Uniaxial mechanical stretch properties correlated with three-dimensional microstructure of human dermal skin.

The intact and healthy skin forms a barrier to the outside world and protects the body from mechanical impact. The skin is a complex structure with unique mechano-elastic properties. To better direct the design of biomimetic materials and induce skin regeneration in wounds with optimal outcome, more insight is required in how the mechano-elastic properties emerge from the skin's main constituents, collagen and elastin fibers. Here, we employed two-photon excited autofluorescence and second harmonic generation microscopy to characterize collagen and elastin fibers in 3D in 24 human dermis skin samples. Through uniaxial stretching experiments, we derive uni-directional mechanical properties from resultant stress-strain curves, including the initial Young's modulus, elastic Young's modulus, maximal stress, and maximal and mid-strain values. The stress-strain curves show a large variation, with an average Young's modules in the toe and linear regions of 0.1 MPa and 21 MPa. We performed a comprehensive analysis of the correlation between the key mechanical properties with age and with microstructural parameters, e.g., fiber density, thickness, and orientation. Age was found to correlate negatively with Young's modulus and collagen density. Moreover, real-time monitoring during uniaxial stretching allowed us to observe changes in collagen and elastin alignment. Elastin fibers aligned significantly in both the heel and linear regions, and the collagen bundles engaged and oriented mainly in the linear region. This research advances our understanding of skin biomechanics and yields input for future first principles full modeling of skin tissue.

A network score-based metric to optimize the quality assurance of automatic radiotherapy target segmentations.

Background and purpose: Existing methods for quality assurance of the radiotherapy auto-segmentations focus on the correlation between the average model entropy and the Dice Similarity Coefficient (DSC) only. We identified a metric directly derived from the output of the network and correlated it with clinically relevant metrics for contour accuracy. Materials and Methods: Magnetic Resonance Imaging auto-segmentations were available for the gross tumor volume for cervical cancer brachytherapy (106 segmentations) and for the clinical target volume for rectal cancer external-beam radiotherapy (77 segmentations). The nnU-Net's output before binarization was taken as a score map. We defined a metric as the mean of the voxels in the score map above a threshold (λ). Comparisons were made with the mean and standard deviation over the score map and with the mean over the entropy map. The DSC, the 95th Hausdorff distance, the mean surface distance (MSD) and the surface DSC were computed for segmentation quality. Correlations between the studied metrics and model quality were assessed with the Pearson correlation coefficient (r). The area under the curve (AUC) was determined for detecting segmentations that require reviewing. Results: For both tasks, our metric (λ = 0.30) correlated more strongly with the segmentation quality than the mean over the entropy map (for surface DSC, r > 0.65 vs. r < 0.60). The AUC was above 0.84 for detecting MSD values above 2 mm. Conclusions: Our metric correlated strongly with clinically relevant segmentation metrics and detected segmentations that required reviewing, indicating its potential for automatic quality assurance of radiotherapy target auto-segmentations.

Deep learning for segmentation of the cervical cancer gross tumor volume on magnetic resonance imaging for brachytherapy.

BACKGROUND: Segmentation of the Gross Tumor Volume (GTV) is a crucial step in the brachytherapy (BT) treatment planning workflow. Currently, radiation oncologists segment the GTV manually, which is time-consuming. The time pressure is particularly critical for BT because during the segmentation process the patient waits immobilized in bed with the applicator in place. Automatic segmentation algorithms can potentially reduce both the clinical workload and the patient burden. Although deep learning based automatic segmentation algorithms have been extensively developed for organs at risk, automatic segmentation of the targets is less common. The aim of this study was to automatically segment the cervical cancer GTV on BT MRI images using a state-of-the-art automatic segmentation framework and assess its performance. METHODS: A cohort of 195 cervical cancer patients treated between August 2012 and December 2021 was retrospectively collected. A total of 524 separate BT fractions were included and the axial T2-weighted (T2w) MRI sequence was used for this project. The 3D nnU-Net was used as the automatic segmentation framework. The automatic segmentations were compared with the manual segmentations used for clinical practice with Sørensen-Dice coefficient (Dice), 95th Hausdorff distance (95th HD) and mean surface distance (MSD). The dosimetric impact was defined as the difference in D98 (ΔD98) and D90 (ΔD90) between the manual segmentations and the automatic segmentations, evaluated using the clinical dose distribution. The performance of the network was also compared separately depending on FIGO stage and on GTV volume. RESULTS: The network achieved a median Dice of 0.73 (interquartile range (IQR) = 0.50-0.80), median 95th HD of 6.8 mm (IQR = 4.2-12.5 mm) and median MSD of 1.4 mm (IQR = 0.90-2.8 mm). The median ΔD90 and ΔD98 were 0.18 Gy (IQR = -1.38-1.19 Gy) and 0.20 Gy (IQR =-1.10-0.95 Gy) respectively. No significant differences in geometric or dosimetric performance were observed between tumors with different FIGO stages, however significantly improved Dice and dosimetric performance was found for larger tumors. CONCLUSIONS: The nnU-Net framework achieved state-of-the-art performance in the segmentation of the cervical cancer GTV on BT MRI images. Reasonable median performance was achieved geometrically and dosimetrically but with high variability among patients.

