•Implications and promise of effective usefulness of Lidar metrics for monitoring forest landscape restoration.•Canopy structural attributes were useful to characterize and distinguish different ...forest types.•Canopy structure showed relation with the tree community Shannon Index but not with richness.•A novel Lidar metric (LAHV) showed promising results for qualifying tree cover and assess its functions.•We expect our results can help address realistic restoration goals and actions under a global change context.
Ambitious pledges to restore over 400 million hectares of degraded lands by 2030 have been made by several countries within the Global Partnership for Forest Landscape Restoration (FLR). Monitoring restoration outcomes at this scale requires cost-effective methods to quantify not only forest cover, but also forest structure and the diversity of useful species. Here we obtain and analyze structural attributes of forest canopies undergoing restoration in the Atlantic Forest of Brazil using a portable ground lidar remote sensing device as a proxy for airborne laser scanners. We assess the ability of these attributes to distinguish forest cover types, to estimate aboveground dry woody biomass (AGB) and to estimate tree species diversity (Shannon index and richness). A set of six canopy structure attributes were able to classify five cover types with an overall accuracy of 75%, increasing to 87% when combining two secondary forest classes. Canopy height and the unprecedented “leaf area height volume” (a cumulative product of canopy height and vegetation density) were good predictors of AGB. An index based on the height and evenness of the leaf area density profile was weakly related to the Shannon Index of tree species diversity and showed no relationship to species richness or to change in species composition. These findings illustrate the potential and limitations of lidar remote sensing for monitoring compliance of FLR goals of landscape multifunctionality, beyond a simple assessment of forest cover gain and loss.
•Flawed models may seem reliable if assessing MD, RMSD and R2 alone.•We promote using hypothesis tests in evaluating observed vs. predicted scatterplots.•Overfitting must be included in model ...evaluation and variable selection algorithms.•Assessing overfitting with cross-validation: better compare squares sums than R2.
The evaluation of accuracy is essential for assuring the reliability of ecological models. Usually, the accuracy of above-ground biomass (AGB) predictions obtained from remote sensing is assessed by the mean differences (MD), the root mean squared differences (RMSD), and the coefficient of determination (R2) between observed and predicted values. In this article we propose a more thorough analysis of accuracy, including a hypothesis test to evaluate the agreement between observed and predicted values, and an assessment of the degree of overfitting to the sample employed for model training. Using the estimation of forest AGB from LIDAR and spectral sensors as a case study, we compared alternative prediction and variable selection methods using several statistical measures to evaluate their accuracy. We showed that the hypothesis tests provide an objective method to infer the statistical significance of agreement. We also observed that overfitting can be assessed by comparing the inflation in residual sums of squares experienced when carrying out a cross-validation. Our results suggest that this method may be more effective than analysing the deflation in R2. We proved that overfitting needs to be specifically addressed since, in light of MD, RMSD and R2 alone, predictions may apparently seem reliable even in clearly unrealistic circumstances, for instance when including too many predictor variables. Moreover, Theil’s partial inequality coefficients, which are employed to resolve the proportions of the total errors due to the unexplained variance, the slope and the bias, may become useful to detect averaging effects common in remote sensing predictions of AGB. We concluded that statistical measures of accuracy, precision and agreement are necessary but insufficient for model evaluation. We therefore advocate for incorporating evaluation measures specifically devoted to testing observed-versus-predicted fit, and to assessing the degree of overfitting.
Mesenchymal stem/stromal cells (MSCs) can regenerate tissues through engraftment and differentiation but also via paracrine signalling via extracellular vesicles (EVs). Fetal-derived MSCs (fMSCs) ...have been shown, both in vitro and in animal studies, to be more efficient than adult MSC (aMSCs) in generating bone and muscle but the underlying reason for this difference has not yet been clearly elucidated. In this study, we aimed to systematically investigate the differences between fetal and adult MSCs and MSC-derived EVs at the phenotypic, RNA, and protein levels.
We carried out a detailed and comparative characterization of culture-expanded fetal liver derived MSCs (fMSCs) and adult bone marrow derived MSCs (aMSCs) phenotypically, and the MSCs and MSC-derived EVs were analysed using transcriptomics and proteomics approaches with RNA Sequencing and Mass Spectrometry.
Fetal MSCs were smaller, exhibited increased proliferation and colony-forming capacity, delayed onset of senescence, and demonstrated superior osteoblast differentiation capability compared to their adult counterparts. Gene Ontology analysis revealed that fMSCs displayed upregulated gene sets such as "Positive regulation of stem cell populations", "Maintenance of stemness" and "Muscle cell development/contraction/Myogenesis" in comparison to aMSCs. Conversely, aMSCs displayed upregulated gene sets such as "Complement cascade", "Adipogenesis", "Extracellular matrix glycoproteins" and "Cellular metabolism", and on the protein level, "Epithelial cell differentiation" pathways. Signalling entropy analysis suggested that fMSCs exhibit higher signalling promiscuity and hence, higher potency than aMSCs. Gene ontology comparisons revealed that fetal MSC-derived EVs (fEVs) were enriched for "Collagen fibril organization", "Protein folding", and "Response to transforming growth factor beta" compared to adult MSC-derived EVs (aEVs), whereas no significant difference in protein expression in aEVs compared to fEVs could be detected.
