data from this study to our previous study where cancer patients received unilateral leg cooling (without compression) together with their chemotherapy while the study’s uncooled limbs were treated as the control group .Clinical evaluation using the modified total neuropathy score (TNS) was performed at the same time points as the NCS . Clinicipre) and averaged across patients.The temporal trend of skin temperature changes, over the duration of hypothermia was summarized as a mean of the recorded temperatures for all cycles of hypothermia for all patients. Similarly, tolerability to limb hypothermia was analysed as an average of all patients’ tolerance scores across all cycles of hypothermia. Sensory and motor nerve parameters of amplitude and velocity, at every NCS visit, were analysed as relative percentage changes with respect to the first NCS visit .Continuous variables are shown as mean ± S.D. (standard deviation). A paired 2 of either paclitaxel or docetaxel.Thirteen cancer patients  scheduled to receive taxane-based chemotherapy participated in the study. The patient characteristics are detailed in Table Cryocompression (at 16 °C and dynamic compression) was overall well tolerated as measured by a well-defined range of tolerability measures Skin temperature changes on the limbs were recorded and averaged across all patients over all 142 cycles. At the end of duration of cryocompression, a mean skin temperature drop of 2.9 ± 2.3 °C and 3.8 ± 1.7 °C was achieved on the arms and legs, respectively. On an average, cryocompression achieved a skin temperature decrease of 2.32 °C lower than continuous-flow cooling . NCS analysis following cryocompression showed preservation of cMAP amplitudes at NCSpost and NCS3m   . Limb hypothermia did not seem to significantly change outcomes compared to the control group (without hypothermia)  subjects who received paclitaxel chemotherapy . Changes at NCS subjectsrs) Fig. . On the pre Fig. . Moreove3m showed a correlation with skin temperature changes (r = 0.4).Changes in motor amplitudes  allows for delivery of significantly lower degrees of hypothermia, resulting in lower skin temperatures. This mode of delivery of regional hypothermia is well tolerated and can be administered over multiple cycles of chemotherapy. Moreover, cryocompression may potentially improve the efficacy of preventing CIPN compared to continuous-flow limb cooling, however, this remains to be demonstrated in further studies.Studies describe compression contributing to improved tolerability of pain over long durations of hypothermia administered post-surgery by reducing the formation of oedema . In partCompression, often used in combination with cryotherapy for care of acute injuries appears to improve tolerability, magnitude and depth of hypothermia. Several studies have repeatedly and meticulously documented that cryocompression results in significantly higher magnitude of surface and even intramuscular temperatures than cryotherapy alone , 27. TheWe also observed that the efficacy of cryocompression in preservation of peroneal motor nerve function was better than cryotherapy and control Fig. –3. HowevAnother crucial factor influencing the therapeutic efficacy of hypothermia for chemo-induced toxicities is the therapy protocol. Studies of scalp cooling have shown that initiating cooling 30 to 45 min prior to the administration of chemotherapy is crucial for vasoconstriction-induced neuroprotection . Our temSeveral studies have employed frozen gloves as a mode of cryotherapy in preventing CIPN , 31, 32.Our study serves as a pilot to demonstrate that cryocompression is a safe and tolerable therapeutic strategy for alleviating CIPN in patients with cancer undergoing taxane treatment. Limitations of the pilot study include a small sample size and the lack of a randomized, prospective control. These findings would need to be validated in a larger prospective study, preferably with randomization. Similar to other supportive care measures such as scalp cooling to reduce CIA, cryocompression may serve to improve the quality of life in cancer patients during and after treatment and also support the delivery of optimal chemotherapy by preventing a dose delay or reduction.ESM 1(DOCX 2380 kb)


