Rattus rattus and Rattus norvegicus. Based on metadata obtained considering 35 selected studies related to observed heterozygosity, measured by nuclear molecular markers , socioeconomic and mobility anthropogenic factors were used as predictors of genetic diversity of R. rattus and R. norvegicus, using the Gini index, principal component analysis and Random Forest Regression as analysis methodology. Population density was on average the best predictor of genetic diversity in the Rattus species analyzed, indicating that the species respond in a particular way to the characteristics present in urban environments because of a combination of life history characteristics and human-mediated migration and colonization processes. To create better management and control strategies for these rodents and their associated diseases, it is necessary to fill the existing information gap in urban landscape genetics studies with more metadata repositories, with emphasis on tropical and subtropical regions of the world.On a planet experiencing constant human population growth, it is necessary to explore the anthropogenic effects on the genetic diversity of species, and specifically invasive species. Using an analysis that integrates comparative phylogeography, urban landscape genetics, macrogenetics and a systematic review, we explore the worldwide genetic diversity of the human commensal and anthropogenic species  Today, some 56% of the world’s population—4.4 billion inhabitants—live in cities. This trend is expected to continue, with the urban population more than doubling its current size by 2050, at which point nearly 7 of 10 people will live in cities [The ecological and evolutionary dynamics that occur in the species that inhabit these modified environments can be explored from a genetic perspective to quantify the degree of impact that urbanization and anthropic effects have on the behavior of genetic diversity and population structure . In the Rattus rattus), native to the India subcontinent, and the brown rat (Rattus norvegicus), native to Siberia and China, are two rodent species that have adapted to urban life, spreading to and invading many cities around the world [The black rat  guidelines . We collR. rattus and R. norvegicus, we conducted a systematic review to evaluate at least one metric of genetic diversity of these species according to a series of socio-economic and human mobility predictors.Based on the absence of a worldwide exploration of the genetic diversity status of two invasive species with cosmopolitan distribution, such as R. rattus, R. norvegicus or both species as study species; (2) articles focused on phylogeographic studies; (3) articles with Studies were selected based on the following inclusion criteria: (1) articles considering The exclusion criteria were as follows: (1) posters, conference papers, abstracts and review papers; and (2) pathogenicity studies associated with viruses and bacteria.Rattus AND genetic* AND geographic”; “Rattus AND genetic* AND phylogeograph*”; “Rattus AND phylogenetic”. In the first step, we eliminated duplicate items. In the second step, we reviewed the titles and abstracts based on the inclusion and exclusion criteria. In the third step, we selected studies carried out on R. rattus and R. norvegicus in urban or semi-urban areas . In the fourth step, all selected articles were reviewed for their respective citations using the statistics generated by Google Scholar. As a fifth step and as a final inclusion criterion, we selected articles that had We searched the Scopus database for information using the following Boolean combinations: “Ho). Furthermore, given that we found deviations from the Hardy–Weinberg Equilibrium (HWE) in several selected studies [Ho was precisely the most appropriate metric to use in our study, considering that the expected heterozygosity (He) assumes HWE. In addition, only nuclear markers were considered for the analyses to reduce the bias of Ho values due to mitochondrial markers’ nature and avoid standardization by marker type as well.From reading the full text of all selected articles that met the inclusion criteria, we identified that the only metric of genetic diversity that met the requirement of being present in all studies was observed heterozygosity  there was much heterogeneity in the use of the diversity metrics used; (2) several studies presented global values of genetic diversity, omitting locality-specific values; (3) several studies did not include geographic coordinates in the data analyzed; (4) several studies were focused on the analysis of population genetic structure, presenting genetic variability values at the interpopulation level but not at the intrapopulation level; (5) several studies used data from other studies, which, when reviewed, could not be used due to lack of information ; and (6) several studies did not use data from other studies, which, when reviewed, could not be used due to lack of information . Therefore, to carry out meta-analyses and macrogenetic analyses from a systematic review, we decided to homogenize the data collection in the studies, increase the number of variables to be considered and provide free access to the information through repositories, tables within the text of the articles and the presence of ample and sufficient Ho) was found across most of the studies recovered by PRISMA methodology, we chose to use anthropic factors under the hypothesis of anthropic factors affecting the genetic diversity of both Rattus analyzed. Thus, this study tried to take into consideration only raster data with the available data. This means that no pixel was imputed to reduce the description and predictability to areas where complete data were found for all the predictors described below. This contributed to reducing the spatial bias and improving the reproducibility of the results, but also allowed a fair comparison by avoiding creating synthetic data or including noisy information. Nevertheless, the spatial distribution of analysis for species was not considered here, since there is a lack of genetic diversity studies of both species worldwide that did not allow the use of spatial sampling to avoid spatial autocorrelation.Since only observed heterozygosity  for the year 2015 [Ho) from a holistic perspective, according to the following criteria: (1) GDP was chosen to observe if economic growth affects the Ho values of the two species; (2) human population density was chosen to observe if the human population density is related to higher Ho values; (3) the relative levels of multidimensional deprivation and poverty affecting rats genetic diversity were chosen to observe how the closeness to main cities or port affects the Ho value of both rat species. All datasets were resampled to 5 km (2.5 arcmins) using the nearest-neighbor method to reduce the computation time and standardize data to the same resolution for the description and prediction of observed heterozygosity via random forest regression.To identify the anthropic factors (economic and human population indicators) affecting the genetic diversity of ear 2015 ; Populatear 2015 ; Global ear 2015 ; travel ear 2015 . These pR. norvegicus and R. rattus localities sampled according to observed heterozygosity and the five predictors described above. Predictor values were extracted for each of the localities extracted from the systematic search using the Raster v3.6-20 R package. Localities were omitted from the analysis if they did not possess values for all the predictors for each species . A principal component analysis (PCA) was performed using FactoMineR v.7 to inspect visual similarities between Ho to identify the influence of mainland and island effect over the presence of both rat species. No regression predictions were performed for island and mainland records separately, since the number of island and mainland records were low for both species: Rattus norvergicus , and R. rattus . PCAs were performed for each species to detect predictors of multicollinearity and perform a hierarchical principal component clustering (HCPC) with the FactoMineR v.7 R package to detect groups of localities per species. On the other hand, random forest regressions were performed to detect the impact of the predictors on the observed heterozygosity. A Jackknife validation strategy (without sample replacement), given the small number of samples per species (<100), was used. Likewise, as a criterion of importance to predict the effect on observed heterozygosity, we used the Gini index. In addition to the Jackknife validation strategy, Wilcoxon tests were performed for A gridded search to find the random forest number of trees from a range from 10 to 10,000 trees was performed. The optimal random forest regression configuration per species was defined as the regression with the minimum value of out-of-bagging value (OOB) root mean square error (RMSE), and it was used for all the subsequent analyses. These analyses were performed in the ranger R package v0.14.1.Rattus species using the random forest regression and the spatial predictors. To obtain an evaluation of possible extrapolation and uncertainty for the predictors used to project the predicted observed heterozygosity spatially, a Multivariate Environmental Similarity Surface (MESS) was calculated with the spatial predictors GDP, Population Density, GRDI, travel time in minutes to the nearest urban area between 5000 and 110 million people and travel time in minutes to any port, extracted from the localities obtained to train the random forest regressions for each species. This approach was preferred, given that the MESS index highlights areas where environmental conditions are outside conditions found in the presence records [Ho values, only observed heterozygosity grids greater than the median of the MESS positive values were considered valid predicted grids to reduce the bias of extreme predicted values in maps. MESS maps are available in A worldwide prediction of observed heterozygosity was obtained for each  records . NegativR. rattus and 18 for R. norvegicus) were systematically reviewed  than for R. norvegicus (0.453 ± 0.181). For R. rattus, the highest values were recorded in Southern India and the lowest in Bowditches, United States , travel time to cities (min) and travel time to ports (min), showed light to medium correlations for R. rattus, but a slight effect could be observed with the travel time to ports for the last species. Thus, none of the predictors for both species had multicollinearity with the Ho values  of the out-of-bag evaluation, RMSE = 0.0229 and RMSE = 0.005 for R. norvergicus and R. rattus, respectively. A similar behavior was observed for the RMSE of observed vs. predicted Ho values, RMSE = 0.095 and RMSE = 0.0422 for R. norvergicus and R. rattus, respectively. This indicates that both regressions accurately predicted the training and validation dataset values used in the Jackknife strategy , but this does not affect our original working hypothesis. The Gini index values using random forest regression methodology  and prinR. rattus and the global Gridded Relative Deprivation Index (GRDI) and GDP per capita for R. norvegicus.Other predictors identified by the analyses in the