Introduction: Baroreflex sensitivity (BRS) is essential to ensure rapid adjustment to variations in blood pressure (BP). Little is known concerning the adaptive responses of BRS during acclimatization to high altitude at rest and during exercise.Methods: Twenty-one healthy sea-level residents were tested near sea level , the 1st (ALT1) and 16th day (ALT16) at 5,260 m using radial artery catheterization. BRS was calculated using the sequence method . At rest, subjects breathed a hyperoxic mixture  to isolate the preponderance of CO2 chemoreceptors. End-tidal CO2 varied from 20 to 50 mmHg to assess peripheral chemoreflex. Rebreathing provoked incremental increase in CO2, increasing BP to assess baroreflex. During incremental cycling exercise to exhaustion, subjects breathed room air.Results: Resting BRS decreased in ALT1 which was exacerbated in ALT16. This decrease in ALT1 was reversible upon additional inspired CO2, but not in ALT16. BRS decrease during exercise was greater and occurred at lower workloads in ALT1 compared to SL. At ALT16, this decrease returned toward SL values.Discussion/Conclusion: This study is the first to report attenuated BRS in acute hypoxia, exacerbated in chronic hypoxia. In ALT1, hypocapnia triggered BRS reduction whilst in ALT16 resetting of chemoreceptor triggered BRS reduction. The exercise BRS resetting was impaired in ALT1 but normalized in ALT16. These BRS decreases indicate decreased control of BP and may explain deteriorations of cardiovascular status during exposure to high altitude. High altitude hypoxia challenges blood pressure (BP) homeostasis in humans. In the short term, baroreceptor afferents principally counteract the stress of hypoxia on BP homeostasis by affecting the activity of the parasympathetic and sympathetic divisions of the autonomic nervous system (ANS). Hypoxemia induces vasodilation and therefore hypotension, which stimulates the baroreflex function. The baroreceptor-mediated sympathetic excitation in hypoxia results in an increase in set point , whereas the central chemoreceptors, located in the CNS, are primarily sensitive to variations in CO2 (pHCSF), but not in O2, unless arterial O2 saturation (SaO2) falls below 50%  is a measure of baroreflex function where the faster the response to small changes in BP, the more sensitive the autonomic control of BP and the higher the BRS. The baroreceptors, located in the aortic arch and the carotid bodies, signal to the medulla via the cranial nerves IX and X  and changes in systemic vascular resistance (SVR), renal and gastrointestinal vasoconstriction, along with working muscle vasodilatation, cause an increase in blood pressure that activates the arterial baroreceptors  BRS would be attenuated in acute hypoxia and further decreased after acclimatization at rest and during exercise; (2) and that additional inspired CO2 would have distinct effects at sea level, and during acute through chronic hypoxia, that would indicate a resetting of BRS toward low PaCO2 values after acclimatization.To reveal the role of changes in BRS over time in hypoxia on BP homeostasis, BRS was quantified with intra-arterial pressure measurements in humans during acute and chronic hypobaric hypoxia at rest and during exercise, and whilst breathing various OThis study is part of the AltitudeOmics project  to assess the aerobic fitness of the subjects and to ensure that the inclusion criteria were met. After familiarization, the subjects underwent experimental trials near sea level  and on the 1st (ALT1) and 16th day (ALT16) at 5,260 m (barometric pressure 406 mmHg). For each subject, all ALT measurements were carried out around the same time of day to minimize any confounding effects of the circadian rhythm. During ascent  the subjects breathed supplemental oxygen . Administration of O2 was ceased just before ALT1 measurements. This ensured standardized acute exposure at ALT1 and minimized any influence of early acute mountain sickness (AMS) during ALT1. Likewise, no symptoms of AMS were observed at ALT16 because of successful acclimatization. An overview of the entire experimental design of the AltitudeOmics project is given elsewhere  into a radial artery  under local anesthesia (2% lidocaine). Arterial blood pressure was measured using this catheter and a calibrated pressure transducer  connected to an amplifier . After ~30 min of instrumentation, the subjects underwent the resting protocol, followed by the exercise protocol.2 pressure (PETCO2) clamped at 40 mmHg (cl-40); (4) 3 min of voluntary hyperventilation to lower PETCO2 to ~20 mmHg (HVE); and (5) a modified rebreathing test . Stages 3 to 5 of the protocol were carried out in a background of hyperoxia  so that the input from O2 chemoreceptors was reduced, so that the vast majority of the input would come from CO2 chemoreceptors. Clamping CO2 at 40 mmHg normalized the conditions to look at the influence of the peripheral chemoreflex. Rebreathing was used to provoke an incremental increase in CO2, consequently increasing BP, and thus bringing the baroreflex into play. Figure Following 10–15 min of quiet rest in a seated position, each experimental testing session consisted of (1) instrumentation; (2) 10 min in room air for baseline; (3) 10 min with end-tidal partial CO2 at 40 mmHg by constantly adding a varying portion of CO2 into the inspired gas mixture. Throughout the end-tidal PCO2 clamping, we maintained PETO2 at ~250 mmHg by titrating 50%  or 100% O2 into the inspiratory reservoir, at SL and ALT, respectively.Throughout the protocol, the subjects sat upright and breathed through a mouthpiece attached to a two-way, non-rebreathing valve . The breathing circuit allowed switching from room air to either an end-tidal clamping system or a rebreathing system. The end-tidal clamping setup used in the present study was a modified version of the system previously described by Olin et al. . The set2 and PO2 of 0 mmHg and 300 mmHg, respectively, at each altitude. Subjects were instructed to hyperventilate for 3 min (part 4) to lower and then maintain PETCO2 at 20 mmHg at both sea level and 5,260 m (in a background PETO2 of ~250 mmHg). Subjects were then switched to the rebreathing bag and following two initial deep breaths to mix the gas from the bag with that in the respiratory system, they were instructed to breathe ad libitum (part 5). The rebreathing tests were terminated when PETCO2 reached 50 mmHg, PETO2 dropped below 200 mmHg, or the subject reached the end of his or her hypercapnia tolerance.The rebreathing bag was filled with gas to achieve inspired PCOSubjects were seated on an electrically-braked cycle ergometer . The protocol began with a three-min resting baseline in pedaling position on the ergometer. The subjects then completed four 3-min stages at 70, 100, 130, and 160 Watts, followed by 15 Watts/min increments until they could no longer maintain pedaling >50 rpm despite strong verbal encouragement. No specific pedaling frequency was required. Maximal power output (Watts) was calculated as: work rate of last stage completed + [(work rate increment) × ] , PCO2 (PaCO2), and pHa in triplicate . The blood gas analyzer was calibrated daily with tonometered whole blood samples.During the exercise protocol, arterial blood samples (2 ml) were taken from the catheter in a radial artery during the resting baseline, at the end of each of the four three-min stages and immediately before the cessation of exercise . Core body temperature was telemetrically recorded from an ingested pill  and used to correct the results from the blood gas analyzer. All samples were analyzed immediately for arterial PO2 and PETCO2 were measured using fast-responding gas analyzers . The pneumotachograph was calibrated using a 3-liter syringe  and the gas analyzers were calibrated using gas mixtures of known concentrations of O2 and CO2 prior to each testing session.Throughout the exercise protocol, the subjects breathed through a mouthpiece connected to a two-way, non-rebreathing valve . Ventilation and respiratory frequency (Rf) were measured using a pneumotachograph , expressed in units adjusted to body temperature and pressure, saturated (BTPS) and averaged over windows of 30 s. PETOAll analog data were sampled and recorded at 200 Hz on a personal computer for off-line analysis .Heart beat-to-beat time intervals were extracted directly from BP recordings. Initially, systolic blood pressure (SBP) peaks were extracted from the BP waveform with heartbeats representing the time of their occurrence. However, low sampling rates (<250 Hz) may produce jitter in the estimation of peaks  of BRS are reported in Table Two-way repeated measures ANOVAs were performed to assess the effect of time (SL vs. ALT1 vs. ALT16) and the effect of condition (Rest vs. cl-40 vs. hyperventilation vs. rebreathe) in the rest protocol, and the effect of time and exercise intensity  in the exercise protocol. One-way repeated measures ANOVA was performed to assess the effect of condition when time was not available (time decay). The Tukey-Kramer post hoc test was performed when appropriate. The alpha level for significance was set at 0.05 and is reported rounded to three digits after the decimal. All analyses were completed using MATLAB® . The coefficient of variation (CV) and the inferior and superior 95% confidence interval (CITable p = 0.048 vs. SL), this decrease was exacerbated at ALT16 (p < 0.001 vs. ALT1) as shown on Figure BRS decreased at ALT1 during seated rest (p = 0.499). BRS did not change between SL and ALT1 (p = 0.341) but decreased at ALT16 (p = 0.021 vs. SL and ALT1) as shown on Figure p = 0.031 vs. rest ALT1) back to SL values. At ALT16, it did not change compared to rest (p = 0.213). In short, clamping CO2 at 40 mmHg did not affect BRS at SL whilst at ALT1 it restored it to SL values.During cl-40, BRS did not change compared to rest at SL (p = 0.002) and ALT16 (p = 0.002) but there was no difference between ALT1 and ALT16 (p = 0.792), as shown on Figure During REB, BRS decreased from SL to ALT1 (p > 0.050). At SL and at ALT1, there was a significant decrease in BRS during HVE compared to rest (both p < 0.001 vs. rest SL and ALT1) but not at ALT16 (p = 0.770).No significant differences across time were found during HVE  to ALT1  and further to ALT16 . An inverse relationship was found between pHa and BRS. As illustrated on Figure p < 0.001 vs. SL) and further to ALT16 .Figure p = 0.003) and ALT16 (p = 0.004). Time decay was not different between SL and ALT16 (p = 0.718). These results indicate that the kinetics of the decrease in BRS when exercise intensity increases were not different between SL and ALT16 although the pre-exercise BRS value was lower in ALT16 compared to SL (p = 0.002). At ALT1, the kinetics were different compared to SL and ALT16, notably because of a greater decrease between pre-exercise and 70W and a BRS roughly stable at the subsequent workloads. The decrease in BRS during exercise appeared to be more progressive during SL and ALT16 than during ALT1.Figure p = 0.001, p = 0.032, p = 0.021, respectively). The decrease was not significant between the subsequent exercise stages . Pre-exercise BRS decreased from SL to ALT1 (p = 0.003) and to ALT16 (p = 0.003); there was a tendency between ALT1 and ALT16 (p = 0.083). BRS at 70W decreased at ALT1 compared to SL (p = 0.001) and ALT16 (p = 0.016), but there was no significant difference between SL and ALT16 (p = 0.125). BRS at 100W decreased from SL to ALT1 (p = 0.035) and ALT16 (p = 0.025), whilst there was no significant difference between ALT1 and ALT16 (p = 0.682). At 130W, 160W, and Max, there were no differences between SL, ALT1, and ALT16 .At SL, ALT1, and ALT16, BRS decreased between pre-exercise and 70W ; at SL and ALT16 during cl-40 ; at SL, ALT1 and ALT16 during REB ; and at SL during HVE (p = 0.002). BRS also decreased at ALT1 and ALT16 compared to SL after removal of respiration.Table p < 0.001, p = 0.003, p = 0.022, p = 0.001, respectively). At 100W, 130W, 160W and Max, removing respiration did not significantly alter BRS .Table 2 at 40 mmHg, but not after acclimatization to hypoxia; and (3) the decrease in BRS during exercise was greater at low exercise intensities only, in acute hypoxia compared to normoxia; after acclimatization to hypoxia, the decrease in BRS during exercise returned toward SL values.This study is the first to report BRS systematically, during acute and chronic exposure to 5,260 