There was no significant gain in effect with the 3 × 106 ahMNCs dose. Histological analysis suggested that ahMNCs exert their effects by modulating glial scar formation, neuroprotection, and/or angiogenesis. These data indicate that ahMNCs from patients with hemorrhagic stroke could be used to develop stem cell therapies for SCI and that the indirect injection route could be clinically relevant. Moreover, the optimal transplantation dose of ahMNCs defined in this preclinical study might be helpful in calculating its optimal injection dose for patients with SCI in the future.Stem cell-based therapeutics are amongst the most promising next-generation therapeutic approaches for the treatment of spinal cord injury (SCI), as they may promote the repair or regeneration of damaged spinal cord tissues. However, preclinical optimization should be performed before clinical application to guarantee safety and therapeutic effect. Here, we investigated the optimal injection route and dose for adult human multipotent neural cells (ahMNCs) from patients with hemorrhagic stroke using an SCI animal model. ahMNCs demonstrate several characteristics associated with neural stem cells (NSCs), including the expression of NSC-specific markers, self-renewal, and multi neural cell lineage differentiation potential. When ahMNCs were transplanted into the lateral ventricle of the SCI animal model, they specifically migrated within 24 h of injection to the damaged spinal cord, where they survived for at least 5 weeks after injection. Although ahMNC transplantation promoted significant locomotor recovery, the injection dose was shown to influence treatment outcomes, with a 1 × 10 Traumatic spinal cord injury (SCI) results in the loss of both cells and connecting axons at the injury site, which interrupts the flow of information to and from the brain. Since transected axons cannot regrow or replenish these lost neurons, there is no naturally occurring pathway to repair the mature human central nervous system (CNS) . This meTransplantation of neural stem cells (NSCs) has been shown to promote repair or regeneration of damaged CNS. Implanted NSCs replace lost neurons ,3, creatIn a previous study , ahMNCs This study was designed to address these issues and evaluate the use of cultured ahMNCs from patients with hemorrhagic stroke, and design and evaluate an indirect delivery route for ahMNCs to treat SCI animals. We determined that an intracerebroventricular (ICV) transplantation protocol using stem cells produced the best in vivo migration of injected cells into the disease sites of SCI via the cerebrospinal fluid (CSF) ,14,15. aahMNCs were isolated from the surgically excised brain tissues of patients with hemorrhagic stroke. These ahMNCs were primarily cultured under adherent culture conditions as previously described  and demo5 (low), 1 × 106 (medium), or 3 × 106 (high) were transplanted into the LV of the SCI rats  or ahMNCs in 30 μL HBSS at a dose of 3 × 10SCI rats A. To assSCI rats A. LocomoSCI rats . NotablySCI rats . These rTissue loss within the injured spinal cords was evaluated using histology at 6 weeks post injury A. The loTo elucidate the underlying mechanisms facilitating the ahMNCs tissue-protective functions, we went on to compare the glial scar formation in each of the experimental groups at 6 weeks post injury A. AstrocThe in vivo neuroprotective effects of ahMNC transplantation were evaluated using immunohistochemistry against NeuN and Tuj1 on tissues collected 6 weeks post injury A. First,We evaluated Tuj1 expression to determine the effects of ahMNC transplant on both the axons and dendrites in injured rats D,E. Tuj1Taken together these results support the hypothesis that ahMNCs exert their SCI therapeutic effect via their protection of the neurons and fibers near the injury site.Olig2-positive oligodendrocytes were identified and quantified at the rostral and caudal points A. AlthouWe previously reported that transplantation of ahMNCs significantly increased vessel density in damaged spinal cords . To evaln = 3 for each group). Immunohistochemistry  type IIIa ,10,11. ISecond, ahMNCs transplanted into the LV of SCI animals successfully migrated to the lesion in the thoracic spinal cord and survived in this niche for at least 5 weeks post transplantation. Direct injection of stem cells into the injured spinal cord is common in preclinical evaluations ,22, howeThird, the in vivo migration of ahMNCs into the damaged spinal cord was specific. In mammalian CNS, both human and non-human NSCs have the innate ability to proliferate and move to injured and/or degenerating brain areas . This isFourth, primary cultured ahMNCs exhibit significant preclinical therapeutic effects for SCI. The histological analysis revealed four important alterations to these lesions linked to the presence of the ahMNCs. (1) Gliosis: Glial scars have long been considered the major barrier to axonal regeneration ,31. Howe5), medium (1 × 106), and high (3 × 106) dose and observed a maximum therapeutic effect in the medium dose. Since cell suspensions at high concentrations (3 × 106 cells in 30 μL HBSS) are relatively sticky, we hypothesize that the ahMNCs in this injection line might not be transplanted properly. A recent study suggested that a higher dose of fetal NSCs increases successful engraftment, neuronal survival, and axonal regeneration in animal models of SCI leading to better functional recovery . C. C45]. C® Blood and Tissue Kit (250)  according to the manufacturer’s instructions. Alu PCR was completed using the following primers: forward = 5′-TCAGGAGATCGAG-ACCATCCC-3′′and reverse = 5′-TCCTGCCTCAGCCTCCCAAG-3′ [Genomic DNA was extracted using the DNeasyCCAAG-3′ . PCR wasAll data are presented as the mean ± standard error (SEM). Data were analyzed using the two-tailed Student’s t-test and P-values of less than 0.05 were considered significant.


Salmo salar L.) is seen as one of the best solutions to solve key issues in the salmon farming industry, such as the impact of escapees on wild stocks and pre-harvest sexual maturation. However, the effects of triploidy on salmon smoltification are poorly understood at the molecular level, even though smoltification is a very sensitive period that has a major influence on survival rate and performance of farmed salmon. In this study, we have compared the liver transcriptomes of diploid and triploid Atlantic salmon at three ontogeny stages: fry, parr and smolt. In diploid fish, a total of 2,655 genes were differentially expressed between fry and parr, whereas 506 genes had significantly different transcript levels between parr and smolts. In triploids, 1,507 and 974 genes were differentially expressed between fry and parr, and between parr and smolts, respectively. Most of these genes were down-regulated and 34 genes were differentially expressed between ploidies at the same stage. In both ploidy groups, the top differentially expressed genes with ontogeny stage belonged to common functional categories that can be related to smoltification. Nucleotide and energy metabolism were significantly down-regulated in fry when compared to parr, while immune system processes were significantly down-regulated in parr when compared to smolts. The close resemblance of enriched biological processes and pathways between ploidy groups suggests that triploidy is regulated by genome dosage compensation in Atlantic salmon. Histological analysis revealed that areas of vacuolization (steatosis) were present only in fry and parr stages, in contrast to a compact cellular histology with glycogen granules after smoltification. There was no significant difference in vacuolization between ploidy groups at the fry stage but the liver of diploid parr had a 33.5% higher vacuolization area compared to their triploid counterparts. Taken together, our data provide novel insights into the changes that occur at the molecular and histological level in the liver of both diploid and triploid Atlantic salmon prior to and during smoltification.Triploid Atlantic salmon ( Sterility may also minimize the risk of genetic and ecological interactions between farmed and wild stocks in the event of accidental farmed fish escapees2. In Atlantic salmon  aquaculture, escaped fish from sea cages represent one of the most persistent issues for sustainable development of the industry and have generated considerable concerns across all producing regions3. In Norway, farmed escapees are one of the main anthropogenic impact factors identified as an expanding threat to wild Atlantic salmon populations, besides sea lice infestations4. Genetic introgression from farmed salmon is reported to vary across rivers but remains widespread in Norwegian wild salmon populations, encouraging additional efforts to implement more robust production systems, escape prevention plans and other mitigation measures5.The use of functionally sterile triploid fish has the potential to improve post-pubertal growth, survival and carcass quality under culture conditions2. Regardless of the theoretical advantages that triploids might have over their diploid counterparts, comparative studies assessing their culture performance under different settings have produced “conflicting” results. For instance, a large and comprehensive study assessing the growth performance and sexual maturation in diploid and triploid Atlantic salmon of both sexes in seawater tanks under natural or continuous photoperiod regimes resulted in growth performance of triploid groups being equally or better in some cases when