Antimicrobial protein REG3A regulates glucose homeostasis and insulin resistance in obese diabetic mice.

Innate immune mediators of pathogen clearance, including the secreted C-type lectins REG3 of the antimicrobial peptide (AMP) family, are known to be involved in the regulation of tissue repair and homeostasis. Their role in metabolic homeostasis remains unknown. Here we show that an increase in human REG3A improves glucose and lipid homeostasis in nutritional and genetic mouse models of obesity and type 2 diabetes. Mice overexpressing REG3A in the liver show improved glucose homeostasis, which is reflected in better insulin sensitivity in normal weight and obese states. Delivery of recombinant REG3A protein to leptin-deficient ob/ob mice or wild-type mice on a high-fat diet also improves glucose homeostasis. This is accompanied by reduced oxidative protein damage, increased AMPK phosphorylation and insulin-stimulated glucose uptake in skeletal muscle tissue. Oxidative damage in differentiated C2C12 myotubes is greatly attenuated by REG3A, as is the increase in gp130-mediated AMPK activation. In contrast, Akt-mediated insulin action, which is impaired by oxidative stress, is not restored by REG3A. These data highlight the importance of REG3A in controlling oxidative protein damage involved in energy and metabolic pathways during obesity and diabetes, and provide additional insight into the dual function of host-immune defense and metabolic regulation for AMP.

Explaining the dosimetric impact of contouring errors in head and neck radiotherapy.

Objective. Auto-contouring of organs at risk (OAR) is becoming more common in radiotherapy. An important issue in clinical decision making is judging the quality of the auto-contours. While recent studies considered contour quality by looking at geometric errors only, this does not capture the dosimetric impact of the errors. In this work, we studied the relationship between geometrical errors, the local dose and the dosimetric impact of the geometrical errors.Approach. For 94 head and neck patients, unmodified atlas-based auto-contours and clinically used delineations of the parotid glands and brainstem were retrieved. VMAT plans were automatically optimized on the auto-contours and evaluated on both contours. We defined the dosimetric impact on evaluation (DIE) as the difference in the dosimetric parameter of interest between the two contours. We developed three linear regression models to predict the DIE using: (1) global geometric metrics, (2) global dosimetric metrics, (3) combined local geometric and dosimetric metrics. For model (3), we next determined the minimal amount of editing information required to produce a reliable prediction. Performance was assessed by the root mean squared error (RMSE) of the predicted DIE using 5-fold cross-validation.Main results. In model (3), the median RMSE of the left parotid was 0.4 Gy using 5% of the largest editing vectors. For the right parotid and brainstem the results were 0.5 Gy using 10% and 0.4 Gy using 1% respectively. The median RMS of the DIE was 0.6 Gy, 0.7 Gy and 0.9 Gy for the left parotid, the right parotid and the brainstem, respectively. Model (3), combining local dosimetric and geometric quantities, outperformed the models that used only geometric or dosimetric information.Significance. We showed that the largest local errors plus the local dose suffice to accurately predict the dosimetric impact, opening the door to automated dosimetric QA of auto-contours.

An anomaly detector as a clinical decision support system for parotid gland delineations.

