A screen for regeneration-associated silencer regulatory elements in zebrafish.

Regeneration involves gene expression changes explained in part by context-dependent recruitment of transcriptional activators to distal enhancers. Silencers that engage repressive transcriptional complexes are less studied than enhancers and more technically challenging to validate, but they potentially have profound biological importance for regeneration. Here, we identified candidate silencers through a screening process that examined the ability of DNA sequences to limit injury-induced gene expression in larval zebrafish after fin amputation. A short sequence (s1) on chromosome 5 near several genes that reduce expression during adult fin regeneration could suppress promoter activity in stable transgenic lines and diminish nearby gene expression in knockin lines. High-resolution analysis of chromatin organization identified physical associations of s1 with gene promoters occurring preferentially during fin regeneration, and genomic deletion of s1 elevated the expression of these genes after fin amputation. Our study provides methods to identify "tissue regeneration silencer elements" (TRSEs) with the potential to reduce unnecessary or deleterious gene expression during regeneration.

Towards AI-augmented radiology education: a web-based application for perception training in chest X-ray nodule detection.

OBJECTIVES: Artificial intelligence (AI)-based applications for augmenting radiological education are underexplored. Prior studies have demonstrated the effectiveness of simulation in radiological perception training. This study aimed to develop and make available a pure web-based application called Perception Trainer for perception training in lung nodule detection in chest X-rays. METHODS: Based on open-access data, we trained a deep-learning model for lung segmentation in chest X-rays. Subsequently, an algorithm for artificial lung nodule generation was implemented and combined with the segmentation model to allow on-the-fly procedural insertion of lung nodules in chest X-rays. This functionality was integrated into an existing zero-footprint web-based DICOM viewer, and a dynamic HTML page was created to specify case generation parameters. RESULTS: The result is an easily accessible platform-agnostic web application available at: https://castlemountain.dk/mulrecon/perceptionTrainer.html.The application allows the user to specify the characteristics of lung nodules to be inserted into chest X-rays, and it produces automated feedback regarding nodule detection performance. Generated cases can be shared through a uniform resource locator. CONCLUSION: We anticipate that the description and availability of our developed solution with open-sourced codes may help facilitate radiological education and stimulate the development of similar AI-augmented educational tools. ADVANCES IN KNOWLEDGE: A web-based application applying AI-based techniques for radiological perception training was developed. The application demonstrates a novel approach for on-the-fly generation of cases in chest X-ray lung nodule detection employing deep-learning-based segmentation and lung nodule simulation.

Missed Cystic Fibrosis Newborn Screening Cases due to Immunoreactive Trypsinogen Levels below Program Cutoffs: A National Survey of Risk Factors.

Testing immunoreactive trypsinogen (IRT) is the first step in cystic fibrosis (CF) newborn screening. While high IRT is associated with CF, some cases are missed. This survey aimed to find factors associated with missed CF cases due to IRT levels below program cutoffs. Twenty-nine states responded to a U.S-wide survey and 13 supplied program-related data for low IRT false screen negative cases (CFFN) and CF true screen positive cases (CFTP) for analysis. Rates of missed CF cases and odds ratios were derived for each factor in CFFNs, and two CFFN subgroups, IRT above ("high") and below ("low") the CFFN median (39 ng/mL) compared to CFTPs for this entire sample set. Factors associated with "high" CFFN subgroup were Black race, higher IRT cutoff, fixed IRT cutoff, genotypes without two known CF-causing variants, and meconium ileus. Factors associated with "low" CFFN subgroup were older age at specimen collection, Saturday birth, hotter season of newborn dried blood spot collection, maximum ≥ 3 days laboratories could be closed, preterm birth, and formula feeding newborns. Lowering IRT cutoffs may reduce "high" IRT CFFNs. Addressing hospital and laboratory factors (like training staff in collection of blood spots, using insulated containers during transport and reducing consecutive days screening laboratories are closed) may reduce "low" IRT CFFNs.

From genotype to phenotype: Genetic redundancy and the maintenance of an adaptive polymorphism in the context of high gene flow.

