DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate  Types of genetic changes that can be induced include gene disruption, gene excision, sequence substitution and targeted integration of exogenous sequence  the generation and phenotypic screening of many independent transgenic events, each with different random transgene insertions, (ii) introgression of transgenic loci from selected events to elite germplasm and (iii) repeated monitoring of transgenes for maintained expression and efficacy. Genome editing technologies promise to improve this paradigm by enabling targeted transgene integration into genomic loci known to support robust and stable gene expression and known not to interfere with agronomic characteristics, thus facilitating the identification of events that perform as desired . Engineered nucleases currently in use include meganucleases, TALENs , ZFNs and a collection of related CRISPR/Cas  ribonucleoproteins  is a globally important oilseed crop, most current commercial varieties of which are transgenic  site, with donors designed to select for targeted integration . Specifically, primers were designed to bind just outside the identical homology regions present in both the target and the donor. The use of these primers thus yields the shortest and most easily amplified products capable of detecting targeted transgene integrations, but will detect both full‐length and partial transgene integrations with internal deletions. Furthermore, although the sequence of each primer binding site was unique in the soybean genome, primers were not designed specifically to maximize differences with the corresponding sites in paralogous loci, and thus some apparent positives may be from targeted integrations at a locus paralogous to As shown in Table Having obtained preliminary evidence for HDR‐mediated targeting, we next tested whether the regions of sequence identity flanking the desired donor region (i.e. the homology arms) are required for targeted transgene integration. Specifically, we aimed to determine whether donor DNA could be delivered to a target locus via NHEJ, as this DNA repair mechanism (but not HDR) is thought to be operable in most cells at most times Puchta, , and thuFAD2‐1a. In addition, we incorporated a second transgene  into the NHEJ donor construct  such that they would detect small‐scale deletions or rearrangements near the site of the ZFN‐induced DSB. In addition, donor primers were designed and verified to bind to an internal region of the HPT gene, such that the entire sequence of an intact targeted donor (either HDR or NHEJ) would be contained within an overlapping pair of genome‐donor junction amplicons.From the amplicon‐positive, candidate targeted events described in the previous two sections, we selected 95 low or single copy events for additional molecular analysis (62 from NHEJ‐based targeting and 33 from HDR‐based targeting). First, genomic DNA from each clone was analysed by junction PCR using a second set of genome‐ and donor‐specific primers that had been designed for higher stringency. Specifically, genomic primers were designed and verified to generate amplicons from HPT  was in the same direction as the transcription of FAD2‐1a (denoted “forward” orientation). This orientation is also such that in the case of perfect NHEJ‐mediated integration, two functional ZFN target sites are reconstituted flanking the donor. Interestingly, several integration events for the HDR donor were apparently in reverse orientation, suggesting that despite the presence of homology arms, these donors were integrated at the target locus via NHEJ.From the 95 clones analysed under these more stringent conditions, a total of 56  gave rise to an amplicon of the expected size for at least one genome‐donor junction Fig a. The orOf the 56 events giving rise to at least one amplicon of expected size, 29 , gave rise to a single 5′ and a single 3′ amplicon of expected size for a full‐length, targeted integration. The remainder variously yielded no amplicons, a single amplicon, amplicons of unexpected size, or two or more amplicons associated with conflicting donor orientations, indicating that donors were fragmented, concatemerized, or otherwise aberrant, or that the initial PCR was a false positive.FAD2‐1a. Donor intactness could be directly demonstrated in the four of these 21 events containing only a single copy of the donor, as reads covering the entire donor can be identified and uniquely attributed to the single integration at FAD2‐1a. For the remaining 17 events, the presence of multiple donors (either on‐ or off‐target) prevents the assignment of internal donor reads to any particular copy of the donor, and thus prevents donor intactness from being directly assessed via NGS in these events.In tandem with stringent junction PCR, all 95 clones were also analysed by Illumina‐based Next Generation Sequencing. Prior to paired‐end sequencing, DNA elements of interest  and adjacent sequences were enriched using sequence capture—isolation of selected DNA fragments via hybridization to bead‐bound oligonucleotides complementary to these sequences. To identify events in which targeted integration had occurred in the NHEJ‐based experiment, we searched the set of all reads from each sample for individual reads that spanned the novel genome‐donor junction created by targeted integration Fig. b. Of theThe results of NGS and high stringency PCR of the NHEJ events were in close agreement with one another Table . Of the FAD2‐1a. The donor integrations in these five events can thus be inferred to be intact, and the lack of detectable integration at other sites in the genome can be taken as indirect evidence for integration at FAD2‐1a. It should be noted, however, that because the region of shared sequence in the donor and the target site (1000 bp) was longer than the fragments sequenced by NGS (~800 bp) no individual reads contained sequence that was unambiguously attributable to both the genome and the donor.NGS analysis of HDR‐derived clones was used to determine the existence of off‐target insertions, as well as to address donor intactness in the case of clones where all genome‐donor junctions mapped to a single genomic locus. These analyses showed that five of the ten clones exhibiting single 5′ and 3′ junction amplicons of expected size in stringent junction PCR had no detectable off‐target integrations, and no evidence for fragmentation of the donor at FAD2‐1a locus , and the remaining two events had integrated the donor in reverse orientation.To build on the results we obtained with the 7.1 kb, two‐transgene donor described above, and to attempt to deliver a donor of a size more typical for commercial molecular transgene stacks currently being developed  confirmed the presence of a full‐length targeted insertion in each of the three events Fig. b. Finallant Fig. c. From tFAD2‐1a, together with a circular DNA donor bearing the same target site, is sufficient to drive precise integration of the donor into the target locus. Targeted integration was achieved in both tissue culture and in immature embryos, followed in the latter by somatic embryogenesis, regeneration of fertile plants and Mendelian transmission of the targeted insertion from primary transformants to their progeny. The largest donor integrated was approximately 16.2 kb, carried four transgenes, lacked any shared sequence with the genome besides the ZFN target site and was integrated in either of the two possible orientations relative to the target. For all configurations of donor and ZFN expression construct tested, bombardment of 5 000 to 10 000 immature embryos was sufficient to yield several mature plants with fully intact targeted integrations of the donor. These results show that homology arms are dispensable for targeted transgene integration in plants, that donors sufficiently large to accommodate multiple transgenes can be delivered to an endogenous locus of a crop plant using ZFNs and that the frequency with which mature targeted soybean plants can be generated with this system is compatible with commercial production.We have reported here first use of an engineered, site‐specific endonuclease to deliver large, multigene donors to a target genomic locus of a major crop via NHEJ. Specifically, we have shown that biolistic bombardment of soybean cells with a ZFN expression construct targeting a site in et al., et al., et al. is attributable to species, transformation system, nuclease activity or another variable remains an open question.The 7.1 and 16.2 kb multigene donors we have reported here lacked the ~1 kb homology arms that are typically used in HDR‐based gene targeting experiments in both plants and animals. The decision to exclude these homology arms from constructs delivered to immature embryos was based on our initial experiments with tissue cultures, in which events with putative junction amplicons were recovered at a higher frequency with donors lacking homology arms than with donors possessing homology arms , nuclease‐dead Cas9 pairs fused to FokI , with a required input of 5 000 to 10 000 bombarded embryos in each case. This number of bombardments, though substantial, is well within the number that might occur for a commercial or larger scale research soybean transformation campaign, and would be reduced further by future improvements increasing transformation or regeneration frequency, or reducing donor fragmentation. The purported error‐prone nature of NHEJ did not present a significant problem in this system, as the great majority of genome‐donor junctions we observed were either perfect or near‐perfect.Of critical importance to any future application of gene targeting technology in plants is the ability to generate mature, fertile plants with the desired genome modifications, at a cost not prohibitive in time and resources. From the work reported here, we have obtained several mature, targeted plants for each of three configurations of donor and ZFN expression constructs  transformation methods, (ii) a suite of engineered nucleases to generate DSBs at desired loci, (iii) validated donor configurations for either HDR‐ or NHEJ‐mediated integration, (iv) analytical methods for identifying and characterizing targeted events and (v) a bioinformatics‐derived set of potentially attractive targeting sites  and Agrobacterium tumefaciens ORF23 (AtuORF23) under the control of AtUBI10 and AtuORF23 elements. The 16.2 kb NHEJ vector contains HPT under the control of CsVMV  regulatory elements, and glufosinate and 2,4‐D tolerance markers (dsm‐2 and aad‐1 respectively) under the control of AtUBI10 regulatory elements.The characterization of the ZFN monomer pair used in this study, and its selection from several candidate ZFN monomer pairs targeting Soybean embryogenic tissue cultures from two cultivars, Westag 97 and X5 (Agriculture and Agri‐Food Canada breeding line X2650‐7‐2‐3) were initiated, maintained and bombarded as previously described  and maintained in a Conviron growth chamber  for all remaining culture steps. After 4 h on plasmolysis medium, explants were subjected to biolistic bombardment. After 1 d recovery, explants were cultured 1 week on SE40 medium  until roots grew and apical meristem had formed. After the shoots had elongated to about 7.5 cm and had a sufficient root system, they were transferred to soil and grown for 2–3 weeks in a Conviron growth chamber. Surviving plants were transferred to large pots and self‐fertilized to produce T1 progeny.After selection and induction on SE40 medium, the explants were placed on somatic embyrogenesis germination medium  for 6 weeks (subcultured every 2 weeks). By the end of the 6 weeks, the somatic embryos were isolated from the immature embryos from which they had developed and were allowed to mature for another 2 weeks on SEGM. Matured somatic embryos were dehydrated for 5 days in a 15 × 100 mm Petri dish with a 1 cm® Real‐Time PCR System  according to the manufacturer's instructions. About 10 ng of genomic DNA was used as template for each reaction, with the following programme: 95 °C 10 min; 35 cycles of 95 °C 10 s, 60 °C 1 min, plate read. Previously identified hemi‐ or homozygous plants were used as standards for each transgene.qPCR analyses were performed with PerfeCTa qPCR Supermix  on a LightCycler 480http://bioinfo.ut.ee/primer3). Amplicon sequences were aligned and analysed with the software tool Sequencher™ .PCR reactions were set up using Takara ExTaq DNA polymerase  according to the manufacturer's instructions. Approximately 4 ng of genomic DNA was used as template for each reaction, with the following thermalcycler programme: 95 °C for 3 min; 35 cycles of 95 °C 30 s, 60 °C 30 s, 72 °C 4 min. Sequencing was provided by Eurofins Genomics . PCR and sequencing primers were designed using the online Primer3 tool (v. 4.1.0) (et al., et al., Illumina library production from sonicated genomic DNA, size‐selected to ~800 bp was performed as described (Guttikonda Table S1 Summary of stringent junction PCR and NGS on 62 candidate targeted events from NHEJ‐based targeting experiments with embryogenic suspension cells.Click here for additional data file.