characterization of genetic diversity were travel time to ports and Gross Domestic Product (GDP) per capita for R. rattus and R. norvegicus  species dispersal; (2) heterogeneity and scale of urban environments; (3) migration and colonization processes of invasive species; (4) species behavior defined by life history traits; and (5) availability of metadata-type information.Rattus indicate that dispersal is low or very local, at the scale of cities and even neighborhoods within the same city, indicating the effect that the urban environment may have on the distribution of genetic diversity [Ho; gridded poverty/deprivation value vs. Ho; travel time to cities/ports vs. Ho), we were able to identify some form of relationship  and the social behavioral characteristics per se of the species, such as philopatry and their level of territoriality and aggressiveness [Likewise, the genetic patterns of siveness ,29,30,31R. norvegicus (Gini index = 1) and the second for R. rattus (Gini index = 0.74). In this regard, there is no study that has systematically addressed this predictor in the distribution of genetic variability in invasive rodents. The only study that tangentially approached it was [R. rattus data points; and (3) most of the points correspond to the same localities (same georeferenced points).High population density can also create barriers to gene flow and dispersal, which can reduce the genetic diversity of invasive species populations. For example, urban areas can be surrounded by barriers such as highways, rivers or mountains, which can restrict the movement of invasive species and lead to genetic isolation. In our study, population density was the main predictor identified for d it was , which ed it was  should bR. rattus and R. norvegicus are arguably the most successful invasive species on the planet, and human migrations have had a major influence on the spread and dispersal of invasive species populations within and between urban areas through processes such as genetic drift and gene flow [R. norvegicus in New York City at a scale of less than 600 m between colonies, Ref. [R. norvegicus individuals from eastern North America, South America, Africa, Australasia and western Europe. ene flow ,34. Humaene flow ,35,36,37ene flow , using Ses, Ref.  found coR. rattus. The history of Rattus colonization in the world is the history of human migrations; consequently, the origin of commensalism in these rodents inevitably reflects the history of human settlement and associated anthropogenic factors. Although further studies are needed, the more recent dispersal of R. norvegicus from its center of origin in Siberia to Europe in the 16th century [R. rattus, which is linked to the beginning of Indian civilization (approximately 5000 years BC) and subsequent dispersal dynamics, mainly by maritime routes [R. rattus than in R. norvegicus was found in two studies with a comparative phylogeography approach to that reported in our study. The first corresponds to a regional scale across the United States [Moreover, due to the genetic variability detected between different inheritance types of molecular markers in the same study, such as ,38,39 in century  may be oe routes . Other ed States , and thed States .R. rattus and R. norvegicus, human commensal and anthrodependent species, it is vital to explore the spatiotemporal dynamics of genetic diversity in current and emerging urban areas around the world  to design management and control strategies for these pests and their associated diseases. For instance, it would be interesting to observe if the changes in Ho values are related to changes in the human population to provide tracking systems of which strategies can influence and lead to the mitigation of the dispersal of these invasive species worldwide.While high human population density can create conditions that favor the establishment and spread of invasive species, it can also create barriers to gene flow and dispersal and increase selective pressures that reduce genetic diversity. Understanding these factors is crucial to managing and mitigating the impact of invasive species in urban areas. In the case of Although we identified several studies in regions other than developed countries and with tropical or subtropical distribution, e.g., Nigeria , Benin , SenegalRattus, both those covered in this study and others not considered. Following the study by Fusco et al. 2021 mentioned in the introduction, we conducted a search between 2021 and 2023 with the keywords “urban landscape genetics”, considering its appearance anywhere in the body of a paper, finding only 20 studies, of which none were conducted in rodents. This shows that the information gap persists today and needs to be filled.Our analyses reveal critical data and theory gaps and call for increased efforts to monitor global genetic diversity. To increase the data matrices of invasive species in the world, and thus strengthen management strategies based on empirical evidence, it is necessary to expand the focus on urban landscape genetics in species of the genus Human activities can also affect intraspecific genetic diversity at the natural level , henTo compare the genetic diversity of invasive species at different spatial and temporal scales, it is necessary to strengthen the metadata related to nuclear molecular markers  to desiR. rattus and R. norvegicus are considered among the most important invasive species in the world for reasons such as their cosmopolitan distribution, high reproductive capacity, adaptive capacity, negative impact on ecosystems, disease vectors and economic impacts. In this scenario, control strategies are a challenge. Macrogenetic-scale studies such as the one presented here can provide the following recommendations to mitigate the negative effects of these species on the invaded ecosystems: (1) By analyzing the genetic composition of rat populations in different areas using metadata, scientists can determine the origin of introductions, colonization patterns and the degree of genetic mixing between populations more precisely. This information could help understand how these species spread and establish new populations.(2) Macrogenetic studies can help identify the routes and pathways by which rats are introduced into new regions. Through analysis of the genetic signatures of rat populations, the origins and possible sources of invasion can be traced. This knowledge is crucial for implementing targeted prevention measures and biosecurity protocols at points of entry to reduce the risk of new introductions.(3) Examination of large-scale genetic variation can identify patterns in genetic traits or markers associated with traits such as reproductive capacity, survival, behavior and resistance to control measures. This information can guide the development of more effective control strategies that target specific traits or exploit vulnerabilities in invasive populations.(4) Different rat populations throughout the world may vary in their behavior, their response to particular control methods or their susceptibility to specific toxins. By accounting for genetic diversity on a global scale, control strategies can be tailored to the unique characteristics of specific populations, increasing the chances of success and minimizing unintended consequences.(5) Large-scale genetic diversity studies can provide a more robust basis for monitoring the effectiveness of control strategies over time. By comparing the genetic composition of rat populations before and after control efforts, it is feasible to assess the impact of interventions, detect any changes in population structure or genetic diversity and make necessary adjustments to control strategies.


Novel alleles can now be directly written into related genomes by sequence identity, thus removing the resource-intensive process of trait introduction by genetic introgression and eliminate the loss of genetic diversity that typically results from introgression bottlenecks.Genome editing technology encompasses a revolutionary set of molecular tools that holds the potential to improve human health, unlocking improvements in medicine and agriculture. Here, we discuss a specific case study of the use of this technology in agriculture: direct germplasm editing of the same loci in multiple plant lines, varieties, or germplasms to produce the same or similar alleles and traits to accelerate crop improvement. The application of direct editing can eliminate the complex and costly process of trait introduction by genetic introgression. The use of direct editing would also eliminate the barriers to accessing genetic diversity that typically arise from introgression bottlenecks associated with backcrossing.in situ exists, and these changes can also be recapitulated precisely in distinct host plants , the two heterotic groups  are highly divergent and are on separate genomic trajectories (Vitus vinifera (grape) and Actinidia deliciosa (kiwifruit) have lengthy generation times so that breeding and field testing can take decades , which eliminates productivity from established orchards (Citrus sinensis), and then introgress and backcross the edit into the other commercial varieties of citrus such as Citrus limon (lemon), Citrus limettioides (sweet lime), Citrus × paradisi (grapefruit), Citrus margarita (kumquat), and others , Musa acuminata (banana), Coffea arabica or robusta (coffee) and other fruits, as well as vegetable and grain crops (A trait-based approach is one possible way of addressing the regulatory status of edits generated through direct editing. This approach makes the sequence or trait outcome of a desirable allele in any crop variety the unit of commerce and regulation ; Fig. 1.One key consideration for trait-based regulation of genome edits is a rigorous product development pipeline. As different germplasm backgrounds are unique both genotypically and phenotypically, stringent gRNA design through computational approaches .In summary, the application of direct editing into diverse germplasm backgrounds can fundamentally change the path for crop development. Genetic improvement no longer needs to be a matter of patience and chance. Discoveries of novel alleles are no longer once-in-a-lifetime events. Genomes are no longer mysterious repositories of desirable traits whose identity can only be ensured by transmission from parent to offspring through traditional breeding methods including introgression. Novel alleles can now be directly written into related genomes by sequence identity. From a technical standpoint, direct editing can eliminate the resource-intensive process of trait introduction by genetic introgression and eliminate the loss of genetic diversity that typically results from introgression bottlenecks. From a regulatory standpoint, a shift towards trait-based regulatory status determination from the current event-based regulation scheme would be required to enable the acceleration of product development that direct editing can offer. Making these changes will also require a shift towards acceptance of the new genetic technologies that have arisen in the past few decades and, along with these changes, some revision of our concepts and language around genetic traits.