Purpose. Auto-contouring (AC) is rapidly becoming standard practice for OAR contouring. However, in clinical practice, clinicians still need to manually check and correct contours. Anomaly detection systems (ADS) can aid the clinical decision process by suggesting which structures require corrections or not, greatly enhancing the value of AC. The purpose of this work is to develop and evaluate a decision support system for detecting anomalies in the case of parotid gland delineations. METHODS: Head and neck parotid gland delineations (1037 right, 1038 left), were retrieved from the Netherlands Cancer Institute (NKI) database. Morphological and image-based features were extracted from each patient's CT and structure set. An isolation forest model was initially trained on 70% of the data, of which 10% had synthetically generated anomalies and validated on the remaining 30% of clinical data. The ADS was tested on an independent set of 250 patients (Normal: 174, Anomalies: 76) and on a clinical autocontouring software. RESULTS: Applied to the validation set, the ADS system resulted in area under the curve (AUC) values of 0.93 and 0.94 for the parotid left and right respectively. Image features appeared more important than morphological, but using all features resulted marginally in the best model. Applied to the test set the ADS system reached an accuracy level of 0.83 and 0.81 for the parotid left and right respectively. The ADS was particularly sensitive to uniform expansions/contractions, misplacements, extra/missing slices and anisotropic over-contouring. CONCLUSION: Anomaly detection can serve as a powerful contour quality assurance tool, especially for cases of organ misplacement and over-contouring.

Portal dosimetry of small unflattened beams.

We developed and validated a dedicated small field back-projection portal dosimetry model for pretreatment andin vivoverification of stereotactic plans entailing small unflattened photon beams. For this purpose an aSi-EPID was commissioned as a small field dosimeter. Small field output factors for 6 MV FFF beams were measured using the PTW microDiamond detector and the Agility 160-leaf MLC from Elekta. The back-projection algorithm developed in our department was modified to better model the small field physics. The feasibility of small field portal dosimetry was validated via absolute point dose differences w.r.t. small static beams, and 5 hypofractionated stereotactic VMAT clinical plans measured with the OCTAVIUS 1000 SRS array dosimeter and computed with the treatment planning system Pinnacle v16.2. Dose reconstructions using the currently clinically applied back-projection model were also computed for comparison. We found that the latter yields underdosage of about -8% for square beams with cross section near 10 mm x 10 mm and about -6% for VMAT treatments with PTV volumes smaller than about 2cm3. With the methods described in this work such errors can be reduced to less than the ±3.0% recommendations for clinical use. Our results indicate that aSi-EPIDs can be used as accurate small field radiation dosimeters, offering advantages over point dose detectors, the correct positioning and orientation of which is challenging for routine clinical QA.

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium. The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

Forest and woodland replacement patterns following drought-related mortality.

Forest vulnerability to drought is expected to increase under anthropogenic climate change, and drought-induced mortality and community dynamics following drought have major ecological and societal impacts. Here, we show that tree mortality concomitant with drought has led to short-term (mean 5 y, range 1 to 23 y after mortality) vegetation-type conversion in multiple biomes across the world (131 sites). Self-replacement of the dominant tree species was only prevalent in 21% of the examined cases and forests and woodlands shifted to nonwoody vegetation in 10% of them. The ultimate temporal persistence of such changes remains unknown but, given the key role of biological legacies in long-term ecological succession, this emerging picture of postdrought ecological trajectories highlights the potential for major ecosystem reorganization in the coming decades. Community changes were less pronounced under wetter postmortality conditions. Replacement was also influenced by management intensity, and postdrought shrub dominance was higher when pathogens acted as codrivers of tree mortality. Early change in community composition indicates that forests dominated by mesic species generally shifted toward more xeric communities, with replacing tree and shrub species exhibiting drier bioclimatic optima and distribution ranges. However, shifts toward more mesic communities also occurred and multiple pathways of forest replacement were observed for some species. Drought characteristics, species-specific environmental preferences, plant traits, and ecosystem legacies govern postdrought species turnover and subsequent ecological trajectories, with potential far-reaching implications for forest biodiversity and ecosystem services.