A central question in evolution is how several adaptive phenotypes are maintained within a species. Theory predicts that the genetic determination of a trait, and in particular the amounts of redundancy in the mapping of genotypes to phenotypes, mediates evolutionary outcomes of phenotypic selection. In Mediterranean wild thyme, numerous discrete chemical phenotypes (chemotypes) occur in close geographic proximity. Chemotypes are defined by the predominant monoterpene produced by individual plants in their essential oil. In this study, we analyze the ecological genetics of six chemotypes nested within two well-established chemical families (hereafter ecotypes). Ecotypes, and chemotypes within ecotypes, are spatially segregated, and their distributions track local differences in the abiotic environment. By combining population genomic, phenotypic, and environmental data from 700 individuals, we show how the genetics of ecotype determination mediates this evolutionary response. Variation in three terpene-synthase loci explains variation in ecotype identity, with one single locus accounting for as much as 78% of this variation. Phenotypic selection combined with low segregating genotypic redundancy of ecotypes leaves a clear footprint at the genomic level: alleles associated with ecotype identity track environmental variation despite extensive gene flow. Different chemotypes within each ecotype differentially track environmental variation. Their identity is determined by multiple loci and displays a wider range of genotypic redundancy that dilutes phenotypic selection on their characteristic alleles. Our study thus provides a novel illustration of how genetic redundancy of a phenotype modulates the ability of selection to maintain adaptive differentiation. Identifying the precise genetics of the chemical polymorphism in thyme is the next crucial step for our understanding of the origin and maintenance of a polymorphism that is present in many aromatic plants.

Niche Variation in Endemic Lilium pomponium on a Wide Altitudinal Gradient in the Maritime Alps.

The relationship between altitudinal and ecological gradients has long been a dominant theme in plant ecology; moreover, how species respond to climate change has renewed this interest. Mediterranean mountains are often hotspots of endemism, and some endemic species have local distributions that span different climatic belts; hence, local variations in topography and fine-scaled niche conditions may play crucial roles in their persistence along such gradients. Studies of the fine-scaled niche are, however, very rare; most studies involve broad-scale variations in climatic parameters. The Turban lily, Lilium pomponium L. is endemic to the Maritime and Ligurian Alps, where it occurs across a wide altitudinal gradient. Previous work has shown no link between climatic marginality and geographic range limits on morphological traits and genetic variability; however, possible variations of local topographic and ecological parameters have not yet been examined. The objective of this paper is to characterise local ecological niche conditions of L. pomponium populations in the different bioclimatic zones it occupies along the altitudinal gradient. The species occurs in four main types of microecological niches. One of these niche types, with a high mineral cover, is the most abundant­type 2: it was detected in 39% of sampled quadrats and occurs across the whole bioclimatic gradient. Other niche types are more limited to subsections of the gradient: type 3 (in 19% of sampled quadrats) is restricted to high-altitude sites (>1070 m.a.s.l.) and is characterised by high vegetation and litter cover; type 4 (26%) corresponds to more forested habitats on substrates with low water retention capacities, in more inland zones close to the centre of L. pomponium distribution and across a range of altitudes; and type 1 (16% of quadrat) only occurs in the Mediterranean part of the gradient, close to distribution limits in pockets of soil among large blocks of rocks, mainly found at mid-altitudes. Despite heterogeneity in the spatial locations of niche types, there is no correspondence between ecological gradients and the distribution limits of this species. Knowledge of the fine-scaled ecological conditions that determine niche types is thus essential for conservation management of the habitats of this species and for the exploration of its possible response to ongoing climate change.

Predictors of radiation dose for uterine artery embolisation are angiography system-dependent.