compared to their diploid counterparts6. In addition, a study assessing the performance of diploids and triploids in freshwater from hatching until smolt stages showed no significant differences in survival rate, but triploids were smaller in size at earlier stages7. Welfare issues such as high prevalence of skeletal deformities in addition to other potential limitations in triploid Atlantic salmon, including abnormal heart morphology and abdominal lesions has been highlighted to limit its performance and subsequent adoption by producers9. In addition, escaped triploid males have been shown to interfere with natural reproduction in the wild by competing with wild males for wild fertile females by stimulating spawning in Atlantic salmon10 and in Atlantic cod11 as well.Despite the potential of sterile triploid fish to contribute towards the development of a more sustainable and environmentally-friendly Atlantic salmon industry, uncertainties regarding the performance of cultured triploids have hindered their widespread adoption by most producers12. Contrary to expectations, the use of dietary hydrolysed fish proteins rich in free amino acids and low molecular peptides that may ease feed absorption had no positive influence on nutrient utilization and growth in triploids as in diploids13. Reports at the molecular level with emphasis on understanding the physiology and impact of triploidy on Atlantic salmon during ontogeny until post-smoltification are quite scarce. A study comparing growth-related gene expression profiles of diploid and triploid bighead catfish  showed similar liver transcriptome responses in both ploidy groups14. In contrast, a molecular study showed that early nutritional programming for up to 6 weeks affected metabolic processes in the liver of diploid and triploid Atlantic salmon, where ploidy differences indicated the need for different ploidy-specific nutritional requirements for optimal performance15. A more recent study has shown that increase in dietary micronutrient levels led to vertebral expression of bone biomarker genes associated with reduced skeletal malformations in parr diploids and triploids but was found to be significant in the former16. Findings from these studies16 suggest that morphological, physiological and ploidy differences could play a role in determining digestive efficiency, nutritional requirements and subsequent growth and welfare of these fish.From a nutritional perspective, a gut morphology study comparing triploid and diploid Atlantic salmon revealed a lower pyloric caeca number and shorter relative gut length in triploid post-smolts compared to its diploid counterparts17. Little is known about changes in the liver transcriptome of diploid and triploid Atlantic salmon prior to and during smoltification. Hence, we used RNA-seq to investigate potential differences in the liver transcriptome of diploid and triploid individuals at three ontogeny stages . This molecular approach was complemented by histological observations aimed at examining liver morphology and degree of steatosis in relation to ontogeny and ploidy over the same developmental stages.Fish liver is involved in numerous functions including metabolism and immune system processes, to mention just a few. A study assessing the liver transcriptome response in Atlantic salmon fed diets with either fish oil or vegetable oil for up to 55 weeks revealed that there are genotype-specific metabolic responses at the molecular levelA similar number of raw reads was obtained for the three ontogeny groups, ranging from 7.9 to 40.9 million sequences  . There were 69 significantly enriched GO terms associated with down-regulated DEGs in diploid fry when compared to their parr counterparts  was the most up-regulated in fry, with a fold-change of 44.2 when compared to smolt fish. Other up-regulated DEGs in this comparison were related to immune system and cellular processes , lipid metabolism (hepatic triacylglycerol lipase-like) and carbohydrate metabolism (Table ornithine decarboxylase 1-like) was down-regulated in both diploid and triploid fry when compared to their parr counterparts.Within diploids, most down-regulated DEGs for fry compared to parr were associated with either carbohydrate, lipid or other metabolic processes Table . Notablesm Table . A gene ornithine decarboxylase 1. On the other hand, DEGs up-regulated for triploid parr when compared to smolt were linked to cellular processes, polyamine biosynthesis (ornithine decarboxylase 1-like), genetic information processes (eukaryotic translation initiation factor 4E binding protein 3–1),heme