The small LOV/F-box/Kelch family of E3 ubiquitin ligases plays an essential role in the regulation of plant circadian clocks and flowering time by sensing dusk. The family consists of three members, ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2), and FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1), which share a unique protein domain architecture allowing them to act as photoreceptors that transduce light signals via altering stability of target proteins. Despite intensive study of this protein family we still lack important knowledge about the biochemical and functional roles of the protein domains that comprise these unique photoreceptors. Here, we perform comparative analyses of transgenic lines constitutively expressing the photoreceptor LOV domain or the Kelch repeat protein-protein interaction domains of ZTL, FKF1, and LKP2. Expression of each domain alone is sufficient to disrupt circadian rhythms and flowering time, but each domain differs in the magnitude of effect. Immunoprecipitation followed by mass spectrometry with the ZTL Kelch repeat domain identified a suite of potential interacting partners. Furthermore, the ZTL Kelch repeat domain can interact with the ZTL homologs, LKP2 and FKF1, and the LOV domain of ZTL itself. This suggests a hypothesis that the Kelch repeat domain of ZTL may mediate inter- and intra-molecular interactions of the three LOV/F-box/Kelch proteins and provides added insight into the composition of the protein complexes and an additional role for the Kelch repeat domain. Sensing day-night transitions is essential for proper adaptation of an organism to its environment. In plants, dusk is particularly important as it can communicate photoperiod duration and thereby the season to the plant. A multitude of biological processes depend on seasonal timing, such as reproduction, energy production, and starch biosynthesis and degradation rates that balance plant growth with night-time survival –4.ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) [A small family of blue-light photoreceptors, the LOV/F-box/Kelch proteins, communicates the dusk transition to the photoperiodic flowering time pathway and the circadian clock in plants. In Arabidopsis, this family consists of three members, 2 (LKP2) –7. These2 (LKP2) , 9.ZTL plays a major role in the regulation of the circadian clock and has only a minor role in the regulation of flowering time, while FKF1 is an essential regulator of photoperiodic flowering that has minimal impact on the circadian clock [LKP2 is redundant with ZTL and FKF1, as single knockout mutations in this gene lead to minimal phenotypic consequences but exaggerate ZTL and FKF1 mutant phenotypes [Despite the similarities in primary amino acid sequence between these three proteins, they display a complex pattern of genetic redundancy. For example, an clock , 10–13. enotypes , 12, 14.enotypes .In order to fully understand the overlapping and distinct functions of this important gene family, intense research has begun to investigate the structures and biochemical functions of the LOV and Kelch repeat domains , 14–24. In addition to its role in promoting protein interactions with regulatory proteins such as GI, the LOV domain is also involved in directly binding substrate proteins that are stabilized or destabilized. For example, the FKF1 LOV domain interacts with the floral activator CONSTANS (CO) in a GI-dependent manner, stabilizing CO in the light . In contZTL and FKF1 that lack the F-box domain mimics published loss-of-function mutant phenotypes [Adjacent to the LOV domain is a typical F-box domain and at the C-terminus of the protein is a Kelch repeat type protein-protein interaction domain. The F-box domain is a critical component of the SKP1/CULLIN/F-BOX (SCF) multi-subunit E3 ubiquitin ligase that is required for interaction with the other components of this complex –34. We henotypes . One rolenotypes . Interesenotypes , 26.ZTL which contains only the LOV and F-box domains lengthens period similarly to a ztl loss-of-function mutant rather than shortening period as is observed in plants overexpressing full-length ZTL [In contrast, the ZTL Kelch repeat domain has not been demonstrated to interact with known ZTL substrates , 29–31. ngth ZTL , 10, 15.ngth ZTL . TogetheZTL, LKP2, and FKF1 LOV and Kelch repeat domains, we demonstrate that both the LOV and Kelch repeat domains are independently sufficient to disrupt the circadian clock and flowering time when expressed in plants. We then focus further studies on the biochemical role of the ZTL Kelch repeat domain using immunoprecipitation followed by mass spectrometry to identify a list of putative protein interacting partners. We find that FKF1 and LKP2 as well as the native ZTL protein are part of a complex with the ZTL Kelch repeat domain, supporting and extending previous studies. Using yeast-two-hybrid analyses, we determine that the Kelch repeat domain has the potential to interact with the LOV domain of ZTL allowing us to hypothesize two possibilities: an antiparallel conformation for intermolecular homodimers or intramolecular interaction between the two domains. Our results suggest that a biochemical role of the ZTL Kelch repeat domain may be promoting interaction within or between LOV/F-box/Kelch proteins, supporting previous data and providing a possible mechanism for the role of ZTL in promoting auto-ubiquitylation and also mediating stability of FKF1. Furthermore, our genetic analyses allow us to hypothesize that the Kelch repeat domain may modulate the formation of higher order protein complexes that are essential for the function of LOV/F-box/Kelch proteins.In this study, we further investigate the genetic and biochemical functions of the ZTL Kelch repeat domain. By performing comparative genetic analyses of plants overexpressing the FKF1, LKP2, and ZTL in the CCA1p::Luciferase (CCA1 promoter driving expression of firefly Luciferase) background, and monitored circadian clock period and flowering time. We included CCA1p::Luciferase plants that express FKF1, LKP2, and ZTL decoys (LOV-Kelch fusion proteins which lack the F-box domain), which we have analyzed previously [CCA1p::Luciferase parental plants, as controls. In order to compare results from experiments performed separately, we use the difference between the period or flowering time of the individual T1 transgenic and the average period or flowering time of the concurrent wild type control plants for our statistical analyses [We have previously shown that expressing ZTL, LKP2, and FKF1 without the F-box domain results in disruption of circadian clock function and flowering time . We nexteviously , and wilanalyses , 37. TheZTL, LKP2, and FKF1 and monitored circadian clock period and flowering time , the period defect of the smallest magnitude in the plants expressing the FKF1 LOV domain or FKF1 decoy , and a period defect of intermediate magnitude in the plants expressing the LKP2 LOV domain or LKP2 decoy .ZTL, LKP2, and FKF1 have all been shown to regulate clock function , 13, 30.FKF1 LOV domain or FKF1 decoy , the smallest delay in flowering observed in plants expressing the ZTL LOV domain or ZTL decoy , and an intermediate delay in plants expressing the LKP2 LOV domain or LKP2 decoy . Interestingly, expressing the FKF1 LOV and FKF1 decoy do not have the same effect on flowering time (4.1 days versus 6.0 days). This is consistent with the known role of the FKF1 Kelch domain in promoting the degradation of the CDF flowering time regulators [The relationship was inverted with regards to flowering time, with the longest delay in flowering observed in plants expressing the gulators , 26.ZTL LOV domain or ZTL decoy, we observe no statistical difference in the period from wild type, and extreme delayed flowering , consistent with what had previously observed in plants expressing the ZTL decoy (LKP2 decoy in our previous study [LKP2 decoy in both period and flowering time phenotypes and subpopulations for the plants that express the LKP2 LOV domain in the flowering time phenotypes (LKP2 LOV domain (17.0 days delayed), and a more extreme delay in flowering in the minority subpopulation of plants expressing the LKP2 decoy (29.5 days delayed). In the minority subpopulation of plants expressing the LKP2 decoy, we also observe a small, but statistically significant, shortening of the circadian period. These results are consistent with published data that the LKP2 Kelch repeat domain may regulate CDF proteins and be important for the role of LKP2 in flowering time control [FKF1 LOV domain, and instead observe a positive association between delayed flowering and a lengthened circadian period in these plants  and three hours after dusk (ZT15)  suggesting that we were effectively immunoprecipitating the appropriate protein domain.Our genetic results indicate that the ZTL Kelch repeat domain plays an important role in the regulation of the circadian clock and flowering. We hypothesize that the ZTL Kelch repeat domain may interact with unknown substrates or regulatory partners that may help elucidate its biochemical function. Thus, we performed an immunoprecipitation followed by mass spectrometry (IP-MS) experiment in our transgenic plants constitutively expressing a HIS-FLAG tagged k (ZT15) . As contin planta. Thus, we searched our IP-MS results for peptides corresponding to the known ZTL interactors. We were unable to identify peptides corresponding to the majority of the known ZTL substrates and interacting partners  Col-0 expressing the circadian reporter CCA1p::Luciferase [Agrobacterium tumefaciens GV3101.The creation of the ZTL, LKP2, and FKF1 decoy was described previously . PCR wasciferase  by the fciferase  using AgpCCA1∷Luciferase and transgenic seeds were surface sterilized in 70% ethanol and 0.01% Triton X-100 for 20 minutes prior to being sown on ½ MS plates  with or without appropriate antibiotics ). Seeds were stratified for two days at 4°C, then transferred to 12 hours light/12 hours dark conditions for seven days. Twenty seven-day old seedlings from each genotype were arrayed on 100 mm square ½ MS plates in a 10x10 grid, then treated with 5 mM D-luciferin  dissolved in 0.01% TritonX-100. Imaging was performed at 22°C under constant 100 μmol m−2 s−1 white light provided by two LED light panels (Heliospectra L1). Hourly images were acquired for approximately six and a half days. Each hour, lights are turned off for a total of eight minutes in order to capture a 5 minute exposure on an Andor iKon-M CCD camera; lights are off two minutes prior to the exposure and remain off for one minute after the exposure is completed. After imaging is complete, the lights return to the normal lighting regime. The CCD camera was controlled using Micromanager, using the following settings: binning of 2, pre-amp gain of 2, and a 0.05 MHz readout mode [Control out mode . Data coThe mean intensity of each seedling at each time point was calculated using ImageJ . The cal-2 s-1. Plants were monitored daily for flowering status, recording the dates upon which each individual reached 1 cm inflorescence height. Each experiment was performed twice with new independent T1 insertion transgenics in order to demonstrate repeatability. Data presented in figures and tables represents all experimental repeats, and raw values can be found in Following luciferase imaging, seedlings were transferred to soil (Fafard II) and grown at 22°C in inductive 16 hours light/8 hours dark conditions with a light fluence rate of 135 μmol mTo allow for comparison across independent imaging experiments, data was normalized to the individual wild type control performed concurrently. The average value of the wild type control was calculated for every experiment, then this average was subtracted from the value of each individual T1 insertion or control wild type plant done concurrently. This normalized value was used for statistical analyses.Welch’s t-test was used to compare each normalized T1 insertion population or subpopulation to the population of normalized control plants. In order to decrease the number of false positives caused by multiple testing, we utilized a Bonferroni corrected α as the p-value threshold. The α applied differs between experiments, and is noted throughout.pB7-HFN-ZTL-Kelch transgenics in a Col-0 background and control Col-0 and pB7-HFC-GFP were grown as described for phenotype analysis. Seven-day old seedlings were transferred to soil and grown under 16 hours light/8 hours dark at 22°C for 2–3 weeks. Prior to harvest, plants were entrained to 12 hours light/12 hours dark at 22°C for 1 week. 2–3 mature leaves were collected from 20 T1 or control plants and flash frozen in liquid nitrogen. Tissue samples were ground in liquid nitrogen using the Mixer Mill MM400 system (Retsch). Immunoprecipitation was performed as described previously [® M-270 Epoxy  for immunoprecipitation. Immunoprecipitation was performed by incubation of protein extracts with beads for 1 hour at 4°C on a rocker. Beads were washed with SII buffer three times, then twice in F2H buffer . Beads were eluted twice at 4°C and twice at 30°C in F2H buffer with 100 μg/mL FLAG peptide, then incubated with TALON magnetic beads  for 20 min at 4°C, then washed twice in F2H buffer and three times in 25 mM Ammonium Bicarbonate. Samples were subjected to trypsin digestion  at 37°C overnight, then vacuum dried using a SpeedVac before being dissolved in 5% formic acid/0.1% trifluoroacetic acid (TFA). Protein concentration was determined by nanodrop measurement (A260/A280)(Thermo Scientific Nanodrop 2000 UV-Vis Spectrophotometer). An aliquot of each sample was further diluted with 0.1% TFA to 0.1μg/μl and 0.5μg was injected for LC-MS/MS analysis at the Keck MS & Proteomics Resource Laboratory at Yale University.Individual T1 eviously , 49, 55.LC-MS/MS analysis was performed on a Thermo Scientific Orbitrap Elite mass spectrometer equipped with a Waters nanoACQUITY UPLC system utilizing a binary solvent system . Trapping was performed at 5μl/min, 97% Buffer A for 3 min using a Waters Symmetry® C18 180μm x 20mm trap column. Peptides were separated using an ACQUITY UPLC PST (BEH) C18 nanoACQUITY Column 1.7 μm, 75 μm x 250 mm (37°C) and eluted at 300 nl/min with the following gradient: 3% buffer B at initial conditions; 5% B at 3 minutes; 35% B at 140 minutes; 50% B at 155 minutes; 85% B at 160–165 min; then returned to initial conditions at 166 minutes. MS were acquired in the Orbitrap in profile mode over the 300–1,700 m/z range using 1 microscan, 30,000 resolution, AGC target of 1E6, and a full max ion time of 50 ms. Up to 15 MS/MS were collected per MS scan using collision induced dissociation (CID) on species with an intensity threshold of 5,000 and charge states 2 and above. Data dependent MS/MS were acquired in centroid mode in the ion trap using 1 microscan, AGC target of 2E4, full max IT of 100 ms, 2.0 m/z isolation window, and normalized collision energy of 35. Dynamic exclusion was enabled with a repeat count of 1, repeat duration of 30s, exclusion list size of 500, and exclusion duration of 60s.Arabidopsis thaliana database with oxidation set as a variable modification. The peptide mass tolerance was set to 10 ppm, the fragment mass tolerance to 0.5 Da, and the maximum number of allowable missed cleavages was set to 2.The MS/MS spectra were searched by the Keck MS & Proteomics Resource Laboratory at Yale University using MASCOT . Data waTo determine statistically significant interactors, we removed all proteins that only occurred in the controls, then performed SAINTexpress using interface available on the CRAPome website , 39. ProZTL full length, decoy, LOV, and Kelch repeat coding sequences in pENTR/D-TOPO vectors were recombined into the pGBKT7-GW destination vector (Gateway-compatible pGBKT7 vector). This resulted in a translational fusion of the ZTL domains to the GAL4 DNA-binding domain [Saccharomyces cerevisiae) Y187 strain. Similarly ZTL Kelch repeat coding sequences in pENTR/D-TOPO vectors were recombined into the pGADT7-GW vector (Gateway-compatible pGADT7 vector), resulting in a translational fusion to the GAL4 activation domain . These were transformed into the yeast AH109 strain. To test protein-protein interactions, diploid yeast was generated by yeast mating of Y187 and AH109 strains bearing pGBKT7 and pGADT7 vectors, respectively, and tested on synthetic dropout/-Leu-Trp and synthetic dropout/-Leu-Trp-His-Ade plates. The empty pGBKT7-GW and pGADT7-GW vectors were included as negative controls.Yeast two-hybrid assays were performed according to the Yeast Protocol Handbook . Briefly, g domain . These cS1 TableAll results from the plants expressing the HIS-FLAG-tagged ZTL Kelch repeat domain, the plants expressing the HIS-FLAG-tagged GFP, and wild type plants are presented here.(XLSX)Click here for additional data file.S2 TablePeptide counts and SAINT scores are presented for all published ZTL interactors. Please note that the peptides belonging to ZTL listed in this table include both the peptides belonging to the bait and the native ZTL protein.(XLSX)Click here for additional data file.S3 TableThe results of statistically significant interactors of the ZTL Kelch repeat domain as produced by SAINTexpress.(XLSX)Click here for additional data file.S4 TableIP/MS results from this study were compared with previously generated results from the ZTL decoy .(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableRaw period and flowering time data for each individual plant presented in Figs (XLSX)Click here for additional data file.


The prior slip system is determined by the combined effect of the Schmid factor and the critical resolved shear stresses (CRSS). Twinning plays a crucial role during plastic deformation since basal and prismatic slips are limited. The The deformation mechanisms of Mg, Zr, and Ti single crystals with different orientations are systematically studied by using molecular dynamics simulations. The affecting factors for the plasticity of hexagonal close-packed (hcp) metals are investigated. The results show that the basal < In hcp metals, the most popular plastic deformation mechanisms are slip and twinning. In general, slip is always along the lattice close-packed direction on the close-packed plane. Thus in hcp metals, slip system varies with the change of the c/a ratio. For example, basal <n hcp Mg . Howeveri and Zr ,3. Underpossible ,5,6.c/a ratios, lattice packing densities, interplanar spacings, stacking fault energies, and so on. In Mg and its alloys, Twinning is popularly observed in hcp metals. Compared to metals with cubic crystal structure which have limited numbers of twinning systems, there are at least seven twinning modes in hcp metals, such as ng modes ,8,9,10. nd 101¯1 ,14. Twinnd 101¯1 . In Zr a1¯2 twin , whereason in Zr ,18. Moreg alloys , while 1recently . 