This study sought to achieve radiation dose reductions for patients receiving uterine artery embolisation (UAE) by evaluating radiation dose measurements for the preceding generation (Allura) and upgraded (Azurion) angiography system. Previous UAE regression models in the literature could not be applied to this centre's practice due to being based on different angiography systems and radiation dose predictor variables. The aims of this study were to establish whether radiation dose is reduced with the upgraded angiography system and to develop a regression model to determine predictors of radiation dose specific to the upgraded angiography system. A comparison between Group I (Allura,n= 95) and Group II (Azurion,n= 95) demonstrated a significant reduction in kerma-area product (KAP) and Ka, r (reference air kerma) by 63% (143.2 Gy cm2vs 52.9 Gy cm2;P< 0.001,d= 0.8) and 67% (0.6 Gy vs 0.2 Gy;P< 0.001,d= 0.8), respectively. The multivariable linear regression (MLR) model identified the UAE radiation dose predictors for KAP on the upgraded angiography system as total fluoroscopy dose, Ka, r, and total uterus volume. The predictive accuracy of the MLR model was assessed using a Bland-Altman plot. The mean difference was 0.39 Gy cm2and the limits of agreement were +28.49 and -27.71 Gy cm2, and thus illustrated no proportional bias. The resultant MLR model was considered system-dependent and validated the upgraded angiography system and its advance capabilities to significantly reduce radiation dose. Interventional radiologist and interventional radiographer familiarisation of the system's features and the implementation of the newly established MLR model would further facilitate dose optimisation for all centres performing UAE procedures using the upgraded angiography system.

Niche Differentiation at Multiple Spatial Scales on Large and Small Mediterranean Islands for the Endemic Silene velutina Pourr. ex Loisel. (Caryophyllaceae).

The aim of this work is to investigate niche variations in endemic Silene velutina (Caryophyllaceae, Angiosperms) on Mediterranean islands that differ in size. Six populations on both large and small islands were sampled across the geographic range of the species. For each population, 10 plots (1 × 2 m, with a 25 cm grill) were randomly placed to quantify environmental (abiotic and biotic factors and disturbance) and population (demographic structure and reproductive success) parameters. Niche parameters related to substrate, plant cover, community diversity and composition and disturbance showed significant variation in relation to island size. At the regional scale, we detected a broader niche on large islands associated with spatial heterogeneity and island size. In contrast, at the local scale, populations on small islands showed a broader niche, potentially due to a release from competition (low diversity and plant cover and absence of phanerophytes). Populations on large islands had a demographic structure biased towards vegetative individuals (seedlings and juveniles) with few reproductive individuals, while those on small islands had a majority of adults. Together, the results on niche breadth and demographic structure concord with the idea of a strategy based on adult persistence on small islands.

Ecological niche differentiation among six annual Lythrum species in Mediterranean temporary pools.

The ecological niche defines the favourable range of a species in a multidimensional space of ecological factors that determine the presence and function of individuals. This fundamental concept in ecology is widely used to understand plant species coexistence and segregation. In this study, we test for ecological differentiation among six annual Lythrum species that are characteristic of temporary pools in the South of France, where they either coexist or occur separately. We first analysed the co-occurrence of species at two different geographical scales: cluster analyses of species presence in 10 km grid cells and coexistence in 0.25 m2 quadrats within populations of each species. Second, for three to nine populations of each species, we quantified a range of biotic and abiotic parameters using point contacts and soil measurements in five 0.25 m2 quadrats per population. We performed PCA on all variables and analysed each variable separately to compare the ecological niche features of the six species. A phenological index was assessed for the plant community of each site. We detected highly localized niche differentiation in terms of soil pH (all species) and for a range of variables among pairs of species. The six species also showed marked differences in the flowering period relative to the mean and variability of flowering time in their local community. These fine-scaled niche differences are associated with phylogenetic distances among species and may contribute to species' coexistence. These results are integrated in a conservation management plan for the habitat of the rarest species in this group.

3D Guided Dental Implant Placement: Impact on Surgical Accuracy and Collateral Damage to the Inferior Alveolar Nerve.