Mean nebulized dose was 95% ± 6%. For dry conditions, the mean respiratory tract deposited fractions reached 18% (± 4%) next to ventilator and 53% (± 4%) for proximal position. For humidified conditions, it reached 25% (± 3%) prior humidifier, 57% (± 8%) before Y-piece and 43% (± 11%) after this latter. Optimal nebulizer position is proximal before the Y-piece adapter showing a more than two-fold higher lung dose than positions next to the ventilator. Dry conditions are more likely to cause peripheral deposition of aerosols in the lungs. But gas humidification appears hard to interrupt efficiently and safely in clinical use. Considering the impact of optimized positioning, this study argues to maintain humidification.Successful aerosol therapy in mechanically ventilated patients depends on multiple factors. Among these, position of nebulizer in ventilator circuit and humidification of inhaled gases can strongly influence the amount of drug deposited in airways. Indeed, the main objective was to preclinically evaluate impact of gas humidification and nebulizer position during invasive mechanical ventilation on whole lung and regional aerosol deposition and losses. Ex vivo porcine respiratory tracts were ventilated in controlled volumetric mode. Two conditions of relative humidity and temperature of inhaled gases were investigated. For each condition, four different positions of vibrating mesh nebulizer were studied: (i) next to the ventilator, (ii) right before humidifier, (iii) 15 cm to the Y-piece adapter and (iv) right after the Y-piece. Aerosol size distribution were calculated using cascade impactor. Nebulized dose, lung regional deposition and losses were assessed by scintigraphy using  Despite this tremendous success, some of clinical practices in intensive care units (ICU) are still led by empirical choices instead of evidence-based decisions. Among these, nebulization practices in ICU rest on main data from old in vitro studies6. Influence of these parameters, in term of aerosol delivery to the patient, are mainly assessed by in vitro studies widely performed by measure of the aerosol impaction on a filter at the end of the ventilator circuit. This point could lack of relevance because it differs from administration in the desired location in the respiratory tract (RT). Moreover, expiratory loss, cannot be assessed due to the use of filters to collect aerosol8. Besides, clinical studies are mainly focused on patients’ outcomes, such as the therapeutic efficacy of treatment13. Indeed, literature is scarce concerning the impact of humidification and nebulizer position on the regional aerosol deposition in patients under mechanical ventilation15. Moreover, these rare published data lack of systematic assessment and should be cautiously extrapolated due to the wide range of clinical practices, for both ventilator tuning, nebulization devices used and aerosolized drug delivery17.Different parameters affect lung deposition of therapeutic aerosol during invasive mechanical ventilation. First, it can be related to the patient , but also to the couple drug-nebulizer , and lastly to the ventilator and its circuit 1. However, technical constraints and ethical restrictions limit clinical research on ICU patients. Moreover, existing in vivo preclinical models sometimes lack of relevance (e.g. ventilation physiology of rodents is very different from humans22) or are difficult to compare with in vivo human data.Therefore, a data gap needs to be bridged regarding the specific knowledge devoted to regional aerosol deposition in mechanically ventilated patients24 and mechanical ventilation conditions25. Considering reproducibility, ethical, anatomical and physiological interests of this preclinical model, useful data can be produced to implement aerosol therapy practices in ICUs. Consequently, the present study aims to assess the impact of gas humidification and of the nebulizer position in the ventilator circuit on the regional aerosol deposition as well as in the different elements composing the ventilation set up with this ex vivo model.To overcome these limitations, we previously developed and validated an ex vivo preclinical model of porcine RT for aerosol deposition studies in spontaneous25 and allowing a scintigraphic regional aerosol deposition study of each part of the ventilation set up. Reproducibility has been checked and consequently each experimental condition was tested at least in 3 replicates with different porcine RT. This study is composed of two categories of experiments: part D corresponds to dry and cold conditions i.e. no humidification nor heating of gases in the circuit. Part H corresponds to humidified and heated conditions. This latter was achieved thanks to a heated humidifier (HH). Figure This aerosol deposition study is based on an ex vivo RT model previously validated for mechanical ventilation applications25. It was composed of a porcine RT collected from slaughterhouses as waste of food industry. Hence, they succeed all the sanitary controls in accordance with French regulation with strict veterinarian controls of animal welfare and any animal was specifically sacrificed for this work, in accordance with the 3R guidelines26. For sanitary reasons, each respiratory tract was used within 24 h. Otherwise, they were frozen and thereafter thawed at ambient temperature few hours before the experiment. Relevant sutures were realized on each significant cut and their number was recorded. A systematic bronchoscopy allowed to ensure the absence of major obstruction in reachable bronchi. Respiratory tracts were placed in a sealed enclosure. The latter allows both positive and negative pressure ventilations. A 7.5 mm inner diameter ETT  with a 34 cm length was connected to the trachea with a standardized trachea length of 15 cm to avoid selective intubation. The model was set in a 30° proclive position, as close as possible to the patient position for nebulization in ICU and mechanically ventilated with positive airway pressure.Aerosol delivery during invasive mechanical ventilation using a preclinical ex vivo respiratory model was previously fully validated1 and is widely used in ICUs27. The ventilation parameters were identical for each condition and adapted to be comparable to Ehrmann et al.28 with a tidal volume of 540 mL, respiratory rate of 20/min, inspiratory flow of 35 L/min with a constant flow pattern, inspiratory to expiratory ratio of 1/2, positive end expiratory pressure of 9 cmH2O and inspired oxygen fraction of 21%. These parameters were chosen in accordance with data from a large cohort study in ICUs over the world27. For this work, no spontaneous breathing was used. The ETT was set with an angulation of 90°, determined thanks to data from CT scans of intubated patients.The ex vivo model was used under controlled volumetric ventilation without triggering, thanks to a medical ventilator [ICU Evita 4 ventilator ]. Systematic security tests were performed before experiments. An important data is the absence of bias flow for this ventilator according to manufacturer. This ventilation mode was selected because it is currently recommended to optimize RT deposition fraction of nebulization28.The ventilator was connected to the endotracheal tube with a medical 2 limbs circuit  with or without heated humidifier  in the inspiratory limb according to each experimental condition studied. The first part of this study corresponds to dry and cold conditions (conditions D) i.e. without HH. These were tested each time with a different circuit, without any humidifier in line. The second part corresponds to humidified and heated conditions (conditions H) thanks to the HH placed on the circuit as recommended by manufacturer. The humidifier was turned on at least 15 min before nebulization. An expiratory filter (DAR™ Heat and Moisture Exchangers (HME) Filter, ref. 354/5876, Covidien™, Dublin, Ireland) was systematically placed on the expiratory limb, close to the expiratory valve to avoid damages and obstructions, as recommended for nebulization®, Aerogen Limited, Galway, Ireland) because among all nebulization devices; vibrating mesh are those leading to the best percentage of nominal dose nebulized30. The minimal duration of nebulization was standardized to 15 min based on preliminary test run, in good accordance with previous study from Dugernier et al.15 showing an average 9 min (± 3 min) nebulization. The ending of nebulization was checked systematically before acquisitions. According to each experimental condition, the nebulizer was placed at four different positions on the respiratory circuit: next to the ventilator, right before HH, 15 cm from Y-piece adapter and right after the latter  for acquisition of planar scintigraphic images. The latter (matrix 256 × 256) were recorded with a variable angle dual detector Single Photon Emission Computed Tomography with a Computed Tomography scan  equipped with a low-energy, high-resolution collimator (FWHM 8.3 mm at 10 cm); tested weekly for uniformity . First, the initial radioactive dose filled in the nebulizer was quantified . Nominal dose stood for the total amount of radioactivity introduced into the nebulizer, allowing to determine the nebulized fraction of nominal dose, i.e. the output of the nebulizer. Then, the nebulization procedure was performed. Finally, 180-s anterior/posterior images of each part of the experimental setup were acquired for each element: empty nebulizer, inspiratory limb, heated humidifier, endotracheal tube, Y-piece associated to expiratory limb and RT. Regarding the latter element, two different regions of interest (ROIs) were identified on scintigraphies within each lung to divide lungs into central and peripheral zones. A nuclear medicine physician used a semi-automatic delineation method with a variable threshold based on adaptative signal to background ratio and manual outlines the central region (trachea ad stem bronchi). This allowed to calculate the relative uptake of central and peripheral regions for both left and right lung. ROIs were delimited on the images with a correction considering the background radiation, the physical decay of radioactivity and tissue attenuation correction factors. Consequently, as stated by Newman et al.33, some limitations of data quantification in planar imaging are inherent to its two-dimensional nature. It is accepted that errors of no more than 10% in the quantification of whole-lung deposition are tolerated and that mass balance close to 100% validate the method used for attenuation and scatter correction. Hence, the results were expressed as a percentage of activity in relation to the nebulized dose, adjusted to 100%.The nebulizer was filled with 3 mL of 34). The ACI holds 8 stages from 0.4 to > 9 µm and is operated at 28.3 L/min with a vacuum pump . 