and ion binding and oxidoreductase activity   in both ploidy groups at the parr stage. This was more pronounced in triploid parr, which had a degree of vacuolization significantly lower (P < 0.001) than their diploid counterparts  in triploid than in diploid fish at all three developmental stages  were significantly enriched for biological processes and pathways in both diploid and triploid fish.19. Therefore, a down-regulation of nucleotide metabolism can potentially decrease energy metabolism. It has been reported that nucleotide deficiency negatively impacts the performance of key organs and processes, such as liver, heart, intestine and immune functions20. The higher levels of transcripts related to nucleotide and energy metabolism in parr when compared to fry are likely related to a surge in energy metabolism needed for subsequent smolt transformation, according to a previous report21.There were several down-regulated pathways in both diploid and triploid fry when compared to parr, namely genetic information processes, cellular processes and organismal systems. Some pathways involved in metabolism  and environmental processes  were exclusively down-regulated between diploid fry and diploid parr. Nevertheless, the biological processes observed for the top 10 GO terms significantly enriched and down-regulated between fry and parr were identical in diploid and triploid fish. Most biological processes significantly down-regulated in fry when compared to parr were involved in either energy or nucleotide metabolism. Nucleotides are known to be involved in a variety of chemical functions, including energy metabolism23. It has been reported that decline in lipid metabolism of salmonids during smoltification helps mobilise energy reserves (triglycerols) required for environment and ontogeny-associated changes21. Also, Wedemeyer et al.24 highlighted the contribution of lipid decline as salmonid develops into smolts towards silvering, increased growth rate, salinity tolerance and increased pituitary growth hormones in order to adapt to environmental-associated changes at sea. These findings demonstrate the importance of down-regulating lipid metabolism during ontogeny and environmental-associated changes encountered by salmonids during smoltification. In addition, the interactions between lipid, carbohydrate and amino acid metabolism decline in Atlantic salmon facilitate smoltification25. Lipid and carbohydrate depletion during smoltification are the results of increased lipolytic/glygenolytic enzyme activity, decrease in lipid/glycogen synthesis and increase in the rate of plasma fatty acid/glucose turn over21. Hence, the observed down-regulation of genes involved in lipid and carbohydrate metabolism in the liver of fry compared to parr probably favour mobilisation of energy required to reach the parr stage. Common to both ploidy groups for the same comparison was the down-regulation of ornithine decarboxylase 1-like, which is known to be involved in immune and inflammatory responses through directing arginine flux away from nitric oxide synthase and nitric oxide production by existing as a free radical toxic to bacteria and a signalling molecule26. Down-regulation of this gene in fry when compared to parr stages may be related to the susceptibility of Atlantic salmon fry to bacterial infections27 during transition to parr. Some genes involved in various cellular processes  were down-regulated exclusively in triploid fry when compared to their parr counterparts. This could be due to the fact that triploidy leads to an overall reduced cell number in comparison to diploids, which limits their normal cellular functions and organ system processes to some extent28. We did not observe significant differences in hepatocyte number between diploid and triploid fish but our histology analysis was limited to the mid-portion of the liver.Compared to parr, fry had lower transcript levels of genes related to lipid, carbohydrate and amino acid metabolism. In fish, a lower lipid content is known to favour increase in body water content and vice versaadh1, a gene involved in alcohol metabolism and oxidative stress. This may be linked to the fact that triploid salmonids tend to be more prone to oxidative stress than their diploid counterparts at juvenile29 and post-smolt stages30.Both diploid and triploid fry had relatively few up-regulated DEGs when compared to their parr counterparts and these DEGs belonged to similar functional categories, mostly related to cellular processes. A notable difference between ploidy groups was the 44-fold up-regulation exclusively