Models of cerebrospinal fluid (CSF) circulation have been mainly proposed in the last century: CSF goes from the ventricles to the subarachnoidal space (SAS), passing through the aqueduct and the foramen of Luschka and Magendie. Indeed, new models, involving the Virchow-Robin space (VRS) and the perivascular space (PVS), have been proposed. We critically reviewed the literature, in order to clarify the “classical” errors and to discuss the “new” models that are evolving currently. Conclusions of past experiments are often not justified, due to lack of reproducibility and methodological issues. On the other hand, investigation on the microanatomy of Virchow-Robin spaces (VRS) and several new experiments showed a potential pathway for a more complex CSF “circulation,” with chaotic and unpredictable flows. It seems reasonable to elaborate a new model of CSF physiology, including new findings and questioning old certainties. However, proved data are still missing and it is hazardous to come to final conclusions. More studies are needed. All of us studied that cerebrospinal fluid (CSF) is produced by choroid plexuses (CP) (80%) and by ependyma (20%) and then circulates from the ventricles to the subarachnoidal space (SAS), passing through the aqueduct and the foramina of Luschka and Magendie. CSF is then reabsorbed into the venous sinuses, thanks to the arachnoidal granulations of Pacchioni. This movement is meant a real one, with a bulk flow originated by a mass transfer of molecules in a preferential direction.Although anatomical description and functioning of ventricles date back to ancient Greece and Leonardo da Vinci, the actual model of CSF physiology is based on a series of experiments carried out, mainly in the last century, by famous neurosurgeons or neurophysiologists like Dandy, Cushing, or Retzius.Nowadays, evidences not coherent with the classical paradigm are emerging. Several authors have arisen doubts on all steps of CSF circulation: the historical experiments present several methodological issues, and many researchers recently failed in reproducing those results.In this dissertation, we review the literature to clarify the “classical” errors and to discuss the “new” model that is evolving by now. spiritus animalis.In 1764 Cotugno et al. [In 1825 Magendie [Experiments on CSF circulation that build our actual model of conceiving have been carried out over the past 150 years .In 1875 Key and Retzius demonstrated that the CSF is absorbed by the arachnoid villi by injecting coloured gelatine into the SAS. The injection pressure was above 60 mmHg, causing the rupture of the villi with passage of gelatine in the lumen of venous sinuses .In 1913, Dandy and Blackfan  induced In 1914, Weed  repeatedIn 1919, Dandy  demonstrDandy introduced the choroid plexectomy as a treatment for hydrocephalus. The clinical results were so poor that in the 1950 most neurosurgeons abandoned this technique .proves to be in continual movement in a definite direction through a highly specialized pathway that cuts across the blood circle to envelop an organ in which a lymphatic apparatus of the usual type does not exist”, serving as a vehicle for metabolites.In 1925, Cushing published “The third circulation and its channels” . He intr in vivo with ventriculography and ex vivo by analysing the specimen's brain.In 1970, Milhorat  reproduc obvious regions of CSF drainage into the vascular system, “from a purely anatomic point of view”.In 1987, Davson  stated tOver the last thirty years, the classical paradigm of CSF circulation has been questioned .Hassin et al.  in 1961 Magnus  and MilhOreskovic et al.  investigThey also injected a hyperosmolar solution of saccharose, observing an increased outflow. This proved that also osmolarity influences CSF physiology .The common bias of exploring CSF circulation by injecting tracers in the SAS is that tracers' density is higher than CSF. Once injected, tracers would reach the lowest position pushed by gravitational force, i.e., the aqueduct.For this reason, Bulat et al.  infused During a three-hours infusion, the venous concentration of 3H water sharply increased, while arterial and CSF concentration remained stable. Their explanation consisted of a continuous turnover of water across microvascular walls, with a continuity of IF and CSF . 3H-wateThe same procedure was performed with 3H-Inuline, which poorly passes across cerebral microvascular walls. 3H-inuline increased in CSF compartments and remained stable in arterial and venous compartments. They observed a bidirectional 3H-inuline distribution between cisternal CSF and lateral ventricle and between cisterna magna and cortical CSF. They assumed that substances slowly eliminated into microvessels spread not only along CSF spaces, but also between CSF and brain parenchyma. Subsequently they are slowly eliminated across capillary walls into bloodstream.They concluded that neither the net formation of CSF volume in brain ventricles nor its unidirectional flow in subarachnoid space is present.This is coherent with the observation that intrathecal infusion of drugs leads to a wide diffusion of the therapeutic molecules over the parenchyma .In 1851 and in 1859, Virchow and Robin described the pia mater surrounding the vessels. Originally, it was thought that this space served as a communication to the subarachnoidal space (SAS) and as an alternative CSF draining towards the arachnoid villi .Then, the Virchow-Robin space (VRS) was shown to be a virtual space, enlarged only in pathological processes .Nowadays, the original idea about the function of this perivascular space (PVS) has been restored .The PVS can be identified on larger cerebral blood vessels in both the subarachnoid spaces and within the parenchyma . It is aThe first evidence of an intraparenchymal bulk flow along this PVS was provided by Cserr . They reA consistent movement of fluid was also demonstrated by Rennels , by injeIn its complexity, the perivascular flow could consist of a sort “lymphatic system” . The intThe Aquaporin-4 (AQP4) water channels are involved in this glia-mediated convective transport of fluids and solutes through the brain extracellular space .Notably, AQP4 shows a polarized expression along CSF and parenchyma interface: pial surface-facing glia limitans, ventricle-facing ependymal cells, and perivascular end feet of astrocyte .AQP4-null mice demonstrated an increased brain extracellular space volume fraction  and incrMany evidences question the proposed ‘glymphatic' system and the role of AQP4.These molecules are not able to transport macromolecules  and therAquaporins clearly have a central role in brain water balance, both in health and disease , 36, butOur knowledge on CSF circulation is based on historical experiments. The major issue with those experiments is that all these studies involved rather drastic experimental procedures. A classical model of CSF circulation is no longer acceptable, since almost all its steps have been rebutted : CSF pro.CSF production in CP was demonstrated by Dandy with only one experiment. Hassin et al. , revealiThe belief that CSF is absorbed from the subarachnoid space to sagittal sinus through arachnoid villi arises from the experiments of Key and Retzius. Weed et al.  perfecteThe unidirectional CSF flow, described by Cushing, has evidences in the experiments of Dandy and Blackfan (1913) and Milhorat (1970). These authors obstructed the sylvian aqueduct of dogs (Dandy) and monkeys (Milhorat) and observed that animals developed hydrocephalus. Dandy experiment was very traumatic. Milhorat experiments (an inflated Foley) determined an intense parenchymal stretching of the aqueduct, causing a lumen dilatation more than twice .Indeed, stenosis/obstruction of the sylvian aqueduct represents a known cause of hydrocephalus.It is possible that aqueductal narrowing/closure may occur as a result and not as a cause of hydrocephalus. Experimental studies  have shoMoreover, cine-MRI in children younger than 2 years old, or patients with hydrocephalus, often curiously reports an inverse flow, from the fourth to the third ventricle . The algThe recent experiments, on the one hand, allow refuting the classical hypothesis, while on the other hand, they constitute the basis for a new model of “CSF noncirculation”. It would be more appropriate to talk about fluids movements, fluids exchanges among different intracranial compartments. These exchanges are represented by a dynamic equilibrium among parenchyma, interstitial space, SAS, vascular compartment, and ventricles . The hydIf this model corresponds to what happens physiologically, several pathophysiological phenomena leading to pathological conditions must be reconsidered.Hydrocephalus is not an imbalance between CSF production and resorption, but an imbalance between IF (decreased) and ventricular fluid (increased), while benign intracranial hypertension is an opposite imbalance (increased IF and decreased ventricular fluid).Hydrocephalus caused by tumoral aqueduct obstruction could be due to an increased osmolarity.Post-SAH hydrocephalus could be caused by an obstruction of IF and/or a hyperosmolar intraventricular/subarachnoid fluid.We could hypothesize the following phenomena: This model could also provide a possible explanation for neurodegenerative disorders: could these pathologies have a correlation or a trigger in the interstitial fluid imbalance? In cerebral amyloid angiopathy, beta-amyloid is deposited around and in the vascular wall of arteries and arterioles. A dysfunction and obstruction of this drainage could be part of the aetiology of disease such as Alzheimer . Indeed,More experiments are needed to better clarify the mechanisms determining the imbalance of fluids dynamic equilibrium in different pathological conditions.The methods of historical papers, which have been used to determine the classical CSF circulation hypothesis, have very frequently been performed under nonphysiological conditions. The obtained results are therefore of questionable reliability.New findings are emerging regarding the behaviour and the function of CSF. de facto the necessity of a real flow as classically described.The consequence of this conceptual evolution is that CSF physiology should not be considered as a unidirectional flow, through a unique anatomical well-defined pathway. More likely, it is a complex and dynamic equilibrium of fluids between vascular, neural, and cisternal spaces. In addition, the neurovascular interface is involved in the CSF production and resorption. This activity seems to be driven both by hydrostatic and osmotic forces, avoidingThese considerations make the classic model of CSF circulation no longer acceptable.