m, at rest and during dynamic exercise to exhaustion. The main findings were (1) BRS decreased in acute hypoxia and this decrease was exacerbated after acclimatization to hypoxia; (2) the decrease in acute hypoxia was reversible when clamping end-tidal CO2 chemoreceptors, therefore leaving the majority of the input of the chemoreflex to CO2 receptors. The immediate reversal of the decrease in BRS during cl-40 at ALT1  indicated that reduced CO2 is likely the main trigger for BRS decrease in acute hypoxia. The reduction in BRS in acute hypoxia is probably partially mediated by the carotid body chemoreceptors .The decrease in BRS at rest in acute hypoxia confirmed previous findings in the literature , therefore evidencing the reset of the CO2 chemoreceptors toward low PaCO2.In normoxia HVE toward a PETCOe Figure . In chro2 and concomitant rapid drop in blood pHa. To be able to correct such changes in BP, BRS should have augmented by a very large margin, probably more than it is physically able to do, hence the enormous increase in BP. Additionally, in hypoxic conditions BRS decreases, which is not in favor of the ability to correct large increases in BP.Rebreathing did not affect BRS but provoked large increases in BP at ALT1 and ALT16, as if the capacity of the baroreflex was overwhelmed by the very high levels of COBRS decreases when HR increases. Intrinsic HR does not seem to change significantly with acute hypoxia  suggesting that the ANS mechanisms must be responsible for HR (and BRS) changes at altitude  The Clamp-40 condition normalizes CO2, however the respiratory frequency is increased, which we address further in a dedicated section; (3) The REB condition shows BRS with high levels of CO2 and increased respiratory frequency; (4) Finally, the HVE condition shows BRS with low levels of CO2 and an effect from central command on breathing pattern. The results obtained in these conditions clearly show the effects of CO2 on the chemoreflex, but cannot exclude a partial role of respiratory frequency, tidal volume (lung stretch) or central command. A previous study tightly controlling ventilation for tidal volume and respiratory frequency showed that hyperpnoea did not influence BRS whilst hypoxia and hypocapnia did  which is independent of the hypoxic and hypocapnic conditions inherent to altitude exposure .Our results can be summarized as follows: (1) the rest condition shows BRS with low O2 were targeted for hypo and hypercapnic conditions in SL, ALT1, and ALT16, for example making cl-40 a normocapnic condition in SL but a hypercapnic condition in ALT1 and ALT16. The goal of this experimental design was to demonstrate an absolute reset of the chemoreceptors after acclimatization. That is, after prolonged exposure to low PaCO2, the CO2 chemoreceptors activity would be centered on a low PaCO2. Suddenly returning to a PaCO2 of 40 mmHg immediately restores BRS in ALT1 therefore indicating no resetting, whilst the same sudden exposure at ALT16 does not restore BRS to SL value indicating a resetting. Choosing what values to target is not easy. Depending on the question asked, different targets would be appropriate, for example, using same delta PaCO2 from normocapnia in each condition , was the only way to demonstrate the absolute reset.Similar PaCO2 available in the present study allows us to assess how much the baroreflex can control BP from HVE to REB, notably by taking the slope of this relationship, which can be used to assess baroreflex gain. Figure 2 changes. Accordingly, a previous study showed that respiratory-induced variations in blood pressure are greater in hypoxic conditions  whilst in ALT1 there was a sudden drop between rest and 70W, BRS remaining around its 70W value for the subsequent exercise stages  whilst it greatly diminished at SL  therefore decreasing BRS. Even if removing respiration modified BRS response in HVE, we still contend that PaCO2 is essential, whilst respiratory frequency probably comes as a second factor. Overall it seems that the effects of the rest protocol on BRS were mainly due to modifications occurring in the arc reflex controlling the baroreflex rather than to an artifact linked to the respiratory frequency.One of the main physiological factors affecting BRS is the respiratory frequency . Comparable trends between those methods have been reported (Laude et al., Also, using the sequence method, positive and negative sequences were isolated and treated separately, which did not significantly change the results. When more than three consecutive points constituting a sequence were found BRS was calculated with and without overlap of those points, which again did not significantly change the results. The sequence method has a number of limits concerning the criteria about what sequences can be used, but we have taken as many precautions as we could to ensure that the reported BRS represent as fairly as possible what is actually going on during acclimatization to altitude.Finally, neural and molecular mechanisms of the BRS reduction in acute and chronic hypoxia were also beyond the scope of the present work. Future studies are needed to further explore the mechanisms.2 at sea level values in acute hypoxia, but not in chronic hypoxia, we contend the following mechanisms to explain our findings: during acute exposure, a hypoxia-induced heightened activation of the CO2 chemoreceptors would be the main trigger, whilst in chronic hypoxia an increase in CO2 chemoreceptor sensitivity might be the principal cause of the attenuated BRS. The increased sensitivity during acclimatization is not immediately reversible with acute hyperoxia. Future studies, using a tight control of the respiratory pattern, are needed to determine the nature of the neural and molecular changes underlying those decreases in BRS. During incremental exercise, the BRS decrease is similar in normoxia and chronic hypoxia whilst it happens at lower intensity and with larger drop in acute hypoxia.This study is the first to report a decrease in BRS in acute hypoxia that was exacerbated after acclimatization to 5,260 m. Because this decrease in acute hypoxia was reversible when clamping COAL, AS, BK, and RR conceived and designed the experiments. NB and AS performed the experiments NB, SY, and J-MV analyzed data. NB interpreted the data, wrote the first version of the manuscript, and prepared the figures. SY, AS, AL, RR, J-MV, and BK revised the manuscript. All authors approved final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Four natural variants of PFN1 were described as related to ALS, the most common adult-onset motor neuron disorder. However, the pathological mechanism of PFN1 in ALS is not yet completely understood. The goal of this work is to thoroughly analyze the effects of the ALS-related mutations on PFN1 structure and function using computational simulations. Here, PhD-SNP, PMUT, PolyPhen-2, SIFT, SNAP, SNPS&GO, SAAP, nsSNPAnalyzer, SNPeffect4.0 and I-Mutant2.0 were used to predict the functional and stability effects of PFN1 mutations. ConSurf was used for the evolutionary conservation analysis, and GROMACS was used to perform the MD simulations. The mutations C71G, M114T, and G118V, but not E117G, were predicted as deleterious by most of the functional prediction algorithms that were used. The stability prediction indicated that the ALS-related mutations could destabilize PFN1. The ConSurf analysis indicated that the mutation C71G, M114T, E117G, and G118V occur in highly conserved positions. The MD results indicated that the studied mutations could affect the PFN1 flexibility at the actin and PLP-binding domains, and consequently, their intermolecular interactions. It may be therefore related to the functional impairment of PFN1 upon C71G, M114T, E117G and G118V mutations, and their involvement in ALS development. We also developed a database, SNPMOL ( Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that progressively affects the upper and lower motor neurons, leading to muscular atrophy and paralysis due to neuron injury and death . ALS is PFN1 gene were described as being involved with fALS development [PFN1 gene encodes profilin 1 (PFN1), a 140-residues ubiquitously expressed [Four non-synonymous single nucleotide variants (nsSNVs) in the elopment ,7. Interelopment . The PFNxpressed  cytosolixpressed  that plaxpressed .PFN1 is crucial for monomeric actin conversion into filamentous actin, as it sequestrates cytosolic actin monomers and catalyzes the assembly of monomers into filamentous-actin . PFN1 alMoreover, PFN1 is involved in many cellular processes  through ALS-related mutations in PFN1 are known to cause cytoskeletal disruption in neurons , resultiin silico analysis, allows the prediction of SNV effects in a faster, cheaper and efficient way [in silico to identify pharmacological targets for relevant treatments and to gain insight into their molecular mechanisms of pathology [Next-generation sequencing experiments reveal millions of novel SNVs . Howeverient way . The comient way  to be naient way . Moreoveathology . In thisathology  and an eathology ,23.Optimal protein-drug binding is crucial to achieving the desired therapeutic effects, as well as to minimizing associated side effects and toxicity of drugs. Protein-drug interactions are determined by local biochemical and structural features of drug-binding cavities . ResidueIn this work, we applied computational simulations, following the methodology previously established by our group ,27,28, tSince these nsSNVs may influence drug selection, dosing, and adverse effects, understanding their effects on PFN1 structure and function may help the development of new drugs and personalized therapies for ALS .The sequence and natural variants of PFN1 were retrieved from the UniProt database (UniProt ID: P07737) .The functional and stability effects of the PFN1 nsSNVs were predicted using the following algorithms: PhD-SNP , PMUT 3, PolyPheThe evolutionary conservation analysis of PFN1 was performed using the ConSurf server, which determined the degree of evolutionary conservation of each amino-acid of PFN1 . The folMD simulations of the wild-type PNF1 and its natural variants: C71G, M114T, E117G and G118V, were performed using the GROMACS 2018.2 package . MutatorFollowing the methodology previously established by our group , we sele+ and Cl− ions and minimized for 5000 steps using the steepest descent method.The structures were solvated using the TIP3P water model inside a dodecahedral box of dimensions 44 x 37 x 34 Å. The systems were neutralized by adding Nanumber of particles, volume, and temperature), NPT  and production. The NVT ensemble was followed by the NPT ensemble at 1 atmosphere and temperature of 300 K for the duration of 100 ps [After system minimization, three other steps were carried out in the MD simulations: NVT (constant f 100 ps . ParrineThe production simulations were performed in triplicates at 300 K for the duration of 100 ns for the wild-type PFN1 and its variants. The LINCS (linear constraint solver) algorithm was applied to constrain covalent bonds , and theStructural parameters of the wild-type PFN1 and its variants were accessed through the root-mean-square-deviation (RMSD), root-mean square-fluctuation (RMSF), radius of gyration (Rg), intramolecular hydrogen bonds (Hb) and B-factor analyses. These parameters were calculated separately for each triplicate trajectory. The means for each triplicate in the RMSD, RMSF, RG and intramolecular Hb analyses were calculated and plotted using the ggplot2 package in R software .gmx hbond, gmx rms, gmx rmsf, and gmx gyrate.The following GROMACS distribution programs were used to perform the MD analyses: The results presented in this paper were compiled and stored on SNPMOL, an online database. The human-curated database of PFN1 was developed using JSmol, an HTML5-based equivalent of Jmol .PFN1 gene [PFN1 is a 140-amino acid cytoskeletal protein that is coded by the FN1 gene , which iFN1 gene . Four naFN1 gene  structurFN1 gene ,29.PFN1 protein has two important domains: an actin-binding domain and a poly-L-proline (PLP) binding domain , which aA schematic representation of PFN1 containing its natural variants and important domains are shown in The functional and structural consequences of nsSNVs at the protein level can be predicted using computational simulations . The effIn the test case we performed, the algorithms: SAAP, SIFT, SNAP, and