3 mL of Sodium Fluoride solution at 2.5% (m/V) were introduced into the tank of the nebulizer. The nebulization process continues until the volume left in the nebulizer is over, which basically leads the nebulizer to cease functioning continuously and begins to sputter. For D and H experimental conditions, the ACI was placed after the Y piece, right before ETT. After nebulization, deposited fractions on each stage of the ACI were collected and re-suspended in 5 mL of deionized water, then 250 µL of TISAB IV solution  were added. Concentrations of fluoride ions were determined with a ionometer . Calibration of the apparatus was performed before each experiment. Mathematical determination of MMAD (Mass Median Aerodynamic Diameter) was performed with Excel software .To determine the aerodynamic size distribution of the aerosol, the European Pharmacopoeia indicates to use the ACI .Results are expressed as mean percentage (%) ± standard deviation (SD) of the nebulized dose of radioactivity. Statistical analyses were performed using GraphPad PrismComparison of respiratory tracts used, in terms of mass and cuts, for each position was performed with a Kruskall–Wallis’ test and post-hoc Dunn’s multiple comparison test.To compare the deposited fractions on each part of the model, a 2-way analysis of variance (ANOVA) was carried out with a post-hoc Tukey’s multiple comparison test. Comparison of central and peripheral deposited fractions were carried out with a 2-way ANOVA with post-hoc Tukey’s multiple comparison tests. Comparison of MMAD were performed with 1-way ANOVA with post-hoc Tukey’s multiple comparison test.Ethical approval is not applicable according to French regulation as RT were collected from slaughterhouses as waste of food industry. Hence, any animal was specifically sacrificed for this work and they succeed all the sanitary controls in accordance with French regulation with strict veterinarian controls of animal welfare.The model was set up 18 times with different RT achieving successful ventilation and nebulization. 6 experiments were realized for Position D-1, and 3 experiments for conditions . Nebulization ended successfully before acquisitions for all experiments..The nebulized fraction of nominal dose was 94 ± 7% for position D-1, 98 ± 1% for position D-2, 91 ± 12% for position H-1, 97 ± 3% for position H-2, 96 ± 4% for position H-3. Comparisons showed no significant difference in nebulizer output  according to the condition. Nebulized dose for experimental condition are displayed in Table 1The scintigraphic images performed allowed to calculate the deposited fractions for each component of the setting, leading to comparative results as shown in Fig. For heated and humidified conditions, i.e. positions H-1, H-2 and H-3, deposited fractions were respectively 46% (± 6%), 23% (± 16%) and 5% (± 1%) for inspiratory limb, 2% (± 0%), 2% (± 0%) and 3% (± 1%) for endotracheal tube. For the RT they reached respectively 25% (± 3%), 57% (± 8%) and 43% (± 11%). Finally, for expiratory limb values were respectively 15% (± 1%), 18% (± 8%) and 49% (± 10%).For H-1, H-2 and H-3 experiments in the heated and humidified condition, deposited fractions were respectively 46 ± 6%, 23 ± 16% and 5 ± 1% for inspiratory limb, 2 ± 0%, 2 ± 0% and 3 ± 1% for endotracheal tube. For the RT they reached respectively 25 ± 3%, 57 ± 8% and 43 ± 11%. Finally, for expiratory limb values were respectively 15 ± 1%, 18 ± 8% and 49 ± 10%.As presented in Table Results of regional aerosol distribution are displayed in Table . The MMAD measured for the nebulizer used in this work was 1.94 ± 0.34 µm. For dry conditions D-1 and D-2 the MMAD reached respectively 1.69 ± 0.00 µm and 1.92 ± 0.09 µm. For humidified conditions H-1, H-2 and H-3 the calculated MMAD were significantly higher with respectively 4.12 ± 0.04 µm, 4.79 ± 0.34 µm and 4.94 ± 0.27 µm.MMAD for each experimental condition are displayed in Table 3The comparison of MMAD for the nebulizer alone and both dry conditions (D-1 and D-2) showed no significant statistical differences. In contrast, each humidified condition had a MMAD significantly greater from nebulizer alone and dry conditions. Moreover, the condition H-1 showed a MMAD significantly lower from H-2 and H-3, although no significant statistical difference was observed between conditions H-2 and H-3. The Supplementary Table 2. Moreover, this study was performed with a new generation vibrating mesh nebulizer showing a mean aerosol output higher than 90%. This point agrees with previous data35. This is of high relevance because epidemiological studies show a majority use in ICU of less efficient jet nebulizers36. For example Ehrmann et al. observed 10% of vibrating mesh nebulizer and 56% of jet nebulizers use in an international study2. Despite economic considerations, their output is largely lower, ranging from 30 to 55% according to different studies37. Consequently, considering wide differences in aerosol output depending on nebulizer technologies and the great impact of the aerosol device position on the ventilator circuit and gas humidification, the potential cumulative differences could lead to clinical treatment failure independently of drug efficiency. Finally, the type of nebulizer has been suspected to impact ICU patients outcome as a trends to shorter length of stay for asthmatic patients10.To the best of our knowledge, this is the first study comparing the impact of nebulizer position and gas humidification on the regional aerosol deposition using an innovative and reproducible preclinical respiratory model. This work showed with comparative conditions that these two experimental parameters can greatly change the mean deposited fractions in the RT from 18 to 55%, leading to potential wide differences in drug delivery to mechanically ventilated patients. This finding raises a major concern about practices in ICU as some international epidemiological studies highlighted disparate clinical practices38. Indeed, despite a manufacturer recommendation39, position upstream the heated humidifier (HH), (conditions D-1 and H-1) led in this study, to a mean RT deposited fraction ranging from 18 to 25% while a position right before the Y-piece adapter (conditions D-2 and H-2) produced a mean RT deposited fraction ranging from 53 to 57%. Obviously, this difference is largely significant and can lead to wide differences in the treatment efficacy. However, Moraine et al. failed to find a clinical difference while measuring ipratropium in urine after nebulization in mechanically ventilated patients40. According to our results, evaluating separately the impact of gas humidification and of nebulizer position on a standardized preclinical respiratory model, the lake of significant difference for Moraine et al. could be due to the interaction of both factors. Indeed, they compared whether nebulizer prior the HH or nebulizer right before the Y-piece adapter in dry conditions, leading to potential interaction of these factors. Even if the difference is not significant, the nebulizer position beyond the Y-piece adapter tend to a higher loss in expiratory limb and lower RT deposited fraction than the position 15 cm before Y-piece adapter (p = 0.0508). These data are in accordance with the in vitro study from Ari et al.7 which compared the three same positions with and without gas humidification on an in vitro model. Nevertheless, due to a design with deposition on a filter instead of a RT, the expiratory loss and regional deposition could not be evaluated on their model. The expiratory loss is described as the major drawback for positions closed to expiratory limb38 this is consequently an important interrogation. On the other hand, position 2 is characterized by the 15 cm before the Y-piece, serving as a reservoir for aerosol continuously generated during expiration. This position is often recommended for this effect46. Finally, this study finds an optimal position at 15 cm before the Y-piece adapter for the vibrating mesh nebulizer independently from humidification, and this result is confirmed even considering the expiratory loss. As this is different from manufacturer recommendations39 for this specific nebulizer, the intensivists have to be aware of this point.The location of the nebulizer in ventilator circuit is a major determinant for the drug dose reaching the RT47. This could be due to a difference between nebulizer types, as vibrating mesh nebulizers are relatively new technologies. Moreover, Ari et al., using the same nebulizer than us, found a significant decrease with humidification for the positions before the Y-piece adapter but no difference after this latter7. More recently, Ashraf et al., comparing conditions very similar to D-1 and H-1 in our study, i.e. the same vibrating mesh nebulizer close to the ventilator with or without humidification, found very similar results with inhalable doses around 20% equally35. Nevertheless, a difference arose in our results for RT distribution when the position upstream HH and dry condition interacts. Indeed, these parameters (condition D-1) allowed a more peripheral deposition compared to each other condition, as previously described25. However, this finding must be first balanced with an absolute RT deposited fraction lower than other conditions and then with the risks for patients when interrupting the humidification for nebulization. Indeed, there are two principal ways of humidification for patients under mechanical ventilation: heat and moisture exchangers needing to be removed for the duration of nebulization to allow the passing of aerosol48 and HH. The cessation of HH just before nebulization showed an important residual humidity in limbs, being consequently ineffective to obtain “dry conditions”29 and the removal of humidification was reported as dangerous for patients if prolonged50. The humidification question has consequently to be seen as security factor for patients mechanically ventilated. Our finding is in accordance with a clinical study on asthmatic patients showing no significant difference for clinical endpoints according to humidification or not10. This point raises questions about evidence to interrupt humidification during nebulization with this vibrating mesh nebulizer.No difference was shown for humidification in terms of absolute RT deposited dose. This point differs from previous data on the humidification impact on aerosol delivery, as an important decrease, around 40%, is widely described35. Indeed, they found no significant difference in MMAD between dry and humid conditions with the same type of vibrating mesh nebulizer (MMAD ranging from 1.90 ± 0.14 µm for humid condition to 1.57 ± 0.05 µm). These discrepancies, while puzzling at first, could be explained by the sampling choices made that differ from our own sampling method. Indeed, as Ashraf et al. chose to sample at the distal extremity of the endotracheal tube, we decided to sample the aerosol at the extremity of the Y-piece. However, this is not sufficient to explain these discrepancies. Another point that differs between the studies is the vacuum parameters applied to sample the aerosol. To determine the size distribution, Ashraf et al. decided to apply physiological-like sampling of the aerosol with a vacuum parameter similar to standard breathing pattern found in humans. In this study, we decided to follow our bench protocol for aerosol size distribution according to European Pharmacopeia monography, with flows less based on physiology than in optimized conditions for physical bench comparisons. Therefore, these discrepancies are explained by the fact that we did not