Reporte de un caso: Fractura expuesta del mediopie por accidente de tránsito en paciente VIH/SIDA / Case Report: Exposed Midfoot Fracture Due To Traffic Accident In HIV/AIDS Patient

Actualmente hay una alta incidencia de accidentes de tránsito en el mundo, muchos de ellos provocan una discapacidad grave en las personas. Estas lesiones en las extremidades provocan alta morbilidad llegando incluso a una amputación. Esto se agrava en pacientes VIH/SIDA, sobre todo en el tratamiento y la evolución. Se presenta un caso clínico de un paciente con lesión severa del mediopie que termina en amputación y se hace una revisión del manejo quirúrgico del paciente traumatológico con VIH/SIDA

Currently there is a high incidence of traffic accidents in the world, many of them cause severe disability in people. These limb injuries cause high morbidity even reaching an amputation. This is aggravated in HIV/AIDS patients, especially in treatment and evolution. A clinical case of a patient with severe midfoot injury that ends in amputation is presented and a surgical management review of the trauma patient with HIV/AIDS is made.

Identification of Four Immune Subtypes Characterized by Distinct Composition and Functions of Tumor Microenvironment in Intrahepatic Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a severe malignant tumor in which the standard therapies are mostly ineffective. The biological significance of the desmoplastic tumor microenvironment (TME) of ICC has been stressed but was insufficiently taken into account in the search for classifications of ICC adapted to clinical trial design. We investigated the heterogeneous tumor stroma composition and built a TME-based classification of ICC tumors that detects potentially targetable ICC subtypes. APPROACH AND RESULTS: We established the bulk gene expression profiles of 78 ICCs. Epithelial and stromal compartments of 23 ICCs were laser microdissected. We quantified 14 gene expression signatures of the TME and those of 3 functional indicators (liver activity, inflammation, immune resistance). The cell population abundances were quantified using the microenvironment cell population-counter package and compared with immunohistochemistry. We performed an unsupervised TME-based classification of 198 ICCs (training set) and 368 ICCs (validation set). We determined immune response and signaling features of the different immune subtypes by functional annotations. We showed that a set of 198 ICCs could be classified into 4 TME-based subtypes related to distinct immune escape mechanisms and patient outcomes. The validity of these immune subtypes was confirmed over an independent set of 368 ICCs and by immunohistochemical analysis of 64 ICC tissue samples. About 45% of ICCs displayed an immune desert phenotype. The other subtypes differed in nature (lymphoid, myeloid, mesenchymal) and abundance of tumor-infiltrating cells. The inflamed subtype (11%) presented a massive T lymphocyte infiltration, an activation of inflammatory and immune checkpoint pathways, and was associated with the longest patient survival. CONCLUSION: We showed the existence of an inflamed ICC subtype, which is potentially treatable with checkpoint blockade immunotherapy.

Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis.

BACKGROUND & AIMS: Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. METHODS: We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. RESULTS: The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). CONCLUSIONS: Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

Merging paleobiology with conservation biology to guide the future of terrestrial ecosystems.

Conservation of species and ecosystems is increasingly difficult because anthropogenic impacts are pervasive and accelerating. Under this rapid global change, maximizing conservation success requires a paradigm shift from maintaining ecosystems in idealized past states toward facilitating their adaptive and functional capacities, even as species ebb and flow individually. Developing effective strategies under this new paradigm will require deeper understanding of the long-term dynamics that govern ecosystem persistence and reconciliation of conflicts among approaches to conserving historical versus novel ecosystems. Integrating emerging information from conservation biology, paleobiology, and the Earth sciences is an important step forward on the path to success. Maintaining nature in all its aspects will also entail immediately addressing the overarching threats of growing human population, overconsumption, pollution, and climate change.

Global threats from invasive alien species in the twenty-first century and national response capacities.

Invasive alien species (IAS) threaten human livelihoods and biodiversity globally. Increasing globalization facilitates IAS arrival, and environmental changes, including climate change, facilitate IAS establishment. Here we provide the first global, spatial analysis of the terrestrial threat from IAS in light of twenty-first century globalization and environmental change, and evaluate national capacities to prevent and manage species invasions. We find that one-sixth of the global land surface is highly vulnerable to invasion, including substantial areas in developing economies and biodiversity hotspots. The dominant invasion vectors differ between high-income countries (imports, particularly of plants and pets) and low-income countries (air travel). Uniting data on the causes of introduction and establishment can improve early-warning and eradication schemes. Most countries have limited capacity to act against invasions. In particular, we reveal a clear need for proactive invasion strategies in areas with high poverty levels, high biodiversity and low historical levels of invasion.