An increase in the number of implants placed has led to a corresponding increase in the number of complications reported. The complications can vary from restorative complications due to poor placement to damage to collateral structures such as nerves and adjacent teeth. A large majority of these complications can be avoided if the implant has been placed accurately in the optimal position. Therefore, the aim of this in vitro pilot study was to investigate the effect of freehand (FH) and fully guided (FG) surgery on the accuracy of implants placed in close proximity to vital structures such as the inferior alveolar nerve (IAN). Cone-beam computed tomography (CBCT) and intraoral scans of six patients who have had previous dental implants in the posterior mandible were used in this study. The ideal implant position was planned. FG surgical guides were manufactured for each case. In this study, the three-dimensional 3D printed resin models of each of the cases were produced and the implants placed using FG and FH methods on the respective models. The outcome variables of the study, angular deviations were calculated and the distance to the IAN was measured. The mean deviations for the planned position observed were 1.10 mm coronally, 1.88 mm apically with up to 6.3 degrees' angular deviation for FH surgery. For FG surgical technique the mean deviation was found to be at 0.35 mm coronally, 0.43 mm apically with 0.78 degrees angularly respectively. The maximum deviation from the planned position for the apex of the implant to the IAN was 2.55 mm using FH and 0.63 mm FG. This bench study, within its limitations, demonstrated surgically acceptable accuracy for both FH and FG techniques that would allow safe placement of implants to vital structures such as the IAN when a safety zone of 3 mm is allowed. Nevertheless, a better margin of error was observed for FG surgery with respect to the angular deviation and controlling the distance of the implant to the IAN using R2 Gate® system.

When ecological marginality is not geographically peripheral: exploring genetic predictions of the centre-periphery hypothesis in the endemic plant Lilium pomponium.

BACKGROUND: Quantifying variation of genetic traits over the geographical range of species is crucial for understanding the factors driving their range dynamics. The center-periphery hypothesis postulates, and many studies support, the idea that genetic diversity decreases and genetic differentiation increases toward the geographical periphery due to population isolation. The effects of environmental marginality on genetic variation has however received much less attention. METHODS: We tested the concordance between geographical and environmental gradients and the genetic predictions of center-periphery hypothesis for endemic Lilium pomponium in the southern Alps. RESULTS: We found little evidence for concordance between genetic variation and both geographical and environmental gradients. Although the prediction of increased differentiation at range limits is met, genetic diversity does not decrease towards the geographical periphery. Increased differentiation among peripheral populations, that are not ecologically marginal, may be explained by a decrease in habitat availability that reduces population connectivity. In contrast, a decrease of genetic diversity along environmental but not geographical gradients may be due to the presence of low quality habitats in the different parts of the range of a species that reduce effective population size or increase environmental constraints. As a result, environmental factors may affect population dynamics irrespective of distance from the geographical center of the range. In such situations of discordance between geographical and environmental gradients, the predictions of decreasing genetic diversity and increasing differentiation toward the geographical periphery may not be respected.

Effects of environmental heterogeneity on phenotypic variation of the endemic plant Lilium pomponium in the Maritime and Ligurian Alps.

Geographical limits of species' distributions are assumed to be coincident with ecological margins, although this assumption might not always be true. Indeed, harsh environments such as Alpine and Mediterranean ecosystems may favour high phenotypic variability among populations, especially those in peripheral sites. Floral traits are often found to be less variable and less affected by environmental heterogeneity than vegetative traits because variation in the former may have negative effects on fitness. For this reason, it is important to quantify variation in floral traits and plant fecundity in study range limits. The objective of the study is to examine phenotypic variation and differences in reproduction in endemic Lilium pomponium in the Maritime and Ligurian Alps in relation to environmental variation across its distribution range. In this species, marginal climatic populations occur both in the peripheral and central geographical locations of the distribution range; hence, geographical and ecological gradients are not concordant. Floral trait variation is related to local environmental conditions with an array of interactions among resource availability, potential pollen limitation and population size that are differentially related to floral traits. Contrary to the general expectation, all central and peripheral populations had similar, moderate seed production with each group limited by different factors acting on different stages of the life-history strategy. Our results are in line with the idea that general expectations are confirmed only when its assumptions are met and that the differences in pollination environment along an environmental gradient may not be the main determinant of the distribution limit.

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways.

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1+EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals.

Considering Proximal Urea Cycle Disorders in Expanded Newborn Screening.