Normal mammalian development and homeostasis are dependent upon the gut microbiota. Antibiotics, essential for the treatment and prophylaxis of bacterial infections, can have collateral effects on the gut microbiota composition, which can in turn have far-reaching and potentially deleterious consequences for the host. However, the magnitude and duration of such collateral effects appear to vary between individuals. Furthermore, the degree to which such perturbations affect the host response is currently unclear. We aimed to test the hypothesis that different human microbiomes have different responses to a commonly prescribed antibiotic and that these differences may impact the host response.n = 30) humanized with the microbiota of two unrelated donors (A and B) were subjected to a 7-day antibiotic challenge with amoxicillin-clavulanate (“co-amoxiclav”). Microbiome and colonic transcriptome analysis was performed, pre (day 0) and post antibiotics (day 8) and subsequently into recovery (days 11 and 18).Germ-free mice  contains supplementary material, which is available to authorized users. The mammalian microbiome incorporates a vast and diverse range of microorganisms that have co-evolved to live with us, contributing essential functions through their collective genetic and metabolic repertoire . EcologiEnterococcus (VRE)[Clostridium difficile infection (CDI)[Helicobacter pylori infection [Antibiotic treatment, one of the fundamental medical achievements of the past century, represents a notable example of microbiome perturbation, with rapid and sometimes enduring changes to the community structure . Antibiocus (VRE), recurreion (CDI), and theion (CDI). Notablyion (CDI) and the nfection . WhetherDue to the importance of the microbiota for host homeostasis and the marked collateral effects of antibiotics on the microbiota, we posed the questions: (a) can antibiotic administration result in correlated fluctuations of the microbiota and host colonic transcriptome? and (b) if so, is there evidence of a personalized response? To answer these questions, we characterized the microbiota and the colonic transcriptome of germ-free mice (GFM) humanized with fecal samples from two unrelated healthy human donors, before and after treatment with the broad-spectrum antibiotic amoxicillin-clavulanate , commonly used to treat various infections in clinical practice Fig. a.Fig. 1adada2 pipeline [In total, samples from 30 mice passed quality controls, 15 in each group. One animal from the donor B group at day 0 (D0) was excluded due to low microarray signal intensity values. Four hundred fifty-four OTUs , were iPrevotella (mean 26.6% (SD 13.6%)) and Faecalibacterium (mean 24.9% (SD 12.5%)), with Bacteroides (mean 17.3% (SD 7.8%)) being the third most dominant genus ) and Parabacteroides (mean 19.6% (SD 7.6%)), with less than 1% mean abundance of both Faecalibacterium and Prevotella  had a markedly different community compared to mice from donor B. Donor A mice had a microbiota composition dominated by 26.6% SD 3.6%) and26.6% SD 3.6%) androtyping  similarlPrevotella sequences from baseline (D0) (from mean 27.4% (SD 5.9%) to mean 44.9% (SD 3.7%)) with a significant reduction in this genus by late recovery (mean 13.9% (SD 7.7%)) and a corresponding increase in Faecalibacterium OTUs at these time points (D0—mean 13.1% (SD 10.5%), D8—mean 21.3% (SD 5%), D11 (day 11)—mean 23.6% (SD 13%), D18 (day 18)—mean 38.5% (SD 7.4%); Fig. Bacteroides throughout the study in the donor B group, although this did not reach significance at any time point (D0—49.3% (SD 17.5%), D8—40.9% (SD 9.7%), D11—31.9% (SD 12%), D18—32.2% (SD 4%)), while Parabacteroides remained largely stable throughout , a significant reduction in Roseburia at D8 from D0, a significant reduction in Prevotella between D8 and D18, a significant increase in Blautia at D18 compared to D8, and a significant increase in Dorea at D18 compared to D8 , donor A mice demonstrated an increase in 44.9% SD .7%) withRuminococcus, increased significantly in donor B mice at D18 . Correlation of the PCoA scores with genera demonstrated a correlation between a number of key taxa, including Prevotella and Faecalibacterium, associated with the changes in donor A mice  > 5 were plotted. This analysis demonstrated significant changes in many terms, prominently biological processes concerned with extracellular matrix organization, cellular adhesion, signaling, cell migration, developmental processes, and circadian rhythm  microbiota, the degree of change appeared to be more marked in mice humanized by donor A than by donor B. As these were the mice that had a more marked alteration in the microbiota composition in response to co-amoxiclav, we next investigated whether correlation between the transcriptome and microbiota was evident.Prevotella, Dorea, Faecalibacterium, Blautia, and Roseburia). Figure Due to the large number of differentially expressed genes across the different contrasts, we used Gene Set Variation Analysis (GSVA) to identify gene pathways associated with antibiotic treatment. To perform correlation with microbial taxa, we used Hierarchical All-against-All significance testing , a recenTaken together, these results demonstrate correlation between host pathways and microbiome composition was unique to donor A mice. However, as the antibiotic-induced changes in donor B mice were considerably weaker, such correlations are harder to detect and higher numbers of mice would probably be required in this case.Prevotella and Faecalibacterium, with a lesser contribution from Bacteroidetes and underwent marked fluctuations in terms of their dominant genera, most prominently Prevotella and Faecalibacterium, as well as in other lower abundance genera within the community. It is notable that by D18, the mean abundance of Prevotella was approximately half that of baseline (D0), while the mean proportional abundance of Faecalibacterium was almost three times the baseline (D0)  Fig. , suggestRuminococcus sequences at D18. Bacteroides was the dominant genus in this group with Parabacteroides, the second most dominant genus. As mentioned, there was a trend of decreasing proportional abundance in Bacteroides throughout the study in the donor B group, although this did not reach significance. Notably, most dramatic changes in microbiota composition occurred in the recovery period.In contrast to donor A, donor B mice were much more resilient to antibiotic administration, with the notable exception of a late bloom in α-diversity over the duration of the study. We note that there were only three baseline (D0) mice in each group and this may have accounted for our inability to detect a significant change. Furthermore, as we did not detect changes in general in the donor B group, it is consistent that we did not detect a change in α-diversity either. In terms of donor A mice, we did note a drop-off in the Simpson diversity at D18  and not in donor B. However, conclusions from both the microbiota and host transcriptome must take into account the lack of a no-antibiotic control group.Finally, recent work has demonstrated the importance of bacterial load in characterizing the microbiome, and changes in total bacterial counts in both donor groups may have contributed to some of the effects seen .In summary, we have demonstrated that a commonly prescribed antibiotic can exert significant effects on the gut microbiota, in concert with colonic gene expression. Notably, while the host response appeared to be conserved, the degree of activation was related to the degree of microbiota perturbation, resulting in correlations between host transcriptome and the microbiota unique to one donor community. These findings offer a window into the role that our microbiome plays in mediating our personalized response to medications such as antibiotics.n = 31) were transferred into two sterile separate isolators  with no history of intestinal problems or intake of antibiotics 3 months prior to the beginning of the study, resulting in a single donor fecal microbiota transplantation per isolator (donor A (n = 15) and donor B (n = 16)). Mice in each isolator were housed in two separate cages (Table n = 3), donor B (n = 4)), followed by the administration of the penicillin antibiotic amoxicillin in combination with the β-lactamase inhibitor clavulanic acid once a day (80 mg and 10 mg/kg) to all remaining mice. We chose this antibiotic as it is a commonly prescribed, broad-spectrum human antibiotic, and treatment was continued for a total of 7 days, similar to the duration of a typical treatment for a community infection [All procedures in relation to the care and use of laboratory animals were carried out in accordance with European guidelines and the local ethics committee approved this study. Germ-free male 6-week-old mice  according to the manufacturer’s instructions. RNA 6000 Nano chips on a Bioanalyzer 2100  were used to assess RNA integrity. Gene expression profiling was performed with the SurePrint G3 Mouse GE 8x60 K Microarray . Further details are provided in supplementary methods in Additional file N-lauroyl sarcosine (Sigma) for 10 min at room temperature. Five hundred microliters of 5% N-lauroyl sarcosine in 0.1 M phosphate buffer (pH 8.0) was then added and incubated at 70 °C for 1 h. A mixture of sterilized silica beads (0.1- and 0.6-mm diameter) was added and the tube shaken three times at 6.5 m/s for 30 s in a FastPrep apparatus . Fifteen milligrams of polyvinylpolypyrrolidone was then added to the tube, and this was vortexed and centrifuged at 20,000×g for 5 min. Following the recovery of the supernatant, the pellets were washed with 500 μl of TENP (50 mM Tris (pH 8), 20 mM EDTA (pH 8), 100 mM NaCl, 1% polyvinylpolypyrrolidone) and centrifuged for 5 min at 20,000×g and the new supernatant was added to the first supernatant. The washing step was repeated twice. The pooled supernatant was centrifuged briefly to remove particles and then split into two 2-ml tubes. Precipitation of nucleic acids was performed by adding one volume of isopropanol for 10 min at room temperature with centrifugation at 20,000×g for 10 min. Pellets were then resuspended and pooled in 450 μl of 100 mM phosphate buffer at pH 8 and 50 ml of 5-M potassium acetate. The tube was placed on ice overnight and then centrifuged for 30 min at 20,000×g. The supernatant was subsequently transferred to a new tube containing 20 μl of RNase (1 mg/ml) and incubated at 37 °C for 30 min. Nucleic acid precipitation was performed by adding 50 μl of 3-M sodium acetate and 1 ml of absolute ethanol. The tube was incubated for 10 min at room temperature and the nucleic acids were recovered by centrifugation at 20,000×g for 15 min. The resulting DNA pellet was finally washed with 70% ethanol and then dried and resuspended in 100 μl of Tris–EDTA (TE) buffer.Fecal samples were weighed and resuspended in 250 μl of 4 M guanidine thiocyanate in 0.1 M Tris at pH 7.5 (Sigma) and 40 μl of 10% Amplicon libraries for barcoded 454 pyrosequencing were generated by PCR of the V3–V4 hypervariable region of the 16S gene with the primers 343F 5′-TACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACCAGGGTATCTAAT-3′. Barcodes are available in the supplementary methods  and the eBayes test [P values < 0.05). Differentially enriched pathways in the Gene Ontology (GO) databases were identified using the goana function in the limma package. To remove redundancy, GO terms that were significantly enriched  were submitted to REVIGO [P value).Principal component analysis was performed using the ade4 (v1.7-13)  and factyes test . Multiplo REVIGO  and the Demultiplexing of pyrosequencing fasta and quality files was performed with QIIME (v1.9.1)  and FASTDESeq2 (v1.24.0)  and significance was confirmed by Mann-Whitney testing [RVAideMemoire package (v 0.9-73) [P values were adjusted with the Benjamini-Hochberg method and correlations with adjusted P values < 0.1 were retained. Graphs were made using ggplot2 (v3.1.1) [Alpha diversity was presented using the Simpson diversity index. Differential abundance between donor groups and between different time points within donor groups was assessed using  testing . Beta di testing  on propo 0.9-73) . Spearma(v3.1.1)   and differentially expressed genes (3630) to 182 differentially expressed pathways across all contrasts. Due to the extreme differences in microbiota composition between the two donor groups, pathway scores were correlated with genus-level bacterial abundance for each group individually. Correlation was performed using the Hierarchical All-against-All (HAllA) approach developed for multiomics data sets [P values of < 0.1 were retained from the results and were plotted using ggraph and igraph [To determine if there was evidence of the correlation between antibiotic-induced changes in the microbiota and the transcriptome, we firstly used Gene Set Variation Analysis (GSVA) to identify enriched GO pathways across the entire transcriptome data set, using the R package GSVA (v1.32.0) . GO path genes 36 to 182 dd igraph  Additional file 2:Tables of proportional abundance of different genera in the different donor groups. (XLSX 18 kb)Additional file 3:Results from statistical tests of significance for different diversity metrics. (XLSX 8 kb)Additional file 4:Figure S2. Distance-based analysis examining the effects of antibiotics within and between cages. Schematic of the weighted unifrac distances between time points within individual cages for a donor group (S2A) and the distances at individual time points, including those between cages (S2B). In S2C, these distances are plotted for both donor groups, indicating a significant shrinking in distance post antibiotics (D8) in donor A mice, while there is no significant difference for donor B mice. (TIFF 1150 kb)Additional file 5:Figure S3. Heatmap of differentially abundant genes relating to the circadian rhythm at the contrasts for both donor groups  combined. (TIFF 943 kb)Additional file 6:Output from HAllA correlations of GSVA and genus abundance. (XLSX 43 kb)Additional file 7:Figure S4. Heatmap output from Hierarchical All-against-All significance procedure for donor A. (PDF 138 kb)Additional file 8:Supplementary methods and pyrosequencing barcodes. (DOCX 118 kb)Additional file 9:Output describing the code used for microbiota analysis. (HTML 3490 kb)Additional file 10:Output describing the code used for transcriptome analysis. (HTML 2420 kb)