Online 3D EPID-based dose verification: Proof of concept.

PURPOSE: Delivery errors during radiotherapy may lead to medical harm and reduced life expectancy for patients. Such serious incidents can be avoided by performing dose verification online, i.e., while the patient is being irradiated, creating the possibility of halting the linac in case of a large overdosage or underdosage. The offline EPID-based 3D in vivo dosimetry system clinically employed at our institute is in principle suited for online treatment verification, provided the system is able to complete 3D dose reconstruction and verification within 420 ms, the present acquisition time of a single EPID frame. It is the aim of this study to show that our EPID-based dosimetry system can be made fast enough to achieve online 3D in vivo dose verification. METHODS: The current dose verification system was sped up in two ways. First, a new software package was developed to perform all computations that are not dependent on portal image acquisition separately, thus removing the need for doing these calculations in real time. Second, the 3D dose reconstruction algorithm was sped up via a new, multithreaded implementation. Dose verification was implemented by comparing planned with reconstructed 3D dose distributions delivered to two regions in a patient: the target volume and the nontarget volume receiving at least 10 cGy. In both volumes, the mean dose is compared, while in the nontarget volume, the near-maximum dose (D2) is compared as well. The real-time dosimetry system was tested by irradiating an anthropomorphic phantom with three VMAT plans: a 6 MV head-and-neck treatment plan, a 10 MV rectum treatment plan, and a 10 MV prostate treatment plan. In all plans, two types of serious delivery errors were introduced. The functionality of automatically halting the linac was also implemented and tested. RESULTS: The precomputation time per treatment was ∼180 s/treatment arc, depending on gantry angle resolution. The complete processing of a single portal frame, including dose verification, took 266 ± 11 ms on a dual octocore Intel Xeon E5-2630 CPU running at 2.40 GHz. The introduced delivery errors were detected after 5-10 s irradiation time. CONCLUSIONS: A prototype online 3D dose verification tool using portal imaging has been developed and successfully tested for two different kinds of gross delivery errors. Thus, online 3D dose verification has been technologically achieved.

Overview of 3-year experience with large-scale electronic portal imaging device-based 3-dimensional transit dosimetry.

PURPOSE: To assess the usefulness of electronic portal imaging device (EPID)-based 3-dimensional (3D) transit dosimetry in a radiation therapy department by analyzing a large set of dose verification results. METHODS AND MATERIALS: In our institution, routine in vivo dose verification of all treatments is performed by means of 3D transit dosimetry using amorphous silicon EPIDs. The total 3D dose distribution is reconstructed using a back-projection algorithm and compared with the planned dose distribution using 3D gamma evaluation. Dose reconstruction and gamma evaluation software runs automatically in our clinic, and analysis results are (almost) immediately available. If a deviation exceeds our alert criteria, manual inspection is required. If necessary, additional phantom measurements are performed to separate patient-related errors from planning or delivery errors. Three-dimensional transit dosimetry results were analyzed per treatment site between 2012 and 2014 and the origin of the deviations was assessed. RESULTS: In total, 4689 of 15,076 plans (31%) exceeded the alert criteria between 2012 and 2014. These alerts were patient-related and attributable to limitations of our back-projection and dose calculation algorithm or to external sources. Clinically relevant deviations were detected for approximately 1 of 430 patient treatments. Most of these errors were because of anatomical changes or deviations from the routine clinical procedure and would not have been detected by pretreatment verification. Although cone beam computed tomography scans yielded information about anatomical changes, their effect on the dose delivery was assessed quantitatively by means of 3D in vivo dosimetry. CONCLUSIONS: EPID-based transit dosimetry is a fast and efficient dose verification technique. It provides more useful information and is less time-consuming than pretreatment verification measurements of intensity modulated radiation therapy and volumetric modulated arc therapy. Large-scale implementation of 3D transit dosimetry is therefore a powerful method to guarantee safe dose delivery during radiation therapy.

Vulnerability of ecosystems to climate change moderated by habitat intactness.

The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network.