112¯1−1n hcp Ti .In addition to slip and twinning, phase transformation plays an important role on the plasticity of hcp metals. It is generally known that allotropic transformation occurs at 882 °C in pure Ti from hcp ɑ-Ti to bcc β-Ti . In addic + a> slips are activated when the basal slip is restricted, and different tension and compression twins appear under the c-axis tension and compression, separately. By using molecular dynamic simulations, Zu et al. [In general, the hcp metals display strong anisotropic mechanical behavior because of their asymmetric crystallographic structure. The orientation effect has a great effect on deformation behavior of such asymmetric structure ,30,31,32u et al.  have repc/a ratios under different loading conditions. By comparing the deformation mechanisms of different hcp metals and analyzing the orientation effect on deformation behaviors, the affecting factors for the plasticity of hcp metals are further discussed.Because of the complexity of deformation mechanisms in hcp metals, clarifying the affecting factors for the strength and plasticity is of great importance for improving the mechanical properties of hcp materials. In this paper, molecular dynamics simulations are employed to investigate the deformation mechanisms of different hcp single crystals, such as Mg, Zr, and Ti, with different In order to study the plastic deformation mechanisms under different loading conditions, two single crystal models (A and B) with different orientations are established. . for Mg , Mendele. for Mg , Ackland. for Mg  which is. for Mg ,38 and t. for Mg  is appli. for Mg  is used . for Mg  and Comm. for Mg .y-direction with a constant strain rate of about At first, the perfect crystal is relaxed for 30 ps (5000 time steps) at zero force to minimize the potential energy. Then a uniaxial tension is applied along the a> dislocation (BD <a>), prismatic <a> dislocation (PrD <a>), pyramidal <c + a> dislocation (PyD <c + a>), shear band (SB), basal stacking fault (BSF), and the cross-slip of prismatic <a>, and pyramidal <a> dislocation. Crystallographic reorientation includes twinning and basal/prismatic (BP) transformation. Several twinning modes are observed, including <a> and pyramidal <a> dislocation (dark brown), The plastic deformation of Mg, Zr, and Ti single crystals under uniaxial tension with different orientations are shown in c-axis, the pyramidal <c + a> dislocations are favored because the Schmid factors of basal and prismatic slips are zero. With the increase of θ or β, the Schmid factor of basal slip becomes larger, thus basal slips gradually dominate the initial plastic deformation. When θ or β approaches to 90°, the prismatic <a> slip is favored than the basal <a> slip according to the Schmid factor, thus the prismatic <a> dislocations are the main plastic deformation mechanism when θ ≥ 70°.In hcp metals, the dominant slip direction is always the lattice close-packed direction us works ,44. The us works , which ia> dislocation dominates the initial plasticity of Zr. As shown in a> dislocation. Then the basal <a> dislocation moves on and extends to the free surface, with a slip step left on the surface (a> slip (f = 0.433) is primarily activated  generated in a> dislocation is easier to activate than dislocations in other plane. The magnitude of SFE determines the ease of dissociation into partials. The basal dislocation is easier to split into partials because of its lower SFE [a> dislocation into two partials and a BSF are usually observed.In ower SFE . Therefoa> dislocations in Ti can cross-slip into an intersecting pyramidal plane. The activation of secondary slip system is helpful for achieving a complete three-dimensional deformation, which also promotes nucleation of subsequent a> dislocations in Ti was also observed by Caillard et al. [The simulation results of the Mg single crystals are primarily consistent with those of the Zr for model A, whereas the simulation result of Ti single crystal is somewhat different when θ ≥ 70°. The prismatic <d et al. .a> slip (f = 0.125) in Mg and Ti. However, the basal <a> slip occurs prior to the pyramidal <c + a> slip because of the higher CRSS for the <c + a> slip at low temperatures. The CRSS is a material parameter which is dependent on deformation temperature, strain rate, impurity, etc. Chapuis et al. found that at the temperature of 298 K for Mg, the CRSS values are 5 MPa for the basal <a> slip and 110 MPa for the first order pyramidal <c + a> slip [c + a> slip is about 22 times larger than that for basal <a> slip. Thus the basal slip is easier to be activated than the pyramidal slip.We notice that in  a> slip . It indic + a> dislocation first during c-axis loading by sequential nucleation of leading and trailing partial dislocations. Subsequently, the formation of c + a> dislocations on c + a> dislocation is energetically preferable on c + a> slip should dominate on the c + a> dislocations in Ti and its alloys [c-axis compression at high temperatures. In these experiments, the activation of In our simulations, except for the SBs caused by the g et al.  have exprmations . The enes alloys ,52, espec-axis tension. Twinning is a major deformation mode in hcp metals. Four twinning modes are most commonly observed, including two extension twins c-axis tension) for Mg, Zr, and Ti. The CRSS of c-axis tension. The pyramidal <c + a> dislocation occurs first, then the basal slip follows  are left on the TB. Kou et al. revealed that the sessile steps can act as stable sources for the nucleation of b2 twinning dislocations, while the 2 [In order to understand the nucleation and growth mechanisms of n embryo . Kou et pe (TEM) . A twin de of b2 . Moreovea> dislocations is observed in the simulations of Mg and Ti samples in The nucleation of the ¯1 twins ,57,58, i¯1 twins . Our calTwo types of DTs are observed in our simulation, u et al. . After ta-axis, c-axis. The a> dislocation nucleates first at the free surface, then the Beside,  (HRTEM) . Figure SB grows b and extSB grows c throughin forms d. AccordFor the stress-induced phase transformation, it is hard to occur in Mg and Zr at low temperatures. In Ti single crystals, phase transformation from hcp to fcc structure is observed at θ = 0°, θ ≥ 70°, and β = 15°–58.6°. In our simulation, the hcp-fcc phase transformation is accomplished via successive glide of basal partial dislocations, which has been observed in previous studies ,54,60.hcp||{111}fcc and hcp ||fcc. The hcp-fcc transformation induced by the accumulation of BSFs is also observed in the interior of As shown in When β = 35.9° a, the fcocations b, the fcc/a ratio, and etc. The CRSS is related to the crystal structure, which describes the critical stress for plastic deformation to occur on a given deformation mode. Different metals have different CRSS values for one given slip system or twinning mode. We can analyze the ease or complexity of the activation of one deformation mode by comparing the CRSS values of different metals. The basal <a> slip is preferred in Mg while the prism <a> slip is favored in Ti and Zr, which are determined by the c/a ratio and reflected in the magnitude of the CRSS values. The basal <a> dislocation has a much lower CRSS value (5 MPa) than that of other slip systems (> 90 MPa) in Mg [a> dislocation is the most active defect with the minimum value for Zr (15 MPa) and Ti (60 MPa) [As we know, the plastic deformation mechanisms of Mg, Zr, and Ti show a distinct difference with each other. It is closely associated with the crystal structure, a variety of the a) in Mg . The pri(60 MPa) ,54. The 260 MPa) ,53,54. Tc/a ratio. For a given deformation mechanism, the Schmid factors for different hcp metals  have a tiny difference. The slip system with a larger Schmid factor has priority to be activated during the deformation. Generally, comprehensive analysis of Schmid factors and CRSSs of slips is almost matched with the consequence of the deformation mechanism in our simulation. For an example, the basal <a> slip occurs prior to the pyramidal <c + a> slip when β = 10° in Mg and Ti, although the Schmid factor of the a> slip (f = 0.125). This is due to the higher CRSS for the <c + a> slip than the basal <a> slip at low temperatures. The combined effect of CRSS and the Schmid factor determines the prior activated slip system. Moreover, when the loading is applied near basal plane, the Because of the asymmetric and anisotropy crystallographic structure, the orientation effect is very important for the deformation of hcp metals. When the load is applied in different direction, the yield stress is rather changeable as the low symmetry and the limited slip systems of hcp metals. The Schmid factors are used to analyze the orientation effect, which are determined by the loading directions and es in Zr ,18.Based on the systematic analysis of the deformation mechanisms in hcp metals, the affecting factors for the plasticity of hcp materials are discussed. In order to improve the mechanical properties of hcp materials, several methods can be applied according to the above analysis. At first, alloying with some elements might lead to changes in the CRSSs of the different deformation modes with respect to pure hcp metals. Many researchers have observed the alloying effect on the CRSS in hcp metals. Qian et al. have reported that the addition of Li to Mg increases the CRSS for basal slip, while the CRSS for prism slip is lowered . Ojha et(1)The slips dominate the plasticity of hcp single crystals. The basal slip is preferred in Mg while the prism slip is favored in Ti and Zr, which are characterized by c/a ratio and the CRSS values. The slip mechanisms observed in our simulation match well with the comprehensive analysis of the Schmid factor and CRSS of slip systems.(2)When the basal and prismatic slips are restricted, the twinning is activated. The (3)The stress-induced phase transformation from hcp to fcc structure in Ti is observed, which is achieved by the accumulation of BSFs. For samples with different orientations, the fcc phase can be nucleated at the defects or the free surface via the activation and successive glide of basal partial dislocations.(4)The lower CRSSs of slip and twinning in Mg result in the strength of Mg is much lower than that of Ti and Zr. Moreover, there are more types of plastic deformation mechanisms in Ti than in Mg and Zr. Multiple deformation mechanisms coordinate with each other, resulting in high strength and ductility of Ti.The plastic deformation mechanisms of Mg, Zr, and Ti single crystals with different orientations are systematically investigated by using molecular dynamics simulations. For different orientation samples with θ or β ranging from 0° to 90°, different plastic deformation mechanisms are exhibited. By comparing the deformation mechanisms of different hcp metals and analyzing the orientation effect on deformation behaviors, the affecting factors for the plasticity of hcp metals are analyzed.Based on the comprehensive analysis of the deformation mechanisms in hcp single crystals, the strengthening mechanisms for the hcp materials are discussed. Alloying with the elements might lead to the change of the CRSS with respect to pure hcp metals, resulting in the improvement of the mechanical properties. Moreover, the processing technique related with the orientation effect can effectively affect the deformation mechanisms, which can be implemented to design novel hcp materials.