SNPs&GO, showed the best accuracy amongst the used functional prediction algorithms. They were able to detect the known deleterious effects of the studied PFN1 mutations . The PhDDespite the high accuracy in detecting the known deleterious effects of C71G, M114T and G118V, the algorithms that were used showed low accuracy in predicting the known deleterious effect of the E117G variant of PFN1. These algorithms apply different strategies to make predictions . Moreoveet al. [in vitro [The effects of amino acid substitutions on PFN1 stability were further analyzed using the FoldX  and I-Muet al.  showed tin vitro ,16.The divergent results presented in the stability prediction analysis may occur due to the different prediction strategies applied by I-Mutant2.0 and FoldX ,38. Whilin vitro [Lastly, the effects of amino acid substitutions on PFN1 aggregation tendency (TANGO), amyloid propensity (WALTZ), and chaperone binding tendency (LIMBO) were analyzed using the SNPeffect4.0 algorithm . Accordiin vitro .ConSurf is a bioinformatics tool that analyzes the evolutionary conservation of protein regions and calculates the conservation score of each amino acid based on statistical inference methods, machine learning, and multiple sequence alignments. The conservation scores are associated with a coloring scheme and projected on the protein’s surface. ConSurf is widely used to detect functional regions on proteins as important residues are usually conserved throughout evolution .The evolutionary conservation score of each amino acid of PFN1 was calculated by ConSurf . Highly In addition to showing the conservation scores of PFN1 mutated sites, the ConSurf analysis also provided an interesting graphical representation in which the conservation scores for amino acid of PFN1 is plotted on its three-dimensional protein structure, highlighting its conserved regions and structural proximities.in silico method of solving Newtonian equations of motions for a given set of atoms [et al., 2014).MD is an of atoms . This meof atoms , the molof atoms . As chanof atoms ,58,59, tTo further analyze the effects of PFN1 nsSNVs we carried out MD simulations of the wild-type PFN1 and its four natural variants  using thRMSD is a useful parameter to analyze the structure motions over time and to determine its spatial convergence throughout the simulation ,56,60. AThe RMSD analysis, however, only provides information about the overall structure fluctuations . We thenThe C71G variant presented increased flexibility at the actin-binding domain and adjacent regions, especially at the region comprised between the residues 50–56 and 75–79. It also had an increased flexibility at the N and C-terminal helices of the PLP-binding domain. In addition, this variant presented increased flexibility especially at the coils regions.The M114T variant, in turn, presented reduced flexibility at the actin-binding domain and adjacent regions, especially at the region comprised between the residues 73–82 and 92–94. It also had increased flexibility at the N-terminal helix of the PLP-binding domain and decreased flexibility at the C-terminal helix of the PLP-binding domain. Moreover, this variant presented decreased flexibility especially at the coil and helices regions.The E117G variant presented reduced flexibility at the actin-binding domain and adjacent regions, especially at the region comprised between the residues 64–68 and 77–81. It also had increased flexibility at the N-terminal helix of the PLP-binding domain and an increased flexibility in a region adjacent to the C-terminal helix of the PLP-binding domain (residues 116–120). In addition, this variant presented decreased flexibility especially at the coil regions, except for the region comprised between the residues 36–42, which had an increased flexibility when compared to the wild-type.The G118V, in turn, presented decreased flexibility in adjacent regions to the actin-binding domains, except for the region comprised between the residues (93–96). It also had an increased flexibility at the N-terminal helix of the PLP-binding domain. Moreover, this variant presented decreased flexibility especially at the coil regions, except for the region comprised between the residues 36–50, which had an increased flexibility when compared to the wild-type PFN1.Since protein flexibility has a wide influence on the thermodynamics of binding ,63 the fThe structural flexibility can also be assessed throughout the simulation by analyzing the B-factor . As wellThe C71G variant presented increased flexibility at adjacent regions to the actin-binding domain of PFN1. The M114T variant, in turn, presented decreased flexibility at the actin-binding domain and adjacent regions, as well as increased flexibility at the PLP-binding domain and adjacent regions. The E117G variant presented decreased flexibility in adjacent regions of the actin-binding domain, as well as increased flexibility in adjacent regions of the PLP-binding domain. The G118V variant, in turn, presented decreased flexibility at the actin binding domain and adjacent regions, except for the loop that connects the fifth and sixth beta-strands, which presented increased flexibility when compared to the wild-type. In addition to reaffirming the flexibility alterations observed in the RMSF analysis, B-factor analysis also provided an interesting graphical representation of structural flexibility.The Rg analysis is useful for describing the overall dimensions of protein structures throughout the simulation ,52,61. AThe stability of protein structures can be assessed throughout the simulation by analyzing the formation of intramolecular hydrogen bonds . As showThe MD analyzes therefore suggested that the studied mutations could affect the PFN1 flexibility at the actin and PLP-binding domains, and, consequently, their intermolecular interactions. It may explain the known altered binding ability of the C71G, M114T, E117G and G118V variants . MoreoveVisualization and analysis of intricate 3D structures of macromolecules, such as proteins, are essential to provide insights into their biological processes . For suchttp://www.snpmol.org/). The database is freely available for biologists and clinicians to exploit the PFN1 variants described here and their functional and structural alterations. SNPMOL interface allows users to quickly retrieve and analyze the predicted effects and theoretical models of PFN1 variants. Understanding their effects on PFN1 structure and function may help the development of new drugs and treatments for ALS [The PFN1 results presented in this paper are stored in SNPMOL, the human-curated database developed by our group , containing the results presented in this paper for biologists and clinicians to exploit PFN1 and its natural variants. Furthermore, we can conclude that computational simulations are an effective approach for the study of disease-related mutations, as well as an important ally of the experimental methods.In this paper, we analyzed the effects of PFN1 nsSNVs using ten functional and stability prediction algorithms, an evolutionary algorithm, and MD simulations. The functional prediction algorithms used here showed high accuracy in detecting the known deleterious potential of the C71G, M114T, and G118V mutations, but not E117G. The functional prediction analysis also showed that it is important to use a variety of algorithms to determine the deleterious effects of mutations. The stability prediction suggested that the ALS-related mutations could destabilize PFN1. The evolutionary conservation analysis indicated that the mutations C71G, M114T, E117G, and G118V occur in highly conserved positions. The MD analyses suggested that the studied mutations could affect the PFN1 flexibility at the actin and PLP-binding domains, and consequently, their intermolecular interactions. It may be therefore related to the functional impairment of PFN1 upon C71G, M114T, E117G and G118V mutations, and their involvement in ALS development. We also developed a human-curated database, SNPMOL (


These findings were confirmed for the specific diseases: third-generation cephalosporin use decreased for both UTI  and LRTI ; the duration of intravenous therapy decreased . Median LRTI treatment duration was 9.2 days in 2008 and 6.6 in 2015 (p < 0.001); penicillin prescriptions were more narrow in spectrum (p = 0.02).There is growing attention for antimicrobial stewardship in paediatrics. Currently, little is known about secondary care antibiotic practice. We analysed trends in time with respect to inpatient antibiotic use in a secondary paediatric care setting. Total inpatient antibiotic consumption per year (2010–2015) and antibiotic prescriptions for urinary tract infection (UTI) and lower respiratory tract infection (LRTI) were analysed. Variables were total, antibiotic-specific, and intravenous days of therapy (DOT/100PD) and for UTI/LRTI treatment type, route and duration. Third-generation cephalosporin use decreased  contains supplementary material, which is available to authorized users. Given the emergence of antimicrobial resistance and the impact on morbidity and mortality rates, as well as increased health care costs, appropriate antimicrobial use has become extremely important . AntimicASP are also important in paediatrics. Children and neonates receive antibiotics frequently, because of the nature of childhood diseases , 13, 21.There has been limited development of ASP in paediatrics, due to the lack of a standardised method of comparing antimicrobial use between hospitals . Days ofCurrently, little is known about paediatric antibiotic use in the Netherlands and official ASP in paediatrics have not been introduced yet. Compared to other European countries, the numbers of antimicrobial prescriptions are low, but the number of inappropriate prescriptions is still worrying and antibiotic resistance is rising steadily . PatternThis study was performed in the paediatric and neonatal ward of a secondary care urban teaching hospital in Rotterdam, the Netherlands . About 55% of the admissions on the neonatology wards are neonates born < 37 weeks of gestational age, but all are > 30 weeks of postmenstrual age. The most common infectious syndromes on the paediatric ward that require antibiotics are fever of unknown origin, lower respiratory tract infection, and urinary tract infection. In this hospital; since 2014, two consultant paediatricians regularly discuss antibiotic management and local guidelines with both the senior and junior doctors prior and after initiation of antimicrobial therapy. Moreover, in 2014, new protocols on lower respiratory and urinary tract infection were introduced. Two specific topics were studied: the total inpatient antibiotic use per year (2010 to 2015) and antibiotic prescriptions for two important infectious discharge diagnoses on the paediatric ward, namely lower respiratory tract infection (LRTI) and urinary tract infection (UTI) (2008 and 2015). The local ethics review board deemed formal ethical approval of the study unnecessary because of the absence of risk for patients. The data were stored anonymously.Admission data for the paediatric and neonatology wards were obtained from the hospital database. Admission data from before 2010 could unfortunately not be reliably obtained. Single day admissions for diagnostic tests, chemotherapy courses, or blood transfusions were excluded. Children admitted for a non-paediatric speciality (e.g. surgery or ENT) were excluded because the paediatricians were not the primary responsible doctors. Since 2008, the pharmacy has stored all prescription data electronically. All prescriptions with the Anatomical Therapeutic Chemical classification-code (ATC) J01 for antibacterials were extracted from its computer system.For the first part of the study, both databases were matched and the patient medical file was manually checked in case of differences in admission and antibiotic therapy duration. All files of children on co-trimoxazole (TMP/SMX) and trimethoprim therapy were checked manually; prophylactic prescriptions were excluded. Paediatric oncology patients (mainly leukaemia) were excluded from the analyses because in 2013, oncology treatment was discontinued in this hospital because of local policies. For the second part of the study, the pharmacy database was used to identify the children who were prescribed antibiotics for a LRTI or UTI in both 2008 (the first year data available) and 2015. Data were then retrieved from the medical files of these children.For the estimation of the total inpatient antibiotic use, variables were obtained per year and included age at admission, total number of admission days per year, days of antibiotic therapy . The DOT per patient was calculated as the sum of antibiotics used per patient per day. Antibiotic consumption for the specific wards was expressed as DOT per 100 (in) patient-days (DOT/100PD) and estimated separately for every year. For the second part of the study, the following variables were noted: patient age, diagnostic parameters , comorbidity, admission duration, antibiotic prescriptions . The total antibiotic prescription was studied, including discharge medication.Data were stored in an online research database (Castor EDC). Statistical analysis was performed with SPSS 23 (IBM SPSS Statistics).U test, the chi-square, or Fisher’s exact test. p values < 0.05 were considered statistically significant.Demographical data were analysed by descriptive statistics, including median and mean values with interquartile range or standard deviation for numerical data and proportions for categorical data. The Shapiro-Wilk test was used to test for normality of the distribution. Trends in DOT/100PD  for the period of 2010–2015 were analysed by time series analysis (ARIMA) for non-independent data. The data of the 2008/2015 patient cohorts were compared by using the Mann-Whitney The number of admissions and total admission duration per year are shown in Table R2 = 0.17, p = 0.42). The combined penicillin and cephalosporin DOT/100PD increased from 20 to 32 but no significant differences in the DOT/100PD were calculated because of strong fluctuations. Antibiotics were mainly given intravenously .The total DOT/100PD varied over the years from 22.9 to 34.4 , antibiotic therapy was discontinued within 48 h because of negative blood culture, re-assuring clinical condition and inflammatory parameters.R2 = 2.7E−6, p = 0.99), although 2015 seems to show a decrease in DOT/100PD compared to the previous years. The beta-lactam penicillins (J01C) and cephalosporins (J01D) represent 73–84% of the total yearly DOT. Most frequently prescribed penicillins were amoxicillin and amoxicillin/clavulanic acid; no significant differences in the DOT were seen in this group of antibiotics over the years . Most frequently prescribed cephalosporins were cefotaxime and ceftriaxone. Cephalosporin prescriptions showed a significant decrease . The macrolide/lincosamide (J01F) group is the third most often prescribed group of antibiotics. No decrease was observed for this group .The total DOT/100PD varied over the years from 28.6 to 44.9 .The intravenous DOT/100PD varied from 9.9 to 20.5 and the oral DOT/100PD varied from 13.0 to 26.8 per year .All patients admitted in 2008 and 2015 with a UTI or a LRTI were reviewed. The latter group consisted of patients with the discharge codes of pneumonia or bronchiolitis.E. coli resistance for penicillins, cephalosporins, and TMP/SMX occurred more often in 2010 compared to 2015.The baseline and microbiological characteristics of the patients included for urinary tract infection are shown in supplementary Table p = 0.02).In Table 93 vs. 45%, p = 0.002) and replaced by penicillin antibiotics (amoxicillin/clavulanic acid). De-escalation and oral switch therapy were performed in 87 and 88% of patients in 2008, for 100% of patients in 2015 when deemed appropriate.The empiric antibiotic therapy for both years is shown in Fig. The demographic and clinical characteristics of the patients included for LRTI are shown in supplementary Table The 2008 cohort had higher median inflammatory parameters; however, the number of patients tested was significantly lower in 2015. The number of additional investigations  for LRTI decreased significantly over the years. Despite the decrease in additional investigations for LRTI, no difference was seen in the percentage of patients that started on antibiotics for LRTI.p < 0.001). A significant decrease was also noticed in total duration of intravenous treatment (p < 0.001). Intravenous-to-oral switch was performed for 96% of patients in 2008 and 100% in 2015 when deemed appropriate. In 2015, the majority of patients initiated with oral antibiotics (91.8 vs. 64.4% in 2008).In Table p = 0.18). However, the penicillin prescriptions were significantly more often smaller in spectrum in 2015 (51% amoxicillin and 49% amoxicillin/clavulanic acid in 2008 vs. 72% amoxicillin and 28% amoxicillin/clavulanic acid in 2015 (p = 0.02)).Figure This study is the first that focused on the trend of antibiotic prescriptions in time in a secondary paediatric care setting in Northern Europe. Although no significant trend in total DOT/100PD could be observed on the paediatric and neonatology ward, trends regarding specific antibiotic classes, route, and duration were observed. This setting differs from a tertiary setting because of the absence of haemato-oncology and intensive care patients: a population with a high antibiotic consumption. However, since antibiotics are frequently prescribed in all fields of paediatrics, it is important to investigate all possibilities for reducing antibiotic use in the light of antimicrobial stewardship.The average DOT/100PD for the paediatric ward in our study was between 35 and 45. Recent data from smaller and larger American community hospitals showed the median DOT/100PD over 2011–2013 for paediatric wards including surgery to be 49, somewhat higher compared to our figures . IdeallyThe year 2015 had a somewhat lower, although not statistically significant, paediatric DOT/100PD compared to the other years. Since 2014, antibiotic case rounds and teaching have been installed in this hospital to make the staff and junior doctors more aware of the possible consequences of antimicrobial treatment. During the case rounds, all infectious patients are reviewed and indications and choices for antimicrobial therapy are discussed. Additionally, antibiotic prescriptions are discussed at the morning report. Whether this will lead to a sustained trend or is the result of temporal changes in case mix should be analysed in the future. The neonatology department showed a strongly fluctuating DOT/100PD with a non-significant increase from 24 to 34; this might be explained by case mix (e.g. higher patient turnover in 2015).Third-generation cephalosporin consumption and the intravenous route for the paediatric ward were significantly lower over the years; this was shown both in the global DOT study and the supporting UTI/LRTI study.Unfortunately, admission figures to calculate the patient days from before 2010 could not be reliably obtained.E. coli isolates was still low (< 20%) in this hospital, but might change in the future. Additionally, a national childhood community-acquired pneumonia (CAP) protocol was published, emphasising the limited value of additional investigations and the shorter duration of antibiotics (5 days) needed for non-complicated LRTI. In our setting, regular feedback and local and national protocols have definitely created awareness amongst doctors regarding the preferred spectrum, route, and duration of antibiotics for LRTI. Moreover, significantly less diagnostic tests were requested. In other studies, identical results were observed [This study identified that the total duration of antibiotic therapy in patients with LRTI significantly decreased. In addition, intravenous antibiotic treatment decreased in both patients with LRTI and UTI. A recent review emphasised the importance of switching to oral agents as soon as clinical improvement has occurred . The obsobserved , 22. AftStrikingly, the percentage of patients who were administered any antibiotic for LRTI did not decrease in our study, but the number of additional investigations for LRTI decreased significantly. Education about the aetiology of LRTI in infants could possibly further reduce the percentage of infants receiving antibiotics for a LRTI, which are mostly viral in nature.The strength and originality of this study is that it focuses on both total and detailed aspects of antibiotic prescriptions. Apart from analysing inpatient antibiotic use by DOT, this study also evaluated the in- and outpatient antibiotic use for one infectious disease episode, which together reflect the full antibiotic prescription. Both analyses showed a decrease in third-generation cephalosporin use over the years. Although the specific diseases sub study only compared 2 years, the outcomes are supported by the DOT study and the interventions in the context of ASP. This study was restricted to one centre; consequently, implementations and outcomes might not be extrapolated to other clinical centres. Ideally DOT’s should be calculated (semi)-automatically by hospital software programs. This allows for comparing DOT’s easily between time periods and hospitals. However, using DOT’s in paediatrics also has some limitations. DOT’s include the assumption that antibiotic dosing is appropriate, and the quality of the prescriptions cannot be assessed, e.g. broad-spectrum monotherapy leading to fewer DOT’s compared to narrow-spectrum combination therapy.Future research should be aimed towards evaluating comparable care units with comparable resistance rates and understanding differences in antibiotic prescribing and barriers to rational prescribing. This will aid in developing national antimicrobial stewardship priorities and allow for more selective antimicrobial use.Concluding, in this secondary setting, a significant decrease in overall inpatient cephalosporin use and cephalosporin use for UTI and LRTI was identified. No trend in total DOT/100PD could be observed for both the neonatal and paediatric wards. However, the total intravenous administration was shortened in time. The total treatment duration and proportion of broad-spectrum beta-lactam antibiotics significantly decreased for LRTI. Increased awareness of antimicrobial resistance and a selection of interventions in the context of antimicrobial stewardship could explain this decrease in antibiotic use. This decrease in antibiotic use is feasible and important in non-tertiary paediatric wards and outpatient clinics.ESM 1(DOCX 159 kb)ESM 2(DOCX 79 kb)ESM 3(DOCX 80.9 kb)


The organic/inorganic hybrid materials from polyhedral oligomeric silsesquioxane  and polybenzoxazine (PBZ) have received much interesting recently due to their excellent thermal and mechanical properties, flame retardance, low dielectric constant, well-defined inorganic framework at nanosized scale level, and higher performance relative to those of non-hybrid PBZs. This review describes the synthesis, dielectric constants, and thermal, rheological, and mechanical properties of covalently bonded mono- and multifunctionalized benzoxazine POSS hybrids, other functionalized benzoxazine POSS derivatives, and non-covalently (hydrogen) bonded benzoxazine POSS composites. T. T109]. We also developed a new PBZ/POSS nanocomposite prepared from the reactions of a multifunctional vinyl-terminated POSS derivative (VBa-POSS) and the VBa BZ monomer at various compositions  110]. T. T110]. In addition, these poly(VBa/VBa-POSS) hybrid materials displayed enhanced mechanical and thermal properties after curing  because Moreover, scanning and transmission electron microscopy of cured VBa/VBa-POSS (70/30) revealed uniformly dispersed spherical POSS NPs throughout the PBz matrix, without any discernible phase separation a,b.et al. ,11749,11Tg) of PA-T/OBA-POSS nanocomposites were higher than that of neat OBA-POSS and (ii) the value of Tg of the hybrid materials decreased with the increase of OBA-POSS contents, because of the aggregation of POSS units and the occurrence of macro-phase separation. In addition, TGA results revealed that upon incorporation 20 wt % of OBA-POSS, the thermal stability of PA-T was greater than those neat PA-T and OBA-POSS, presumably because of intermolecular hydrogen-bonding interaction and formation of network structure PA-T and inorganic POSS. The TEM image and selected area electron diffraction (SAED) pattern of pure PA-T and the PA-T/OBA-POSS nanocomposites ((i) the glass transition temperatures  linked throughout T-functionalized PBZ matrix, stabilized via the multiple hydrogen bonding and the π–π stacking interaction  as presented in k materials .Many different kinds of PBZ/POSS nanocomposites have been prepared displaying superior thermal stability, higher glass transition and degradation temperatures, and lower dielectric constants than those of pristine BZ monomers and PBZ polymers. POSS NPs have several attractive features that make them attractive alternatives to traditional fillers or clay, including well-defined structures, an absence of trace metals, highly uniform dispersion within polymer matrices, and good interfacial interactions with the polymer segments. The incorporation POSS NPs into PBZ matrix has been performed through both chemical crosslinking and physical blending. In this review, we have discussed several different classes of PBZ/POSS hybrid materials formed from mono- and multi-functionalized POSS through both covalent and noncovalent bonding. According to literature reviews, they reported that the incorporation POSS nanoparticles and DNA bases into polybenzoxazine could be enhanced the thermal and mechanical properties of PBZ. The preparation of such organic/inorganic hybrid materials containing POSS and PBZs remains one of the hottest topics in academic and industrial research because of their potential applications in, for example, drug delivery and microelectronic devices, and for their use as low-