measure the same aerosol distribution. On one hand, Ashraf et al. presented granulometry data more focused on the granulometry of droplets that would efficiently reach out the distal extremities of the endotracheal tube. This method is likely to exclude the droplets which would be sedimented during the expiratory phase of the breathing-like sampling pattern, which would decrease aerosol size distribution as the biggest droplets would be excluded from the analysis. On the other hand, the data presented in this study are more focused on assessment of aerosol size distribution on bench with standardized parameters allowing the robust comparison of different nebulizers without exclusion of the biggest droplets composing the aerosol. One could argue on the advantages of one method over the other. Such assertion should be made with caution as the two sampling methods differ on the focus of the analysis. Ashraf et al. method is more patient-based, as measuring fraction reaching the respiratory tract while the method presented in the study is more device-based, not considering deposition in the ETT.Interestingly, the findings in this study differ in terms of aerosol size distribution from Ashraf et al. data14 where with an ultrasound nebulizer (MMAD 4.0 µm) and gas humidification, the central fraction of RT deposited fraction had a median of 62.8%. Moreover, considering that a vast majority of data for aerosol therapy provide from in vitro studies, this work adds important new information. Indeed, by the adjunction of a RT with an anatomy very close to human’s, data on regional deposition in the RT and exhaled particles are considered. Second, the model used is based on porcine RT collected from slaughterhouses and any animal was specifically sacrificed for this study. This point is equally a limitation because they presented cuts that generated leakage. This particular point was discussed on the previously published data and did not influence the homogeneity of the ventilation25. Moreover, improvements were realized to minimize the potential leakage using cyanoacrylate glue and stitches and by a better sealing of the enclosure.Despite its interesting results, this work presents some limitations. First, as all studies using models, our ex vivo RT will never be able to mimic the complexity of a whole organism. Therefore, as preclinical data, they need to be confirmed by clinical studies. To date, results from this study seem in good agreement with the clinical study on ICU patients of Klockare et al.51. Moreover, most of the studies were conducted on patients under non-invasive ventilation, which is a different condition. Hence, more than 30 years after the development of ICU and aerosoltherapy, the nebulizer position in the ventilation circuit is mainly driven by empirical practices, as well as the practices concerning the humidification during nebulization. Moreover, nebulizer technologies have changed as well as ventilators and ventilation parameters. Therefore, there is a need for preclinical studies with a good reproducibility as observed here, allowing to compare humidification and position as isolated factors but equally with their interaction. Finally, these comparisons are quantitative but equally qualitative on the repartition in the RT. To our knowledge, this is the first study allowing all these comparisons.As previously stated, there is a lack of published clinical literature concerning the impact of the inhaled gas humidification and the nebulizer position on the deposition pattern of an aerosol when administered during mechanical ventilation. Among the few clinical studies, the endpoints mainly concern patients’ outcomes, such as improvement of the general state, weaning of mechanical ventilation, etc. or have difficulties to show differences, probably because of the multiplicity of influencing factorsInhaled gas humidification and nebulizer position in the ventilator circuit are two major determinants to optimize aerosol deposition by nebulization under mechanical ventilation. The position 15 cm before the Y-piece adapter allows a more than two-fold higher dose reaching the RT. This result, even needing additional clinical data, is of importance to standardize practices for aerosol delivery in ICU with optimal conditions.Gas humidification tends to a more central aerosol deposition in the RT but as the interruption can be dangerous for patients and considering the low impact compared to the nebulizer positioning, the removal of HH has to be examined carefully before considering its recommendation. Moreover, considering the impact of nebulizer technology and this optimized position in the ventilator circuit, this study leads to think about pursuit of humidification as a security recommendation.To conclude, these data are preclinical and consequently need clinical confirmation. Yet, the standardized ex vivo respiratory model used with comparative data of each parameter i.e. humidification and nebulizer position, leads to consider these results for further works on aerosol therapy in ICU and need to be acknowledged by ICU clinicians.Supplementary Information.


While most voids in Class-II restorations were observed in C4 (p ≤ 0.0031), no significant differences were found between the other groups (p > 0.05). The percentage of voids showed no differences in E1-E5 (p > 0.05). C4 showed a significantly higher percentage of voids compared to C2 (p < 0.001). There is no benefit in applying sonic vibration when filling Class-II or endodontic access cavities.This pilot study investigated whether sonic-powered application of a bulk-fill resin-based composite (RBC) in Class-II or endodontic access cavities reduces void formation. The crowns and roots of 60 bovine teeth with Class-II cavities (C) and endodontic access cavities (E) respectively, were assigned to ten groups . Cavities were filled with RBC  using different application techniques: no adaptation (C1 + E1), spreading of RBC on the cavity surfaces with a dental explorer tip (C2 + E2), low (C3 + E3) or high frequency (C4 + E4) direct activation by inserting a sonic-powered tip into RBC and high frequency indirect activation with an ultrasonic insertion tip (C5 + E5). The restorations were light-cured and investigated for voids using microtomography. The number of voids and percentage of voids related to the volume were statistically analysed ( For example, tooth-colored RBC can be used to seal and fill endodontic access cavities (EAC) after root canal treatment, allowing for esthetic rehabilitation and reducing the need of dental crowns, particularly in anterior teeth3.Resin-based composites (RBC) have become the material of first choice for direct tooth restoration in modern dentistry, covering a wide range of indications and restorative treatment options. They are commonly used for all types of tooth restorations in both the anterior and posterior region4.Conventional firm, packable, high viscosity RBC, as well as low viscosity, flowable RBC are frequently used in this context. However, they require multiple increments of ≤ 2 mm thickness, to ensure adequate polymerization, minimize polymerization shrinkage and stress. Proper application requires more time and for packable RBC, there is a risk of insufficient adaptation to the cavity surfaces and also between the increments Fig. a4.Figure5. It can easily be applied and adequately polymerized in increments of up to 3–5 mm layer thickness, while maintaining low polymerization shrinkage and stress6. These properties are enabled by novel resins, specific fillers, polymerization modulators and optimized photo-initiator systems7. Superior flow and adaptation properties are claimed by some manufacturers. However, an increased number of voids in the cured material . Standardized Class-II cavities were prepared in the crowns and the root canals were shaped, obturated with gutta-percha and prepared with standardized access cavities as follows:An illustration of sample preparation and cavity design is given in Fig. An illustration of the sample preparation and cavity design is shown in Fig. 22. The bovine teeth are classified as slaughter by-products and are therefore free from ethical and animal welfare concerns.Finally, the 60 tooth roots with shaped and obturated root canals and prepared EACs (E) and the 60 crowns with prepared and matrix-fitted Class-II cavities (C) were randomly assigned to ten groups . All specimens were stored at 5 °C in 0.1% thymol . The experimental protocols and methods were conducted in accordance with ARRIVE guidelines2, as verified using a radiometer . The cavities were then filled with a bulk-fill RBC  in one increment, with the cannula of the compule slowly withdrawn from the bottom of the cavity while consistently keeping the tip within the applicated material. For each sample, a new compule of RBC was used without disposing the first amount of RBC before dispensing it into the cavity. In the control groups (C1 and E1), the applied material was not manipulated before light-curing. In C2 and E2, the RBC was manually adapted to the cavity surfaces using a sharp dental explorer tip .All specimens were sprayed with water for 5 s and dried with paper tips (E) or gently air dried (C). Afterwards, a universal adhesive  was applied with a microbrush  for 20 s in self-etch mode. Excess adhesive was removed with a paper tip and the solvent was gently air dried for 5 s before the adhesive was light-cured for 10 s using a polywave LED curing unit  with a 385–515 nm emission spectrum and an output irradiance of at least 1.1 mW/cmIn C3 and E3 (low frequency) and C4 and E4 (high frequency), a sonic-powered tip  was shortened to 5 mm, the thickness of a conventional dental explorer (0.6 mm), and inserted into the uncured RBC material that was initially filled into the cavity, as described for C1 and E1. Subsequently, the tip was activated for 5 s at a sonic intensity of 160,000 units (C3 and E3) or 230,000 units (C4 and C5), as displayed on the treatment unit . The small amount of RBC remaining on the tip of the compule after deactivation was disposed of and refilled without additional activation before light-curing.2. The distance between the curing unit tip and the uncured RBC was approximately 1 mm. The filling procedures of C and E were each performed by one investigator. The study design is illustrated in Fig. In C5 and E5, sonic vibrations were indirectly transmitted to the RBC by placing an ultrasonic insertion tip  on the tooth surface next to the cavity margin  based on slice-by-slice reconstruction of the µCT scans, allowing for quantitative assessment of the total volume and the void volume of each filling. The scan resolution predefined the minimal ascertainable inhomogeneity (air inclusion) within a filling. While all measurable inhomogeneities were included in the assessment of the percentage of voids related to the overall volume of the filling, only air inclusions ≥ 50 µm were counted for the assessment of the number and the location of the voids. This was due to the exceedingly high and innumerable number of miniscule voids observed in some restorations. An example for the µCT analysis of a Class-II filling and an EAC filling, including a three-dimensional