N-linked glycans play an important role in immunity. Although the role of N-linked glycans in the Fragment crystallizable (Fc) region of immunoglobulins has been thoroughly described, the function of N-linked glycans present in Ig-variable domains is only just being appreciated. Most of the N-linked glycans harbored by immunoglobulin variable domain are of the complex biantennary type and are found as a result of the presence of N-linked glycosylation that most often have been introduced by somatic hypermutation. Furthermore, these glycans are ubiquitously present on autoantibodies observed in some autoimmune diseases as well as certain B-cell lymphomas. For example, variable domain glycans are abundantly found by anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) as well as by the B-cell receptors of follicular lymphoma (FL). In FL, variable domain glycans are postulated to convey a selective advantage through interaction with lectins and/or microbiota, whereas the contribution of variable domain glycans on autoantibodies is not known. To aid the understanding how these seemingly comparable phenomena contribute to a variety of deranged B-responses in such different diseases this study summarizes the characteristics of ACPA and other auto-antibodies with FL and healthy donor immunoglobulins, to identify the commonalities and differences between variable domain glycans in autoimmune and malignant settings. Our finding indicate intriguing differences in variable domain glycan distribution, frequency and glycan composition in different conditions. These findings underline that variable domain glycosylation is a heterogeneous process that may lead to a number of pathogenic outcomes. Based on the current body of knowledge, we postulate three disease groups with distinct variable domain glycosylation patterns, which might correspond with distinct underlying pathogenic processes. B cell antigen receptors (BCR) are rearranged at the pre-B cell stage during B-cell development, and may be present as membrane-bound B-cell receptors or as soluble immunoglobulins. Through distinction between foreign and self-antigens, immunoglobulins play a key role in the defense against infections. Immunoglobulins can initiate a variety of inflammatory effects throughout the organism, depending on e.g., the recognized target, the isotype used or the properties of the Fragment crystallizable (Fc) tail. Once a B cell has encountered antigen, the B cell becomes activated and is able to secrete the B-cell receptor, particularly after maturation to antibody-secreting plasma blasts and plasma cells, in soluble form as IgM. Upon further activation and acquisition of T cell-help, class switching from IgM to IgG, IgA, or IgE can occur, leading to the presence of antigen-specific, isotype-switched antibodies in serum and other bodily fluids . Of all en bloc (dolichol-P-P-GlcNAc2Man9Glc3). When the protein is subsequently transferred to the Golgi complex, glycosyl-hydrolases and transferases further diversity the attached glycan . This process is primed by the presence of N-linked glycosylation motifs, which consist of an asparagine (N), followed by any amino acid but proline, followed by serine (S) or threonine (T) . Initially, a pre-formed lipid-linked glycan consisting of two N-acetlyglucosamines (GlcNAc) linked to nine mannose (Man) and three glucose (Glc) residues is attached d glycan  11). Fi. Fien bld glycan  12). Fi. Fien bld glycan  (12). Cod glycan . Third, Immunoglobulins are glycoproteins with conserved N-linked glycans in their Fc tail. For example, all IgG subclasses harbor N-linked glycans at heavy chain position N297 . It is bIn contrast to the ubiquitous N-linked glycosylation of the Fc-tail, only a proportion of immunoglobulins contains additional N-linked glycans in the variable domain. In the naïve human B-cell repertoire, N-linked glycosylation motifs are only present in the variable domain when rearrangements contain the V-segments IGHV1-8, IGHV4-34, or the more rarely used IGHV5-10, IGKV5-2, IGLV3-12, and IGLV3-22 , 35–37, The type of glycans present in the Fc-part and the Fab-part can differ considerably. Fc glycans show increased degrees of fucosylation as compared to variable domain glycans. Conversely, variable domain glycans are more often galactosylated, bisected, and sialylated, putatively due to the Fc glycan being shielded in between two heavy chains in the quaternary immunoglobulin protein structure, decreasing its accessibility for the glycosyltransferases responsible for the complex glycan structures . In contin vivo selection mechanism for these antibodies)  in healthy individuals. In general, estimates from genetic analyses have been slightly lower than from protein-based strategies, such as the measurement of sialic acids to determine the presence of variable domain glycans , 51–54. ibodies) . As thisVariable domain glycans were found to cluster around antigen-binding sites, suggesting that they are not randomly acquired, but rather lead to selective outgrowth of B cells , 53. A pOver 90% of healthy donor IgG N-linked glycans contain a core fucose, of which the fucosylated digalactosylated form is the most abundant , 56. TheWithin immunoglobulins, there are key differences found between subclasses. Recently, both IgE and IgG4 were shown to contain significantly more variable domain glycans than other isotypes , 54, 58.Autoimmune diseases are characterized by an adaptive immune response against host tissues. A growing body of evidence shows an increased prevalence of autoantibody variable domain glycosylation in the context of many autoimmune diseases .+ plasma cells may be found in a number of anatomical locations, particularly the bile ducts  represents a particularly interesting disease, as it is characterized by increased IgG4 titers. IgG4-RD is a multisystem fibroinflammatory condition, in which tumor-like masses related to organ dysfunction and tissue infiltration by IgG4le ducts , 61. Recle ducts . In partle ducts . This oble ducts . Althougle ducts .Rheumatoid Arthritis (RA) is a destructive, autoantibody-mediated inflammatory disease of the joints which affects ~0.5–1% of the population , 64. TheWe were the first to observe the abundant presence of N-linked glycans in the variable domain of ACPA-IgG  and provThe ACPA-IgG variable domain glycans show high degrees of sialylation, could impart an immune-modulatory function , 68, 69.Anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (AAV), is characterized by inflammatory lesions of the blood vessels , 71. Of Systemic Lupus Erythematosus (SLE) is an autoantibody-mediated chronic disease characterized by multi-organ involvement that typically affects women , 75. In Primary Sjogren's Syndrome (pSS), is characterized by lymphocytic infiltrates in the exocrine glands . A hallmin vivo observations rather than genetic analyses, as IgG4 may undergo Fab-arm exchange with the potential of radically changing the antibody's properties (In Myasthenia Gravis (MG), autoantibodies against neuromuscular junction protein impair neuromuscular transmission, resulting in skeletal muscle weakness . Althougoperties .In summary, variable domain glycans are present in high prevalence in a number of autoimmune diseases with disease-specific autoantibodies. For some (such as RA and AAV), the ubiquitous presence of variable domain glycans, suggest a mechanistic role, for example in the breach of immunological tolerance of variable domain glycans in these diseases. In other autoimmune diseases, variable domain glycosylation is less abundant . The reason for these differences is not known, but might relate to the type of antigens recognized or the chronicity/abundance of antigen-exposure. In comparison with healthy donor IgG, auto-antigen IgG from patients with autoimmune diseases can show an enhanced variable domain glycosylation . NonetheIntriguingly, the abundant presence of variable domain glycans has not only been observed in autoimmune disease, but also in the case of certain lymphomas. These findings are interesting as both disease groups may result from poor immune regulation and could therefore point to an unappreciated shared pathogenic mechanism.Follicular lymphoma (FL) is the most common indolent B-cell lymphoma, representing roughly 40% of all non-Hodgkin lymphomas . Immortain vivo glycosylation, remains to be confirmed (Primary Cutaneous Follicle Center Cell Lymphoma (PCFCL) is an uncommon indolent B-cell lymphoma of unknown etiology that is found preferentially on the skin of the scalp and trunk . As to ionfirmed .Burkitt's lymphoma (BL) is an aggressive B-cell lymphoma associated with Epstein Barr virus and Human immunodeficiency virus which accounts for ~1–5% of the non-Hodgkin lymphomas . Like FLDiffuse large B-cell lymphoma (DCBCL) is a common lymphoid malignancy and accounts for 40% of non-Hodgkin lymphoma. DLBCL is clinically heterogenous, but generally aggressive and characterized by the presence of mature B cells . One stuSummarizing, B-cell lymphomas with a follicular growth pattern have a strong proclivity to acquire variable domain glycans. Indeed, the variable domain glycans may in fact determine their follicular architecture as it links the cell to the germinal center microenvironment . Since nThe position of an N-linked glycosylation motif in the variable region may prove informative for its functional consequences. Indeed, depending on the variable region position, glycans may block antigen-binding, contribute to antigen-specificity or interact with other molecules without interfering with the antigen-binding site. An analysis of structural regions containing the newly acquired glycans may help to make a rough distinction between these possibilities. The majority of the N-linked glycosylation motifs in patients with pSS, RA and AAV were observed in framework region 3 (FR3) , Table 1In contrast to acquired variable domain glycans in autoimmune diseases, 87% of heavy chain variable domain glycosylation motifs of FL patients were observed in the CDR. Moreover, rather than clustering on particular regions, FL BCR acquired N-linked glycosylation motifs in a number of very specific hotspots , with thThese differences in distribution indicate an interesting difference in variable domain glycosylation between lymphoma and autoimmune disease. The variable domain glycans typically found in lymphoma CDR, could prove to be detrimental to autoantibodies. Indeed since the CDR is intimately involved in antigen binding, blocking the antigen-binding site with a large glycan molecule may be expected to interfere with autoantibody recognition, a requirement for B-cell growth and selection. Conversely, the strong hotspot clustering of variable domain glycans in lymphoma BCR could also be explained as a result of glycans contributing to or “mimicking” antigen-binding, especially when those antigens are environmental or bacterial lectins. Whether those antigens are indeed lectins, other microenvironmental antigens or the substrate of physiological B-cell activity before malignant transformation, is one of the key open questions to understand the importance of this phenomenon. Alternatively, the hotspot clustering may not signify antigen-recognition at all, but could contribute to auto-stimulatory properties due to tertiary formations of the BCR [as described by a different process in for example CLL ].N-linked sugars present on ACPA IgG variable domains form a notable exception with their high sialic acid content of up to 44% . Through interaction of sialic acids with for example SIGLEC CD22, an inhibitory co-receptor of the B-cell receptor , an optiAs mentioned above, in case of FL, it has been proposed that interaction with environmental lectins act as a driving force behind the formation of variable domain glycans. In this case, glycan-lectin-interaction does not modulate or disrupt