in triploid fry of 33. Cell-mediated immunity is particularly important in the elimination of intracellular pathogens31. A recent study showed a strong relationship between MHC and disease resistance of Atlantic salmon to infectious salmon anaemia34. The potential changes in the immune system of smolts is likely important for their survival at sea, where they will be exposed to new pathogens.The enriched GO terms for down-regulated genes between parr and smolt fish were very similar in diploid and triploid fish, and they were related to cell-mediated immunity or general immune system processes. In both ploidy groups, the top significantly enriched GO terms down-regulated in parr when compared to smolts included taxis and chemotaxis, cellular response to chemical stimulus and cell migration, which indicate increased cell-mediated immunity and directional phagocytosis in smolts. Movement of microphages in fishes has already been demonstrated to be by either chemokinesis  or chemotaxis  in response to bacterial antigens in vivo or in vitroornithine decarboxylase 1-like) was up-regulated in parr with fold changes of 6.3 and 4.5 in diploids and triploids, respectively. The enzyme encoded by this gene is a key regulator in growth processes in vertebrates and has been positively correlated with specific growth rate in Atlantic salmon35. Its higher transcript levels in parr may be related to the rapid growth change known to occur in pre-smolts. Up-regulated metabolic rates could also be due to the fish being bigger in size and with a subsequently higher basal metabolic rate. The eukaryotic translation initiation factor 4E binding protein 3–1 was also up-regulated in parr and it can affect growth, since it encodes a vital mRNA cap-binding protein that controls global translation rates36.The most up-regulated DEGs in diploid and triploid parr when compared to smolts included genes involved in metabolism and cellular processes. For example, a gene involved in polyamine biosynthesis  and their triploid offspring showed similar expression levels in both ploidies where expression level dominance (ELD) was found to be biased towards the maternal genome42. In addition, homolog gene expression levels in this study showed the combined role of regulatory functions and epigenetics through a transcriptome network vital for adaptation during growth of triploids42 which is likely to be the case of this study. Generally, triploid salmon shows either a positive or negative dosage compensation when compared to diploid full-sibs counterparts43. Determining dosage compensation with focus on the liver can be questionable because the liver of eukaryotic organisms has been reported to form variable percentages of polyploidy cells during liver growth and under other circumstances44. In mammals, the onset and degree of polyploidization in hepatocytes vary across species and can be observed during normal or pathological conditions but the functional significance of the process remains uncertain46. To date, polyploidization events have not been reported for the liver of fish47, including salmonids48, irrespective of their ploidy status but information on Atlantic salmon is still lacking. In our study, we compared the nuclear size of hepatocytes in the liver of diploid and triploid salmon13 as indicator of their ploidy status. Cell size measurements (e.g. minor axis of erythrocytes) are frequently used as indirect measurements of DNA content in artificially induced triploids49. Overall, hepatocyte cell nuclei in triploids measured 1.2–1.4 times those of diploids and contained by definition correspondingly higher levels of DNA than diploid nuclei. Overall, these quantitative histological findings validate the use of liver as central organ regulating energy metabolism and several other functions to assess the possibility of dosage compensation in our study. This is in line with the work of Christensen et al.50 where liver tissue was chosen to measure gene expression levels in coho salmon under different metabolic states.A recent study has shown that performance and growth of triploid Atlantic salmon is linked to the second maternal chromosome set49. For example, variability in terms of nuclear differences between these two ploidies, possibly induced by specific cellular and nuclear adjustments, has been reported across tissues and organs of the marine medaka, Oryzias dancena51. In our work, cell measurements performed on histological sections showed that the nuclear size (minor and major axis) of triploid over diploid hepatocytes of juvenile Atlantic salmon was below the