The compressive stress of concrete is used as a design variable for reinforced concrete structures in design standards. However, as the performance-based design is being used with increasing varieties and strengths of concrete and reinforcement bars, mechanical properties other than the compressive stress of concrete are sometimes used as major design variables. In particular, the evaluation of the mechanical properties of concrete is crucial when using fiber-reinforced concrete. Studies of high volume fractions in established compressive behavior prediction equations are insufficient compared to studies of conventional fiber-reinforced concrete. Furthermore, existing prediction equations for the mechanical properties of high-performance fiber-reinforced cementitious composite and high-strength concrete have limitations in terms of the strength and characteristics of contained fibers  even though the stress-strain relationship is determined by these factors. Therefore, this study developed a high-performance slurry-infiltrated fiber-reinforced cementitious composite that could prevent the fiber ball phenomenon, a disadvantage of conventional fiber-reinforced concrete, and maximize the fiber volume fraction. Then, the behavior characteristics under compressive stress were analyzed for fiber volume fractions of 4%, 5%, and 6%. Conventional concrete, which is commonly used as a construction material, does not have an adequate resistance level in terms of collisions or explosive loads; thus, concerns are arising about human casualties from the brittle fracturing when reinforced concrete (RC) structures explode ,2. With As the compressive stress of concrete increases, the elastic area becomes larger, and the load-bearing capacity decreases sharply after the manifestation of the maximum strength . The strPreviously proposed stress-strain relationship equations have been modified based on the research results of Propovics  and SargHere, A review of existing studies’ results revealed that the mechanical properties of concrete, including the maximum strength, elastic modulus, and strain at maximum strength, need to be defined before the stress-strain relationship of concrete under compressive stress can be defined. Of these, the elastic modulus is an important design variable for a concrete structure and has a significant effect on the stress-strain relationship. The elastic modulus tends to increase with the strength of the matrix and is proportional to the square root or cube root of the compressive stress of the matrix . The strSteel fibers are mixed for increasing the tensile strength of concrete; however, they also change the mechanical properties in compressive stress measurements . The mixThe strengthening effect of fibers is realized as an algebraic sum or a magnification of the fiber reinforcing effects for the elastic modulus of the matrix alone ,17. The Furthermore, a prediction equation for estimating the elastic modulus of concrete is generally proposed on the basis of an empirical formula using regression analysis of experimental data of various ranges. The elastic modulus of conventional concrete can be estimated using the compressive stress, unit weight of concrete, etc. ,10,11,15We examined established prediction equations and found that studies of high volume fractions remained insufficient compared to studies of conventional fiber-reinforced concrete. Therefore, we conducted an experimental study of the behavior characteristics under compressive stress to examine the mechanical properties of SIFRCCs with a high volume fraction of fibers.This study used type 1 ordinary Portland cement (OPC), the physical and chemical properties of which are listed in To realize high-performance and high-strength filling slurry, this study used silica fume , the physical and chemical properties of which are listed in To improve the filling performance of the slurry and reduce material separation, fine aggregates with diameters of 0.5 mm or smaller were used. No coarse aggregates were used.To improve the filling performance of the slurry, a polycarboxylic acid, the high-performance water reducing agent with high dispersion performance, was used. The admixture used in this experiment had high strength and high flow characteristics, as well as excellent unit water quantity reduction property and material separation resistance. 3 and a tensile strength of 1200 MPa. Double-hook steel fibers for conventional concrete with a diameter of 0.75 mm, length of 60 mm, and an aspect ratio of 80 were used. These steel fibers had a density of 7.8 g/cmSIFRCCs that can accommodate a high volume fraction of steel fibers were developed in this study to prevent the fiber ball phenomenon, the main disadvantage of conventional fiber-reinforced concrete, and maximize the fiber volume fraction ,24,25,26OPC, fine aggregate, water, superplasticizer, and additional silica fume were mixed to prepare a slurry. The water binder ratio was 0.35. First, the cylindrical mold with diameters of 100 mm and heights of 200 mm were filled with steel fiber with respect to volume fraction. Randomly sprinkled steel fibers in the mold should not overfill the depth of mold and level up as much as possible ,26. The To analyze the compressive behavior characteristics of the SIFRCCs for fiber volume fractions of 4% (312 kg per cubic meters), 5% (390 kg per cubic meters), and 6% (468 kg per cubic meters), cylindrical specimens with diameters of 100 mm and heights of 200 mm were fabricated with respect to the mixing ratio of each variable, as listed in A universal testing machine (UTM) was used to study the compressive behavior of specimens. The specimens were placed centrally between the two compression plates, such that the center of moving head was vertically above the center of specimen, as shown in Many studies have shown that the mixing of fibers in fiber-reinforced concrete generally does not influence the compressive stress of the matrix itself. The compression experiment results of Shah and Rangan  reportedThe compressive fracture of conventional fiber-reinforced concrete was caused not by the yield or drawing of fibers but by the fracture of a matrix with relatively low strength before fibers play a structural role. Accordingly, the compressive stress was considered to improve with the fiber volume fraction as sufficient adhesion occurred between the fiber and the high-strength cementitious matrix used in this study. Micro-cracks were caused in conventional concrete and fiber-reinforced concrete by the interface properties of the coarse aggregates and the cement paste. For SIFRCCs, a composite made of fine particles with no coarse aggregates, cracks at the interface could be reduced, and the adhesion performance of the filling slurry and steel fibers could be improved. Therefore, the effect of steel fibers, such as the fiber volume fraction, rather than the effect of the compressive stress caused by matrix fracture was considered to be reflected.The elastic modulus experiment results indicated that the elastic modulus showed insignificant differences with the fiber volume fraction; its value remained ~28 GPa. This was in contrast to the compressive stress experiment results. However, as shown in Poisson’s ratio of conventional concrete is known to be 0.16–0.20; that of fiber-reinforced high-strength concrete is expected to be higher. The stress-strain experiment results of SIFRCCs with respect to the fiber volume fraction showed relatively high strain values for all variables compared to that of conventional concrete, with a strain of 0.008 or higher at peak stress. However, the strain at peak stress also increased with the fiber volume fraction. In contrast to the above-mentioned compressive stress experiment results with respect to the fiber volume fraction, the compressive stress for the fiber volume fraction of 6% increased slightly by approximately 1.11 and 1.27 times compared to those for fiber volume fractions of 5% and 4%, respectively. However, the strain at peak stress for the fiber volume fraction of 6% increased significantly by 1.51 and 2.67 times compared to those for fiber volume fractions of 5% and 4%, respectively. The compressive fracture patterns obtained by performing the compressive behavior experiment, shown in (1)The static compressive behavior characteristics with respect to the fiber volume fraction of SIFRCCs showed that the compressive stress increased in proportion to the increasing fiber volume fraction. The micro-cracks in conventional concrete and fiber-reinforced concrete were caused by the interface properties of coarse aggregates and cement paste. By contrast, SIFRCCs could reduce cracks at the interface because it was a composite made of fine particles with no coarse aggregates. Furthermore, the filling slurry and steel fibers improved the adhesion performance and reflected the effects of the fiber volume fraction and steel fibers instead of the compressive stress characteristics caused by matrix fracture. In addition, the high volume fraction of the steel fibers generated a restraining effect in the compressive stress test specimen, thereby affecting the increase in compressive stress.(2)The elastic modulus experiment results showed that the elastic modulus did not increase with the increasing fiber volume fraction, unlike the compressive stress experiment results. However, for strain under compressive stress, the increased rate of the strain at peak stress showed a difference of up to 2.7 times depending on the fiber volume fraction. This means that with increasing fiber volume fraction, the post-peak behavior after peak stress showed a strain hardening behavior, implying that the energy absorption capacity improved at a higher fiber volume fraction.(3)The characteristics of the strain increase rate were attributed to the restraining effect of the high fiber volume fraction in which many steel fibers resisted the expansion force generated vertically to the specimen axis that caused longitudinal split cracks. Furthermore, the reinforcing effect of steel fibers was considered to increase the peak stress and strain at peak stress in all compressive stress areas.(4)Poisson’s ratio experiment results showed insignificant differences in the elastic modulus with the fiber volume fraction. Similarly, Poisson’s ratio showed insignificant differences with the fiber volume fraction. However, Poisson’s ratio was ~0.3 for every variable; this was similar to that of steel. This was caused by the improved interface adhesion performance between the high-strength filling slurry matrix and the steel fibers. Therefore, the SIFRCCs with a high fiber volume fraction could suppress brittle fracture, a disadvantage of conventional concrete, and exhibit sufficient energy absorption capacity owing to its homogeneity.A high-performance SIFRCCs that can prevent the fiber ball phenomenon, a disadvantage of conventional fiber-reinforced concrete, and maximize the fiber volume fraction was developed, and the compressive behavior characteristics with respect to the fiber volume fraction were analyzed. The following conclusions were derived from this study.