an inhibitory receptor, but rather serves as surrogate to mediate BCR-crosslinking. Indeed, it was shown that lectins can bind variable domain glycans introduced during somatic hypermutation, and in this way provide a survival signal for autoreactive B cells from the germinal center and onwards during development . FurtherConcluding, variable domain glycans appear to play an important role in several B-cell diseases, both hemato-oncological and autoimmune in nature. However, in both fields the precise role and consequences of the variable domain glycosylation remain at best incompletely understood. There are, nonetheless, some observed commonalities between N-linked glycans in autoimmunity and in hematology. Lectin interaction is a recurring hypothesis in both autoimmune and malignant settings, although additional evidence is warranted to further support these paradigms.Careful scrutiny of the distribution of variable domain glycans could hold the key to an increased understanding of any of these diseases. Currently, only RA ACPA and FL BCR have been investigated in extensive detail, which has led to corresponding pathogenic postulations of the variable domain glycans. Researchers should aim to compile publicly accessible compendia for all other implied or potentially affected diseases, so side-by-side comparison may clarify important commonalities and differences for each disease.A possible lead to further understanding of the contribution of variable domain glycans in different disease, is the different distribution and composition of variable domain glycans in autoimmunity and malignant hematological disease. Such differences suggest that in these disease groups, whilst both apparently resulting from deficient immune monitoring, variable domain glycosylation may play distinctly different roles.On the other hand, defining common variable domain glycosylation patterns should help cluster diseases by shared underlying pathological mechanisms. Based in the current body of knowledge, we propose three such clusters: (1) near ubiquitous CDR-glycosylation with high-mannose structures, which is typical for certain lymphoma types and may depend on auto-lectin interactions potentially mimicking BCR-triggering; (2) near-ubiquitous FR(3)-glycosylation with extensive sialylation that may facilitate SIGLEC interaction, comprising some well-studied autoimmune diseases; and (3) Increased, but non-ubiquitous (FR) glycosylation in autoimmune diseases, which may well be a result of concurrent immune processes rather than an independent driving mechanism.All in all, variable glycosylation in B-cell diseases is an emerging field of investigation. Through comparison of both commonalities and differences in variable glycosylation of B-cell diseases across a number of fields, diseases may be clustered by shared pathogenesis and currently missing causative mechanisms may be clarified.EV and MK screened, summarized and analyzed available literature, and wrote the manuscript. MK, HS, HV, and RT provided project supervision. All authors have critically read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date.A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation.In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of The online version contains supplementary material available at 10.1186/s12866-021-02138-w. Azorhizobium caulinodans ORS571, as a gram-negative nitrogen-fixing bacterium, has the dual ability to fix nitrogen both under free-living conditions and in a symbiotic interaction with the tropical legume Sesbania rostrata, which forms both stem nodules and root nodules [The alphaproteobacterium  nodules . The intVibrio fischeri luxR and Agrobacterium tumefaciens traR) are activated when they bind to N-acyl homoserine lactones [Sinorhizobium meliloti FixJ [Escherichia coli NarL [Enterobacteria RcsB belong to this category. Other types of RRs contain autonomous effector domains but do not have a regulatory domain (GerE) [Bacteria possess various signalling systems that enable them to sense and respond to diverse signals from external environmental conditions. Two-component system (TCS), a major type of cellular signal transduction system, are widely used by bacteria to sense and adjust their behaviour according to environmental changes . TCS plalactones , 7. The lactones  and haveoti FixJ , Escherioli NarL , UhpA [1oli NarL , and Entn (GerE)  or multin (GerE) .S. meliloti, FixJ contains a phosphorylatable N-terminal REC domain and a C-terminal HTH_LuxR DNA-binding domain. FixJ, the oxygen-sensitive two-component response regulator, plays a pivotal role in symbiotic nitrogen fixation in S. meliloti. FixJ is a positive activator required for the regulation of nitrogen-fixing gene transcription [A. caulinodans ORS571 (https://www.ncbi.nlm.nih.gov/) suggested the existence of a FixJ family response regulator AcfR (A.caulinodansfixJ family regulator) that contains a REC domain in the N-terminus and an HTH_LuxR domain in the C-terminus. In this work, we elucidated the potential role played by AcfR in A. caulinodans under free-living and symbiotic conditions.In the symbiotic bacterium cription . Genome http://smart.embl.de/), which indicated that it contains an N-terminal signal receiver domain REC (SM00448) and an HTH_LuxR DNA-binding domain (SM00421) , we analysed the domain architecture and phylogenetic relationship of AcfR and some proteins containing an HTH DNA-binding domain. First, the protein sequence was entered in SMART , FixJ of S. meliloti and A. caulinodans ORS571 was 97.20, 41.20, 44.02%, respectively. These results suggested that AcfR belongs to the FixJ family of two-component response regulators.Furthermore, the evolutionary relationship between AcfR and several proteins that contain REC + HTH_LuxR domains was analysed. For phylogenetic reconstruction, these proteins were aligned with the ClustalW program and the phylogenetic tree was created with MEGA-X software by the neighbor-joining method. As shown in Fig. ned Fig. . Amino ahttps://string-db.org/) [A. caulinodans, including FixL, two AAA family ATPase proteins, two PAS domain S-box proteins, a two-component sensor histidine kinase, a GHKL domain-containing protein, and a magnesium-translocating P-type ATPase. Interestingly, six of eight proteins were predicted to be histidine kinases with HisKA and HATPase_c domains  . As showacfR deletion mutant strain (ΔacfR) of A. caulinodans by gene homologous recombination and constructed its complemented strain (ΔacfR-C). To confirm whether this mutation affects bacterial growth under normal conditions, the dynamic growth curves of the wild-type (WT), mutant, and complemented strains were tested at various stages of growth. The results indicated that the relative growth rates of the mutant and complemented strains were not significantly different from that of the WT strain , and complemented (ΔacfR-C) strains was tested on 0.3% soft agar plates. Bacterial cultures of these strains were inoculated in the middle of soft agar plates. The plates were incubated for 2–3 days at 37 °C. As shown in Fig. acfR-C. These results indicated that AcfR positively modulates the cells swimming motility behaviour of A. caulinodans ORS571.To further investigate the functions of AcfR in bacterial motility behaviour, the swimming activity of the WT, mutant  between the ΔacfR and WT cells. The ΔacfR mutant produced less “black pigmented”  production in bacteria . Figure ed” Fig. a and toted” Fig. b than diacfR mutant also affect symbiosis characteristics with the host plant, competitive nodulation assays were performed by analysing the levels of nodulation on the host plant. To test whether ΔacfR possessed a competitive disadvantage when competing with WT, cultures of WT and ΔacfR were mixed at 1:1, 1:5, and 1:10 ratios and subsequently incubated with S. rostrata roots and stems. The ratio of the number of wild-type cells to the number of mutant cells in the inoculum was determined by cell counts performed before mixing. At the end of each experiment , the ratio of nodules induced by the wild-type or the mutant strain was determined by PCR to detect the colonies that grew from surface-sterilized crushed nodules on TY agar plates. The results shown in Fig. acfR = 1:5 or 1:10), the nodule occupancy also increased. These results indicated that the competitive nodulation ability of the mutant strain was dramatically weakened compared with that of the WT. However, the number of nodules induced by ΔacfR-C was not significantly different compared with that of the WT when inoculated at a 1:1 ratio, indicating that the competitive nodulation ability was restored in the complemented strain. Therefore, we conclude that the AcfR regulator is essential for normal nodulation competitiveness during symbiosis on S. rostrata.To investigate whether the motility and EPS formation phenotypic defects of the ΔB. subtilis and Lactobacillus plantarum [AcfR, a typical two-component response regulator, includes an N-terminal REC domain and a C-terminal HTH_LuxR DNA-binding domain. Previous studies have shown that among the 991 LuxR-associated protein sequences (containing at least one HTH_LuxR domain) of Actinobacteria, the REC domain appears to be associated with LuxR in 53% of cases . These Rlantarum  that is E. coli [S. meliloti, the FixJ and FixL are two-component regulators that are widely spread among prokaryotes. FixL, as a transmembrane protein, senses and transduces environmental signals to FixJ. Next, the transcriptional regulator FixJ activated the expression of nif and fix genes [A. caulinodans ORS571, seven REC + HTH_LuxR regulators were identified. Our previous results showed that the REC + HTH_LuxR regulator AclR1 (WP_012169103) is involved in the c-di-GMP signalling pathway and initiates a downstream related signal transcription pathway [A. caulinodans, which showed multiple affected phenotypes: (i) AcfR positively regulates the swimming motility behaviour of A. caulinodans under free-living conditions but does not affect the normal growth of cells; (ii) the acfR gene deletion mutant strain produced less “black pigmentation” and EPS than the WT when glycine was used as the sole carbon source; and (iii) AcfR positively regulated the competitive nodulation ability of S. rostrata roots and stems. These pleiotropic phenotypes were attributable to the deletion of the acfR gene, which encodes a REC + HTH_LuxR response regulator of the NarL/FixJ family. Rhizobial motility, chemotaxis and EPS are considered to play essential roles in the early stages of competitive colonization and nodulation [A. caulinodans ORS571 regulates competitive nodulation ability and may directly affect the symbiotic pathway or indirectly regulate symbiosis by affecting bacterial motility behaviour and EPS production. Further research is warranted to investigate the signalling pathway of symbiosis between A. caulinodans and its host plant.In gram-negative bacteria, NarL with the REC + HTH_LuxR domain regulates the nitrate/nitrite signal transduction pathway in E. coli . In S. mix genes . In the  pathway . In thislR1 WP_0169103 is dulation . AcfR ofRhizobium-legume host symbiosis.Our results indicated that AcfR, as a FixJ family response regulator, is involved in the regulation of gram-negative bacterial motility, exopolysaccharide production and competitive symbiosis processes with the host. These findings indicate that AcfR is both a response regulator of the TCS and a transcription factor that plays an important role in regulating A. caulinodans ORS571 was used throughout this study as the wild-type strain. All strains and plasmids used in this study are listed in Table E. coli) were routinely grown overnight in Luria-Bertani (LB) broth or on LB agar at 37 °C for the amplification of recombinant clones and plasmid isolation. The A. caulinodans ORS571 WT strain and mutant ΔacfR were grown in L3 