reconstruction of voids, is presented in Fig. For each group, a blinded and calibrated investigator assessed the number, location and percentage of voids related to the overall volume of the filling using µCT . The location of voids was categorized as internal (within the filling), marginal (at the interface of the filling and the tooth) and superficial (on the surface of the filling). In accordance with a previously established method25, including the packages tidyverse26, rcompagnion27, ggplot228 and PMCMR29. A power analysis was not possible since no comparable data were available prior to the study. However, in a similar study conducted at the institute30, a total of 60 cavities were prepared and allocated to 10 experimental groups. Based on that study, it was decided to use a slightly higher number, to ensure a sufficient number of samples. Nevertheless, this study must still be regarded as a pilot study.Descriptive statistics were computed for the target variables . Additionally, the target variables “number of voids “ and “percentage of voids “ were investigated using statistical inference. The non-parametric omnibus test Kruskal–Wallis one-way analysis of variance was used to assess if the test groups originated in a common population. The non-parametric Conover test was carried out as a post-hoc test to check for significant differences between the test groups and the target variables. To account for multiple testing, corrections were made according to the Bonferroni-Holm method. The level of significance was set at 5%. One blinded statistician performed the entire statistical analysis and plots using the statistical software R (2015) Overall, air entrapment was detected in 93.3% of the Class-II and in 95.0% of the EAC restorations.p > 0.05), the number of voids was significantly higher after high frequency direct activation (C4) (p ≤ 0.0031). Within the EAC restorations, no significant differences (p > 0.05) were found between the test groups (E1–E5): E1 [1.5 (1.2)], E2 [2.0 (1.2)], E3 [1.5 (4.0)], E4 [4.0 (3.2)], E5 [2.0 (1.2)] ] were observed: C1 [1.0 1.5)], C2 [2.0 (2.0)], C3 [2.5 (4.8)], C4 [8.0 (1.2)], C5 [1.0 (3.0)]   [median (interquartile range)] were recorded: C1 [2.7 (0.8)], C2 [1.7 0.6)], C3 [2.8 (2.1)], C4 [4.9 (5.0)], C5 [2.7 (0.8)]. No significant differences were found between the test groups C1, C2, C3 and C5 and between C1, C3, C4 and C5. The test group C2 showed a significantly lower percentage of voids compared to C4   [median (interquartile range)] were found between the five test groups E1 [1.1 (1.0)], E2 [1.1 (1.8)], E3 [1.5 (2.0)], E4 [1.8 (2.5)], E5 [1.0 (1.1)] (05) Fig. b.An overview on the location of voids is given in Table 31.Partly rejecting the null hypothesis, the results showed that sonic vibration had a significant effect on voids in flowable bulk-fill RBC restorations of Class-II cavities, with significantly higher numbers and percentages of voids observed when high frequency, direct sonic vibration was applied. In contrast, no significant differences in the number or percentage of voids related to the volume of the restorations of EACs were found between the different application techniques30 a total of 60 cavities were prepared and allocated to 6 experimental groups. Based on this, it was decided to use two additional samples in each group. However, when looking at the results of the present study, it becomes apparent that the sample size may still have been insufficient. For example, Fig. Prior to the study, no power analysis was performed as no comparable data were available. In a study with a similar design32. The cavity dimensions aimed to reflect realistic conditions which may occur during root canal treatments of anterior teeth and premolars, as well as Class-II cavity restorations. It is worth noting that while the definition of a Class-II cavity originally refers to posterior teeth according to Black33, the cavities in the present study were prepared in modified anterior bovine teeth, due to the inadequate shape and anatomy of posterior bovine teeth, which are generally not suitable for standardized Class-II cavities.The Class-II cavities and EACs in this study were prepared in extracted lower bovine incisors. Bovine teeth are easily accessible, have been previously used and discussed in multiple studies and can therefore be regarded as suitable substitutes for human teeth, within the scope of this study34. A new compule of RBC was used for each sample to avoid changing the compule while filling up the cavity, thus minimizing the risk of incorporating additional air between non-polymerized increments. However, in groups where a dental explorer or ultrasonic tips were inserted into the applied RBC , the cavities were carefully refilled if RBC was removed from the cavity while withdrawing the instruments. Therefore, it is not feasible to exclude the possibility of additional air being entrapped in the filling, which might be a limitation of the study. Furthermore, it should be noted that the experiments were performed by two master students, with limited clinical experience, but under the guidance and supervision of experienced clinicians.All cavities were treated with a universal adhesive applied in the self-etch mode i.e. without additional phosphoric acid etching. Aiming to minimize potential errors and facilitate the application, this technique was found to be the most suitable, within the scope of this study, as it has been shown that there are hardly any differences compared to current multi-step etch-and-rinse adhesives21. This can be considered a strength of this study. The µCT analysis and assessment of voids was based on established and proven methodical approaches conducted in previous studies at the institute and was performed by one blinded investigator who was instructed and calibrated by an experienced colleague with superior expertise in µCT analysis. As this process was implemented during data analysis and no further calibration was performed using independent samples, there is still an inherent risk of calibration bias, although this would have affected all test groups equally as the investigator was blinded.The three-dimensional evaluation of the samples using µCT is generally regarded as a suitable tool for non-destructive porosity assessment of RBC restorations. This method not only provides high-resolution images and a detailed assessment of the number and size of voids, but also allows for precise determination of the void volumes, including the percentage related to the volume of the respective filling31.The aim of this study was to evaluate the potential of sonic-powered application of a flowable bulk-fill RBC in the restorations of Class-II cavities or EACs, to reduce voids. In theory, the vibrations would create an upward flow within the uncured RBC, thus loosening air inclusions and vibrating them to the surface. Additionally, the enhanced thixotropy of the RBC during activation would promote better adaptation to the surfaces10. Inevitably, questions arise about the reason for these high numbers. It should be taken into account that due to the particularly high resolution (12 µm) of the µCT, even the tiniest air inclusion, which normally cannot be seen on an X-ray or without magnification, could be assessed in this study, which might explain these high numbers. While all measurable inhomogeneities were counted in the assessment of the percentage of voids related to the overall volume of the filling, only air inclusions ≥ 50 µm were counted for the assessment of the number and location of the voids in this study. While this may be considered a limitation of the study, this decision was made due to the occasionally innumerable amounts of miniscule voids or inhomogeneities in various restorations, as illustrated in Fig. Overall, voids were detected in 93.3% (Class-II) and 95.0% (EAC) of all restorations, which is surprisingly high. Even in the best performing group (E1), 83.3% of the restorations were affected. Another study that investigated voids in Class-II cavities using optical coherence tomography reported similar high percentages of voids (79.2%) in restorations filled with a flowable bulk-fill RBC35. Additionally, air may be aspirated through the cannula of the compule-tip during inconsistent or intermittent composite application. In such cases, it might be helpful to squeeze out and dispose of a small amount of RBC before dispensing it into the cavity. However, as a new compule of RBC was used for each filling and no benefits of this technique have been described in literature, the RBC was applied into the cavity without disposing of the first drop.An attempt was made to identify the sources or causes of the inhomogeneities. It is conceivable that air inclusions may be present in the RBC compules prior to their use, due to the manufacturing processes. These air inclusions could then in turn transfer to the filling as soon as the RBC is applied36. In particular, in small, deep and tapered cavities, inhomogeneous distribution of low viscous RBC flowing down the lateral cavity surface might trap and enclose air at the bottom of or within the cavity, thus forming voids37. As confirmed in this study, a considerable number of voids are present in restorations without any further manipulation of the RBC after application. It was therefore of interest to investigate if these voids could be eliminated through the use of additional adaptation techniques including the manual spreading of the applied RBC to the cavity surfaces with a sharp dental explorer, the use of low or high frequency direct activation by putting a sonic-powered tip into the applied RBC, or the use of high frequency indirect activation by setting the whole tooth under vibration using an ultrasonic insertion tip.Previous studies confirm that the way a flowable RBC is applied into the cavity has an influence on the formation of voids21. It is conceivable that the vibrating tips might displace the honey-like flowable RBC for a fraction of a second, leading to a sort of air crater along the tip that rapidly collapses on the surface, thus incorporating air within the RBC and forming new air inclusions. With increased frequency, this “foaming effect” seems to intensify, which might explain the results observed. When comparing the Class-II cavities with the EACs, differences can mainly be found in the high frequency, direct sonic-powered application groups. It might be speculated that the reason for the higher numbers and percentage of voids in the Class-II cavities could also be due to the above described “foaming effect” taking place in different cavity geometries. As the Class-II cavities had a larger width of 4 mm (compared to the EACs with 1.8–2.1 mm diameter), this could have allowed for a greater vibration amplitude of the tip without contact to the lateral cavity walls. Thus, more RBC might have been displaced, resulting in more extended collapsing air craters and hence more air inclusions.It was found that none of the investigated additional adaptation techniques provided a benefit in terms of void-reduction. On the contrary, high frequency, direct sonic-powered application resulted in significantly higher numbers of voids in