Proximal urea cycle disorders (PUCDs) have adverse outcomes such as intellectual disability and death, which may benefit from newborn screening (NBS) through early detection and prevention with early treatment. Ornithine transcarbamylase deficiency (OTCD) and carbamoyl phosphate synthetase 1 deficiency (CPS1D) are screened in six and eight states in the United States. We analyzed current evidence to see if it supports inclusion of PUCDs in the NBS panels based upon prevention potential, medical, diagnostic, treatment, and public health rationales. A literature review was performed in PubMed using MESH terms for OTCD, CPS1D, and NAGSD. A systematic review was performed in the hallmark of NBS inclusion criteria. We reviewed 31 articles. Molecular and biochemical diagnosis is available to provide diagnostic evidence. Untreated PUCDs have a significant burden with considerable developmental delay and mortality that may improve with early treatment. Tandem mass spectrometry can be used for NBS for PUCDs; however, citrulline and glutamine alone are not specific. Medical treatments currently available for PUCDs meet existing medical, diagnostic, treatment, and public health rationales. Improvement in NBS algorithms to increase sensitivity and specificity will allow earlier diagnosis and treatment to potentially improve disability and mortality rates.

Identification and requirements of enhancers that direct gene expression during zebrafish fin regeneration.

To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

The value of preliminary clinical evaluation for decision making in injuries of the hand and wrist.

Introduction: Recent research suggests that up to 20% of minor trauma patients admitted to the emergency department (ED) will suffer from non-specific chronic conditions over the subsequent several months. Thus, the present study assessed the correlates of symptoms that persisted at 4 months after an ED visit and, in particular, evaluated the associations between these symptoms and self-reported stress levels at ED admission and discharge. Method: This study was a prospective observational investigation conducted in the ED of Bordeaux University Hospital that included patients admitted for minor trauma. All participants were contacted by phone 4 months after presentation at the ED to assess the occurrence of post-concussion-like symptoms (PCLS). Results: A total of 193 patients completed the follow-up assessment at 4 months; 5.2% of the participants suffered from post-traumatic stress disorder (PTSD) and 24.5% suffered from PCLS. A multivariate analysis revealed an association between PCLS and stress level at discharge from the ED (odds ratios [OR]: 2.85, 95% confidence interval [CI]: 1.10­7.40). Conclusions: The risk of PCLS at 4 months after an ED visit for a minor injury increased in association with the level of stress at discharge from the ED. These results may improve the quality of life for the millions of patients who experience a stressful injury event every year.

The impact of focused training on abnormality detection and provision of accurate preliminary clinical evaluation in newly qualified radiographers.

INTRODUCTION: Preliminary clinical evaluation (PCE) can be a useful initial assessment of traumatic abnormalities by frontline radiographers; new graduates are expected to have the skills and knowledge required to provide this initial interpretation. This study evaluates the abnormality detection performance and accuracy of PCE commenting in newly qualified radiographers. METHOD: Four newly qualified radiographers completed a fracture/dislocation detection task consisting of 58 cases, including providing a PCE for each suspicious area. Following this, an 8-week training program was completed to improve competence in recognizing abnormalities and providing an accurate PCE. Equally weighted jackknife alternative free-response receiver operating characteristic (wJAFROC) analysis was performed; a difference between pre- and post-training would be considered significant at a test alpha of less than 0.05. RESULTS: Fracture/dislocation detection was significantly better in the post-training evaluation for fixed observers and random cases (F (1,57) = 4.48, p = 0.0387). The reader averaged wJAFROC FOM and 95% CIs for pre- and post-training were 0.619 (0.516, 0.737) and 0.703 (0.622, 0.852). A paired t-test demonstrated a significant difference in PCE scores in favour of the post-training evaluation p = 0.0006. This small cohort demonstrated difficulty in recognising undisplaced fractures and buckle fractures. CONCLUSION: An 8-week training program had a positive impact on participants' ability to localise and accurately describe fractures. Implementation of abnormality detection training should be considered during preceptorship periods. Due to the small sample size, it is inappropriate to suggest these findings are representative of all graduate radiographers.

Live fate-mapping of joint-associated fibroblasts visualizes expansion of cell contributions during zebrafish fin regeneration.