medium containing 100 μg/mL ampicillin and 25 μg/mL nalidixic acid antibiotics at 37 °C [acfR-C was grown in L3 medium containing 100 μg/mL ampicillin, 25 μg/mL nalidixic acid and 50 μg/mL kanamycin. The L3 medium was supplemented with 10 mM NH4Cl as nitrogen source and with 10 mM glycine or succinate as the sole carbon sources.at 37 °C . The comAzorhizobium sp. AG788, FixJ of S. meliloti, FixJ of A. caulinodans, NarL, CitB, RcsB, and UhpA). QS-LuxR of Vibrio fischeri as an outgroup was analysed to construct a phylogenetic tree.Amino acid sequences were aligned and analysed with the multiple sequence alignment program ClustalW of BioEdit software. Phylogenetic and molecular evolutionary analyses were conducted using MEGA-X version. A neighbour-joining phylogenetic tree was constructed by using amino acid sequences with 1000 bootstrap replications. These sequences include AcfR and NarL/FixJ family proteins  based on kanamycin resistance.To construct the complemented strain of ΔBBR-MCS2 , which h600 of 0.02 and grown at 37 °C with rotary shaking at 180 rpm. Growth kinetics were determined by monitoring turbidity at 600 nm. The experiments were performed in triplicate, and the data shown are the means and standard deviation.The growth kinetics of WT and mutant strains were grown in TY medium with 25 μg/ml nalidixic acid and 100 μg/ml ampicillin antibiotics. The overnight cultures were diluted into fresh L3 medium to an initial OD4Cl. Briefly, 5 μl of overnight grown cultures were added to plates and incubated at 37 °C for 2–3 days. The swimming diameter at the agar surface was utilized to assess the swimming motility ability.Motility assays were performed on 0.3% soft agar L3 plates according to previous publications . The L3 4Cl and 10 mM carbon sources (sodium lactate and glycine) were used to determine the Congo red-binding properties of colonies. To examine EPS production, mid-logarithmic phase cultures were adjusted at an OD600 of 1.0, and 15 μl bacterial cultures were spotted onto the L3 plates (0.8% agar). Morphological observations were made after an incubation period of 2–3 days at 37 °C. The total EPS was quantified by referring to the method described by Liu et al. [L3 plates supplemented with 40 μg/ml Congo red, 10 mM NHu et al. .acfR at approximately 1:1, 1:5 and 1:10 and WT:ΔacfR-C in approximately 1:1. For competitive nodulation on roots, surface-sterilized seedlings of S. rostrata were inoculated with bacterial cultures corresponding to each treatment at an OD600 of 0.5 for 30 min. For competitive nodulation on stems, cell suspensions corresponding to each treatment were painted onto S. rostrata stems after 35 days of plant growth. Ten plants were used for each of the three treatments. Bacterial endophytes were then isolated from surface-sterilized root or stem nodules at 35 days postinoculation and analysed. The WT and ΔacfR reisolated from nodules were determined by PCR using the primer pair AcfR-F and AcfR-R. The WT and ΔacfR-C reisolated from nodules were distinguished based on kanamycin resistance markers.Plant cultivation and nodulation tests were performed as described by previously , 28 withp-values. P-values < 0.05 and < 0.01 were tested. Each experiment was repeated in at least three independent experiments.All statistical analyses were performed using the SPSS 17.0 software package. We determined average values from at least three independent experiments and performed one-way analysis of variance followed by pairwise two-sample t-test assuming equal variances. Student’s t-test assuming equal variances was used to calculate the Additional file 1: Figure S1. Domain architecture and interaction network of AcfR. (a) Domain architecture of AcfR predicted by SMART. AcfR was identified encoding a protein of 214 amino acids that containing a REC domain and a HTH_LuxR domain. (b) The interactive protein network of the AcfR predicted by STRING. Eight proteins  predicted to interact with A. caulinodans AcfR. (c) The predicted functional partners based on the neighborhood evidence, cooccurrence evidence, and text-mining evidence. Figure S2. Domain structures of proteins that predicted in Fig. S1. The protein domains were predicted by using the SMART program. There are eight predicted interaction proteins . Six of eight proteins with HATPase_c and HisKA domains. Abbreviations: PAS, Per-Arnt-Sim domain; PAC, Motif C-terminal to PAS motifs; GAF, Domain present in phytochromes and cGMP-specific phosphodiesterases; HATPase_c, Histidine kinase-like ATPases; HisKA, His Kinase A (phospho acceptor) domain. Figure S3. Growth rates of the wild-type, mutant, and complemented strain are similar. Growth curves of the WT, ΔacfR, and ΔacfR-C in L3 liquid medium with 10 mM sodium lactate as sole carbon source and 10 mM NH4Cl as nitrogen source.


Macaronichnus burrows, an ichnotaxon usually related to hydrocarbon exploration due to its high concentration in rock notably affecting petrophysical properties. Macaronichnus, a subhorizontal cylindrical burrow, is characterized by a well-defined and developed outer rim surrounding the tube core. Our data indicates a clear zonation in porosity according to burrow structure, with the lowest porosity in the tube core and higher values associated with the surrounded rim. This duality is determined by the tracemaker grain selective feeding activity and the consequent concentrated cementation. The organism concentrates the lighter minerals in the tube core fill during feeding, favoring post-depositional cementation during diagenesis and this results in lower porosity than the host rock. However, heavy minerals, mainly glauconite, are located in the rim, showing higher porosity. Our results support the view that ichnological analyses are essential to determine reservoir quality in bioturbated reservoirs, evidencing that other ichnological properties in addition to those traditionally considered must be evaluated.Bioturbation is an important factor for reservoir quality due to the modification of host rock petrophysical properties . However, there is no predictable relationship between bioturbation and its effect on rock properties, due to the variability of the involved ichnological features. A detailed ichnological analysis is necessary to determine how bioturbation affects petrophysical properties in a bioturbated reservoir. Traditionally, ichnological features such as density, tiering, size, orientation, architecture, and fill, have been considered. However, other properties have been undervalued as is the case of lining. Here, we present a detailed study on the effects of  This influence on rock properties can be either positive or negative . Detailed ichnological analyses should be conducted in every bioturbated reservoir to quantify its effect and estimate the reservoir quality4. Traditionally, attention has been paid to particular characteristics of burrows impacting rock properties and reservoir quality such as density, tiering, size, orientation, architecture, fill, and lining5. With respect to lining, analyses have focused on its presence or absence7, and rarely on the petrophysical properties9. However, thickness and composition of the lining/mantle can be a major feature to be considered for some burrows, as is the case for Macaronichnus10.Bioturbation is an important factor for reservoir quality estimation in the oil and gas industry. Biogenic activities may affect some petrophysical properties such as porosity, permeability, and connectivityMacaronichnus has been commonly associated with reservoir exploitation and then its effect has been analyzed in detail for some deposits13. Thus, Gingras et al.11 briefly pointed out that porosity distribution in sandstones dominated by Macaronichnus is complex and may affect the reservoir quality, but they suggest more research had to be done. Later, Gordon et al.13 analyzed the presence of Macaronichnus in the Bluesky Formation  to determine that permeability was enhanced in those zones where Macaronichnus were abundant compared to similar non-bioturbated intervals. Then, Greene et al.8 pointed to differences between lining and filling, and Quaye et al.9 found that porosity and permeability in the burrow fill of Macaronichnus were higher than in the host sediment in the Funing Formation . These studies have shown the effect of Macaronichnus and its positive impact in spite of the general idea that bioturbation reduces permeability4. Moreover, Macaronichnus usually occurs in dense concentrations, commonly occupying more than 70% of the rock4, and it just takes very little time for producers  to do it14. At that concentration, it is an important biogenic structure affecting petrophysical properties, fluid flow, and, consequently, oil production. For all these reasons, we decided to analyze the effect of Macaronichnus in more detail.Macaronichnus segregatis15 can be found from Permian to Holocene2, mostly in shallow foreshore and shallow subtidal deposits, but it has been also observed in deep-sea sediments such as shelf or slope deposits18. Macaronichnus is usually identified as non-branching, cylindrical, and sub-horizontal burrows from just a few millimeters up to 15 mm diameter, characterized by a clear mineralogical segregation between the cylinder tube core and the surrounding rim15. Rim composition shows a concentration of darker and heavy minerals re-sorted by the producer during feeding, while lighter minerals commonly characterize the tube core11. This rim can be relatively thick, especially in the subichnospecies Macaronichnus segregatis degiberti17. However, the incidence of the rim on petrophysical properties has rarely been considered separately but as part of the entire infilling material (tube core and rim)9. Here, we analyze in detail the composition of infilling material from Macaronichnus segregatis degiberti differentiating between tube core and rim, focusing on its respective incidence on porosity and then on their particular impact in reservoir quality. This will help us to understand better the relative effect of every single part in porosity, which is essential to provide an accurate estimation of porosity in bioturbated reservoirs. For this study we investigate rock samples including specimens of Macaronichnus segregatis degiberti that were collected from two outcrops in northern Morocco: El Adergha  and Kirmta 20  and are Late Miocene in age23. These locations were selected as sampling allows us to evaluate nice examples where Macaronichnus specimens are clearly identified with different degrees of bioturbation and overlapping between burrows  and quartz  and the host rock , which represent the primary porosity Table , values 21. Previous studies have demonstrated that Macaronichnus is one of the trace fossils that can play an important role in bioturbated reservoirs13, generally having a positive impact. However, the previous studies mostly compared petrophysical properties from the host sediment and the entire burrow, without distinguishing between burrow tube core and outer rim. Our results demonstrate that the presence of Macaronichnus segregatis degiberti have, in general, a slightly negative impact on porosity, reducing values less than 1%. However, going deeper, our data show a relevant fact, that is the marked difference between tube core and rim, not only in terms of composition, as is well-known, but also in porosity.It is well established that bioturbation may affect reservoir qualityMacaronichnus15 and is determined by trace-makers during selective feeding activity17. In regards to porosity, a clear zonation is revealed. Mean porosity in the rims is two times higher than in the host sediment, reaching up to 8.8% porosity in some areas of the rock samples; however, porosity is very low in tube cores. This porosity zonation could be caused by original mineralogical heterogeneity combined with the associated