Large sheaves, such as crosshead sheaves, are foundation parts in the heavy industry. Counter-roller spinning steel sheaves are utilized to replace traditional casting iron parts. Few studies exist on the groove shape of these spinning sheaves. Consequently, it is significant to explore the spinning groove shape variation rule and confirm the appropriate spinning parameters. Both experimental and numerical methods were utilized to study the groove shape variation of Q235 steel sheaves and their results were well matched. Spring-back phenomena were considered in this study. The groove depth was lower than the spinning depth and the last formed groove was the deepest. The former groove depth would be affected by the adjacent following spinning process. The single groove spinning result was linearly dependent on the multiple spinning groove depth. Certain equations were used to calculate the groove depth. The bottom–middle–top spinning sequence, which was better than other spinning sequences, should be used in the sheave spinning method. A sheave spinning process could be designed based on the study to obtain a fine groove shape. Large sheaves are used in various domains, such as crosshead sheaves for elevators and traction wheels for cranes. The counter-roller spinning method, which was developed to produce high-quality large sheaves, is of interest to many suppliers and consumers . CompareOnly a few studies exist on groove shape variation during counter-roller spinning. The groove shape is significant for sheaves ,5. An exThe traditional metal spinning method has been widely studied. Elknabeery et al. observed that through the decrease of the roller angle and feeding rate, the spinning quality of an aluminum cup could be improved . Jia et In order to explore the spinning shape to provide the design foundation for large sheave manufacturing, certain items should be studied. Firstly, it was required to affirm the groove shape subsequently to spinning. Next, it was necessary to explore the groove shape variation rule during spinning and discover the relationship between the groove shape and the spinning parameters. Finally, the appropriate spinning procedure for large sheaves should be ascertained.Based on the numerical and experimental methods, the groove shapes of the Q235 steel sheave was studied. The numerical model was established with ABAQUS software , which was satisfactory for the metal forming simulation ,17. The The groove shapes for large sheaves are presented in During spinning, all grooves were formed one by one. In a general bottom to top spinning procedure, all grooves are formed well with rectangular shapes. Consequently, the groove depth could be visualized. Therefore, the depth value was the core research object in this paper. The common groove size of 5.2 mm was selected as the basic spinning depth.The groove depth in this study was the average value of four points measurement data acquired from the circumferential direction of the sheave. This was used to avoid the measurement errors.Three additional feeding depths were designed to explore the relationship between the spinning parameters and the groove depth. The three feeding depths were 2 mm, 4 mm, and 6 mm. The single-groove sheave and the three-groove sheave were studied. The single-groove spinning could clearly show the spring-back effect on the groove depth, while the three-groove sheave was utilized to study the groove depths of the multiple-groove sheaves.The relationship between the forming sequence and the groove shape was studied through the numerical method. Three typical spinning sequences were considered, the bottom to top, the top to bottom, and the bottom to top to middle. The thickness, stress, and strain of the products were used to explore the spinning sequence character.Two numerical models were built with ABAQUS. The three-dimensional (3D) simulation for the general study and the two-dimensional (2D) model for the spring-back study were produced. All geometric models were built in CAD software and loaded into ABAQUS. The blank was a Q235 tube 1000 mm in diameter and 1 mm in thickness. The radius of the circular bead of roller was 5.2 mm. This study is about the cold deformation at room temperature.Both 3D and 2D models assumed a common continuous medium for the metal forming study. To save time, the mass scaling method was used in the dynamic explicit process and the scaling factor was 1000. The elastic–plastic material model was based on the Mises yield criterion, which is appropriate for metal forming. The coulomb friction method was used and the factor was set to 0.1 as the common friction factor of the spinning method. Since all rollers and mandrels were made of die steel with high hardness, they could keep a good shape during the spinning process. Therefore, the tube blank was set as the deformable part and other parts were set as discrete rigid parts. By this method, the model size could be effectively decreased.The 3D model is presented in In ideal circumstances, the groove depth should be the same as the spinning depth, 5.2 mm. Since this was a typical sheet forming method, the spring-back phenomenon could apparently change the product shape ,19. ConsConsidering that the sheave was too large to obtain a nice spring-back result in a 3D numerical simulation, the 2D model was selected to research the spring-back. As is well known, the metal spinning method is executed at the plain strain condition . Also, tIn the 2D model, the element size of the blank was 1 mm. The axisymmetric element CAX4R was used. Nine layers elements were set in the radial direction to improve the simulation accuracy. The roller and mandrel were meshed with the RAX2 element with a 1 mm size. The dynamic explicit method was utilized for the spinning process simulation and the standard method was selected to calculate the spring-back value.The material properties of the Q235 steel were obtained through the material performance measurements presented in The experiments were conducted with a computer-controlled counter-roller spinning machine. The experimental model is presented in The results of numerical simulations and experiments are presented in The final grooves are presented in The depth measurement method is presented in The depths of the three grooves are presented in The results of the three measurements in the 2D simulation are presented in The spring-back of G1 was approximately 0.40 mm and 7.69% of the ideal depth. The spinning affected the groove depth. The latest formed groove was the deepest. G3 was nearly 0.16 mm deeper than the G1 and G2.Actually, the dynamic explicit simulation could also demonstrate the spring-back phenomenon, but it required significant running time to obtain the final result. Considering the time consumption and data accuracy, the 2D simulation was better than the 3D simulation for the groove shape study. The 2D numerical method could produce results that exactly matched the experiments. Therefore, the 2D simulation was regarded as the major numerical method.The experimental results following each groove spinning step are presented in The results of each numerical simulation process step were the same as in the experiment. The numerical groove depth during spinning is presented in The experimental results are presented in Based on the numerical simulation, it could be observed that three main reasons existed for the former groove depth’s decrease. The first reason was the material flow in the axial direction and the second reason was the warping of the blank. The material flow and blank warping could be observed in Since the groove between the forming zone and the routine could restrict both the material flow and warping from routine groove, the routine groove depth would not change during the following spinning. Consequently, the G1 depth significantly decreased during G2 spinning, but was nearly constant during G3 spinning. The final groove depth could be regarded as a random groove depth subsequent to the adjacent groove spinning.The spinning depth is the key parameter, which determines the ideal groove shape in the sheave spinning method. The spring-back is the phenomenon leading to the difference between the ideal spinning groove and the final groove.The theory model of the spring-back phenomenon is shown in The single-groove sheave is a widely utilized part such as pulleys. Also, the single-groove study could directly produce the groove spinning characteristics without the effects of other grooves. Consequently, it was necessary to study the single-groove spinning method.The relationship between the groove and spinning depths is presented in The general spring-back empirical equation for stretch bending, which includes the similar stress states of groove spinning, often has apparent distortions in practical situations, and it is hard to make the coefficients conform . TherefoThe spring-back phenomenon of the spinning groove was similar to the stretch bending process. Therefore, the assumption of the spring-back was accurate to analyze the spring-back phenomenon of the spinning groove. Moreover, certain methods for the spring-back decrease of the spinning depth could also be used in groove spinning. The single-groove depth could be obtained through these equations.The groove depth of a multiple-groove sheave is affected by both the common spring-back phenomenon and the following spinning. The three-groove sheave was selected as an example of multiple-groove sheaves. If the 2 mm, 4 mm, and 6 mm spinning depths were used to form the three-groove sheaves, the numerical results would be as presented in The final and initial groove depths under different conditions are presented in If the experimental result from the 5.2 mm spinning depth situation was inserted into Through a comparison of the results of single-groove and three-groove spinning, their groove depth change rules were similar, as linearly dependent on the spinning depth. Since the final multiple-groove depth variation was the superposition of the common spring-back of the single groove and affected the following groove spinning, the following groove spinning effect was also linearly dependent on the spinning depth.The final groove depth of the single-groove spinning was slightly higher than that of the three-groove spinning. Based on the four previous equations, The three-groove sheave had a fixed side (bottom side), as presented in The results of the three spinning methods are presented in The groove depth of each spinning result differed. The top–top groove depths of these spinning sequences are presented in The stress, strain, and thickness results of these spinning processes are presented in In all spinning results, the maximum stress and strain were produced at the mandrel upper surface fillets. The stress and strain of the B-M-T were slightly higher than in the other two methods, but at a low level compared to the material properties. Also, both the stress and strain during spinning were tensile. Consequently, all these spinning processes could spin the grooves almost without cracking, wrinkles, or other defects.Considering all the aforementioned conditions, in the B-M-T spinning sequence with the best groove shape and acceptable strain, stress, the thickness result was satisfied for the sheave spinning process. The B-M-T sequence should be considered the first choice for sheave spinning.The grooves of all 3D/ 2D numerical simulations and experiments were well formed. Also, the 2D numerical simulation, when the spring-back phenomenon was considered, matched the experimental results well. Therefore, the numerical method was efficient for the groove spinning study.The last formed groove was always the deepest of all grooves, while the former spun grooves had similar depths. When the spinning depth was 5.2 mm, the top–top groove depths of the last and former grooves were approximately 5.28 mm and 4.70 mm, respectively. Moreover, their valid groove depths were approximately 0.25 mm shallower than the top–top groove.The groove depth changed during spinning. The former groove depth was almost constant subsequent to the following spinning. The depth of Groove 1 was highly decreased during the second groove spinning, and changed slightly during the third groove spinning. The Groove 2 depth became lower, with a value equal to the final Groove 1 depth, following the third groove spinning. Therefore, the final groove depth of the spinning sheave could be regarded as the groove depth following the next groove spinning.The groove depth was nearly linearly dependent on the spinning depth. The groove depth of the single-groove spinning was slightly higher than the three-groove spinning. In addition, the single-groove depth could be used to calculate the multiple grooves’ depths. Equations for the relationship between the groove depth and the spinning depth as well as between the single and multiple groove depths were obtained. The groove processing could be designed with these equations.The groove spinning sequences had an apparent effect on the spinning process. The B-M-T sequence was the most suitable for the sheave spinning. The B-M-T results were better groove shapes and higher groove depths than the other results. Also, the B-M-T result was a well-retained wall thickness, including similar stress and strain values to the other results. Therefore, B-M-T should be selected in the sheave spinning process.The groove shapes of the spinning sheaves were studied with numerical and experimental methods. The general spinning groove shape characters, the groove depth distribution, the groove depth variation during spinning, the groove depth calculation and the spinning sequence were discussed.