The blastema is a mass of progenitor cells responsible for regeneration of amputated salamander limbs and fish fins. Previous studies have indicated that resident cell sources producing the blastema contribute lineage-restricted progeny to regenerating tissue. However, these studies have labeled general cell types rather than granular cell subpopulations, and they do not explain the developmental transitions that must occur for distal structures to arise from cells with proximal identities in the appendage stump. Here, we find that regulatory sequences of tph1b, which encodes an enzyme that synthesizes serotonin, mark a subpopulation of fibroblast-like cells restricted to the joints of uninjured adult zebrafish fins. Amputation stimulates serotonin production in regenerating fin fibroblasts, yet targeted tph1b mutations abrogating this response do not disrupt fin regeneration. In uninjured animals, tph1b-expressing cells contribute fibroblast progeny that remain restricted to joints throughout life. By contrast, upon amputation, tph1b+ joint cells give rise to fibroblasts that distribute across the entire lengths of regenerating fin rays. Our experiments visualize and quantify how incorporation into an appendage blastema broadens the progeny contributions of a cellular subpopulation that normally has proximodistal restrictions.

The impact of simulated motion blur on lesion detection performance in full-field digital mammography.

OBJECTIVE: Motion blur is a known phenomenon in full-field digital mammography, but the impact on lesion detection is unknown. This is the first study to investigate detection performance with varying magnitudes of simulated motion blur. METHODS: 7 observers (15 ± 5 years' reporting experience) evaluated 248 cases (62 containing malignant masses, 62 containing malignant microcalcifications and 124 normal cases) for 3 conditions: no blurring (0 mm) and 2 magnitudes of simulated blurring (0.7 and 1.5 mm). Abnormal cases were biopsy proven. Mathematical simulation was used to provide a pixel shift in order to simulate motion blur. A free-response observer study was conducted to compare lesion detection performance for the three conditions. The equally weighted jackknife alternative free-response receiver operating characteristic was used as the figure of merit. Test alpha was set at 0.05 to control probability of Type I error. RESULTS: The equally weighted jackknife alternative free-response receiver operating characteristic analysis found a statistically significant difference in lesion detection performance for both masses [F(2,22) = 6.01, p = 0.0084] and microcalcifications [F(2,49) = 23.14, p < 0.0001]. The figures of merit reduced as the magnitude of simulated blurring increased. Statistical differences were found between some of the pairs investigated for the detection of masses (0.0 vs 0.7 and 0.0 vs 1.5 mm) and all pairs for microcalcifications (0.0 vs 0.7, 0.0 vs 1.5 and 0.7 vs 1.5 mm). No difference was detected between 0.7 and 1.5 mm for masses. CONCLUSION: The mathematical simulation of motion blur caused a statistically significant reduction in lesion detection performance. These false-negative decisions could have implications for clinical practice. Advances in knowledge: This research demonstrates for the first time that motion blur has a negative and statistically significant impact on lesion detection performance in digital mammography.

The inclusion of biodiversity in environmental impact assessment: Policy-related progress limited by gaps and semantic confusion.

Natural habitat loss and fragmentation, as a result of development projects, are major causes of biodiversity erosion. Environmental impact assessment (EIA) is the most commonly used site-specific planning tool that takes into account the effects of development projects on biodiversity by integrating potential impacts into the mitigation hierarchy of avoidance, reduction, and offset measures. However, the extent to which EIA fully address the identification of impacts and conservation stakes associated with biodiversity loss has been criticized in recent work. In this paper we examine the extent to which biodiversity criteria have been integrated into 42 EIA from 2006 to 2016 for small development projects in the Montpellier Metropolitan territory in southern France. This study system allowed us to question how EIA integrates biodiversity impacts on a scale relevant to land-use planning. We examine how biodiversity inclusion has changed over time in relation to new policy for EIA and how the mitigation hierarchy is implemented in practice and in comparison with national guidelines. We demonstrate that the inclusion of biodiversity features into EIA has increased significantly in relation to policy change. Several weaknesses nevertheless persist, including the continued absence of substitution solution assessment, a correct analysis of cumulative impacts, the evaluation of impacts on common species, the inclusion of an ecological network scale, and the lack of monitoring and evaluation measures. We also show that measures for mitigation hierarchy are primarily associated with the reduction of impacts rather than their avoidance, and avoidance and offset measures are often misleadingly proposed in EIA. There is in fact marked semantic confusion between avoidance, reduction and offset measures that may impair stakeholders' understanding. All in all, reconsideration of stakeholders routine practices associated with a more strategic approach towards impact anticipation and avoidance at a land-use planning scale is now necessary for the mitigation hierarchy to become a clear and practical hierarchy for "no net loss" objectives based on conservation priorities.