This study provides detailed and systematic insight into the differences between fMSCs and aMSCs, and MSC-derived EVs. The key finding across phenotypic, transcriptomic and proteomic levels is that fMSCs exhibit higher potency than aMSCs, meaning they are in a more undifferentiated state. Additionally, fMSCs and fMSC-derived EVs may possess greater bone forming capacity compared to aMSCs. Therefore, using fMSCs may lead to better treatment efficacy, especially in musculoskeletal diseases.
ABSTRACTNeuron‐derived exosomes (NDEs) were enriched by anti‐L1CAM antibody immunoabsorption from plasmas of subjects ages 18–26 yr within 1 wk after a sports‐related mild traumatic brain injury ...(acute mTBI) (n = 18), 3 mo or longer after the last of 2–4 mTBIs (chronic mTBI) (n = 14) and with no recent history of TBI (controls) (n = 21). Plasma concentrations of NDEs, assessed by counts and levels of extracted exosome marker CD81, were significantly depressed by a mean of 45% in acute mTBI (P < 0.0001), but not chronic mTBI, compared with controls. Mean CD81‐normalized NDE levels of a range of functional brain proteins were significantly abnormal relative to those of controls in acute but not chronic mTBI, including ras‐related small GTPase 10, 73% decrease; annexin VII, 8.8‐fold increase; ubiquitin C‐terminal hydrolase L1, 2.5‐fold increase; AII spectrin fragments, 1.9‐fold increase; claudin‐5, 2.7‐fold increase; sodium‐potassium‐chloride cotransporter‐1, 2.8‐fold increase; aquaporin 4, 8.9‐fold increase (3.6‐fold increase in chronic mTBI); and synaptogyrin‐3, 3.1‐fold increase (1.3‐fold increase in chronic mTBI) (all acute mTBI proteins P < 0.0001). In chronic mTBI, there were elevated CD81‐normalized NDE levels of usually pathologic β‐amyloid peptide 1‐42 (1.6‐fold, P < 0.0001), P‐T181‐tau (2.2‐fold, P < 0.0001), P‐S396‐tau (1.6‐fold, P < 0.01), IL‐6 (16‐fold, P < 0.0001), and prion cellular protein (PRPc) (5.1‐fold, P < 0.0001) with lesser or greater (IL‐6, PRPc) increases in acute mTBI. Increases in NDE levels of most neurofunctional proteins in acute, but not chronic, mTBI, and elevations of most NDE neuropathological proteins in chronic and acute mTBI delineated phase‐specificity. Longitudinal studies of more mTBI subjects may identify biomarkers predictive of and etiologically involved in mTBI‐induced neurodegeneration.—Goetzl, E. J., Elahi, F. M., Mustapic, M., Kapogiannis, D., Pryhoda, M., Gilmore, A., Gorgens, K. A., Davidson, B., Granholm, A.‐C., Ledreux, A. Altered levels of plasma neuron‐derived exosomes and their cargo proteins characterize acute and chronic mild traumatic brain injury. FASEB J. 33, 5082–5088 (2019). www.fasebj.org
Assessing the persistent impacts of fragmentation on aboveground structure of tropical forests is essential to understanding the consequences of land use change for carbon storage and other ecosystem ...functions. We investigated the influence of edge distance and fragment size on canopy structure, aboveground woody biomass (AGB), and AGB turnover in the Biological Dynamics of Forest Fragments Project (BDFFP) in central Amazon, Brazil, after 22+ yr of fragment isolation, by combining canopy variables collected with portable canopy profiling lidar and airborne laser scanning surveys with long-term forest inventories. Forest height decreased by 30% at edges of large fragments (>10 ha) and interiors of small fragments (<3 ha). In larger fragments, canopy height was reduced up to 40 m from edges. Leaf area density profiles differed near edges: the density of understory vegetation was higher and midstory vegetation lower, consistent with canopy reorganization via increased regeneration of pioneers following post-fragmentation mortality of large trees. However, canopy openness and leaf area index remained similar to control plots throughout fragments, while canopy spatial heterogeneity was generally lower at edges. AGB stocks and fluxes were positively related to canopy height and negatively related to spatial heterogeneity. Other forest structure variables typically used to assess the ecological impacts of fragmentation (basal area, density of individuals, and density of pioneer trees) were also related to lidar-derived canopy surface variables. Canopy reorganization through the replacement of edge-sensitive species by disturbance-tolerant ones may have mitigated the biomass loss effects due to fragmentation observed in the earlier years of BDFFP. Lidar technology offered novel insights and observational scales for analysis of theecological impacts of fragmentation on forest structure and function, specifically aboveground biomass storage.
Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been ...purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.
Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within ...the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS‐HAT). We report that PBS‐HAT buffer facilitates clearly improved short‐term and long‐term EV preservation for samples stored at ‐80°C, stability throughout several freeze‐thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.
The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and ...trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs.
Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even ...impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.
Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a ...robust cargo loading strategy. To address the paucity of such scaffold proteins, we leverage a simple and reliable assay that can distinguish intravesicular cargo proteins from surface- as well as non-vesicular proteins and compare the EV-sorting potential of 244 candidate proteins. We identify 24 proteins with conserved EV-sorting abilities across five types of producer cells. TSPAN2 and TSPAN3 emerge as lead candidates and outperform the well-studied CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles in cell cultures and mice as demonstrated by efficient transfer of luminal cargo proteins as well as surface display of different functional entities. The discovery of these scaffolds provides a platform for EV-based engineering.
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