Class-II cavities. Overall, there was a clear tendency towards increased void formation with increased frequency of direct sonic activation for the restorations of Class-II cavities and EACs. Similarly, other studies indicate that sonic-activated application might lead to increased air inclusions or void formation in RBC restorations21.Analogous to a laboratory plaster vibrator, the indirect activation aimed to loosen air inclusions and vibrate them to the surface by placing an ultrasonic insertion tip on the tooth surface next to the cavity margin. As the results in this study did not show any benefit for this technique, it might be speculated that the rheological properties of the RBC did not allow the voids to move upwards to the surface, or that the sonication process may have caused smaller air inclusions  to coalesce and form consolidated, more obvious voids38.Compared to the control group, manual adaptation of the RBC to the cavity surfaces also did not result in a reduced number of voids in both cavity geometries, nor did it reduce the percentage of voids related to the volume in the restorations of the EACs. However, the overall lowest percentage of voids was found in Class-II cavities with manual adaptation. On the one hand, this technique might help to adapt the RBC to the cavity walls and remove obvious air inclusions within and at the surface of the material, which can be imagined to be more efficient for larger and easily accessible cavity geometries. On the other hand it seems questionable whether voids, especially those located at the bottom of small, deep and visually uncontrollable cavities, can be reliably eliminated or moved to the surface and then eliminated with a sharp dental explorer. Moreover, there is an additional risk of entrapping air in the RBC, thus generating new or even worse air inclusions40.The manufacturer of the flowable bulk-fill RBC used in this study claims that their product has superior flow and self-leveling properties and does not require any additional instrumentation or manipulation. This claim was supported by the low numbers and percentages of air inclusions in the control group. Nevertheless, voids were detected in 83.3% of all restorations, which may be attributed to either air inclusions already present in the RBC compule or the way the RBC was applied into the cavity, especially with regard to air getting enclosed and trapped. To further minimize the risk of air inclusions, it is recommendable to fill up the cavity starting at the bottom and slowly lifting the tip of the compule simultaneously with the rising surface level, especially in small, deep or visually uncontrollable cavitiesThere is no reduction of voids by applying direct or indirect sonic vibration to flowable bulk-fill resin-based composite in Class-II cavities or root canal access cavities.The results suggest a potential concern regarding high frequency, direct activation of uncured, flowable bulk-fill resin-based composite in Class-II cavities. However, the limitations of this study emphasize the need for larger-scale research to validate the results before clinical recommendations can be made.Voids continue to be an issue in flowable, bulk-fill, resin-based composite restorations.For the present pilot study, the following conclusions can be made:


Ganoderma lingzhi culture (GLC) as a fermented feed on growth performance, serum biochemical profile, meat quality, and intestinal morphology and microbiota in Sanhuang broilers. In addition, the association between gut bacteria and metabolites was investigated via untargeted metabolomic analysis.The experiment was conducted to evaluate the effects of A total of 192 Sanhuang broilers (112 days old) with an initial body weight of 1.62 ± 0.19 kg were randomly allocated to four treatments, six replicate pens per treatment with 8 broilers per pen. The four treatments contain a control diet , a positive control diet , and the experimental diets supplemented with 1.5 and 3% of GLC, respectively. The trial includes phase 1 (day 1–28) and phase 2 (day 29–56).P < 0.05) in phase 2 and overall period and a higher ADG (P < 0.05) in phase 2. On day 56, the concentrations of serum SOD (P < 0.05), and HDL (P < 0.05) and cecal SCFA contents (P < 0.05) were increased in broilers fed GLC diets. Broilers fed GLC also showed a higher microbiota diversity and an elevated abundance of SCFA-related bacteria in the caecum. The association between intestinal bacteria and metabolites was investigated via correlation analysis. The differential metabolites in the caecum, such as L-beta-aspartyl-L-aspartic acid and nicotinamide riboside, were identified.The results showed that broilers in PCON and GLC-added treatments showed a lower FCR (In summary, dietary GCL supplementation could increase growth performance to some extent. Moreover, GLC might benefit broilers' health by improving serum HDL content, antioxidant status, SCFAs contents, bacterial diversity, and probiotic proliferation in the caecum. Animal-source foods are central to maintaining global food security, contributing 25% of protein and 18% of calorie consumption worldwide. A rapid-growing population has led to projected increases of 48 and 57% for milk and meat, respectively, in global demand between 2005 and 2050. With the increasing demand for meat products, the livestock feedstuff shortage is inevitable, as the expected expansion of feed supply will be required to improve from 6.0 to 7.3 billion (Ganoderma lingzhi (GL), also named G. lucidum, has been collected, cultivated, and utilized as a traditional edible-medicinal fungus in Asian countries for centuries  is produced via solid-state fermentation with GL strain. It possesses abundant nutrients and bioactive substances, such as protein, polysaccharides, and triterpenoids. We hypothesized that GLC could be an effective feed ingredient that may positively affect the growth and health of broilers. Therefore, the study aimed to evaluate the effects of GLC addition on growth performance, serum biochemical parameters, intestinal function , and meat quality in Sanhuang broilers.The via solid-state fermentation for 35 days to produce GLC. Subsequently, the fermented mixed culture containing 60% moisture was dried at 60°C. The analyzed nutrient of GLC is listed in A sterilized culture media (mainly wheat bran) was inoculated with GL strain and proceeded ad libitum.A total of 192 16-week-old Sanhuang broilers with an initial body weight of 1.62 ± 0.19 kg were purchased from a local commercial market. All the experimental birds were randomly assigned to four treatments containing six replicates of eight birds per replicate. Four treatment diets included the control diet , the positive control diet , and the control diet supplemented with 1.5 and 3% GLC, respectively. The composition of experimental diets is presented in 2, subsequently, slaughtered for sample collection. The ileum sections (about 5 cm) and breast meat samples (about 100 g) were collected. For further morphology analysis, the ileum fragments were stored in the sterile centrifuge tube (50 mL) containing 30 mL 4% paraformaldehyde at 4°C. The breast meat samples were stored at 4°C for meat quality determination. Cecal digesta samples from each bird (one bird from each pen) were collected and kept in liquid nitrogen for further short chain fatty acid (SCFA), microbiome bioinformatics, and untargeted metabolomic analysis.The individual body weight (BW) were weighed on day 0, 28, and 56, and the amount of feed was recorded daily to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). On day 56, the 24 broilers (one bird from each pen) were selected by the BW close to the average BW in every pen. The wing vein blood samples (about 5 mL) were acquired using 10 mL vacuum blood collection tube. The serum sample was then obtained at 3,000 rpm for 15 min and stored at −20°C. These same broilers were then stunned and euthanized by immersing in COThe GLC sample was assessed for dry matter (DM), crude protein (CP), ether extract (EE), and ash . The conThe contents of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) were measured by assay kits  and determined using automatic biochemical analyzer . The concentrations of total triglyceride (TG), total cholesterol (TC), glucose (GLU), high-density Lipoprotein (HDL), and low-density Lipoprotein (LDL) were measured by assay kits  according to the manufacturer's instructions and determined at the wavelength of 450 nm.Intestinal morphology was measured following the method described by Abdelqader and Al-Fataftah . In brieThe cecal content was collected on day 56 and used for SCFAs concentration analysis . In brien = 6) were collected to analyze meat quality, including color, pH, and shear force. Meat color was expressed as lightness (L*), redness (a*), and yellowness (b*) and was measured by Chroma Meter . The pH values at 45 min and 24 h were determined by a pH meter . The pH and meat color were measured in triplicate at three different locations. The shear force was measured following the method described by Ciobanu et al. . The quality of the DNA extract was checked using 1% agarose gel electrophoresis, and the concentration of DNA was determined by spectrophotometer .Total bacterial DNA from cecal contents was extracted according to the method described by instruction of the E.Z.N.A.The V3-V4 region of the 16S was amplified using bacterial primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT). The PCR amplification of each sample was conducted with a 20 μL reaction mixture containing 5 × FastPfu Buffer (4 μL), 2.5 mM dNTPs (2 μL), FastPfu DNA Polymerase , BSA (0.2 μL), template DNA (10 ng), and each primer (0.8 μL). The procedure consisted of an initial denaturation at 95°C for 3 min, the following 30 cycles of temperature gradient , and finally, an extension step at 72°C for 10 min using a thermal cycler . Each sample was performed in triplicate. The triplicate amplicons were pooled, electrophoresed in an agarose gel (2%), and recovered by a DNA Gel Extraction Kit .https://github.com/OpenGene/fastp, version 0.20.0) for quality control, and the FLASH  was used for stitching. The OUT clustering was performed on sequences according to 97% similarity using UPARSE . The silva 16S rRNA gene database (v138) was compared by RDP classifier  for taxonomic annotation of OTU species. The 16S function prediction analysis was determined by PICRUSt2 (version 2.2.0). All the data were analyzed through the free online platform of majorbio cloud platform (cloud.majorbio.com).The purified amplicons were quantified by a Fluorometer . The sequencing libraries were constructed using combining equimolar ratios of amplicons. The resulting libraries for paired-end sequencing were determined on an Illumina MiSeq PE 300 platform by Majorbio Technology Inc . The original sequence was analyzed using FASTP . The mobile phases consisted of solvent A [0.1% formic acid in water: acetonitrile ] and solvent B [0.1% formic acid in acetonitrile: isopropanol: water ]. The separation gradient was as follows: 0–0.1 min, solvent B from 0 to 5%; 0.1–2 min, solvent B from 5 to 25%; 2–9 min, solvent B from 25 to 100%; 9–13 min, the linearity of solvent B was maintained at 