post-depositional cementation1. During producer feeding activity, light minerals (mostly quartz and carbonates) are concentrated in the tube core and darker minerals  are placed on the surrounded rim. This zonation causes the concentration of depositional cementation in the tube core. This concentrated cementation produced during diagenesis, generates a porosity zonation, which plays a determinant role in the effect on petrophysical properties. Then, tube cores are characterized by lower porosity values than the surrounded rims.Regarding composition, the tube core is filled with light minerals that are highly cemented, and dark minerals, mainly glauconite, are concentrated in the rims. This compositional zonation is a diagnostic criterion for 11 reported that porosity distribution in Macaronichnus bioturbated sandstone is complex and acts like a dual porosity–permeability system. That means there would be a preferential flow through the burrows and a secondary flow from surrounding sediment into the burrows. Dual permeability systems are found in reservoirs where permeability contrast between matrix and burrows is high, around three orders of magnitude4. However, when permeability contrast is lower, a dual-porosity system is developed and there is not a preferential flow through the trace fossils4. Based on our results, this can be more complex for Macaronichnus as there is not a single flow system for the whole burrow due to the observed zonation. Petrophysical properties from tube core and rim are completely different and then both parts of the burrow should be considered separately when evaluating the effect on reservoir quality. Then, fluids would preferentially flow through the rim and, to identify the flow system, permeability contrast between host sediment and rim has to be evaluated. Macaronichnus burrow diameters have been reported from just a few millimeters22 to larger sizes up to 15 mm diameter24. However, most of the previous studies do not provide rim thick measurements. Savrda and Uddin10 recorded burrow tube cores and rim measurements and noted that tube core diameters ranged from 4 to 19 mm in large specimens. They reported that the rim thickness was variable along the burrow, ranging from about 1 to 5 mm, being thicker at the bottom part of the burrow. This reveals that the rim occupies 25–30% of the volume of the burrows10. This fact can also be observed in some of the illustrated figures from previous papers. For example, Rodríguez-Tovar and Aguirre16 show large specimens of Macaronichnus segregatis degiberti with ~ 15 mm diameter where rim was around 2–3 mm thickness, and Nara and Seike17 illustrated that the rim can be even thicker representing half the volume of the actual burrow .Gingras et al.Macaronichnus porosity effect. Especially considering that rim porosity can be two or three times higher than host sediment and represent an important volume portion of the whole burrow (up to 50%). Assuming that Macaronichnus are frequently in dense concentrations, commonly representing more than 70% of bioturbation4, we can estimate that in some cases burrow rims can represent 35% of the total volume in a bioturbated sample.Therefore, rim thickness must not be underestimated, revealing as a key point to control Macaronichnus-bearing rocks, porosities from both zones (rim and tube core) and their relative volumes need to be considered. Another aspect that needs to be considered is the frequent burrow crosscutting between specimens. Several theoretical situations involving different variables such as rim/tube core volume ratios, the degree of bioturbation and the overlapping between burrows are illustrated in Fig. Macaronichnus is commonly found representing more than 70% of rock volumes4, where rims could represent up to 50% of the burrows17 and overlapped, they could significantly modify the resulting porosity values of the host rock.Accordingly, to conduct a proper reservoir quality estimation in Macaronichnus has been shown to be a relevant trace fossil for reservoir quality in bioturbated deposits, mainly related to its occurrences as dense concentrations. This study has demonstrated that the porosity effect by Macaronichnus is not homogenous in the entire burrow and its distribution is clearly associated with the trace fossil structure. Porosity in the burrow tube core is different to that from the outer rim.Macaronichnus generate a grain resorting during feeding when they concentrate carbonate components and quartz grains in the tube core fill, and heavy and darker minerals, mainly glauconite, in the rim. Regarding reservoir quality, results reveal that Macaronichnus is a complex case and has a dual behavior. Burrow rims have increased porosity with respect to the host sediment and provide positive effects. Fillings in tube cores have lower porosity and provide negative effects.Producers of Macaronichnus on porosity. Any evaluation of the impact of Macaronichnus on petrophysical properties, and then on reservoir exploitation, based on just the intensity of bioturbation or the study of the whole burrow must induce significant misinterpretations of relevant scientific and economic implications. This complex dual behavior has been observed for the first time in Macaronichnus, but we could not discard that this could also be identified in some other trace fossils with marked internal zonation.On this basis, the relative volume occupied by rims and tube cores should be determined to estimate the real effect of Macaronichnus segregatis degiberti were collected from two outcrops in northern Morocco: El Adergha  and Kirmta 20, , a software commonly used in quantitative petrography26, considering the largest intact rectangular area avoiding any marginal alteration. Mineral compositional analyses of tube cores and rims were undertaken by defining an elliptical area within these burrow parts. Rim modal analyses were not possible in samples from El Adergha due to rim size limitations. Porosity was also measured during modal analyses in host rock, tube core, and rim. Additionally, porosity from all the thin sections was also obtained by blue pixels counting using Photoshop27, obtaining a representative mean value for the whole samples and from every single part . Blue pixels were quantified using Color Range Selection Method28. This method allows selecting all the blue pixels from an area of interest by clicking on some blue pixels and extending the selection to all the pixels with similar values. Once all the blue pixels are quantified, porosity can be calculated considering the number of pixels composing the area of interest. Intensity of bioturbation was quantified considering the bioturbated surface (BS), using Ichnological Digital Images Analysis Package (IDIAP) quantitative method28.Twenty-four rock samples of contourite sandstones including specimens of Theoretical scenarios were simulated considering different rim/tube core ratios, degree of bioturbation and overlapping between burrows Fig. . For tha


Helminthic infections, in particular those caused by gastrointestinal nematodes (GIN), are found worldwide and are among the most economically important diseases of goats. Anthelmintic resistance (AR) in GIN of goats is currently present worldwide, and single- or multidrug resistant species are widespread. The aim of this study was to determine the prevalence of AR to benzimidazoles (BZ), macrocyclic lactones (ML) and imidazothiazoles represented by levamisole (LEV) in the Polish goat herds by using an in vitro larval development test, which is useful especially in large-scale epidemiological surveys.Haemonchus contortus and Trichostrongylus spp. The use of BZ and frequency of anthelmintic treatments were significantly related to the presence of AR to BZ in Polish goat herds.This cross-sectional study was conducted from September 2018 to June 2019 and enrolled 42 dairy goat herds scattered over the entire country. The most commonly used anthelmintic class in goat herds in Poland were BZ (92%), followed by ML (85%) and LEV (13%). BZ-resistant GIN populations were found in 37 herds , ML-resistant GIN populations in 40 herds , and LEV-resistant GIN populations in 5 herds . Multidrug resistance involving all three anthelmintic classes was found in 5 herds . Based on the morphological features of stage 3 larvae the main resistant GIN turned out to be This cross-sectional study demonstrates the existence of AR to BZ, ML and LEV on Polish goat farms. Resistance to BZ and ML is widespread, while AR to LEV is currently at a low level. A considerable proportion of herds harbours multidrug resistant GIN, which requires further consideration. An effective anthelmintic treatment strategy, reasonable preventive measures and better understanding of the resistance-related management practices by farmers and veterinarians may delay further development of AR.The online version contains supplementary material available at 10.1186/s12917-020-02721-9. Parasitic infections, especially those caused by gastrointestinal nematodes (GIN), are one of the main factors responsible for economic losses in goat farming around the world . Their cSeveral factors are responsible for the development of AR in parasites, of which most important are a high treatment frequency , underdoSeveral in vivo and in vitro tests have been developed for detection of AR. An in vivo fecal egg count reduction test (FECRT) is recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP). However, this test requires animals in a herd to be either tested twice which makes it expensive, time-consuming and laborious, or a randomly selected group of animals in a herd be left untreated which, in turn, is impractical and hardly acceptable to farmers. Moreover, high inter-animal variation in the pharmacokinetics of anthelmintics in goats may lower the quality of FECRT results , 6. The Thus far, a number of reports have been published on the occurrence of AR in goat herds in Europe, and the prevalence of AR especially to BZ appears to be very high in some European countries . In France studies have shown a prevalence of AR to BZ to vary between 70 to 100% , 14. VerData on the AR situation in Polish small ruminant population are only fragmentary. In the last two decades, sporadic cases of AR to BZ in sheep, cattle, horses, pigs and goats have been reported –31. ReceOn the other hand, our long cooperation with a number of Polish goat farmers has shown not only that GIN infections constitute a considerable clinical problem but also that the factors predisposing to the development of AR are present in the vast majority of goat herds. Therefore, we hypothesized that those cases of AR reported so far were only the tip of the iceberg, and we decided to carry out a large-scale epidemiological observational study to determine the prevalence of AR to the three basic anthelmintic classes in Polish goat herds and identify the genus or species of resistant larvae.Forty-two dairy goat herds were enrolled in this cross-sectional study (49% of 85 herds invited). They were evenly scattered over the entire country (located in 13 of 16 provinces of Poland). They counted from 4 to 155 adult goats with the median (IQR) of 15 (10 to 23) goats. Only 5 herds (12%) consisted of more than 50 adult goats and only 2 of them counted more than 100 heads. In most of them two Polish local breeds, Polish White Improved and Polish Fawn Improved, were kept. Nine herds (21%) kept only Anglo-Nubian goats and three herds (7%) kept a traditional local goat breed – Carpathian. In the vast majority of herds  goats were grazed from April to October. In 10 herds (24%) goats were grazed together on the same pasture with sheep and in 8 herds (19%) with cattle. Only 13 herds (31%) relied solely on their own replacement, the remaining 29 herds (69%) purchased goats from other herds.n = 26; 67%) dewormed twice a year, 7 herds (18%) once