Based on our results, we propose that the apical actin ring could be a common site of initiation of actin-based protrusions in microvilliated sensory cells. Furthermore, our work provides a set of actors underlying actin-based protrusion elongation shared by different sensory cell types and highlights the critical role of the apical extension shape in sensory detection.Multiple types of microvilliated sensory cells exhibit an apical extension thought to be instrumental in the detection of sensory cues. The investigation of the mechanisms underlying morphogenesis of sensory apparatus is critical to understand the biology of sensation. Most of what we currently know comes from the study of the hair bundle of the inner ear sensory cells, but morphogenesis and function of other sensory microvilliated apical extensions remain poorly understood. We focused on spinal sensory neurons that contact the cerebrospinal fluid (CSF) through the projection of a microvilliated apical process in the central canal, referred to as cerebrospinal fluid-contacting neurons (CSF-cNs). CSF-cNs respond to pH and osmolarity changes as well as mechanical stimuli associated with changes of flow and tail bending. In vivo time-lapse imaging in zebrafish embryos revealed that CSF-cNs are atypical neurons that do not lose their apical attachment and form a ring of actin at the apical junctional complexes (AJCs) that they retain during differentiation. We show that the actin-based protrusions constituting the microvilliated apical extension arise and elongate from this ring of actin, and we identify candidate molecular factors underlying every step of CSF-cN morphogenesis. We demonstrate that Crumbs 1 (Crb1), Myosin 3b (Myo3b), and Espin orchestrate the morphogenesis of CSF-cN apical extension. Using calcium imaging in  A study of differentiating spinal sensory neurons in vivo uncovers critical factors required for the morphogenesis of sensory microvilli and reveals fine modulation of mechanosensory responses by microvillus length. Cells assume a variety of shapes known to be instrumental in their specific functions. Morphologies of neurons are among the most complex and diverse: they extend protrusions, build dendritic arborizations, and project axons in an intricate and stereotypical manner . SensorySpinal CSF-cNs are located along the central canal, into which they extend an apical extension composed of 1 motile cilium surrounded by actin-based protrusions that are in direct contact with the CSF –8. Over The CSF-cN neural progenitors are epithelial cells displaying an apicobasal polarity with their apical side facing the CSF. At this side, CSF-cN precursors form apical junctional complexes (AJCs), including adherens junctions and tight junctions, closely associated with actin filaments organized in a circumferential belt . TogetheIn the case of sensory neurons, specialized protrusions called microvilli can arise at their apical side to constitute the sensory organelle. Our current understanding of the formation of actin-based protrusions comes from studies of filopodia—short-lived thin processes—and involves 2 critical steps: initiation and elongation ,29. The espin and myo3b encode proteins that have been shown to interact and ensure the proper morphogenesis of stereocilia [After their initiation, membrane protrusions lengthen and, in the case of microvilli, stabilize. The elongation of sensory microvilli has been thoroughly studied in hair cells of the inner ear that form rigid microvilli derivatives referred to as stereocilia ,32. Throreocilia –36. Lossreocilia ,37. It icrb1—and several actin cytoskeleton interactors—baiap2a, baiap2l1b, myo3b, and espin. Our observations suggest a model in which these factors cooperate to regulate each step of the formation of the CSF-cN apical extension. Investigating the phenotype of a newly generated crb1 mutant, we describe a critical role of Crumbs 1 in the morphogenesis of CSF-cN apical extensions. Using a dominant-negative (DN) form of Myosin 3b, we demonstrate that this protein is required to address the actin-bundling factor Espin to the apical extension and enable it to reach the proper size. The analysis of a newly generated espin mutant indicates that Espin actin-bundling activity is necessary for the correct elongation of the microvilli constituting the CSF-cN apical extensions. Finally, functional analysis demonstrated that shorter apical extensions were associated to reduced mechanosensory responses in CSF-cNs lacking Crb1 or Espin. All together, these results provide insightful elements to build a mechanistic model of the formation of the apical extension by CSF-cNs and how its structure contributes to CSF-cN sensory function.Here, we monitor CSF-cN differentiation using long-term time-lapse imaging of F-actin and report 3 critical steps in their morphogenesis: (i) the presence of a ring of actin at the level of the AJCs, (ii) the initiation, and (iii) the elongation of actin-based protrusions from this actin ring. We show that, in contrast to other newborn neurons, CSF-cNs do not withdraw from the ependymal surface to migrate and differentiate. Instead, they appear to retain their apical cell polarity throughout differentiation, an original element of their morphogenesis. We describe new molecular factors known to be involved in cell polarity and/or morphogenesis, which are specifically enriched in CSF-cNs in the spinal cord. We identified a polarity factor—pkd2l1 promoter [To investigate the formation of the actin-based apical extension in CSF-cNs, we used the mosaic expression of LifeAct, a marker of F-actin , under tpromoter . We perfOur results indicate that the actin cytoskeleton goes through dynamic and extensive remodeling, suggesting an active participation in CSF-cN morphogenesis. F-actin is first organized at the apical side of the cells to form a ring from which, later on, protrusions seem to arise to form the apical extension.The localization and shape of the ring of actin are reminiscent of the circumferential structure formed by actin and adherens and tight junctions at the AJCs in epithelial cells . These cIn 72-hpf larvae, in contrast to ZO-1 being expressed in all cells lining the central canal . In ventThese results show that a ring of actin is formed early on in CSF-cNs at the level of the AJCs and that both structures persist after the apical extension is formed. This observation highlights a peculiarity of CSF-cNs compared with other neurons  because crb1 gene using clustered regularly interspaced short palindromic repeats/CRISPR/Cas9-mediated genome editing  with immunohistochemistry (IHC) for fluorescent proteins under the control of the  CSF-cNs  in the s microscopy to improve imaging resolution compared with standard confocal microscopy .We then investigated in more details the structure of CSF-cN apical extension in the unctions  and 6 isThese results demonstrate that Espin actin-bundling activity is necessary for the morphogenesis of CSF-cN apical extensions in a dose-dependent manner, through the regulation of microvilli elongation.espin+/− and espin−/− larvae, however, CSF-cN calcium transients in response to tail bends were smaller, correlated with wild-type espin gene copy number (espin+/− and 60% in espin−/−).We previously reported that CSF-cNs respond to mechanical stimuli associated with passive and active tail bending in zebrafish larvae . To invey number   at 24 hpf—urvature ,51. We kurvature . HoweverOur results suggest that polarization factors involved in the establishment of the apical domain and located at the vicinity of AJCs, near the ring of actin, are critical for the formation of the apical extension. One of these factors, Crb1, particularly drew our attention. First, Crb1 is specifically retained in spinal CSF-cNs at 72 hpf, while being down-regulated in the other cells lining the central canal. Second, Crb1 shows distinct patterns of expression in ventral CSF-cNs, where it forms a ring of actin at the vicinity of the AJCs, and dorso-lateral cells, where it covers the entire apical domain and protrusions. Our dominant-negative and knock-out strategies to induce Crb1 loss of function failed to cause early polarization defects in CSF-cNs, a result that can be explained by the high functional redundancy between intracellular domains of the different Crb proteins in zebrafish .However, we find that the spatial restriction of Crb1 to CSF-cN apical domain during differentiation participates in the proper morphogenesis of apical extensions. Our results suggest that the differential localization of Crb1 in the 2 CSF-cN subtypes contributes to the observed difference in size between ventral and dorsolateral cells but does not fully explain the difference in terms of shape . FurtherDrosophila [crb1 gene, mostly in the region encoding the extracellular domain, causes inherited retinal dystrophies [Drosophila Crb plays a critical role in the morphogenesis of microvilli-based structures in the photoreceptor cells, rhabdomeres [Crb proteins have been demonstrated to regulate morphogenesis of sensory protrusions in zebrafish and fly photoreceptors ,54 as weosophila . In humatrophies . It was bdomeres ,53. Togemyo3b and espin during their differentiation. Myo3b is a class III unconventional myosin, whereas Espin is an actin-bundling factor. Actin-bundling factors are able to cross-link actin filaments together and form higher-magnitude actin structures, such as microvilli [We also show that CSF-cNs express crovilli ,56,57. Icrovilli ,59. ESPIcrovilli . Previoucrovilli ,36. Our Our work further shows that suppressing one actin-bundling factor, Espin, shortens but does not suppress the actin-based CSF-cN apical