100%; from 13.0 to 13.1 min, the linearity of solvent B was decreased from 100 to 0%, and from 13.1 to 16 min, the linearity of solvent B was maintained at 0%. The flow rate was 0.4 mL/min. The column temperature was kept at 40°C, and all these samples were stored at 4°C during the analysis period.Metabolites of cecal contents (50 mg) were extracted by 400 ml 80% methanol (aqueous solution) with 0.02 mg/mL L-2-chlorophenylalanin as internal standard. The mixture was treated with a high-throughput tissue crusher Wonbio-96c  at 50 Hz for 6 min at −10°C, then ultrasound was performed at 40 kHz for 30 min at 5°C. The sample was placed at −20°C for 30 min to precipitate the protein. The supernatant was then carefully transferred to the sample bottles for LC-MS/MS analysis after centrifugation at 13,000 × The data of mass spectrometric was collected by UHPLC-Q Exactive Mass Spectrometer equipped with an electrospray ionization source. The detection mass range was 70–1,050 m/z. Full MS resolution was 70,000, and MS/MS resolution was 17,500. The conditions were performed as follows: Capillary temperature, 320°C; heater temperature, 400°C; Aux gas flow rate, 10 arb; sheath gas flow rate, 40 arb; ion-spray voltage floating (ISVF), 3,500 V in positive mode and −2,800 V in negative mode respectively; Normalized collision energy, 20-40-60 V rolling for MS/MS.P < 0.05 was determined as significance, and a value of 0.05 ≤ P < 0.10 was determined as a tendency for significance.All the data were checked for the normality of equal variances and residuals by the UNIVERIATE of SAS , and were subjected to ANOVA by the GLM procedure of SAS. The LSMEANS was used to separate group means with Turkey's test for adjustment. The value of P < 0.05) and decreased FCR (P < 0.05) and showed a tendency toward a reduction of ADFI (P < 0.10). During the overall period, the GLC-fed broilers showed a lower FCR (P < 0.05) compared to those fed control diets. A decreasing trend was found on ADFI (P < 0.10) for broilers fed GLC diets from day 1 to 56. There was no difference between ADG and FCR to broilers in GLC and PCON treatments during the overall period.As shown in P < 0.05) and SOD (P < 0.05) concentrations in serum compared to those fed the control and positive control diets. A decreasing trend of LDL content (P < 0.10) was also observed after GLC addition.As shown in P < 0.05) were revealed after GLC supplementation (As shown in entation .P < 0.10) and did not affect pHat 24 h, meat color, and shear force.As shown in As shown in Firmicutes and Bacteroidota hold most of the community abundance at the phylum level. Compared with CON and PCON treatment, broilers fed GLC showed a higher relative abundance of Bacteroidaceae, and a lower relative abundance of Lactobacillaceae and Barnesiellaceae at the family level.As shown in g__unclassified_f__Coriobacteriaceae, g__Marvinbryantia, g__Coprobacter, g__NK4A214_group, g__Christensenella, g__Faecalicoccus, g__GCA-900066575, g__Lachnospiraceae_NK4A136_group, g__Bifidobacterium, g__Anaerofilum, g__Pseudoflavonifractor, f__unclassified_o__Oscillospirales, and g__unclassified_o__Oscillospirales was increased significantly after GLC supplementation. In the cecum, an increase in the Synergistota populations on the phylum level was found in broilers fed the GLC diet  that 170 metabolites with significant differences were identified between CON and 3% GLC treatments. As shown in Furthermore, the results of differential metabolite analysis between CON and 3% GLC treatments showed , and the abscissa was log 2 (fold change). Each metabolite was represented by a point. The L-beta-aspartyl-L-aspartic acid, nicotinamide riboside, 2-amino-4-[(2-hydroxy-1-oxopropyl) amino] butanoic acid, methionyl-proline, topaquinone, and 8-azaadenosine were selected as biomarkers between the CON and 3% GLC treatments. All metabolites obtained from level-two identification were assigned to the HMDB (Human Urine Metabolome Database) database, 326 of which were matched and classified into 10 HMDB super classes. Meanwhile, “organic acids and derivatives,” which contained 101 metabolites, was the first class (30.98%), followed by “lipids and lipid-like molecules”  and “organoheterocyclic compounds”  glutamine. The hydroxyprolylhydroxyproline and aspartyl-leucine were negatively correlated with the abundances of f__Butyricicoccaceae, f__Rikenellaceae, and f__norank_o__Rhodospirillales. The L-N-(3-Carboxypropyl) glutamine was positively correlated with the relative abundance of f__Synergistaceae, and negatively correlated with the relative abundance of f__norank_o__Rhodospirillales.As shown in The current study detected a notable decrease in FCR of birds-fed GLC during the finisher and overall period, compared to the control diet. Similar studies have shown that the bioactive substance contained in GL could remarkably improve the growth performance of broilers. Dietary supplementation of 200 mg/kg GL spores to aflatoxin B1-contaminated diets could counteract its adverse effects on the growth performance of broilers during the overall period . MoreoveThe mechanism by which GLC reduces FCR has yet to be fully elucidated. It could be partially attributed to the antioxidative characteristics of GLC. During the finisher period, growing broilers produce enhanced oxidative free radicals and endogenous calories due to body weight-correlated changes in endocrine function, body composition, and metabolic rate . We obseWe also found that broilers fed GLC showed a decreasing trend on ADFI. We deduced that the higher dietary fiber content in GLC (NDF: 45.00% and ADF 13.90%) might negatively affect the ADFI of broilers. Other studies reported that broilers fed higher fiber diets had a lower feed intake than control diets , 18.via oxidation processes. We found that GLC could improve SOD levels in broilers, which is probably due to the polysaccharides and triterpenoids in GLC. Evaluating the antioxidant activity of GL polysaccharides or triterpenoids has been done via different methods in vitro or in vivo. All polysaccharides or triterpenoids derived from different parts of GL have confirmed the ROS scavenging abilities  in the serum of pre-diabetic mice . Reactivbilities . A studyin vitro . The polin vitro . In anottic rats .via fermenting dietary fiber in the large intestine. Xie et al.  in GLC might play a vital role. The dietary fiber, such as β-glucan and pectin, could not be digested directly in the upper gastrointestinal tract. The SCFAs are the primary metabolites produced by microbiota e et al.  found thu et al. , dietaryAs is well-known, meat pH value is associated with muscle lactic acid content derived from anaerobic glycolysis . A higheThe intestinal microorganisms are complex communities and have established a close symbiotic relationship with their host that contributes to the maturation of the immune system, prevention of pathogen colonization, synthesis of some vitamins, and metabolism of indigestible components . We assuFirmicute and the ratio of Firmicutes to Bacteroidetes (F/B) decreased in the 3% GLC treatment, compared to the CON treatment (2.84–2.41). Krajmalnik-Brown et al.  could modulate intestinal microbiota composition, mainly by decreasing the F/B ratio in mice.In the current study, the relative abundance of n et al.  have prog et al. , they reSynergistota, in which the bacteria were mainly anaerobic microorganisms  and SCFAs  and NADPH, which are vital for resistance to oxidative stress (L-beta-aspartyl-L-aspartic acid or β-aspartylaspartic acid is a sort of dipeptides and are derivatives of asparagine . Nicotine stress , 46. Thee stress .f__Coriobacteriaceae, f__Synergistaceae, and f__norank_o__Oscillospirales were positively correlated with porric acid A, L-pipecolic acid, and deoxycytidine (The changes in gut microbiota elicited by GLC digestion will inevitably trigger alterations in microbial metabolites. The correlation heat map was established to clarify the potential association between microorganisms and metabolites in broilers treated with 3% GLC. The data showed that the family increased after 3% GLC addition, such as cytidine . The porcytidine . The L-Pcytidine .Coriobacteriaceae family is strictly anaerobic, gram-positive bacteria. The Coriobacteriaceae might play an essential role in regulating glucose homeostasis in animals, and it might be beneficial to host glucose metabolism. The previous studies showed that Coriobacteriaceae had a strong negative correlation between hepatic glucose and glycogen levels. Moreover, positive correlations were found between Coriobacteriaceae and hepatic triglycerides levels in mice and non-high-density lipoprotein cholesterol in the plasma of hamsters (The hamsters , 51.f-Synergistaceae. The previous study revealed that most cultured bacteria from the family Synergistaceae could ferment amino acids into SCFAs. Certain species might also degrade SCFAs with methanogens via syntrophic relationships during anaerobic digestion (We observed that GLC supplementation could increase the relative abundance of igestion .Oscillospirales, a butyrate producer (Rikenellaceae, belonging to the Bacteroidetes phylum, might contribute to decreased visceral fat mass. It might act as an adiposity modulator by affecting the production of acetate and propionate (The producer , is signopionate .Synergistota, Lachnospiraceae, Bifidobacterium, Synergistaceae, Oscillospirales, and Rikenellaceae. Namely, the GLC could influence host health by promoting the relative abundance of SCFAs-related microbe. Despite various links among the metabolism, gut microbiota, and metabolites, this effort only occupied a part portion of the entire metabolic system of the host. Thus, the underlying mechanism of the effects of GLC on broiler's growth and health still needs to be further explored.Based on the results above, we proposed that the elevated SCFAs contents in GLC-treated broilers might be positively correlated with the increased SCFAs-related bacteria, such as via edible and medicinal fungi fermentation.This study determined the effects of GLC supplementation on growth performance, serum biochemical indices, meat quality, intestinal morphology, microbiota, and metabolites. The results revealed that GLC could reduce FCR, increase serum SOD and HDL levels, and enhance cecal SCFA contents. The study also confirmed that GLC could increase bacteria diversity and promote the proliferation of SCFAs-generated bacteria in the gut. Differential metabolites, such as L-beta-aspartyl-L-aspartic acid and nicotinamide riboside, were identified. Overall, GLC could positively affect the health of Sanhuang broilers, and its utilization provides a new target for developing biological feed The data presented in the study are deposited in the Genome Sequence Archive in National Genomics Data Center, accession number CRA010095.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Guangxi Academy of Agricultural Sciences.XL designed the study and analyzed the data. LH, YS, XW, YLuo, SW, YQ, YLu, WZ, and YJ performed the animal experiment and analyzed the tissue samples. YY and YLi revised the manuscript and had primary responsibility for the final content. All authors read and approved the final manuscript.