expected 1.5 ratio and further studies are required to explore the cellular processes occurring in the liver of these two ploidy groups.Triploid cell nuclei are expected to contain one extra haploid set of maternal chromosomes and therefore 50% more DNA than diploid cell nuclei with some degree of variation likely to occur across tissues within the same individual52, functional feeds54 and inclusion of micronutrients in low marine fish diets16 on fish growth, development and health. In the present work, the observed decrease in liver steatosis from fry to parr reflected the hepatic transcriptome profiles, which showed a significant up-regulation of genes involved in lipid metabolism in parr compared to fry, independently from their ploidy status. Such higher levels of relevant transcripts likely supported the mobilisation of energy required to reach this phase of development and in preparation of smoltification. These results are in agreement with Peruzzi et al.13, where the same experimental diploid and triploid fish fed a standard commercial fishmeal diet displayed a surge in somatic growth in the period from fry to parr. It is known that alterations in lipid metabolism during parr-smolt transformation in Atlantic salmon lead to smolt having reduced available liver, as well as muscle, energy reserves compared to parr and these are regarded as an integral part of the process56. In the present study, the lack of vacuolisation in the hepatocytes of both diploid and triploid smolts points indicates additional similarities between these two ploidy groups in the pattern of lipid mobilisation required during the final steps of parr-smolt transformation.Overall, our results indicate clear similarities in the general liver morphology and steatosis between diploid and triploid fish prior to and after smoltification. Quantification of liver steatosis based on the degree of vacuolization in the cytoplasm and the degree of distribution of the vacuolated hepatocytes has been employed in numerous fish studies addressing lipid metabolism. In Atlantic salmon, examples include nutritional and nutrigenomics studies performed to assess the effects of plant meal diets and natural plant extractsaurkb, ima2, cpt1b, cpt1a, slc6a13, odc1, lipc, lipe, dcor1, pla2g3, ppp1r3b, ccl19, gnvip1, scyb7, ifi44, adh1, which are involved in oxidative stress, metabolism, immune system and cellular processes. Most of these genes were common to both ploidy groups, except one gene that plays a role in oxidative stress (adh1) and two genes involved in cellular processes (ima2 and aurkb), which were differentially regulated exclusively in triploids. At each ontogeny stage, the biological processes enriched for down-regulated genes showed a striking resemblance in diploid and triploid fish, which was reflected in a similar liver morphology and level of vacuolisation between ploidy groups.In Atlantic salmon, the liver transcriptome is different between fry, parr and smolts, as expected. The top differentially expressed genes between ontogeny stages are involved in preparation for smoltification. These include 13. Briefly, experimental fish comprising of diploid (2n) and triploid (3n) ova were produced using siblings composed of n = 17 full-sib and half families produced by a commercial hatchery  where triploidy was induced by a pressure shock of 9500 psi applied for 5 min, 300° minutes post-fertilization at 5 °C46. Eyed-ova (~ 400 day-degrees) were then shipped to the Aquaculture Research Station in Kårvika . Prior to start-feeding, the ploidy status of the experimental groups was verified by flow cytometry as previously reported13. The experimental fish were reared in triplicate tanks and fed a commercial standard diet  whilst being reared at low temperature from start-feeding to completion of the parr-smolt transformation. Constant light was used throughout the experiment, except for a period of reduced day length required to simulate winter conditions and induce parr-smolt transformation13. For sampling purposes, fish were euthanized using an anaesthetic overdose of benzocaine . Liver samples were collected from fish at the ontogeny stages below, snap-frozen in liquid nitrogen and transported on dry ice to Nord University  where they were stored at − 80 oC until transcriptome analysis. The average weight of fish at each stage used for this: fry (2n = 3.62 g and 3n = 4.11 g), parr (2n = 27.83 g and 3n = 29.89 g) and smolt (2n = 56.44 g and 3n = 66.56 g). Samples collected for all analyses had weights close to the mean weight.Details on the relationship, origin, production, growth