extension. It has been shown that the development of bundles of actin filaments involves a combination of at least 2 different factors, usually in a sequential manner . The actIt is interesting to note here that Baiap factors contain an SRC Homology 3 (SH3) domain, which was shown to strongly interact with the proline-rich regions present in the longest isoforms of Espin . Our rescrb1 and espin mutations, namely, a reduction of the apical extension size in mutant cells. Because we observe no mislocalization of Espin in crb1 mutant cells and, conversely, no mislocalization of Crb1 in espin mutant cells, it is unlikely that the 2 proteins regulate CSF-cN apical extension morphogenesis through the same pathway. Consequently, the correct morphogenesis of the CSF-cN apical extension appears to rely on at least 2 distinct mechanisms, whose interactions remain unclear. Further investigation of the putative interplay between Crb1, Espin, and their respective interactors, as well as how the 2 pathways converge to support the formation of CSF-cN apical extensions, will bring essential elements to decipher molecular mechanisms underlying the morphogenesis of sensory microvilli organelles.We describe comparable morphological defects resulting from both The functional relevance of Espin has been largely documented in hair cells of the inner ear in which Espin loss is associated with incorrect lengthening of stereocilia, which leads to degeneration of hair cells and deafness ,62,63. Iespin and crb1 mutants provided a unique opportunity to investigate the contribution of microvilli to the process of sensory transduction. We show that the reduction of microvilli length is associated with a reduction in amplitude of CSF-cN mechanosensory response to passive bending of the spinal cord, indicating that the CSF-cN apical extension acts as a sensory antenna where mechanotransduction occurs. This observation is consistent with previous studies from our group and others demonstrating that CSF-cN-mediated detection of spinal bending [The reduction of microvilli length without CSF-cN degeneration in  bending , CSF flo bending ,13 requi bending , which iThe fact that the length of microvilli tunes the amplitude of CSF-cN sensory response while cells retain Pkd2l1 expression suggests that the mechanism underlying mechanotransduction in CSF-cNs is constrained by microvilli length. One possible explanation for such an effect could be that the length of microvilli determines the number of Pkd2l1 channels located at the membrane, which sets the amplitude of CSF-cN response to spinal cord bending. Longer microvilli may, furthermore, enable CSF-cNs to sample changes of CSF flow associated with spinal bending over larger volumes in the central canal and thereby amplify the mechanosensory response. Alternatively, proper lengthening of microvilli may enable the establishment of physical links between microvilli within the apical extension allowing for cohesion and/or mechanotransduction gating in a similar manner to hair cells ,65. The In hair cells of the inner ear, a large body of work has demonstrated the critical role of the staircase organization and patterned elongation of stereocilia for hearing . Our resAnimal handling and protocols were carried out with the validation of the Institut du Cerveau et de la Moelle épinière in agreement with the French National Ethics Committee  and European Communities Council Directive (2010/63/EU). Because experimentation on zebrafish larvae prior to 5 days old does not require approval of a protocol by the ethics committee, our project received the approval from the local ICM health and ethics committee.Zebrafish adults and embryos and/or larvae were reared and maintained in a 14/10-hour light cycle. Embryos and larvae were raised at 28.5°C until the start of experiments conducted at room temperature. When performed below 48 hpf, staging was assessed by counting somites (30 at 24 hpf) according to Kimmel and colleagues .Transgenic lines used in this study are listed in espin resulting in truncated Espin proteins lacking the conserved ABM  and a 10-bp deletion in exon 2 of crb1, resulting in a severely truncated Crb1 protein lacking all domains but one EGF-like extracellular   or Tg eggs at the 1-cell stage. Imaging was initiated as soon as LifeAct-GFP-positive cells were detected in the spinal cord, usually between 20 and 22 hpf. LifeAct-positive embryos were first anaesthetized in 0.02% MS-222 (Sigma-Aldrich), then mounted laterally in 1.5% low-melting agarose in 50-mm glass-bottom dishes (MatTek #P50G-1.5-14-F). Because the imaging took place at early development of the fish, we made sure to set areas to image, containing at least 1 positive cell for LifeAct, large enough to not lose the cell over time because of developmental growth. Z-stacks spanning the entire cell depth were taken every 5 minutes during several consecutive 1-hour-long sessions using Slidebook software . Imaging was performed using Zeiss ×40 or ×63 water-immersion objectives on a custom spinning disk microscope  with a 488-nm laser.To monitor the formation of the CSF-cN apical extension, we performed live imaging of single CSF-cNs labeled with LifeAct-GFP, a marker of F-actin , during aa:Venus  with appWe followed a similar protocol in fluorescent-positive embryos and larvae after screening. First, samples were fixed in 4% PFA at + 4°C for 4 hours, then washed in PBS Tween 0.01%, blocked for 2–5 hours at room temperature in 1% DMSO, 10% NGS, 0.5% Triton X-100 PBS 1×, and incubated with the following primary antibodies: mouse IgG1 anti–ZO-1 , chicken anti-GFP , mouse IgG1 anti-RFP  or rabbit anti-RFP , rabbit anti-Crb1 , rabbit anti-Espin , and rabbit anti-Myo3b  in 1% DMSO, 1% NGS, 0.5% Triton X-100 PBS 1X at +4°C overnight. After washing 3 times for at least 10 minutes in PBS 0.1X Tween 0.01% (PBST), samples were incubated with the secondary antibody  and DAPI  in the dark, overnight, at +4°C. The next day, embryos or larvae were washed again in PBST for at least 3 times for 30 minutes, and proper staining was assessed by checking the pattern of fluorescence of DAPI control staining before proceeding to mounting. Pkd2l1 staining was performed following an adapted protocol using a polyclonal rabbit antibody as described by Sternberg and colleagues [Tg transgenic line crossed to Tg to access the CSF-cN apical extension morphology. To assess the area covered by the CSF-cN apical extension in wild-type or espin and crb1 mutant siblings, we drew polygons outlining the apical extension and the soma on Z-projections using the polygon tool in Fiji.sc/ [Confocal images of the CSF-cN apical extension on spinal cord microtome sections were acquired following the procedure presented in our previous work . We usedFiji.sc/  and usedespin, the selected plasmid was linearized using NotI, and the resulting product was purified and measured for DNA concentration. For myo3b, baipa2a, and baiap2l1b, the product was generated by a second high-fidelity PCR using T7 forward primer, the corresponding reverse primer  or Tg(pkd2l1:TagRFP) prior to fluorescent IHC to reveal GFP or RFP, respectively, to specifically label CSF-cNs. For FISH, samples were prepared by fixation in 4% PFA in PBS overnight at +4°C. At 72 hpf, larvae were treated with Proteinase K in PBST for 20 minutes at 100 μg/mL to ensure the penetration of the probes. Probes were diluted to 1/200 for hybridization overnight at +70°C. To reveal the expression of candidate genes, primary antidigoxigenin-POD coupled antibodies (Sigma-Aldrich) were added to 1/300. Staining was realized using either TAMRA (1/100) in Tg(pkd2l1:GCaMP5G) samples or FITC (1/200) in Tg(pkd12l1:TagRFP) tyramide-fluorophore solution for 30 minutes. Imaging was performed on an Olympus FV1000 confocal microscope equipped with a ×20 water immersion objective.All the probes were generated by amplifying coding fragments from zebrafish 72-hpf larva total cDNA. The resulting PCR product was extracted from gel using the QIAquick Gel extraction kit  and cloned into the pCRII-Blunt-TOPO vector . Clones and orientation were verified by sequencing. For e primer  and Phu att-site tagged primers . The resulting fragment was inserted into pDONR p2rp3 in a BP recombination reaction (Thermo Fisher). The resulting plasmid was injected into 1-cell eggs carrying a combination of Tg and Tg. Confocal images of laterally mounted spinal cords were acquired, and we performed a Z-projection of individual ventral CSF-cNs either expressing Myo3b-DN (GFP and RFP positive) or control (RFP-only positive). The area of the apical extension and the cell body was assessed by drawing a polygon around the structure and measuring the area covered using Fiji.ct/ [To interfere with Myo3b activity, we built a truncated version of the protein devoid of the motor domain, reasoning that it would act as a dominant-negative form by titrating the interactors of the functional Myo3b . The parFiji.ct/  and normespinicm26/icm26 cells and to compare them with previous observations in cells lacking Myo3b function, we proceeded to the same analysis on confocal images of laterally mounted spinal cords at 72 hpf. To further confirm that the loss of Espin function is responsible for the observed phenotype in both conditions (espinicm26/icm26 and Myo3b-DN), we generated a plasmid to rescue the sole expression of Espin. The cDNA of the Espin isoform 1 was amplified by PCR  from zebrafish 72-hpf larva total cDNA using primers containing attB sites for Gateway cloning (forward: 5ʹ-CACCATGGTGGTTGAAAGGACACTTCTCGC-3ʹ and reverse: 5ʹ-TCCGGATCCCTGTTTCGCTATGTCTCC-3ʹ). The stop codon was removed, and a GSG sequence was added to allow efficient