Electroacupuncture (EA) has effective analgesic effects. Our previous study demonstrated that the upregulation of P2X3 receptors in the dorsal root ganglia (DRG) might participate in heroin withdrawal-induced hyperalgesia. The aim of this study is to further explore whether 2 Hz EA reduces heroin relapse associated with its analgesic effect and whether P2X3 receptors in the DRG are involved in this process. 2 Hz EA was adopted to treat the heroin SA rats in the present study. Heroin-seeking and pain sensitivity were evaluated. The expression of P2X3 receptors in the DRG was detected. Our results showed that compared with the control group, the reinstatement, thermal hyperalgesia, and mechanical allodynia of the heroin-addicted group were increased significantly. The expression of P2X3 receptors in the DRG was increased markedly. After being treated using 2 Hz EA, reinstatement was reduced, hyperalgesia was decreased, and the upregulated expression of P2X3 receptors in the DRG had decreased significantly compared to that in the heroin-addicted group. Consequently, our results indicated that 2 Hz EA was an effective method for treating heroin-induced hyperalgesia and helping prevent relapse, and the potential mechanism might be related to the downregulation of P2X3 receptor expression in the DRG. The essential problem in the treatment of opioid (including heroin) addiction is the high rate of relapse . The neuSeveral periphery molecular targets have been proposed to understand the hyperalgesia induced by opioid withdrawal, such as morphine receptors (MORs) , 7, N-mein vivo [Electroacupuncture (EA) is a procedure in which fine needles are inserted into an individual at discrete points, and then electrical stimulation is applied, aiming to relieve chronic pain . Our prein vivo , 19. Thein vivo . Previouin vivo , 22.There is still a lack of evidence to support the notion that 2 Hz EA reduces heroin relapse via its analgesic effect by affecting the expression of P2X3 receptors in the DRG. Therefore, in the present study, 2 Hz EA was adopted to treat heroin SA model rats. The rate of heroin relapse was evaluated with a reinstatement experiment, and the pain sensitivities were examined using paw thermal and mechanical withdrawal tests. The expression of P2X3 receptors in the DRG was observed using immunofluorescence. Our results will partly elucidate the analgesic effect of 2 Hz EA that may assist in suppressing heroin relapse and the potential mechanism through P2X3 receptors in the DRG.ad libitum, and food was restricted to 20 g per day to keep animal weight constant [Male Sprague Dawley rats, weighting 275–300 g at the beginning of the experiments, were purchased from Vital River Laboratory Animal Technology Co., Ltd, Beijing, China. The animals were maintained under a 12 h reversed light/dark cycle (with darkness starting from 8:00 am) with controlled room temperature and humidity. Tap water was made available constant , 24. AllThe heroin self-administration rat model that represented heroin relapse behaviours and was used to induce hyperalgesia was developed with a progressive fixed ratio programme. Experimental apparatus, procedures for surgery, and self-administration training were described in our previous report .μg/kg per infuse) under a fixed ratio 1 (FR 1) schedule of reinforcement, and we observed nose pokes for 3 h each day. The acquisition sessions were carried out until stable heroin intake was reached . Twenty-four rats developed a stable pattern of heroin intake. Thereafter, these rats were randomly divided into three groups (n = 8 per group): a heroin-addicted group, a sham EA group, and a 2 Hz EA group. The same experimental procedures were used for the rats in the control group (n = 8) except the heroin was substituted with the same volume of saline.In short, after a vein intubation operation, heroin SA training was carried out in operant chambers encased in sound- and light-attenuating cubicles, which were equipped with fans that provided ventilation . Animals were allowed to self-administer heroin  for 30 min on the zusanli (ST36) and sanyinjiao (SP6) acupoints every day during the extinction period according to methods reported in the literature . In otheAfter extinction, cue-induced reinstatement was examined to verify whether the addictive behaviour was successfully induced. During testing, the rats were reintroduced to the operant cages for 2 h training. At the beginning of the training, the rats were administered a conditioned cue priming . A single active nose poke was obtained with conditioned cue priming, but no heroin injection. The number of active nose pokes and inactive nose pokes was recorded.Thermal and mechanical nociceptive responses were used to verify whether nociceptive behaviour was induced and assessed using a dynamic plantar instrument with a radiant heat source and von Frey hairs, respectively . Evaluation of paw withdrawal thresholds (PWTs) was performed according to the methods described in the literature . ConcretTesting was conducted before drug exposure , extinction/EA treatment, and after cue-induced reinstatement. The experimental timeline and details are illustrated in μm thick slices. After dewaxing and rehydration, microwave antigen retrieval, and endogenous peroxidase blocking, slices were incubated with rabbit anti-P2X3  and diluted in PBS overnight at 4°C. After 3 rinses in PBS, the sections were incubated with fluorescent secondary antibody  in the dark at 37°C for 40 min. After three washes again in PBS, sections were mounted and covered with glycerol. The sections were examined with fluorescence microscopy , and photographs were taken with a filter set for Cy3 (excitation 540–580 nm/emission 560–620 nm). The optical densities of P2X3 were counted in a blind fashion on 16–20 randomly selected L5-6 DRG sections from 8 animals per condition. The results are expressed as relative optical density from these sections. Negative controls in which PBS was used instead of the primary antibody were processed in the same manner.Immediately after nociceptive behavioural testing, the rats were systemically perfused with 4% paraformaldehyde during 2–3% isoflurane-induced anesthesia. The L5-6 DRG were then isolated and postfixed overnight in 4% paraformaldehyde in PBS. After paraffin embedding, DRG paraffin tissue blocks were cut into 4 p < 0.05 was considered statistically significant.All the results are expressed as the mean ± SEM. The statistical significance of the results was analysed using one-way or two-factor repeated ANOVA. All statistical analyses were performed using SPSS for Windows, version 11.5 . When significance was found using ANOVA procedures, post hoc analyses were conducted using the Fisher LSD test. p < 0.01). The number of active nose pokes significantly decreased in the 2 Hz EA group compared with that in the heroin-addicted group (p < 0.01). In contrast, no significant differences (p > 0.05) were observed in the number of inactive nose pokes during reinstatement among the control, heroin-addicted, sham EA group, and 2 Hz EA groups, which suggested that rats retained good discrimination between active nose pokes and inactive nose pokes. These results indicated that a successful heroin-addicted rat model was verified, and 2 Hz EA, but not sham EA treatment, significantly reduced the conditional cue-induced reinstatement behaviours in heroin-addicted rats.The heroin self-administration rat model represented heroin relapse behaviours, which were developed with a progressive fixed ratio programme. The experimental apparatus, procedures for surgery, and self-administration training followed our previous report . As showHyperalgesic responses were measured on the d −1 prior to heroin self-administration (baseline), on d 10 prior to extinction and 2 Hz EA treatment, and after d 24 cue-induced reinstatements. As shown in p < 0.01), indicating the development of thermal hypersensitivity. Interestingly, the thermal threshold in the 2 Hz EA-treated group increased significantly compared with that in the heroin-addicted rats (p < 0.05). Similar results were observed in mechanical withdrawal threshold measurements. As illustrated in p < 0.01), indicating the development of mechanical hypersensitivity. Similarly, the mechanical thresholds in the 2 Hz EA-treated group also increased significantly compared with those in the heroin-addicted rats (p < 0.01). There was no significant difference in either thermal or mechanical withdrawal threshold between the sham EA group and the heroin-addicted group (p > 0.05). Our results indicated that 2 Hz EA treatment could significantly reduce hyperalgesic behaviours in heroin-addicted rats.As illustrated in p < 0.01) (n = 8 for each group). The average optical density of the P2X3 receptor in the 2 Hz EA therapy group reduced significantly compared with that in the heroin-addicted rats (p < 0.05). The P2X3 downregulation effect in the sham EA group was not obvious (p > 0.05). It was suggested that 2 Hz EA treatment could significantly reduce the upregulated expression of P2X3 receptors in the DRG in heroin-addicted rats.P2X3 receptor expression in the L5∼6 DRG neurons was observed using immunofluorescence . Immunofin vivo [There is still a lack of evidence to support the notion that 2 Hz EA reduces relapse in heroin SA rats and that this relapse reduction is associated with the analgesic effect that occurs via affecting the expression of P2X3 receptors in the DRG. In the present study, 2 Hz EA was used to treat heroin SA model rats. We demonstrated that 2 Hz EA, but not sham EA, reduced the hyperalgesia induced by heroin and contributed to prevention relapse. This effect was partially associated with the downregulation of the expression of P2X3 receptors in the DRG. The neurobiological mechanisms underlying opioid relapse and craving are complex . Accumulin vivo , 19. In in vivo  and behain vivo . Yang etin vivo  and Yoonin vivo  reportedin vivo , 22. Thein vivo , only thIn the present study, the effects of 2 Hz EA on reinstatement and thermal and mechanical hypersensitivity were chosen to explore whether 2 Hz EA could inhibit heroin-induced hyperalgesia and suppress cravings.µ- and δ-opioid receptors, possibly via accelerating the release of enkephalin [In fact, we found that the thermal and mechanical hyperalgesia in the 2 Hz EA-treated group were decreased significantly compared with that in heroin-addicted rats. Additionally, 2 Hz EA treatment significantly reduced conditional cue-induced reinstatement behaviour in heroin-addicted rats. These data suggested that 2 Hz EA could inhibit hyperalgesia induced by heroin withdrawal and reduce relapse behaviours in heroin-addicted rats. P2X3 receptors in the DRG play an important role in nociceptive transduction during chronic neuropathic and inflammatory pain but are seldom discussed in hyperalgesia induced by heroin addiction. Previous work has shown that the process of P2X3 receptor antagonism inhibits inflammatory hyperalgesia and involves the spinal opioid system . In addikephalin . Reportskephalin . Furtherkephalin . The preμm, 20–40 μm, and more than 40 μm, respectively). The current type of recorded slow, intermediate (or mixed), and fast types of ATP-activated current was correlated well with cell size and performed different functions and P2X3 mainly expressed on small nociceptive neurons mediated fast types of ATP-activated current according to others [It is worth noting that the total DRG cell was further divided into three subgroups: small cell, medium cell, and large cell (the diameter of these cell subgroups were about 10–20 o others –41 and oo others  previouso others . Both thIn summary, we showed that heroin SA rats exhibited hyperalgesia and had increased the expression of P2X3 receptors in the DRG. 2 Hz EA could effectively inhibit hyperalgesia induced by heroin withdrawal and reduce relapse behaviours in heroin-addicted rats. The expression of P2X3 receptors in the DRG decreased correspondingly. It was possible that P2X3 receptors were potential peripheral targets related to hyperalgesic states induced by heroin withdrawal. 2 Hz EA might be an effective method for treating hyperalgesia induced by heroin withdrawal and suppressing heroin relapse partly via regulating P2X3 receptor expression in the DRG. Further study is needed to elucidate the potential mechanism of 2 Hz EA's effect on relapse.


C) and strongly monotone operator. One of the advantages of our algorithm is that it does not require the knowledge of the Lipschitz constant of F on any bounded subset of C or the strong monotonicity coefficient a priori. Moreover, the proposed self-adaptive step size rule only adds a small amount of computational effort and hence guarantees fast convergence rate. Strong convergence of the method is proved and a posteriori error estimate of the convergence rate is obtained.In this paper we introduce a new self-adaptive iterative algorithm for solving the variational inequalities in real Hilbert spaces, denoted by Primary numerical results illustrate the behavior of our proposed scheme and also suggest that the convergence rate of the method is comparable with the classical gradient projection method for solving variational inequalities. C be a nonempty, closed and convex subset of Let The variational inequality problem (VIP) was introduced and studied by Fichera , 10 , which approximates λ in → denotes strong convergence.(ii)⇀ denotes weak convergence.(iii)ω-limit set of (iv)In this section, we list some concepts and tools that will be used in the proofs of our main results. In the rest of this paper, we always denote by C be a nonempty closed convex subset of a real Hilbert space z, denoted by x onto C and the operator C. It is well known that the projection operator Let It is well known that cterized , Sect. 3The following inequality holds:Letbe a nonexpansive mapping. Thenis demiclosed at 0 in the sense that ifis a sequence inCsuch thatandasit follows thati.e., Hereis the set of fixed points of T.F is Lipschitz continuous on any bounded subset B of C, i.e., there exists some B) such thatA mapping Assumeis a sequence of nonnegative real numbers such thatwhereis a sequence inandis a sequence of real numbers such that(i)(ii)orThenLetCbe a nonempty closed convex subset of a real Hilbert spaceIfis a strongly monotone and boundedly Lipschitz continuous operator, then the variational inequalityhas a unique solution.C be a nonempty closed convex subset of η and the Lipschitz constant B of C. Also, we always assume that the projection operator Let Now we are ready to present our self-adaptive iterative algorithm for solving Choose Otherwise, setGiven the current iterate Update the next iterate asOtherwise, set It is easy to see by a simple induction that the sequences η be the strong monotonicity coefficient of F. Then the following properties directly follow from the definitions of (i)(ii)F is L-Lipschitz continuous, then (iii)F is L-Lipschitz continuous, then Let We make the following observations for Algorithm Next we present a strong convergence theorem of Algorithm Assume thatFis boundedly Lipschitz continuous and strongly monotone on the feasible set, then any sequencegenerated by Algorithm converges strongly to the unique solutionof problemF is boundedly Lipschitz continuous.First, we verify that ave from  that\doB the closed convex hull of the sequence F restricted to B, respectively. Noting n and m such that Second, we show that ave from  that\do getting  yields\Finally, we prove t}n→∞ in , we obtaTo present a complete convergence analysis of Algorithm Assume thatFis boundedly Lipschitz continuous and strongly monotone on the feasible set and the sequenceis generated by Algorithm Then the following a posteriori error estimate holds:whereηis the strong monotonicity coefficient ofF, and the constantsandMare the same as above.Observe that this estimate can be easily obtained by letting t}m→∞ in . □Since a Lipschitz continuous operator is obviously boundedly Lipschitz continuous, the following results are straightforward.Assume thatFis Lipschitz continuous and strongly monotone on the feasible set, then the sequencegenerated by Algorithm converges strongly to the unique solutionof problemAssume thatFis Lipschitz continuous and strongly monotone on the feasible set and the sequenceis generated by Algorithm Then the following a posteriori error estimate holds:where the constantsandMare the same as above, andLandηis the Lipschitz constant and strong monotonicity coefficient ofF, respectively.In this section, we present three numerical examples which demonstrate the performance of the self-adaptive iterative algorithm   with theF is strongly monotone and Lipschitz continuous with strong monotonicity coefficient nth iterate One can easily verify that mentclass2pt{minimmentclass2pt{minimNext, in Fig. L and η.From Table Consider the variational inequality problem }VI  with theF is strongly monotone and boundedly Lipschitz continuous on C and F is not Lipschitz continuous on C, so GPM and VPGPM are not applicable for this example. Choosing the starting point It is easy to see that Consider the variational inequality problem }VI  with theF is also strongly monotone and boundedly Lipschitz continuous on C, and GPM and VPGPM are not applicable for this example. On the other hand, we definenth iterate Similar to Example The numerical results in Tables In this paper, in the setting of Hilbert spaces, a new self-adaptive iterative algorithm is proposed for solving


Enterobacteriaceae resistant to tetracycline and sulfonamide antibiotics, and the taxonomic composition of the microbiota by sequencing the 16S rRNA genes of one kit per nest. Changing the surrounding microbes of the kits can hinder the transmission of antibiotic resistance genes from one generation to the next, but the three communities widely differed in their ability to orient gut microbes and in their impact on antibiotic resistance genes. The most efficient delivery of the microbial community reduced the proportion of resistant Enterobacteria from 93 to 9%, decreased the relative abundance of eight antibiotic resistance genes, and changed the gut microbes of the kits at weaning. The least efficient did not reduce any ARG or modify the bacterial community. In addition, adding fecal pellets was more efficient than the oral inoculation of the anaerobic suspension derived from these fecal pellets. However, we were unable to predict the outcome of the exclusion from the data of the donor does (species composition and abundance of antibiotic resistance genes). In conclusion, we revealed major differences between microbial communities regarding their ability to exclude antibiotic resistance genes, but more work is needed to understand the components leading to the successful exclusion of antibiotic resistance genes from the gut. As a consequence, studies about the impact of competitive exclusion should use several microbial communities in order to draw general conclusions.Reducing antibiotic use is a necessary step toward less antibiotic resistance in livestock, but many antibiotic resistance genes can persist for years, even in an antibiotic-free environment. In this study, we investigated the potential of three fecal complex microbial communities from antibiotic-naive does to drive the microbiota of kits from antibiotic-exposed dams and outcompete bacteria-carrying antibiotic-resistant genes. The fecal complex microbial communities were either orally delivered or simply added as fresh fecal pellets in four to five nests that were kept clean from maternal feces. Additionally, four nests were cleaned for the maternal feces and five nests were handled according to the common farm practice  as controls. At weaning, we measured the relative abundance of 26 antibiotic resistance genes, the proportion of  Microbes exposed to antibiotics in livestock may develop resistance mechanisms, including a variety of targets such as efflux systems, drug modifiers or changes in the target’s configuration . As a maCampylobacter spontaneously evolved a resistance against macrolides and was not outcompeted by its sensitive kin in an antibiotic-free environment  that were acquired from a French rabbit farm for which the antibiotic history was available. The dams were selected so that they all received diclazuril (coccidiostat), tilmicosin (macrolide), sulfadimethoxine (sulfonamide), oxytetracycline and trimethoprim. They also received a subcutaneous injection of 23 mg/kg of sulfadimethoxine and 5 mg/kg trimethoprim twice a day for three days and 100 mg/kg oxytetracycline by a single subcutaneous injection 25 days before their due date. They arrived in the animal facility of the French National Institute for Agricultural Research (INRA) 18 days before their parturition. In contrast, the three non-gravid “donor” does  producing the competitive microbial communities were from farms that have banned the use of antibiotics for at least 5 years.2. The content was then homogenized using vigorous shaking.The fecal suspensions tested for competitive exclusion were prepared less than 2 h before their oral administration: for each donor doe, five fecal pellets (approx. 5 g) were added to a bottle containing 5 g of 1-mm glass beads and 40 mL 0.9% NaCl. After the bottle was sealed with a septum, oxygen was purged through a 0.1-mm needle via five cycles of pumping at −700 mbar and filling with 500 mbar NEnterobacteriaceae, and 10 colonies per sample were stored at −80°C in order to later test their resistance to antibiotics. At Day 3, 17, and 35, fresh fecal pellets from maternal and the three donor does were recovered and placed in dry ice. A 2-g subsample was homogenized with a bagmixer  for isolation and storage of Enterobacteriaceae, as previously described for kit fecal samples. At Day 35, a fecal pellet from each doe was immediately stored at −80°C for DNA extraction.Gravid dams were fed with a standard diet  until weaning. They were raised in cages measuring 100 × 42 × 60 cm that included 24 × 42 cm nests that could be closed by a sliding door. Suckling was allowed from 8 to 8:30 am for the first 21 days. From Day 1 to 21, dams had no access to their kits other than during the suckling window, which is common practice until Day 7 for meat rabbit farming in France. The litters were equilibrated to 6–8 kits per litter at Day 1. The 37 dams that had already given birth (Day 0) were divided into eight groups. The kits in the control group were raised as usual; the kits from the “No Feces” control group  were raised as usual except that we removed all maternal fecal pellets after suckling up to Day 21. For the kits of the six treated groups, we also removed the maternal fecal pellets after suckling but we put them in contact with the fecal microbes of a “donor” Doe  until Day 29. Each donor doe was kept individually in cages located in different rooms for the whole duration of the experiment. The groups I41, I43, and I44  received the microbial suspensions prepared as described above by letting them suck 100 μl from a syringe every second day between 10 and 11 am from Day 2 to 29. Gloves and syringes were changed between each cage to avoid cross-contamination. For the groups P41, P43, and P44 , we put five fresh fecal pellets from the relevant donor doe in the nest containing the kits. At Day 21, the nests were opened and kits could freely roam in the whole cage and interact with the dam until Day 36. At Day 35, one kit per cage was anesthetized via an intramuscular injection of 80 mg ketamine/kg  followed by an intracardiac injection of 182 mg pentobarbital/kg , causing death. The experiments were conducted under the agreement number APAFIS-2018021215568441 for animal experimentation from the French Ministry of Agriculture (Ethics Committee C2EA-86). The fecal pellets were immediately recovered from the digestive tract and a 100-mg subsample was placed in dry ice and then stored at −80°C for DNA extraction. Another 500-mg subsample was mixed with 3 mL of sterile phosphate-buffered peptone water containing 30% w/w glycerol and immediately mixed with a pestle by hand. The resulting homogenate was plated on MacConkey medium to isolate Enterobacteriaceae isolates collected from the does or the kits was tested by plating a 24-h pure culture onto Mueller-Hilton agar supplemented with 16 μg/mL tetracycline or 128 μg/mL sulfamethoxazole according to the ECOFF E. coli epidemiological cut-off values defined by Eucast . Each test included a resistant and sensitive control strain to confirm that the antibiotic was active and that the incubation conditions allowed growth. The average percentage of antibiotic-resistant Enterobacteriaceae isolates was calculated for each lactating doe (maximum of 30 isolates with 10 isolates per sampling day) and for their nest (maximum of 30 isolates with 10 isolates for one kit per nest). When the number of isolates was too low to calculate an average percentage (n < 10 for the doe or the nest) and/or when the average percentage of antibiotic-resistant Enterobacteriaceae of the lactating doe was less than 50%, we excluded the maternal doe/kit couple from the analysis.The antibiotic susceptibility of TM in which 80 mg of frozen feces were processed according to the manufacturer’s instructions . The microbial DNA was also extracted from the three donor does at 16 days for ARG detection. The DNA concentration was measured using the Quant-iT TM PicoGreen TM dsDNA Assay Kit according to the manufacturer’s protocol  and subsequently diluted to 10 ng/μl.The microbial DNA was extracted at weaning from the three donors, the 37 dams and the 37 kits using the ZR-96 Fungal/Bacterial DNA KitTM Integrated Fluidic Circuit (IFC). Pre-amplification of the samples, chip loading and qPCR reactions in nanoliter volumes were performed according to the manufacturer’s protocol. A pre-amplification step was applied to all samples for all primer sets except the Zhu16S primer set. Briefly, 13 ng of total DNA were submitted to 14 PCR cycles using the PreAmp Master Mix (Fluidigm) and a mix of primers . Pre-amplified samples were 5-fold diluted after an exonuclease treatment. The diluted pre-amplified samples and the primer sets were loaded in a 96.96 IFC using an IFC Controller HX (Fluidigm). The Biomark thermal protocol was as follows: a thermal mix step , a hot start , 35 cycles of PCR , and a final melting phase (60°C to 95°C). The list of primers can be found in High throughput real-time qPCR was performed using the Biomark microfluidic system from Fluidigm  using a 96.96 Dynamic ArrayThe V3V4 region was amplified from the 77 purified genomic DNA with the primers F343 and R784  using 30The statistical analysis was performed with R 3.3.1 . The sigt-test. In other words, the decrease observed in the fecal pellets was compared to the decrease observed in the suspension for each ARG. The difference of the percentage of antibiotic-resistant Enterobacteriaceae between the different groups of lactating dams or groups of kits was also tested using the Kruskal-Wallis test, but the multiple testing was performed with the pgirmess package (version 1.6.9) in order to obtain a two-tailed p-value since we assume that the percentage of antibiotic-resistant genes decreases.The difference in the abundances of ARGs between the dams in the eight groups was tested using the Kruskal–Wallis test with the Benjamini–Hochberg correction of the two-tailed Dunn’s test for multiple testing since ARGs could increase or decrease. The efficiency of fecal pellets vs. the corresponding suspension in decreasing ARGs was tested using a paired The richness was estimated by the Chao1 estimate using the estimateR function (R package vegan 2.4-2), which was then analyzed by a one-way analysis of variance (ANOVA) to detect an impact of the group. The significance of the separation observed on the nMDS plot using the Bray-Curtis dissimilarity was tested using pairwise permutational multivariate analysis of variance with distance matrices on the square-root-transformed abundances of the operational taxonomic units (OTUs) with the adonis function, after confirming the homogeneity of the variance though the betadisper function (R package vegan 2.4-2). Two samples out of 37 were ignored because of aberrant profiles (kit10 and kit28).In order to quantify the immigration rates under the assumptions of the neutral model, we used the script provided by X- and an Y- vector of values by quantifying the non-randomness of points in a two-dimensional space. This index was calculated on all the OTUs with more than 10 counts on average, which represented 104 OTUs. A relationship was considered significant if its MIC value was observed in less than 1% of the 37950 MIC values across 100 randomized datasets. In our case, the MIC threshold values were 0.66365 for the kits and 0.65429 for the dams.The linear and the non-linear relationships between the OTUs and the ARGs were characterized by the maximal information coefficient (MIC) index . BrieflyAs expected, many antibiotic resistance genes are reduced in does from farms that have banned antibiotics for several years (referred to as “donor” does below). Based on the 37 samples from the lactating dams and the six samples from the donor does, half of the ARGs , whereas suspension I44 reduced the abundance of ermG by half (mefA (macrolide efflux pump), which explains why kits fed with suspension I41 had 312-fold more mefA than the control kits.When inoculating the kits with a microbial suspension every second day between day 2 and 29, suspension I43 reduced eight antibiotic resistance genes . In addition, the fecal pellets from Doe44 decreased the carriage of three different ARGs , whereas the corresponding suspension only changed ermG.Adding the fecal pellets to the nest is slightly more efficient than oral ingestion to drive the competitive exclusion. Indeed, six ARGs decreased in both kits exposed to suspension I43 and to the fecal pellets of Doe43, but the decrease was more drastic for kits exposed to pellets , while suspension I44 shows a trend that only becomes significant when performing a second trial  but the fecal communities of the kits in contact with fecal pellets from Doe43 and Doe44 differed from the communities observed in the control kits, unlike the fecal communities of kits in contact with fecal pellets from Doe41 . Finally, tet32 did not coexist with OTU20 belonging to the Lachnospiraceae family  associated with OTU95 belonging to the Clostridales order in which ermB determinants were previously detected in Clostridium .In does that were analyzed separately to avoid spurious relationships , the onlstridium  and in Rnococcus . It shouEnterobacteriaceae are tested for tetracycline and sulfonamide resistance using classic culturing techniques.In our study, we modified the microbial community that colonizes the gut of the kits using microbial fecal communities from foreign does by either providing them as anaerobic suspensions or as fecal pellets in the nest before weaning. The “donor” does are from farms that have banned antibiotics for at least 2 years. The feces are collected from the kits at weaning, at which point 26 antibiotic-resistant genes are evaluated via quantitative PCR, the gene coding for the small ribosomal subunit is sequenced, and the cultivable Enterobacteria in the feces of the kits mirrors the ratio observed in lactating dams. It should be noted that this ratio does not depend on age, unlike the abundance of the OTUs, as previously described in rabbits . Predicting the exclusion of targeted genes from complex communities is a critical milestone that requires additional information about the microbiota performing the exclusion. Indeed, it is challenging to predict the outcome of the exclusion based on the abundance of the ARGs in the donor doe and the lactating dam  or to small amounts of bacteria transmitted before birth, as observed in human . UnfortuSalmonella for decades, which probably also decreased the carriage of E. coli producing extended spectrum beta-lactamase as a side-effect .Enterobacteriaceae isolates for antibiotic resistance. VD, CSA, OZ, and AB-M drafted the manuscript. All authors edited and approved the manuscript.OZ, CSA, VD, and AB-M designed the study. CSA and SS extracted the microbial DNA, chose the primers, and analyzed the microbial sequences. LC uploaded the sequences. CSA and OZ carried out the statistical tests. VD and NA screened the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.