parameters and rearing protocols of the experimental fish are provided by Peruzzi et al.Total RNA was extracted from frozen liver samples following the QIAzol protocol  and further cleaned with the RNeasy MinElute cleanup kit . Quality and quantity of total RNA were determined using the 2200 TapeStation from Agilent (USA). Each RNA sample used had a RINe (RNA integrity) value above 7. RNA was extracted from 5 individual liver samples (n = 5) per ploidy group at selected sampling stages fry , parr (1888 ddPSF) and smolt (2745 ddPSF). RNA-seq libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA Library Prep kit . The barcoded libraries were checked for quality and quantity on a 2200 TapeStation before pooling and sequencing. Library pools were sequenced on the NextSeq500  with the Nextseq 500/550 high output kit v2.5 sequencing kit (150 cycles single-end reads) at Nord University, Norway.57 and quality of the trimmed reads was checked using the FastQC software58. Clean reads were mapped to the Atlantic salmon genome and transcriptome annotation databases (RefSeq accession: GCF_000233375.1 from the NCBI database) using TopHat259. Uniquely mapped transcripts were quantified using HTSeq (high-throughput sequence) counts60 and DEGs were then identified using DESeq2.Data were converted from BCL to FASTA format and demultiplexed using the Illumina script bcl2fastq conversion software v2.17 with default parameters. Adapters were then removed using the Cutadapthttps://www.bioconductor.org/packages/release/bioc/html/DESeq2.html), with an adjusted p-value < 0.05 (Benjamin-Hochberg method)61. The R packages ggplot2 and heatmap were used for graphical representation of the data. DEGs were then subjected to GO enrichment analysis for biological processes using DAVID v6.8 with an EASE score ≤ 0.0163. Selected DEGs obtained from DESeq2 in Entrez gene identifier format were converted to FASTA format for downstream analysis on the Kyoto Encyclopaedia for Genes and Genomes (KEGG) Automatic Annotation Server (KAAS)64. in hepatocytes using a set scale of 7 pixels/ μm. For each section, a 5,000 μm2 rectangular frame was placed over three hepatic regions distant from large vessels and the average area (μm2) occupied by lipid vacuoles within hepatocytes was calculated. Nuclear size (minor and major axis) of hepatocytes was assessed on the above-reported histological sections (ca. n = 10 nuclei/section) captured at a magnification of 40 × and analysed with ImageJ using a set scale of 1.0 μm.Liver samples from different fish belonging to the same group used for transcriptomic analysis were fixed in 10% (v/v) neutral buffered formalin for 48 h before transferred to 70% (v/v) ethanol for storage. Samples were then transferred to standard cassettes, dehydrated, embedded in paraffin wax and sectioned at 5 μm on a RM2255 rotary microtome . The sections were stained with haematoxylin/eosin  according to standard histological procedures and examined on a DM 2000 LED light microscope  equipped with a Leica DFC 295 digital Colour Camera. Photographs of the sections were processed using the Leica software application suite. A total of 41 individual samples were selected for this study: 10 fry (2n = 4 and 3n = 6), 15 (2n = 8 and 3n = 7) parr and 16 (3n and 2n = 8) smolts. Liver steatosis (lipid vacuolization) was assessed on images captured at magnification of 40 × and analysed with ImageJ following a modification of the method by Campos et al.Liver steatosis data  were log-transformed and checked for normality and equality of variance prior to two-way ANOVA with fish ploidy and ontogeny stage as fixed factors. When significant differences were found, the Hochberg’s post-hoc test was used to determine differences between individual fish groups. Hepatocytes’ nuclear size measurements in both ploidies and at different ontogeny stages were compared by Students’ t-test. Ratios of nuclear measurements in hepatocytes of triploid over diploid fish at different stages were analysed by one-way ANOVA with ontogeny stage as fixed factor. All data analyses were performed using SPSS  and the results were considered significant at P < 0.05. Data are reported as means ± standard error of the mean (SEM).All experimental procedures involving Atlantic salmon were in accordance with the Norwegian legislation on animal experimentation and the guidelines of the European Union Council (Directive 2010/EU) and were approved by the Norwegian Committee on Ethics in Animal Experimentation (Project license permit ID: 8180) issued by the Norwegian Food Safety Authority (Mattilsynet).Supplementary file 1