coexpression of T2A-RFP-NLS [Tgespinicm26 incross allowing the sparse expression of the rescue zEspin1 in espin+/+, espin+/icm26, and espinicm26/icm26 embryos and larvae.To assess morphological consequences of the loss of function of Espin in -RFP-NLS . The res-RFP-NLS  linearizTg and Tg transgenic lines carrying the espinicm26 mutation. The original IHC protocol [espin wild-type and mutant fish, we drew polygons outlining the apical extension and fitted an ellipse to extract the “minor” measurement using the polygon tool in Fiji.sc/ [To gain in confocal imaging resolution, we undertook STED microscopy imaging followed by deconvolution treatment on spinal cord microtome sections from a combination of protocol  was adapFiji.sc/ .Tgespinicm26 adult fish were incrossed, and offspring were screened for GCaMP5G and TagRFP fluorescence at 72 hpf after light anesthesia in 0.02% MS-222 (Sigma-Aldrich). At 120 hpf, double positive larvae were embedded in 1.5% low–melting-point agarose and paralyzed by injecting 0.5 nL of α-Bungarotoxin  in the musculature above the swimming bladder. Half of the tail was freed unilaterally to give access to the glass probe. Calcium imaging was performed using a 2-photon microscope (Intelligent Imaging Innovations) equipped with a ×20 water objective. Both GCaMP and RFP signals were recorded simultaneously at 5 Hz for 770 frames, allowing for 11 probe deflections to bend the tail every 70 frames, starting at 10 frames. Each stimulation therefore lasted 14 seconds. A new version of the Matlab script used in the work by Böhm and colleagues [GCaMP(t) × F0-TagRFP) ÷ (FTagRFP(t) × F0-GCaMP) at every time point left with F0 the average fluorescence of the 10 frames preceding the stimulation. The calcium transient amplitude was calculated as the difference (ΔR/R)post − (ΔR/R)pre, with (ΔR/R)post the average of the 3 frames right after stimulation and (ΔR/R)pre the average of the 10 frames before stimulation.To assess the sensory function of the CSF-cNs in mutant versus wild-type siblings, we performed the same passive tail bending assay as described in the work by Böhm and colleagues . Tg or median (black line). When displayed, the bottom and top edges of box plots indicate the 25th and the 75th percentiles, respectively, and whiskers extend to the most extreme data points not considered as outliers . To test the impact of wild-type espin allele numbers on apical extension size and calcium responses, we used multiple regression. More precisely, we relied on the general linear model (GLM) [t test. In all figures, * means that p < 0.05, ** means that p < 0.01, *** means that p < 0.001, and **** means that p < 0.0001.All statistics were run on Matlab . In most cases, student el (GLM) : the desS1 Data(XLSX)Click here for additional data file.S1 Moviepkd2l1 promoter. CSF-cN, cerebrospinal fluid-contacting neuron; hpf, hours post fertilization.Typical time-lapse recording used to obtain the results presented in (AVI)Click here for additional data file.S2 Moviepkd2l1 promoter in both wild-type  or mutant espin−/−  120-hpf larvae. The 2 signals were recorded at 5 Hz with a 2-photon laser-scanning microscope while the spinal cord of paralyzed animals was deflected with a glass probe. TagRFP signal (upper panels) is used as a reference to correct for motion artefact in all 3 dimensions. GCAMP5G (middle panels) fluorescence varies with calcium concentration and sensory activity. The change of ratio (ΔR/R) between the 2 signals was used to quantify neuronal activity . CSF-cN, cerebrospinal fluid-contacting neuron; hpf, hours post fertilization; RFP, red fluorescent protein; ROI, region of interest.Typical recordings supporting the results in (AVI)Click here for additional data file.S1 Figcrb1 locus in zebrafish on Chromosome 22.  Genomic region targeted by the sgRNA in exon 2 (sequence in bold), the earliest compatible target region containing a restriction site, here for RsaI, which is lost when editing occurs and enables a 2-step genotyping with a PCR followed by RsaI digestion. (Bottom) Sequence of the crb1icm31 allele generated showing the 10-bp deletion generated by the CRISPR-Cas9 genome editing technique. The early frameshift results in an amino acid sequence disturbed from early on (green) leading to a premature stop codon. (C) Schematics showing the predicted mutant truncated Crb1 protein obtained with the crb1icm31 10-bp deletion. Green boxes, EGF-like domains; violet boxes, laminin G-like domains. (D) IHC for Crb1 (cyan) showing the loss of immunoreactivity in TagRFP-CAAX-positive CSF-cNs (magenta) in 72-hpf crb1−/− larvae compared with wild-type siblings. Scale bars, 10 μm. Crb1, Crumbs 1;CSF-cN, cerebrospinal fluid-contacting neuron; EGF, epidermal growth factor; IHC, immunohistochemistry; PAM, protospacer adjacent motif; sgRNA, single guide RNA.(A) Localization and genomic structure of the unique (TIF)Click here for additional data file.S2 Fig(A) IHC for Myo3b shows the enrichment of the protein (cyan) at the level of AEs of TagRFP-CAAX-positive CSF-cNs (magenta) in 72-hpf larvae. Scale bar, 10 μm. (B) IHC for Espin was performed on whole-mount zebrafish 72-hpf larvae. Scale bar, 100 μm. Espin is enriched at the apical extension of various microvilliated sensory cell types: olfactory neurons in the olfactory pit (B1), hair cells of the inner ear (B2), lateral line hair cells (B3), and CSF-cNs (B4). Scale bars, 10 μm. AE, apical extension; CSF-cN, cerebrospinal fluid-contacting neuron; hpf, hours post fertilization; IHC, immunohistochemistry.(TIF)Click here for additional data file.S3 Figespin locus in zebrafish on Chromosome 8. The conserved actin-bundling module is encoded by exons 11 to 13.  Genomic region targeted by the sgRNA in exon 11 (sequence in bold), right upstream of the coding sequence for the actin-bundling module (amino acid sequence indicated in blue). The target region contains a BstXI digestion site, upstream of the PAM, which is disabled when editing occurs. (Bottom) Sequence of the espinicm26 allele generated showing the 5-bp deletion generated by the CRISPR-Cas9 genome editing technique. In espinicm26, the coded amino acid sequence of the actin-bundling module is disturbed from the first codon (green). (C) Schematics showing the predicted mutant truncated Espin protein obtained with the espinicm26 5-bp deletion. The actin-bundling module is entirely disabled (white box). Green boxes, ankyrin-like repeats; violet boxes, proline-rich regions; red box, WH2 domain; blue box, actin-bundling module. (D) IHC for Espin (cyan) showing the loss of immunoreactivity in TagRFP-CAAX-positive CSF-cNs (magenta) in 72-hpf espin−/− larvae compared with wild-type siblings. (E) IHC for Espin showing the gradual loss of immunoreactivity in CSF-cNs of espin+/−and espin−/− compared with espin+/+ 72-hpf larvae. Samples were analyzed simultaneously, and images were acquired and treated with the same parameters. Scale bars, 10 μm. CSF-cN, cerebrospinal fluid-contacting neuron; hpf, hours post fertilization; IHC, immunohistochemistry; PAM, protospacer adjacent motif; sgRNA, single guide RNA; WH2, WASP (for Wiskott-Aldrich Syndrom protein) homology 2.(A) Localization and genomic structure of the unique (TIF)Click here for additional data file.S4 Figespin−/− mutant larvae  compared with wild-type siblings . Both CSF-cN subtypes lacking Espin show a significant reduction of the area covered by their apical extension as observed at 72 hpf . (B1) STED confocal images from spinal cross sections of 72-hpf espin+/+ or espin−/− larvae showing apical extensions of ventral TagRFP-CAAX-positive CSF-cNs. The junctional region is highlighted by ZO-1 staining (green). Scale bars, 1 μm. (B2) Quantification of the vertical extension of ventral CSF-cN apical extensions at 72 hpf in espin−/− mutant larvae (light blue) versus wild-type larvae (dark blue) from STED images obtained as in (B1). Mutant cells formed significantly shorter apical extensions (p = 2.5571 × 10−4), suggesting the critical role of Espin actin-bundling activity for the proper lengthening of CSF-cN microvilli. Underlying data can be found in (A) Quantification of the area covered by the CSF-cN apical extension at 144 hpf (6 days) in ventral and dorsolateral cells in (TIF)Click here for additional data file.S5 Figcrb1−/− larvae shows that Espin is similarly expressed and enriched at the apical extension of mutant V and DL CSF-cNs compared with wild-type cells. Scale bars, 10 μm. Crb1, Crumbs 1; CSF-cN, cerebrospinal fluid-contacting neuron; DL, dorso-lateral; hpf, hours post fertilization; IHC, immunohistochemistry; V, ventral; ZO-1, zonula-occludens-1.(A) IHC for Crb1 (cyan) in TagRFP-CAAX-positive CSF-cNs (magenta) of 72-hpf larvae shows that Crb1 is similarly expressed and located in mutant CSF-cNs compared with wild-type cells. (B) IHC for Espin (cyan) and ZO-1 (magenta) to highlight the junctional region in (TIF)Click here for additional data file.S6 Figcrb1−/− 120-hpf animals (data pooled from 3 experiments). (B) The amplitude of CSF-cN calcium transients shown in (A) is represented as the ratio of peak fluorescence over baseline (ΔR/R) and is significantly reduced in crb1−/− mutant compared with wild-type siblings (p = 1.012 × 10−5). (C and D) IHC for Pkd2l1 channel (cyan) in espin−/− (C) or crb1−/− (D) shows that TagRFP-CAAX-positive CSF-cNs (magenta) retain the expression of the channel at their apical extension in mutant larvae similarly to wild-type siblings. Scale bars, 10 μm. Underlying data can be found in (A) Overlay of calcium transients in ipsilateral dorsolateral CSF-cNs in response to tail bending induced by a glass probe in paralyzed wild-type versus (TIF)Click here for additional data file.