a year, and 6 herds (15%) three or four times a year. BZ  were used in 36 herds (92% of herds practicing routine deworming), ML  in 33 herds (85%) and LEV in only 5 herds (13%).Only 3 herds (7%) did not practice routine deworming. In the remaining 39 herds (93%) at least one anthelmintic was routinely used. Most of the herds which practiced routine deworming  and to BZ detected in 37 herds . AR to LEV was detected in 5 herds  , AR to two anthelmintics in 31 herds  and AR to all three anthelmintics (synonymous to MDR) in 5 herds, which yielded the prevalence of MDR to be 12% (CI 95%: 5 to 25%). In most of herds AR to BZ and ML was severe , while AR to LEV was mostly mild (< 10% of larvae developed at the DC)  and ML , and moderate for LEV   at the DC was significantly positively correlated with ECp = 0.883). cPD at the DC in LEV-resistant populations ranged from 4 to 54% with the median of 9%, and was significantly lower than both the former (p = 0.047) and the latter GIN populations (p = 0.010).cPD at the DC in BZ-resistant populations ranged from 6 to 99% with the median (IQR) of 63% (32 to 91%), and was not significantly different from cPD at the DC in ML-resistant populations which ranged from 5 to 100% with the median (IQR) of 71% (52 to 92%) , Trichostrongylus spp. in 37 herds , Oesophagostomum spp. in 22 herds , and Teladorsagia spp. in 14 herds . Median fecal egg count (FEC) in the herds ranged from 75 to 2450 epg.Oesophagostomum larvae developed irrespective of the herd’s AR status. In the case of BZ-resistance H. contortus larvae developed at the DC in 95% of the populations which initially harboured this GIN, Trichostrongylus larvae in 81% of the populations, and Teladorsagia larvae in 36% of the populations. In the case of ML-resistance Trichostrongylus larvae developed at the DC in 91% of the populations, Teladorsagia larvae in 79% of the populations and H. contortus larvae in 74% of the populations. In the case of LEV-resistance Trichostrongylus larvae developed at the DC in 100% of the populations, while H. contortus larvae in 20% of the populations, and Teladorsagia larvae in no population. Of 5 herds resistant to LEV, 3 had Trichostrongylus spp. as the only GIN left in wells with LEV at the DC, and 2 had Trichostrongylus spp. and H. contortus. These results indicated that H. contortus spp. and Trichostrongylus spp. were mainly responsible for AR to BZ, all three GIN were responsible for AR to ML, while Trichostrongylus spp. was the main GIN resistant to LEV , but not to ML (p = 0.643) or LEV (p = 0.849). The use of BZ was significantly related to the presence of AR to BZ (p = 0.002), however there was no significant relationship between the use of ML and LEV and the presence of relevant AR . Interestingly, AR to LEV was observed in 3 herds whose owners declared not to have used LEV in previous 3 years. The same applied to 9 herds with AR to ML, and 3 herds with AR to BZ. None of the following: access to pasture, introducing goats from other herds, and grazing with sheep or cattle on common pasture was significantly linked with AR to any of the anthelmintics.Presence of AR to BZ and ML in virtually all investigated herds reduced the reliability of most statistical analyses. The number of times goats had been dewormed was significantly positively linked with the intensity of AR to BZ  and it is likely that farmers could have simply forgotten the facts of using of anthelmintics on their farms. And thirdly, there could be some goats purchased from other herds in Poland or abroad which had been dewormed with this particular anthelmintic and thus carried resistant GIN population. It is mainly the case in herds with AR to LEV and without previous use of this anthelmintic as in all these herds Anglo-Nubian goats were kept and goats of this breed are usually imported to Poland from abroad, mostly from Germany where AR to all three anthelmintic classes in small ruminants has so far been detected . MoreoveMultidrug resistance of GIN in goats in Europe is not widespread , and onl50 criterion for differentiation between resistant and susceptible GIN as an application of the EC50 may sometimes underestimate the resistance and thus some herds might have been incorrectly classed by this criterion as susceptible [99 values or the DC in the in vitro tests such as the LDT or EHT can substantially increase test sensitivity of the test and identify resistance when only a small proportion of the GIN population is resistant [99, simultaneously.In our study we decided to use the DC rather than the ECceptible . It has esistant , 61, 62.H. contortus, Trichostrongylus spp., and Teladorsagia spp. Only Oesophagostomum spp. showed no potential to become resistant to any anthelmintic which is at odds with some recent studies in which Oesophagostomum spp. was together with H. contortus and Trichostrongylus spp. resistant to BZ and ML [H. contortus. Initially, it had been set at 5.4 ng/ml, however then its elevation to 21.6 ng/ml was suggested [Trichostrongylus spp. or Teladorsagia sp. so we cannot be sure that the development of these GIN at this DC corresponds to their resistance to ML. Even though it is very likely to be so, it undoubtedly warrants further investigation. Interestingly, it seems that Trichostrongylus spp., not H. contortus as we initially expected, is the main culprit responsible for the early stage of development of AR to LEV. We made this observation first in our recent study [Trichostrongylus spp. and H. contortus are responsible for AR to LEV [AR to BZ and ML seems to be conveyed by three main GIN which we identified in our study – Z and ML , 64. Moruggested , and as nt study  and thisR to LEV , 63–69.Our work presents the results of an in vitro test (LDT) which may in some circumstances differ from those obtained in an in vivo method such as FECRT. Such discrepancies have multifactorial background and most of these factors remain beyond researcher’s control , 70, 71.The main drawback to our study is the fact that herds enrolled in the survey were not randomly selected. Their owners had sent fecal samples to our laboratory at least once before so they were probably somehow aware of the importance of parasite control in goats. Given that most of them used to deworm all their goats routinely, usually using the same drug for a long time, they were more likely to promote the development of AR on their farms. Therefore, it is possible that our results may to some extent overestimate AR prevalence in the country, and we hope they do so. Anyway, they allow to realize that this problem already exists in Poland and unless actions are undertaken to stop it, soon no anthelmintic will remain effective in some goat herds. The another drawback to our study is that when we performed LDT with LEV we wrongly used 1.3% DMSO in control wells while LEV was diluted in water (contrary to TBZ and IVM-AG which were diluted in 1.3% DMSO). As a result if 1.3% DMSO had a potential to reduce larval development, this mistake could have falsely increased the corrected percentage of developing larvae in tested wells at the DC. Our previous observations have not indicated that DMSO at such a low concentration impacts on larval development to any noteworthy extent (unpublished data). However, even if it did we hope that the double criterion we used for classification GIN populations as resistant would reduce the risk of falsely positive result to an acceptable minimum.This study provides the first comprehensive data on the prevalence of AR in goat herds in Poland. AR to BZ and ML is widespread, while AR to LEV is currently at a low level. Therefore, an appropriate strategy of GIN control should be applied by farmers and veterinarians as soon as possible not to let the situation slip out of control.This was a cross-sectional study and lasted virtually one year, from September 2018 to June 2019. During this time the owners of 85 private dairy goat herds who had cooperated with our laboratory in previous 4 years (2015–2018), were invited to voluntarily enrol in the study. Those who declared participation were asked to collect fecal samples after at least 8 weeks since the last deworming had passed, and detailed instructions on proper collection and preparation of anaerobically stored fecal samples were sent to them. In herds which consisted of less than 15 adult goats fecal samples were collected from all animals, otherwise samples were collected from 15 to 20 goats selected by the owners. The owners were asked to pick only these goats which had been present on their farm for at least 8 previous weeks to avoid including goats dewormed within this period of time by the former owner. Fecal samples were collected by the farmers (owners of the goats) directly from the rectum. Pooled fecal samples weighing 50–100 g were placed in plastic containers filled with tap water to ensure anaerobic conditions. Samples were delivered to the laboratory within 24 h at room temperature and processed within next 24 h so that the total time which had elapsed between sample collection and examination did not exceed 48 h.In each herd the following information was collected using the questionnaire: the number of adult goats (> 6 month-old) and kids, access to the pasture, co-grazing or rotational grazing with sheep and cattle, purchase of goats from other herds, and the history of deworming including the number of times the herd had been dewormed in previous 3 years and drugs used.According to Polish legal regulations  no formal ethics consent was required for this study except for the informed consent of participants, which we obtained in written from each participating farmer.T) was corrected by the percentage of developing larvae in the control wells (PDC) according to the following formula:Based on the technique defined by Hubert and Kerboeuf , the larT / PDC,cPD = PDwhere cPD stood for corrected percentage of larvae developing in tested wells.50) and 99% (EC99) of larvae was estimated using the 4 parameter logistic curve [The concentration of each anthelmintic inhibiting development of 50%  of anthelmintic agent which was defined as the concentration of anthelmintic at which the development of at least 99% of susceptible larvae (corrected by the larvae developing in control wells as mentioned above) would have been inhibited , 62. The99 > DC.Two criteria had to be simultaneously fulfilled to classify a GIN population from a given goat herd as resistant to the particular anthelmintic: cPD at the DC significantly higher than 1%  was above 1%) and ECAR was arbitrarily classed in terms of severity as follows: cPD at the DC from > 1 to 10% – mild AR, from > 10 to 50% – moderate AR, above 50% – severe AR. Multidrug AR (MDR) was defined as the presence of AR to all three different classes of anthelmintics.50 were assessed using the Spearman’s rank correlation coefficient (Rs). The significance level (α) was set at 0.05 and the Bonferroni correction was applied in the case of multiple comparisons. Statistical analysis was performed in TIBCO Statistica 13.3.0 .Numerical variables were presented as the median, interquartile range (IQR) and range, and compared between groups using the Mann-Whitney U test (unpaired groups) or Wilcoxon signed rank test (paired groups). Categorical variables were given as count and percentage in a group, and compared between groups using the Pearson’s chi-square test or the Fisher exact test. The 95% confidence intervals (CI 95%) for percentages were calculated using the Wilson score method . CorrelaAdditional file 1: Fig. S1. Correlations  between the corrected percentage of larvae developing in tested wells (cPD) at the discriminating concentration (DC) of each anthelmintic agent and the median effective concentration (ED50) of each anthelmintic agent. Scatter plots presenting correlations between cPD at DC and ED50 for each of anthelmintic agents.