The variations of the mechanical properties of the CoCrFeMnNi high entropy alloy (HEA) during groove cold rolling process were investigated with the aim of understanding their correlation relationships with the crystallographic texture. Our study revealed divergences in the variations of the microhardness and yield strength measured from samples deformed by groove cold rolling and conventional cold rolling processes. The crystallographic texture analyzed by electron back scattered diffraction (EBSD) revealed a hybrid texture between those obtained by conventional rolling and drawing processes. Though the groove cold rolling process induced a marked strengthening effect in the CoCrFeMnNi HEA, the mechanical properties were also characterized by an unusual decrease of the Young’s modulus as the applied groove cold rolled deformation increased up to about 0.5 before reaching a stabilized value. This decrease of the Young’s modulus was attributed to the increased density of mobile dislocations induced by work hardening during groove cold rolling processing. High entropy alloys (HEAs), a new class of metallic materials in which the stability of the solid solution is explained by contribution of the configurational entropy, have recently received particular attention, owing to the prospect of developing new systems with tailored properties particularly suitable for a large range of different applications from dies and molding materials to corrosion-resistant coatings in manufacturing, energy, transport, and aeronautical industries ,2,3,4. TThe equi-atomic CoCrFeMnNi composition developed by Cantor and colleagues  is curreThough most of studies are concerned about the mechanical properties, the modifications of the Young’s modulus induced by work hardening have not been intensively studied. However, as seen earlier in the 1950′s, in their theory of work hardening, both Mott  and FrieThis study was thus undertaken to investigate the evolution of the hardness and tensile properties of CoCrFeMnNi HEA alloy with the increasing area reduction achieved by grooved cold rolling. Though this process resulted in a marked strengthening effect, it was also accompanied by a reduction of the Young’s modulus. The variation of the crystallographic texture with the increasing groove rolling deformation was found to be slightly different from that obtained during conventional cold rolling. This study attempted to provide elements enabling the understanding of the observed changes in the mechanical properties.20.1Cr19.9Fe20.6Mn19.5Ni19.9 (in at.%) as analyzed by electron dispersive spectroscopy (EDS) was produced by vacuum arc melting at KIST, Korea, using initial elements with purity above 99.9%. Hot forging treatment and hot rolling at 1000 °C were performed to homogenize the microstructure and to reduce the initial ingot thickness down to 12 mm. After another thickness reduction by conventional cold rolling, 4 samples of dimension 9.3 × 9.3 × 80 mm3 were machined perpendicularly to the rolling direction. Finally an annealing heat treatment was then applied during 3600 s at 900 °C under a protective Argon atmosphere. Following this heat treatment, samples were considered, for this study, in a reference state in term of microstructure and mechanicals properties.A 5 kg ingot of the equi-atomic CoCrFeMnNi alloy of composition CoRoom temperature groove cold rolling process was performed on the annealed square bars. In this process, rollers present V-grooves of different dimensions as illustrated in 2. For each deformation level, a wire was extracted with a minimal length of about 120 mm for tensile test and microhardness measurements. In order to determine the tensile properties of the initial state, the last sample was rolled until 38% area reduction ratio  and annealed under the same conditions as described above. ie and fe correspond to the initial and final thicknesses, respectively.The samples were respectively rolled down to 14% and 33% area reduction ratios, i.e., rolling deformation ε = 0.2 and ε = 0.4, respectively. The obtained square wire-type samples were cut off in order to investigate the tensile properties . The rests of the samples were rolled to successively up to 89% area reduction ratio , corresponding to a final section of about 3.1 × 3.1 mmn(ei/ef) , where e−3 s−1. Values of the Young’s modulus were determined as the mean of the initial slope of each discharging portion of the curves. Values of the yield strength were obtained from the conventionally 0.2% of plastic deformation. A typical obtained curve is shown in the right-hand side inset in Uniaxial tensile tests were carried out on 100 kN universal Zwick tensile test machine, using a 50 kN load cell. With the aim of determining the Young’s modulus, samples were first loaded to 1% strain, then 5 charge/discharge cycles were performed. Strains were measured using an Epsylon extensometer with a gage length of 25 mm, and the samples were deformed at a constant strain rate of 1 × 10For microstructural observations, samples were polished  using SiC papers with grit size about 80 in order to reduce the thickness and to reach approximately the center part of the samples. Then surfacing polishing was performed with grit size from 1000 to 4000. Hardness values were measured by micro-indentation Vickers tests carried out using a Zwick Rowel machine with a load of 0.3 gf, leading to indentation size of about 35 to 70 µm. Distance between adjacent measurements was at least 200 µm. For each sample, the hardness value was averaged from 15 measurements, and the errors bars correspond to the measured standard deviation.2O3 polishing with final particle size of 0.1 µm. Microstructural analyses were carried out on SEM Jeol 6490, at 20 kV with a working distance of 21 mm. Electron back scattered diffraction (EBSD) analyses were performed by Oxford instrument detector with a minimal number of detected Kikuchi bands equal to 8 and a step size of 0.4 µm. EBSD were studied in four cases: after annealing and after section reduction of ε = 0.5, 1.1 and 2.2. EBSD maps, Figure pole (FP), inverse figure pole (IFP) and orientation density function (ODF) were obtained using ATEX software developed at LEM3 [Before tensile testing, pieces of rolled materials were cut off for microstructural analyses. Samples were polished using SiC paper with grit size down to 4000 followed by a Al at LEM3 . Texture at LEM3 ,18. The 0.3, which is in good agreement with values obtained in published articles under the same annealing treatment [0.3 to about 310 HV0.3, characterized by a larger value of the slope (284 kgf/mm2) than those reported by Otto et al. [2) represented by the green dashed line in the 0.3 for a groove rolling deformation ε = 2.2. It is remarkable that the microhardness values followed the same trend under both groove rolled and conventional rolling processes, though the initial slope was larger and that the breakdown in the slope occurred earlier for samples deformed by groove rolled process.reatment ,23. The e slope 24 kgf/mm2The evolution of the yield strength, as shown in As mentioned in the introduction, a part of this study focused on understanding the evolution of the Young’s modulus as function of the groove rolling deformation. By considering the single-phase character of CoCrFeMnNi HEA, the first mentioned reason appeared to be irrelevant. In their study, Benito et al. have suggested that the internal stresses generated during the early stage of cold rolling were not sufficient to describe the variation of the Young’s modulus . ConsequThe different mechanisms in the CoCrFeMnNi HEA are now well documented in the literature for different deformation modes and different degrees of deformation ,23,36,372 = 45°  and Rotated Goss (RtG) texture components were not detected. The S component also seemed to follow the same trend as the α-fiber texture, with a weaker decrease for deformation up to 2.2 in comparison to the B and RtG components. This first features observed during the conventional rolling process, with the α-fiber was found to continuously increased as the deformation increased, as it was reported by Sathiaraj et al. [For the sample processed to a groove rolling deformation ε = 0.5, a slight partial α-fiber appeared between the G and P components, and it increased till ε = 1.1. As the deformation level increased, this fiber seemed to be “broken” for higher deformation states as indicated by the ODF with φj et al.  and Haasj et al. . The <11j et al. . In our To summarize, the texture of CoCrFeMnNi HEA alloy by groove cold rolling may be described as a hybrid texture between the texture obtained by conventional cold rolling and that obtained by swaging or drawing processes. Under high level of deformation, the <111>RD fiber tended to become predominant due to the decrease of the <110>ND fiber. This final state appeared to be very similar to the texture of FCC materials deformed by swaging process.This study firstly focused on the evolution of mechanical properties of the CrCoFeMnNi HEA during deformation by groove cold rolling. Our results revealed two stages in the evolution of the microhardness, yield strength, and Young’s modulus, with a change of slopes for a groove rolling deformation of about ε = 0.5. Both microhardness and yield strength displayed a strong increase with the strain up to ε = 0.5 by groove cold rolling followed by a moderate increase, to reach values of, respectively, around 400 HV and 1400 MPa for an applied groove rolling deformation ε equal to 2.2. In contrast, the Young’s modulus displayed firstly a decrease of about 15% from its initial value as the groove rolling deformation increased to about ε = 0.5, followed by a slight increase from 175 to 179 GPa as the groove rolling deformation was further increased till ε = 2.2.The texture analysis revealed that the as-annealed and deformed microstructures presented similar features to those reported in previous studies on CoCrFeMnNi HEA processed by conventional cold rolling. From these considerations, it has been assumed that the deformation mechanisms involved during groove cold rolling were the same as those already reported for CoCrFeMnNi HEA alloy, i.e., dislocation glide and twinning. In order to interpret the variation of the mechanical properties, identification of the main texture components and their evolutions tended to confirm the emergence of a specific crystallographic texture responsible for the change in the microhardness and yield strength as described above. It has been shown that these textures could be interpreted as a hybrid texture between conventional rolling and swaging texture. Also, under high deformation level, our analyses suggested that the swaging type texture tended to become predominant.The evolution of hardness and yield strength may be regarded through the evolution of the active deformation mechanism. Otto et al. , as wellijkl of the quaternary CoCrFeNi alloy [The variation of the Young’s modulus followed a different pattern that might be more complicated and less intuitive at this current stage of our investigation. Owing to the strong anisotropy of FCC alloys, such as austenitic Fe and Ni alloys, values of the Young’s modulus are expected to be strongly dependent upon the crystallographic texture . Since tNi alloy . WithoutNi alloy  undertakAs it has been advanced in the introduction, changes in the dislocation arrangement could be responsible for the variation of the elastic properties. Otto et al.  explaine-Both the microhardness and the yield strength exhibited similar evolution as the area reduction ratio applied by groove cold rolling increased. A first marked increase until a groove rolling deformation of about 0.5 was followed by a moderate increase under higher strain level. This two-step behavior has been attributed to change in the activated deformation mechanisms, and characterized by a transition from dislocation mechanism and dislocation + twinning deformation mechanism as reported by Otto et al.  and Haas-The Young’s modulus also displayed a two-step variation as the groove cold rolled deformation increased. An approximately 15% decrease from 210 GPa to 175 GPa was observed between annealed HEA samples and those deformed up to about 0.5 by groove rolled process. For higher level of the groove cold rolling deformation, the Young’s modulus value stabilized around 175 GPa. This evolution of the elastic property, which could not be attributed by a modification of the crystallographic texture, has been explained by the transition of operating deformation mechanisms between gliding of non-pinned dislocations within dislocation cell in the first stage, and contribution of twinning for larger values of the groove rolling deformation.-The texture analysis highlighted a complex crystallographic texture induced by groove cold rolling deformation. This texture has been described as a hybrid texture between the texture developed during conventional rolling and that from swaging or drawing process. This texture was found to consist in a combination of <111> fiber texture along the RD, <110> fiber texture along ND and ND’ and <001> texture along RD. At higher strain level, the <110> fiber texture tended to disappear for the benefit of <111> fiber texture along the RD. This texture was found to be consistent with the bi-axial deformation induced by groove cold rolling but could not account for the decrease in the Young’s modulus.In this study, samples of CoCrFeMnNi HEA were successively deformed by groove cold rolling at different strain levels. The mechanical properties and crystallographic texture were evaluated as a function of the increasing cold rolling deformation, leading to the following conclusions: