Petromyzon marinus) during the critical metamorphic period during which sea lamprey increase osmoregulatory capacity and acquire seawater (SW) tolerance. We demonstrate in vivo that increases in circulating [S] and gill CR abundance are associated with increases in osmoregulatory capacity during metamorphosis. We further show that in vivo and ex vivo treatment with S increases activity and expression of gill active ion transporters and improves SW tolerance, and that only S (and not DOC) has regulatory control over active ion transport in the gills. Lastly, we show that the lamprey CR expresses an ancestral, spironolactone-as-agonist structural motif and that spironolactone treatment in vivo increases osmoregulatory capacity. Together, these results demonstrate that S is an osmoregulatory hormone in lamprey and that receptor-mediated discriminative corticosteroid regulation of hydromineral balance is an evolutionarily basal trait among vertebrates.It is unknown whether and how osmoregulation is controlled by corticosteroid signaling in the phylogenetically basal vertebrate group Agnatha, including lampreys and hagfishes. It is known that a truncated steroid biosynthetic pathway in lampreys produces two predominant circulating corticosteroids, 11-deoxycortisol (S) and 11-deoxycorticosterone (DOC). Furthermore, lampreys express only a single, ancestral corticosteroid receptor (CR). Whether S and/or DOC interact with the CR to control osmoregulation in lampreys is still unknown. We examined the role of the endogenous corticosteroids in vivo and ex vivo in sea lamprey ( Still, it remains unclear whether the Agnathan corticosteroid receptor (CR), which is understood to be ancestral to the appearance and divergence of the mineralocorticoid (MR) and glucocorticoid (GR) receptors of later vertebrates4, has affinity for and is activated by S, DOC or both. For instance, one approach using classical ex vivo receptor binding studies demonstrated that the lamprey CR only had affinity for S1, yet a different approach expressing the ligand binding domain of the lamprey CR in mammalian cells in vitro demonstrated the lamprey CR can be activated by several corticosteroids including both endogenous terminal corticosteroids in lamprey, S and DOC2. This raises the question of whether S or DOC or both act as a hormone in the basal vertebrate group, and thus, there is a need to better understand the true physiological role of the lamprey CR and its interaction with the endogenous corticosteroids, S and DOC. Physiological approaches in lamprey will address this gap in knowledge and provide a more complete understanding of the endocrine action, particularly the osmoregulatory role, of corticosteroids and their receptors in the basal vertebrates5.Corticosteroid signaling is central in controlling many physiological functions in vertebrates including metabolism, ion homeostasis and the stress response. Although the role of corticosteroids in controlling physiological function has been described in most vertebrate groups, the physiological role of corticosteroids and their receptor(s) in the phylogenetically basal vertebrate group Agnatha, represented by extant lamprey and hagfish, is not well understood. Recent investigations indicate that the terminal corticosteroids cortisol (F) and aldosterone (A) are absent in lamprey serum, whereas 11-deoxycortisol (S) and 11-deoxycorticosterone (DOC), respective steroid biosynthetic precursors to F and A in more derived vertebrates, are present at physiologically relevant levelsPetromyzon marinus) is an ideal model to study the evolution of hormonal control of osmoregulatory processes due to its basal phylogenetic position and its complex and fascinating life history, which includes a migration from freshwater (FW) to seawater (SW) following a larvae-to-juvenile metamorphosis. After 4–6 years of larval life in FW with no ability to survive in SW, sea lamprey undergo metamorphosis during which major morphological and physiological changes occur, including the development of an eye and a toothy, oral disc6, proliferation of branchial salt-secreting cells called ‘ionocytes’7, upregulation of hypo-osmoregulatory mechanisms in the gill and intestine11, and acquisition of SW tolerance12. After metamorphosis, juvenile sea lamprey migrate downstream into the sea to feed parasitically and grow into adulthood.Among agnathans, only the lampreys are osmoregulators, whereas all hagfish species are osmoconformers. Sea lamprey  and Na+/K+/2Cl–cotransporter (NKCC1)13. Corticosteroid control of salt-secretory mechanisms and SW tolerance in teleost fishes are well-documented14. Numerous studies in euryhaline fishes—those which can survive in both FW and SW—have shown that cortisol has an important role in controlling osmoregulatory processes15. In anadromous salmonids, for instance, circulating concentrations of cortisol increase during the parr-smolt transformation (which includes development of SW tolerance)16, in vivo treatment with cortisol increases gill NKA activity and SW tolerance21, and the action of cortisol on affecting active ion transport in the gills of salmonids is mediated mainly through the GR rather than the MR23. In teleosts, although DOC signaling acting through the MR has a more minor role in osmoregulation, it has been implicated in partially regulating several osmoregulatory processes, including ion transport and paracellular permeability24.In fishes, the transition from FW to SW includes a transformation of the gill epithelium from a site of ion uptake to a site of salt secretion. The transcellular secretion of Cl25. Recentley, discrepant reports from in vitro and ex vivo studies have been published regarding the binding and transactivation of the lamprey CR by the endogenous lamprey corticosteroids, S and DOC. Thus, there is a clear need for more in vivo studies to discern the true physiological role of corticosteroid signaling in controlling hydromineral balance in this basal vertebrate group.Lampreys possess only a basal CR, rather than the diverged GR and MR of later vertebratesIn the present study, we address this conspicuous gap in our current understanding of the role of corticosteroids in Agnatha by using in vivo and ex vivo experimentation to determine the physiological importance and specificity of corticosteroid control of osmoregulation in the sea lamprey. We focus on the critical developmental period of metamorphosis when there are critical changes in osmoregulatory physiology associated with seawater entry. We seek to relate osmoregulatory function to functional properties of the lamprey CR and, specifically, to discern (i) whether circulating S and/or gill CR drive osmoregulatory changes during metamorphosis and (ii) whether S, DOC, or both endogenous corticosteroids control major osmoregulatory changes during sea lamprey metamorphosis. Through this work, our objective was to resolve whether the basal osmoregulating vertebrate, which expresses only a single corticosteroid receptor, has the capacity for discriminative corticosteroid control over physiological processes of hydromineral balance, or if this is an evolved trait among vertebrates which occurred after the MR/GR divergence.cyp11b genes in the lamprey genome , dissociation constant (Kd), and abundance (Bmax) for the lamprey gill CR  for S (15.7 nM) than DOC (89.1 nM), or F and A (no binding detected).The physiological corticosteroids in lamprey, S DOC, are produced from a truncated steroid biosynthesis pathway in lieu of the existence of −1 h−1 and increased to 19.5 ± 1.7 μmol ADP mg−1 h−1 by the final stages and completion of metamorphosis (Nov) , but gill CR abundance in metamorphosing lamprey increased over threefold, from 90 ± 6 in Aug to 196 ± 36 fmol mg−1 in Nov  , circulating [S], and gill CR expression between larval and metamorphic lamprey in vivo, and assessed the relationships of gill NKA activity with gill CR abundance and plasma [S]. Gill NKA activity in early- and mid-metamorphic lamprey (Aug–Sep) remained near larval levels at 1–2 μmol ADP mg−] and [S] in FW control lamprey (transferred from FW to FW for 24 h) remained at baseline levels of ~ 100 mM and ~ 1 ng mL−1, respectively , plasma [Cl−] had risen to ~ 160 mM  were observed in the Veh controls transferred to 25‰ (hereafter referred to as “SW”)  and significantly lower than in the Veh control  over the Veh control  but there was no significant osmoregulatory improvement from prior treatment with S (P = 0.262).In mid-metamorphic (Oct) lamprey, after the significant increase in gill CR but before the endogenous rise in circulating S, maximal gill NKA activity, and acquisition of SW tolerance, we assessed salinity tolerance and evaluated whether treatment with S in vivo  and ex vivo (explanted gill tissue incubated in S for 24 h) would improve osmoregulatory capacity and/or increase expression of key SW gill ion transporters, NKA and NKCC1. We also performed a similar in vivo experiment with larval lamprey, which have very low osmoregulatory capacity and no SW tolerance. Plasma  and gill CR abundance are upregulated during metamorphosis when endogenous increases in salinity tolerance occur; (ii) plasma [S] is upregulated during SW exposure; (iii) ex vivo and in vivo treatment with S upregulates expression of critical active gill ion transporters and increases SW tolerance; (iv) in vivo treatment with the other known circulating corticosteroid in lamprey, DOC, does not upregulate such transcellular ionoregulatory machinery; and (v) treatment with a pharmacological agonist of the lamprey CR, spironolactone, is effective in stimulating lamprey gill ion transporter activity similar to the effects of S.10) as well as gill NKCC1 protein abundance9. An increase in gill CR abundance was detected earlier in metamorphosis (Oct) compared to that of plasma [S] (Nov). It makes intuitive sense that an increase in the intrinsic capacity of the lamprey gill to receive a corticosteroid signal  would occur prior to an elevation of the concentration of circulating corticosteroid, whereas the opposite case (upregulating plasma [S] before increasing gill CR abundance) would less efficiently utilize the circulating hormone. A similar endocrine program is present in anadromous salmonids in which, along with circulating cortisol, GR abundance (as measured by binding studies similar to those used in the present study) is upregulated during the parr-smolt transformation ahead of SW entry30. In teleost fishes, the GR is known to be important in mediating osmoregulatory adaptation31. The hyperbolic relationship between lamprey gill NKA and plasma [S] shown in the present study suggests a saturating potential of the hormone—at some concentration of circulating S, the CRs in the gill become saturated and a further increase in plasma [S] does not further stimulate gill NKA activity. Indeed, the apparent saturating effect of [S] on gill NKA activity occurs around ~ 5–10 ng mL−1 (NKA activity at 50% = 10.7 ± 5.1 ng mL−1), or ~ 15–30 nM, which closely approximates the range of calculated values for IC50 of the gill CR to S of ~ 15 nM. Conversely, gill NKA activity was more directly related to increases in gill CR abundance, suggesting that the receptor is important in directly mediating the corticosteroid signal and affecting an osmoregulatory response in the metamorphic sea lamprey. In anadromous salmonids, increases in GR abundance during the parr-smolt transformation is regulated by growth hormone (GH)32. GH has been identified in sea lamprey33, but more work is needed to determine whether osmoregulation is controlled by a similar endocrine program in sea lamprey.To understand how corticosteroids control osmoregulation in lamprey, it is necessary to observe the nature of endogenous osmoregulatory and endocrine changes that occur naturally during metamorphosis and SW acclimation. In the present study, we show large increases in gill NKA activity during the later stages of metamorphosis (Oct and Nov) occurred along with increases in plasma [S] and gill CR abundance. The increase in gill NKA activity during metamorphosis corresponds with what has been observed previously with gill NKA  is either stressful and/or that circulating [S] increases to promote salt secretion. In either case, S signaling seems important for lamprey to initiate osmoregulatory changes necessary to survive in SW when osmoregulatory capacity is low.Mid-metamorphic (early-Oct) lamprey exposed to elevated salinity elicit a salinity-dependent increase in both plasma [Cl[S] Fig. , whereas14, and so we used similar approaches to characterize the osmoregulatory role of S in sea lamprey. Our hormone treatment and salinity experiments were carried out in October, which we had determined to be the best time to conduct these experiments as it was after the significant increase in gill CR abundance, but prior to the endogenous increases in plasma [S] and peak gill NKA activity. During this time frame, SW tolerance is higher than in larvae (which can only tolerate a maximum of 12‰) but still lower than fully transformed juveniles9. After 24 h in elevated salinity, metamorphosing lamprey in October experienced salinity-dependent loss of osmotic homeostasis, evidenced by salinity-dependent increases in plasma [Cl−] to well above ~ 120 mM [Cl−]  observed, since these transporters also increase during metamorphosis when salinity tolerance increases. Similar increases in gill NKA and NKCC1 have also been observed in conjunction with higher levels of salinity tolerance in teleost fishes treated with cortisol15.Ex vivo and in vivo experimentation with cortisol have proven a useful method for establishing its role in osmoregulatory control in other vertebrates−] Figs. , 4, whic1 and replicated in the present study. It is also possible that in situ inactivation of DOC by a reductase  or some other pathway of steroid catabolism is occurring in the gill, much like how hydroxysteroid dehydrogenases (HSDs) are known to regulate tissue-specific physiologic actions to corticosteroids in other vertebrates34.In addition to characterizing the role of S and CR during metamorphosis, we sought to investigate the specificity of corticosteroid action in vivo by conducting a similar experiment administering both endogenous corticosteroids, S or DOC. We confirmed that our method of intraperitoneal injection with S achieved dose-wise increases in circulating [S] within a physiological range, and replicated the results from Fig. 35. A functional role for TJ complex proteins has more recently been elucidated in the sea lamprey38. Several studies have demonstrated putative corticosteroid control of TJ proteins in cultured teleost fish gill epithelia40, and it is possible that S may thus also control TJ complexing and paracellular permeability in the sea lamprey gill, in addition to its role in controlling active ion transport.It must be acknowledged that, in addition to its role in transcellular active ion transport demonstrated in the present study, S may also have a role in lamprey osmoregulation through other mechanisms such as control of paracellular ion and water transport. It is known that tight junction (TJ) complexes dynamically control paracellular permeability in many vertebrate epithelia, including changes in gill epithelium of teleost fishes from “tight” in FW to “leaky” in SWBmax ~ 100–150 fmol mg−1) to bind the administered S. It could be that cell type-specific expression of the lamprey CR is modulating differences in osmoregulatory action of S between larvae and metamorphic lamprey—that the CR is expressed in Cl− secreting gill ionocytes in metamorphic lamprey but not in larval lamprey. Immunological studies using antibodies raised against the lamprey CR are needed to better understand the physiological role and localization of the lamprey CR. Another explanation may be that signaling from a GH/insulin-like growth factor (IGF) pathway is required for initial ionocyte differentiation and upregulation of CR early in lamprey metamorphosis, after which the corticosteroid can then act to upregulate protein abundance of ion transporters, as has been a suggested model of coordinated endocrine control of osmoregulation in teleosts41. In any case, the role of S in controlling osmoregulation in metamorphic lamprey is clearly apparent—the correspondence of plasma [S] and calculated gill CR Kd and IC50 values, the concordant rise in systemic S and gill CR, and the specific S effects on the gill ionoregulatory apparatus combine to suggest S is an important hormone contributing to development of SW tolerance in the basal sea lamprey.Interestingly, we were unable to stimulate an osmoregulatory response in larval lamprey with an identical in vivo approach of S treatment, despite larvae having apparently adequate gill CR abundance  on helix 8 of the MR of tetrapods confers the switch of progesterone and its derivative, spironolactone, from MR agonists to antagonists, and it has been suggested that this molecular switch may be causally related to the appearance of aldosterone synthesis and mineralocorticoid function of aldosterone in tetrapods51. Analysis of the lamprey CR revealed the key Leu residue was present on helix 8, and thus we should expect that spironolactone would act as a CR agonist. If the osmoregulatory effects of S treatment were indeed acting through the lamprey CR, we would expect to see a similar effect of spironolactone. We observed this expected rise in gill NKA activity after 3 weeks of in vivo treatment with spironolactone  or GR (such as RU486) have been used for more than half a century, due to their anti-aldosterone, -cortisol, and -progesterone activitiesIn conclusion, the present study provides much needed in vivo evidence and physiological context to earlier investigations and conflicting reports regarding corticosteroid function in the basal Agnathans. Our work underscores the importance of in vivo and ex vivo studies and inspires more research into corticosteroid function in Agnathan physiology. We demonstrate that a receptor-mediated signal from S, and not DOC, is a major endocrine process controlling osmoregulatory processes in the sea lamprey. That lamprey exhibit discriminative corticosteroid control of osmoregulation  indicates that such corticosteroid function appeared early in vertebrate evolution, perhaps in association with the appearance of an osmoregulatory strategy, which has been maintained in nearly all later-evolved vertebrate lineages.All animal care and experimentation were carried out using protocols approved by the Internal Animal Care and Use Committee at the University of Massachusetts and the U.S. Geological Survey (Protocol number: 2016-0009) and in accordance with all relevant guidelines and regulation . Wild larval and pre-metamorphic lamprey were caught in July by electrofishing on the Sawmill River, a tributary to the Connecticut River in Western Massachusetts, USA. After capture, animals were held in 1.5 m diameter flow-through tanks supplied with Connecticut River water with 10 cm of sand. For the metamorphic profile, larval and metamorphosing lamprey were randomly selected for sampling each month from August to December. For hormone treatment experiments, mid-metamorphic lamprey were held in aquaria (80 L) containing dechlorinated, filtered, and aerated recirculating municipal fresh water kept at 15 °C. Artificial SW dilutions were made using a commercial sea salt mix .6. The biometric data of the lamprey in these experiments were: length = 14.7 ± 0.3 cm; body mass = 5.4 ± 0.4 g; sex ratio = 50% male, 50% female. In salinity tolerance tests, lamprey were exposed to varying salinities  or a FW control and sampled for blood after 24 h. For hormone treatments, lamprey were anesthetized with MS-222  and injected intraperitoneally with vehicle alone  or Veh containing S, DOC, or spironolactone, then held in freshwater for 12 days (21 days for spironolactone) prior to sampling in FW or after a 24 h exposure to 25‰ (referred to as “SW”). To account for changes which may be occurring naturally during metamorphosis, uninjected lamprey were sampled on the day of injections as a time = 0 control (T0). The doses for S injection were adjusted slightly between experiments −1 penstrep) containing dissolved hormone such that gill tissue from a single fish would be exposed to every hormone treatment (0.05–5.0 μg mL−1 S) and a media-only control (Ctrl). Doses of S for ex vivo culture were chosen according to doses of cortisol used in similar ex vivo organ culture experiments in teleosts54. Excised gill tissue was frozen and stored at − 80 °C before incubation (Pre) and after 24 h in culture at 15 °C. Ex vivo gill cultures after 24 h incubation were considered viable if they presented (i) no loss of color, (ii) no excessive mucus accumulation, and (iii) no decrease in housekeeping gene mRNA expression.For ex vivo hormone treatment, intact whole gill pouches were excised from FW-acclimated mid-metamorphic lamprey and carefully dissected to unfold them from their basket-like arrangement and cleanly remove the afferent and efferent ends to expose the internal filamental vasculature to incubation media. The final ex vivo branchial unit was 4–8 gill filaments (~ 1 mg of wet tissue). Groups of 6–10 intact filament pieces were placed in incubation media . Blood was collected via caudal transection into heparinized glass hematocrit tubes and plasma was separated following centrifugation. Gills were dissected and frozen at − 80 °C. Gill tissue for enzyme analyses were placed in SEI buffer  before freezing. Plasma [Cl−] was measured using a digital chloridometer .Lamprey were euthanized in MS-222  and centrifuged at 3,000g for 5 min. The resulting supernatant was used in an enzyme-linked kinetic assay, which couples ADP production to NADH reduction in a 1:1 ratio to determine ATPase activity. Protein concentration was determined spectrophotometrically using a bovine serum albumin (BSA) standard curve  and the ouabain-sensitive ATPase activity expressed as μmol ADP mg protein−1 h−1.NKA activity was determined by measuring the ouabain-sensitive ADP production of gill homogenates1. The RIA was carried out in glass culture tubes (10 × 75 mm) using a commercial antibody  and commercial 3H-labeled 11-deoxycortisol . Each reaction consisted of 10 μL of plasma or standard sample, 100 μL of PBS assay buffer  containing 5,000 cpm [3H]S, and 50 μL of Ab diluted 1:5,000 in assay buffer (determined to be appropriate for 50% [3H]S binding). The reactions were prepared on ice, incubated for 1 h at 37 °C, then incubated at 4 °C overnight. After overnight incubation, 500 μL of dextran-coated charcoal  was added to each reaction and incubated on ice for 15 min. Unbound 11-deoxycortisol that associates with charcoal was pulled out of solution by centrifugation at 2,000g for 15 min. A 325 μL aliquot of the supernatant containing S and [3H]S bound to Ab was added to 2.5 mL scintillation fluid, then analyzed by a liquid scintillation counter . Final determination of plasma [S] was made by interpolation using a serial dilution standard curve of unlabeled S  ranging from 32 to 0.5 ng mL−1, as well as a 0 ng mL−1 standard, run in triplicate. All necessary precautions were taken regarding the validation of this assay: (i) the CET-M8 Ab used in our assay was validated to have high specificity for S compared to DOC, F, and A (cross-reaction < 1%); (i) lower limits of detection and quantitation of [S] were determined to be 0.25 and 0.5 ng mL−1, respectively; (iii) serially diluted lamprey plasma returned a linear response of calculated [S] ; additions of 1, 2, 4, and 8 ng mL−1 S to lamprey plasma returned calculated [S] within 10% of expected values (m = 1.02).Plasma [S] was measured using a competitive radioimmunoassay (RIA)21. Frozen gill tissue pooled from two individuals (~ 8–10 gill pouches) were placed in 300 μL of ice-cold HEPES assay buffer  and homogenized on ice using a ground glass homogenizer. Homogenates were centrifuged for 10 min at 2,000g at 4 °C and the resulting supernatant (crude cytosolic preparation) was placed on ice for use in receptor binding assay which was carried out on ice in a 96-well plate. An aliquot of supernatant was reserved to determine protein concentration . For each sample, total (BT) and non-specific (BNS) binding curves were obtained by incubating 25 μL of gill supernatant with 25 μL of assay buffer (HEPES) containing various concentrations of [3H]S, either alone (BT) or with a 500-fold excess of [cold]S (BNS). The final assay in each well was 50 μL and contained 4–6 mg mL−1 gill protein and 2.5, 5, 10, or 20 nM [3H]S and was incubated on ice for 2 h to allow the reaction to reach equilibrium. After incubation, 150 μL of an ice-cold dextran-coated charcoal solution  was added to each well and incubated for 10 min on ice, then centrifuged for 10 min at 2,000g at 4 °C to remove any unbound [3H]S. Finally, 100 μL of supernatant was transferred to scintillation vial containing 2 mL of scintillation fluid  and counted in a liquid scintillation counter . Specific binding (BS) was calculated (BS = BT − BNS) and the corticosteroid receptor binding capacity (Bmax) and equilibrium dissociation constant (Kd) were determined by hyperbolic regression analysis. Binding specificity was analyzed identically as above but using 1 nM [3H]S in competition with various concentrations  of unlabeled S, DOC, F, or A.Corticosteroid receptor binding assay was modified from previous methods9. Antibodies used were: mouse monoclonal anti-NKA α-subunit , and mouse monoclonal anti-NKCC . Original images for Western blot analyses are provided in Supplementary Figs. Gill NKA and NKCC1 mRNA and protein abundance were analyzed by standard quantitative real-time polymerase chain reaction (qPCR) and Western blotting using procedures, molecular primers, and antibodies as previously validated and described in our laboratoryhttps://www.ebi.ac.uk/clustalw) implemented by MEGA7 software56 using the neighbor-joining method with 1,000 replicates. The accession numbers for sequences used in our analyses were: Homo sapiens ; Mus musculus ; Gallus gallus ; Xenopus laevis ; Danio rerio ; Oncorhynchus mykiss ; Petromyzon marinus . To understand the nature of the relationships between hormone or receptor and the developmentally regulated rise in gill NKA activity, we compared three theoretical curve-fitting analyses  to determine the simplest model for the relationship (most degrees of freedom) that best fit (highest r2) the data. Normality and homogeneity of variance assumptions were tested using Shapiro–Wilk and Levene’s tests, respectively. Treatment effects and comparisons between treatment groups were analyzed using Student’s t-test, one- and two-way ANOVA, and Tukey’s post hoc analyses, as indicated in figure captions. An α-value of 0.05 was selected to denote statistical significance in all analyses. All regression and statistical analyses and figures were completed using GraphPad Prism 6.0 .Molecular phylogenetic analysis using peptide sequence data obtained from NCBI Genbank were performed using ClustalW alignment (Supplementary Figures.


P10/mmm) was observed in the Zn–Mg–Y system, in addition to an icosahedral quasicrystal and a decagonal quasicrystal (space group P105/mmc) which have been reported previously. This decagonal quasicrystal can be modeled with dense packing of icosahedrons at vertices of fat rhombic and flattened hexagonal tiles.A new kind of phasonic entropy-stabilized decagonal quasicrystal (space group  58Mg40Y2 (at.%) alloy was studied using electron diffraction and atomic resolution Z-contrast imaging techniques. This stable Frank–Kasper Zn–Mg–Y decagonal quasicrystal has an atomic structure which can be modeled with a rhombic/hexagonal tiling decorated with icosahedral units at each vertex. No perfect decagonal clusters were observed in the Zn–Mg–Y decagonal quasicrystal, which differs from the Zn–Mg–Dy decagonal crystal with the same space group P10/mmm. Y atoms occupy the center of ‘dented decagon’ motifs consisting of three fat rhombic and two flattened hexagonal tiles. About 75% of fat rhombic tiles are arranged in groups of five forming star motifs, while the others connect with each other in a ‘zigzag’ configuration. This decagonal quasicrystal has a composition of Zn68.3Mg29.1Y2.6 (at.%) with a valence electron concentration (e/a) of about 2.03, which is in accord with the Hume–Rothery criterion for the formation of the Zn-based quasicrystal phase (e/a = 2.0–2.15).The structure of a decagonal quasicrystal in the Zn The space group and atomic occupation of the ternary quasicrystal were determined based on electron diffraction and atomic resolution Z-contrast imaging investigations. The structure units of this decagonal quasicrystal are simple icosahedrons which are much smaller than those in Al-TM and Zn–Mg–Dy decagonal quasicrystals  as the Zn–Mg–Dy decagonal quasicrystal  alloy was produced by high-frequency induction melting under an argon atmosphere. Samples cut from the ingot were annealed at 673 K for 100 h, then quenched into water. Specimens for transmission electron microscopy investigations were prepared using standard ion-milling techniques. Tilt series of selected area electron diffraction (SAED) patterns were obtained using a Tecnai G2 F30 microscope. Convergent-beam electron diffraction (CBED) patterns were taken on a JEOL 2100 microscope. Atomic resolution HAADF-STEM observations and atomic resolution energy dispersive X-ray (EDX) mapping were carried out on an aberration-corrected Titan 60–300 microscope equipped with a super EDX spectroscopy detector.A Zn3.et al., 1993c axis, indicating clearly on-average long-range quasi-periodic order in this decagonal quasicrystal. The SAED patterns obtained along twofold axes, designated as the 2D and 2P directions, show a regular periodicity of 0.51 nm along the tenfold direction, which is the same as the periodicity of the Zn–Mg–Dy decagonal quasicrystal along the tenfold axis ] and the directions perpendicular to the c axis [Figs. 2b) and 2(c)]. Fig. 2a) shows a CBED pattern with a tenfold rotation symmetry around the c axis and two sets of mirror symmetries parallel to the c axis, i.e. a 10mm symmetry. The CBED pattern recorded along the 2P direction reveals mirror symmetries both parallel and perpendicular to the c axis, as shown in Fig. 2b). Since CBED patterns shown in Figs. 2a) and 2(b) are formed only by zeroth-order Laue zone reflections, a twofold axis perpendicular to the incident beam acts equivalently to a mirror plane parallel to the incident beam  reflections should be obtained in order to determine the point group exclusively. Fig. 2c) shows a CBED pattern containing HOLZ reflections taken along the direction perpendicular to the c axis, but that deviated from the 2P direction. This CBED pattern shows a mirror symmetry perpendicular to the c axis, which means that the Zn–Mg–Y decagonal quasicrystal has a centrosymmetric point group. The point group of the decagonal quasicrystal is thus determined to be 10/mmm. In addition, there are no reflections due to presence of a superlattice structure and no extinction of diffraction as a result of the presence of a 105 screw axis and c-glide plane. Therefore, the space group of this Zn–Mg–Y decagonal quasicrystal should be P10/mmm, which is consistent with the result of the Zn–Mg–Dy decagonal qusicrystal reported by Örs et al.  and 3(b) present atomic resolution HAADF-STEM images obtained along the 2D and tenfold directions, respectively. Periodicity along the tenfold axis and quasi-periodicity perpendicular to this axis can be seen clearly from the image recorded along the 2D direction, as shown in Fig. 3a). When viewed along the tenfold axis, strong bright image spots are arranged into fat rhombic and flattened hexagonal tiles which are distributed aperiodically, as shown in Figs. 3b) and 3(c). Edges of both kinds of tiles have the same length of about 0.45 nm. The intensities of image spots located at the vertices are about twice those at the mid-edges. These characteristic parameters of fat rhombic and flattened hexagonal tiles in the present Zn–Mg–Y quasicrystal are as the same as those in the F-K Mg4Zn7 crystal  and 3(c), which is similar to Mg–Zn nanoprecipitates . The atomic structure model [Fig. 3d)] of the boat tile is proposed, based on atomic resolution HAADF-STEM imaging [Fig. 3b)] and atomic resolution EDX measurements  and 3(c). The boat tile can be decomposed into three fat rhombic tiles and one skinny rhombic tile, as shown schematically in Fig. 3d). The vertex connecting four edges indicated by dashed lines inside the boat tile should correspond to a column of Zn atoms at z = c/4, since it is a part of the icosahedral chain centered at the vertex indicated by an arrow at the boundary. This is consistent with the fact that the brightness of the corresponding intensity spot is similar to those at the mid-edge positions, as seen from Fig. 3b). Given the low number density of the boat tiles, it can be regarded as a kind of defect in this Zn–Mg–Y decagonal quasicrystal.Figs. 3et al., 2014et al., 2014et al., although both crystallize in the same space group P10/mmm . Atomic resolution EDX mapping was carried out in order to obtain chemical information about the substitution. Fig. 4b). It is clear that there are Zn atoms located at the vertices and the middle points of the tile edges, which is the same as in Mg–Zn Laves crystals. Atomic resolution elemental mapping results indicate substitution of Y for Mg at the centers of ‘dented decagon’ motifs, as shown in Fig. 4c). Interestingly, Y atoms preferentially form the pentagon and skinny rhombus which are the constituents of pentagonal Penrose tiling . The distribution of Mg is not shown in Fig. 468.3Mg29.1Y2.6 (at.%).In the case of Mg–Zn Laves phases, atomic columns within fat rhombic and flattened hexagonal units consist purely of Mg atoms; therefore, they could not produce image spots showing intensity high enough in the HAADF-STEM image, due to the low atomic number of Mg  is expected to be small , and the existence of tenfold symmetry in the local area of this decagonal quasicrystal is strongly supported. A perfect Penrose tiling cannot be formed by only these two tiles observed in the present Zn–Mg–Y decagonal quasicrystal  is the diagonal of the 5D unit cell , as shown by the red arrows in Fig. 3c). The corresponding vertex position r⊥ in the E⊥ perpendicular space spanned by r⊥ as a function of the number of vertices N, et al., 1989et al., 1989N is large enough, as shown in Fig. 5b). This suggests that the tiling in the present Zn–Mg–Y quasicrystal should be interpreted as a phason-perturbed Penrose tiling, instead of a random tiling  ], and the vertices in each sub-lattice  are generated by one of the four pentagonal occupation domains. Numerous fat rhombic and skinny rhombic tiles can be seen in the perfect Penrose tiling. However, a very short distance of the short diagonal (a/τ < a) of skinny rhombic tiles will induce overlap of two icosahedrons, which has not been observed in the F-K compounds. Triangular face sharing of icosahedrons in fat rhombi and flattened hexagons is observed frequently in the Mg–Zn Laves phase ] , suggesting that the present Zn–Mg–Y decagonal quasicrystal can be modeled with an intermediate tiling located between the ideal Penrose tiling and random tiling decorated with icosahedral units at each vertex. This kind of tiling is usually referred to as a phason-perturbed Penrose tiling , although local domains showing fivefold rotation were observed in some tiny precipitates in an Mg–Zn alloy   with an e/a value of about 2.03 which is quite close to that for the Zn58Mg40Dy2 decagonal quasicrystal. A previous study showed that an orthorhombic MgYZn4 phase  forms if 50% of Mg in MgZn2 are substituted by Y  plays an important role in the formation of Zn–Mg–RE quasicrystals.Fat rhombic and/or flattened hexagonal tiles are arranged periodically in binary Mg–Zn compound crystals  at 598 K, and increasing the temperature to 648 K would worsen the quasiperiodic ordering  the binary Mg–Zn decagonal quasicrystal was unstable by 1.1 meV per atom, using an artificial approximant model based on a supercell (with 56 atoms) consisting of two hexagons and one star motif; (ii) the substitution of a Y atom for one Mg atom requires an energy cost of ∼90 meV; (iii) the Zn–Mg–Y decagonal quasicrystal becomes thermodynamically stable when the temperature exceeds 705 K  alloy. The composition of this decagonal quasicrystal is estimated to be Zn68.3Mg29.1Y2.6 (at.%). Atomic resolution HAADF-STEM observations reveal that the structure of this decagonal quasicrystal can be modeled with a rhombic/hexagonal tiling decorated with icosahedral units at each vertex. The structure of the present Zn–Mg–Y decagonal quasicrystal is not only different from that of the Zn–Mg–Dy decagonal quasicrystal, but also different from the theoretically predicted structure for the Zn–Mg–Y decagonal quasicrystal in terms of the arrangement of fat rhombic tiles. This decagonal quasicrystal composed of simple icosahedral units may be a good candidate to investigate the formation and growth of quasicrystals theoretically and experimentally.A decagonal quasicrystal with periodicity 0.51 nm along the tenfold axis and space group


The developed biosensors are generally characterized by a high degree of specificity, but unfortunately also by relatively short lifetimes. There are several investigations where biosensor measurement of NO3− and NO2− have given new insight into the functioning of nitrogen transformations in man-made and natural environments such as sediments and biofilms, but the biosensors have not become routine tools. Future modifications resulting in better long-term stability may enable such general use.Inorganic ions that can be redox-transformed by living cells can be sensed by biosensors, where the redox transformation gives rise to a current in a measuring circuit. Such biosensors may be based on enzymes, or they may be based on application of whole cells. In this review focus will be on biosensors for the environmentally important ions NO Various voltammetry techniques are thus widely used to measure ions such as heavy metals that can be redox-transformed at moderate potentials on an electrode . Voltammand Ca2+  and CO32a2+ [32− , and it ectrodes . Advanceectrodes ,7.3−, NO2−, NH4+, HCO3−, HS− and SO42−. Very good purely electrochemical sensors are available for the carbonate system  and for[32− [2S . No pracNitrate can be directly reduced to nitrite at various cathode materials ,16,17,18MK, values for the enzyme [2, implicating that analysis should be performed under anoxic condition [2 scavengers [To facilitate the transfer of electrons between cathode and nitrate and to minimize reduction of other species, several attempts have been made to utilize the catalytic role of nitrate reductase (NR) in conjunction with an electron carrier—a mediator. Nitrate reductases from bacterial ,21 as weondition ,24,25,26avengers  to wateravengers . It was avengers ), UV-absavengers ) and bac2− without the help of biological catalysis can be performed by application of various electrode materials and mediators, and linear calibration curves over large concentration intervals can be obtained [2 and NO3− interfere, invalidating the approach for environmental monitoring. A more promising approach is detection by nitrite oxidation, and this approach has been investigated by several researchers. The apparently best results were obtained by Zhou et al. [2− the signal decreased by 6% within 1 h. It was, however, possible to regenerate the electrode by mechanical polishing.Electrochemical determination of NOobtained . A generu et al.  who deveThe cathodic reduction of nitrite can be catalyzed by organic molecules as is the case for nitrate. Not only nitrite reductase (NiR) ,32,33, b2 is also reduced, and analysis was therefore performed under completely anoxic condition [2 interference [−, Br−, S2O32−, and SO32− [− concentrations approaching 1 mM.A good nitrite biosensor performance was achieved by Hong et al., using hemoglobin and Au-nanoparticles (AuNPs) as electron transfer relays for nitrite reduction. They reported a LOD of 65 nM and a sensor shelf lifetime of 1 month . The appondition ,36,37,40ondition , phenaxiondition , methyl ondition , or M-NMondition  resultedrference . Interfend SO32− , preventThe major factors preventing general use of enzyme-based nitrate or nitrite biosensors are probably short lifetimes and poor stability by continuous use. Maximum shelf lifetimes at 4 °C were thus reported to be 30 days . As the x−; [2O and subsequent reduction of this N2O in a Clark-type N2O sensor  reaction chambers will thus have a smaller dynamic range than long  reaction chambers, but the response time increases with longer chambers.Bacteria-based biosensors for nitrate  and nitx−;  rely on O sensor . The bac2S that reversibly inactivates the sensor, and N2O. The sensors produce linear calibration curves and may have detection limits (LOD) of 0.1 µM. As will be presented later the linear range of NOx− and NO2− sensors can be tailored to just about any range by applying a charge across the front membrane. In contrast to ion-selective sensors based on ionophores, the bacteria-based biosensors also function well at salinities as high as seawater. For NOx− various denitrifying bacteria devoid of N2O reductase may be applied, and by use of cold resistant (psychrotrophic) bacteria [x− profiles at high resolution in terms of both concentration and depth in sediment at 2000 m water depth off the coast of Tokyo, where the water temperature was only 2.5 °C [The response time increases and the dynamic range decreases when the temperature decreases. The 90% response time for a 200 µm reaction chamber is about 30 s, and the dynamic range  is about 0–500 µM at room temperature . There abacteria  such seny 2.5 °C . The psy2O reductase that could be applied in a nitrite biosensor have not been identified. It would be easy to create a nitrate reductase deficient mutant of the psychrotrophic denitrifyer just mentioned, but laws about genetically modified organisms would complicate practical use of sensors based on such bacteria. The only known rich sources of nitrate reductase deficient nitrite reducing bacteria are air filters rinsing airstreams rich in ammonia, such as ventilation air from piggeries. Nitrite may accumulate to concentrations above 100 mM in these filters [Unfortunately, psychrotrophic bacteria devoid of both nitrate and N filters , but due2O interference may have to be taken into account by analysis of some environments such as wastewater treatment plants, where considerable N2O concentrations may be found. It may thus be necessary to perform simultaneous measurements with a N2O sensor [Macroscale sensors have been constructed with flat end  penetratO sensor , so thatO sensor ,47 and lO sensor  . Also, during storage there is a need for frequent  front membrane cleaning as bacterial growth on the outside of the sensor will result in a cut-off of the essential OA common feature for the sensor design shown in A potentiostat is used for applying the charge in It should be noticed, however, that the calibrations were performed at 10‰ salinity (NaCl), and that variations in the imposed ERC potential results in much larger changes in sensitivity at lower salinity such as found in for example limnic waters or wastewater . A 0.01542−, reducing SO42− to H2S. Clark-type H2S sensors have been described [2. For construction of a sulfate biosensor it is thus necessary to apply a mixed culture where aerobes or facultative aerobes remove the O2. In a first attempt a sensor constructed as shown in 2S sensor of 400 µm. The H2S sensor was not the one previously described [2 formed by fermentation of lactate by the mixed microbial community, and the applied Unisense H2S sensor is H2 sensitive.In addition to nitrite and nitrate, bacteria may also respire with SOescribed , and it escribed , but a s− they cannot be used in saline waters. In contrast to the NOx− and NO2− biosensors described above, there should, however, be no problem by bacterial contamination of a sulfate biosensor that works with a mixed bacterial community. The use of a sulfate biosensor will be limited to oxidized environments, as external sulfide will interfere, but future improved versions might enable long-term monitoring in aerobic aquatic environments. Microscale sulfate biosensors may also be applied in freshwater sediments to obtain new knowledge about sulfate cycling. Interfering free sulfide is often not present in freshwater sediments as it is bound to iron minerals.Potentiometric ion-selective sensors have been described for sulfate , and for2S, but few publications mention this interferent, and it is therefore not included in the table except for the sulfate biosensor.The published characteristics of various sensor types are summarized in 3− and NO2− measurement in environmental waters. Problems with interferences preclude the use of NO3− and NO2− potentiometric ion-selective electrodes in saline waters, and the use of biosensors may here be an attractive alternative. No records could, however, be found of long-term monitoring or detailed environmental investigations by use of enzyme-based sensors. Bacteria-based sensors for NOx− (NO3− + NO2−) and NO2− have been extensively used for environmental analysis and have given new insights into nitrogen transformations in nature, but limited lifetime has precluded extensive use in for example wastewater treatment. It should be possible to extend the lifetime of such sensors to several months by using immotile bacteria which produces sticky extracellular polymer for more robust physical entrapment. The biosensor monitoring of many aquatic environments may then become an attractive alternative to use of potentiometric ion-selective electrodes. A new biosensor for environmental analysis of SO42− is demonstrated, and it may be used to obtain new knowledge about sulfur cycling.Ionophore-based potentiometric ion-selective electrodes or optical sensing are generally preferred over alternative electrode- or sensor-based methods for NO


The Soderberg approach is successfully employed to compute the factor of safety Nf > 1.1. Fully porous stems without dense shells are concluded to fail under fatigue load. It is thus safe to use the porous stems with a shell thickness of 1.5 and 2 mm for all porosities (18–90%), 1 mm shell with 18 and 30% porosities, and 0.5 mm shell with 18% porosity. The reduction in stress shielding was achieved by 28%. Porous stems incorporated BCC structures with dense shells and beads were successfully printed.The current study is proposing a design envelope for porous Ti-6Al-4V alloy femoral stems to survive under fatigue loads. Numerical computational analysis of these stems with a body-centered-cube (BCC) structure is conducted in ABAQUS. Femoral stems without shell and with various outer dense shell thicknesses  and inner cores  are analyzed. A design space (envelope) is derived by using stem stiffnesses close to that of the femur bone, maximum fatigue stresses of 0.3σ Hence, reducing stiffness through porous structures is a desired feature, however, the challenge is to ensure the design has a good fatigue limit (>5 × 106 cycles) [Cementless femoral stems are traditionally used for total hip replacement (THR). The stems are inserted proximally in the medullary canal of the femur and firmly attached to the bone to transfer the physiological load to the bone during daily activities. Ti-6Al-4V alloy (Ti) is proven as an excellent biomaterial –4, that  cycles) .6 cycles simultaneously. 3D finite element models of BCC porous structures were constructed and simulated to investigate the mechanical properties. The BCC porous structures with porosities of 18–90% were simulated under compression to yield the effective elastic moduli and the effective yield strengths. The mechanical properties obtained from simulated compression tests of BCC porous structures were used to model solid stems which are named as effective porous stems. 3D finite element models of stems were constructed with various outer dense shell thicknesses and inner sections with different elastic moduli. The ISO 7206-4 [6 cycles). Soderberg approach was also used to compute the factor of safety [The objective of this work is to derive a design envelope for cementless Ti-6Al-4V alloy porous stems see Fig.  to reducO 7206-4  standardf safety  under fa3 each cube that resulted the BCC structures with dimensions of 4 × 4 × 4, 8 × 8 × 8, 12 × 12 × 12, 16 × 16 × 16 mm3 in our previous study [3 (four cubes in each direction) did not change the effective mechanical properties further increasing the number of cubes. Therefore, BCC structure with dimensions of 16 × 16 × 16 mm3 was modeled that mimics the macro structural behavior as shown in Fig. In the present study, 3D FE models of the BCC microstructures were constructed using computer-aided design (CAD) software  and simulated in ABAQUS software . Porosities of 18–90% were designed using a strut thickness of 0.33–1.25 mm as listed in Table us study . This an3D models of porous and effective porous stem were constructed in computer-aided design (CAD) software  and Materialise Magics® , and simulated in ABAQUS software . The aim of constructing these models was to validate our finite element models. Therefore, a porous stem with a porosity of 63% is simulated and the elastic-plastic mechanical properties of bulk Ti-6Al-4V alloy was assigned to a porous section as shown in Fig. 3D finite element models of the effective porous stem stems were constructed computer-aided design (CAD) software . The effective elastic moduli and effective yield strengths were obtained from simulated compression tests of BCC porous structures for all porosities 18–90%. The 3D finite element models of stems were simulated in ABAQUS software . The porous stems were modeled with and without outer dense shells made of dense Ti-6Al-4V alloy and an inner core was modeled with different effective material properties of porosities in the range of 18–90%, as shown in Fig. The stems were designed to withstand walking loads (cyclic), and thus the ISO 7206-4 standard was followed to test the fatigue behavior of the stems Fig. . Finite The effective elastic moduli and effective yield strengths of BCC porous structures with porosities of 18–90% were assigned to the porous section of the stems. Elastic moduli of 114 GPa  and 3.7 GPa (linear elastic) were used for the outer dense shell and epoxy, respectively. In this study, the body-weight was assumed as 800 N with contact forces on the hip joint as 250–300% of the body-weight , 37. Theys stresses in the porous sections with regards to the fatigue limit of porous structures [σm) and alternating (σa) stresses in computing the factor of safety (Eqs. (σmin.) and maximum stresses (σmax.) in the porous section and outer dense shell were calculated using the minimum (230 N) and maximum (2300 N):Finite element analyses (FEA) was performed to estimate the axial stiffnesses of the stems. The design envelope was developed by considering (a) the effective femur bone stiffness of the stem, (b) 0.3σructures –35, 38 aructures . Overlapructures . This thty Eqs. –4). Mini. Miniys The Soderberg equation is used to calculate the factor of safety in Eq.  as folloNf is as follows:Se, Sys, and N denote the endurance limit of the material, yield strength of the material, and factor of safety, respectively. For the dense outer shell, endurance limit and yield strength of 3D printed direct metal laser sintering (DMLS) Ti-6Al-4V alloy correspond to 500 and 1180 MPa [The fatigue factor of safety 1180 MPa , respect1180 MPa .Fig. 3ReThis study aimed to analyze the design of dense and porous Ti-6Al-4V alloy stems and print using the DMLS technique to reduce the stiffness and provide adequate fatigue life. Therefore, porotypes of dense and porous stems were printed using the EOS M280 printer. The original EOS parameters of performance-based were selected to print the stems. The layer thickness was set to 30 µm and laser power was set to 200 watts. Default scanning speed and laser parameters were set for the printing of Ti-6Al-4V alloy. Argon gas was supplied to the printing chamber throughout the printing session.6 cycles) for orthopedic implants [Different porosities of BCC microstructures were simulated under the uniaxial compression test to compute the effective mechanical properties. The effective elastic moduli and effective yield strengths of porosities in the range of 18–90% were 76.7–3.8 GPa and 760–37 MPa, respectively, as shown in Fig. implants . The valTo investigate the fatigue life of the porous structures, the stresses and the strains were obtained at different load points below the yield point. These points included the data of 30% stresses of the yield strength for a specific porosity. As the porosity was changed, the yield point and 30% stresses of yield strength were changed. Figure Porous and effective porous stems were validated as shown in Fig. As shown in Fig. ys as shown in Fig. ys exhibits the desired limit of porous structures under fatigue loads [ys which are 228 MPa as shown in Fig. ys 159 and 228 MPa, respectively. However, effective porous sections of all effective elastic moduli stems with dense shells of 1.5- and 2-mm thickness shows stresses less than 0.3σys as shown in Fig. As shown in Fig. ue loads , 34. ThuTo estimate the fatigue limit of the outer dense shell, the maximum von Mises stresses are obtained from simulated results as shown in Fig. Stresses in all sections of the stem are computed for all effective elastic moduli as shown in Fig. Dense stems and porous stems with outer dense sleeve . Here, FE models of porous stems with various porosities were constructed. To obtain the mechanical properties, the structures were subjected to uniaxial compressive loads. It was found that the modulus and effective yield strengths corresponded to 3.8–76.7 GPa and 37–760 MPa, respectively. Stress–strain data from compression tests were used for 3D finite element models of the stems. ISO 7206-4 standard was used in developing these FE models along with appropriate fatigue theory. The following conclusions were obtained from this study:


The explants were established on the basal MS medium supplemented with BAP (2.20–8.90 μM) and 0.049 μM NAA, or TDZ  with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 μM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 μmol m–2 s–1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 μM BAP, or 6.0 μM TDZ were optimal cytokinin concentrations for micropropagation. The explants of ‘Alpinist,’ ‘Ay-Nor,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Nevesta,’ and ‘Serenada Kryma’ cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 μmol m–2 s–1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 μM BAP with 0.09 μM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in ‘Crystal Fountain’ (100%), ‘Crimson Star’ (100%), ‘Nevesta’ (97%), and ‘Ay-Nor’ (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars ‘Alpinist,’ ‘Ay-Nor,’ ‘Bal Tsvetov,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Madame Julia Correvon,’ ‘Nevesta,’ ‘Nikitsky Rosovyi,’ ‘Nikolay Rubtsov,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’ were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3–0.4% Cl Clematis L. belongs to the buttercup family (Ranunculaceae Juss.) and includes ca. 300 species and over 3000 cultivars , a collection of clematis plants has been created that includes 24 species and 236 cultivars of native and foreign origin . This vade novo formation of adventitious shoots and roots from an unorganized growing callus mass  and directly from leaf, stem, germ, or flower cells   . Anothereration) . In studogenesis . Genome ogenesis . Using Iogenesis . Indirecogenesis . Earlierogenesis . Among celopment . Chloropnditions . More reresented , and thetization . Finally section .in vitro under the combined influences from multiple factors, namely, genotype, plant growth regulators, light intensity, and temperature (among others). Therefore, this study’s main objective was to assess the morphogenetic potential of explants from 13 clematis cultivars, under the influence of various culture factors, at stages of formation induction of morphogenic structures and plant regeneration. The obtained results could be used for the subsequent propagation of healthy plants and the creation of an in vitro gene bank.In sum, as evinced by the foregoing, the morphogenetic capacity of certain clematis cultivars and species has been mainly studied, albeit to various extents. However, we still lack a thorough and robust evaluation of the regenerative capacity of clematis plants cultured The mother plants of 13 clematis cultivars—‘Alpinist,’ ‘Ay-Nor,’ ‘Bal Tsvetov,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Madame Julia Correvon,’ ‘Nevesta,’ ‘Nikitsky Rosovyi,’ ‘Nikolay Rubtsov,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’—grown at the collection plot of ornamental plants in the Nikita Botanical Gardens  were used as explant sources .The investigations were carried out in the Laboratory of Plant Biotechnology and Virology of the Plant Developmental Biology, Biotechnology and Biosafety Department, Federal State Funded Institution of Science “The Nikita Botanical Gardens—National Scientific Center of the RAS.” To obtain an aseptic culture, plant material isolated in January–February 2017–2019 underwent sequential sterilization in this way: 1 min in 70% ethanol, followed by 15 min in 0.3–0.4% chlorine-containing solution  and 10 min in 1% thimerosal solution  with 1–2 drops of Tween 20. After adding each reagent, the shoot segments 3 cm long were washed three times in sterile distilled water. After shoot segments had been surface-sterilized with a contamination frequency not greater than 10%, the nodal explants 1 cm long with axillary buds were established in test tubes on the MS  inductio® GP filters (0.22 μm) and then added to the media after the autoclaving. Culture vessels (100 or 250 ml jars) with explants were maintained in “BIOTRON” growth chambers and in a plant growth chamber  at 24 ± 1°C, with a 16-h photoperiod under cool-white light fluorescent lamps . Subculturing of each cultivar was carried out at 3–4 week intervals.To induce organogenesis and plant regeneration, 1 cm long microshoots and microcuttings of 13 cultivars were used. Explants were placed on the MS culture medium with MS vitamins and various concentrations of plant growth regulators, 2.20–8.90 μM BAP in combination with 0.049 μM α-naphthylacetic acid  or 3.0–9.0 μM thidiazuron , supplemented with 100 mg/L myo-inositol , 30 g/L sucrose, and 9 g/L of agar. As the control, the MS medium with 0.89 μM BAP was used. Medium pH was 5.7–5.8 for all culture media, which were autoclaved at 120°C for 7–12 min in a LAC 5060S sterilizer . Plant growth regulators and vitamins were first sterilized by cold filtration through MILLEX–2 s–1 light intensity). In an experiment with somatic embryogenesis induction, the culture vessels with calli were placed in growth chambers with temperature (20–30°C) and light intensity (5–60 μmol m–2 s–1) regulated under a 16-h photoperiod. Subculturing of each cultivar was carried out at 3–4 week intervals.For the induction experiments of indirect somatic embryogenesis, clematis microshoots or microcuttings with one or two internodes of 13 cultivars were cultured on a solidified MS medium with MS vitamins, 100 mg/L of myo-inositol, and different plant growth regulators. Firstly, microcuttings of 13 cultivars with one or two internodes were placed on an MS culture medium supplemented with 1.8 μM zeatin and 0.04 μM indole-3-butyric acid  to induce callus formation. To induce embryogenic callus formation, 0.9–6.8 μM zeatin  or 0.9–6.8 μM 2,4-D  was used. Callus was placed on an MS medium with 0.4–4.6 μM zeatin in order to induced indirect somatic embryogenesis. To induce the secondary somatic embryo formation, primary somatic embryos derived from callus were transferred to media with 0.4–4.6 μM IBA and 1.8 μM zeatin. All culture media were supplemented with 30 g/L sucrose, and the media were solidified with 9 g/L agar. The plant growth regulator-free medium served as the control. Culture vessels (100 or 250 ml jars) with microshoots, microcuttings, and somatic embryos were maintained at 24 ± 1°C, with a 16-h photoperiod under cool-white light fluorescent lamps . Subculturing of each cultivar was carried out at 3–4 week intervals.For the direct somatic embryogenesis induction experiments, firstly, vegetative buds of 13 clematis cultivars were cultured on an MS medium with 0.44–8.9 μM BAP and 0.09 μM IBA. The plant growth regulator-free medium served as the control. Due to the low rate of direct somatic embryogenesis induction in five cultivars, the main experiments with ‘Ay-Nor,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Nevesta,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’ cultivars were followed. For the secondary somatic embryogenesis induction, the primary somatic embryos were cultured on an MS medium with 0.89 or 2.22 μM BAP. All culture media were supplemented with 30 g/L sucrose and media solidified with 9 g/L agar. Cultures were incubated at 24 ± 1°C, 16-h photoperiod under cool-white light fluorescent lamps  were analyzed: ‘Alpinist’ and ‘Madame Julia Correvon’—bud conglomerates, ‘Crystal Fountain’—callus with somatic embryos, and ‘Nevesta’ and ‘Crystal Fountain’—somatic embryos on different stages. Slides for this analysis were prepared according to the commonly used methods refer to . BrieflyP < 0.05).Each treatment consisted of five glass vessels with four explants of each clematis cultivar , repeated in triplicate. The number of vegetative buds regenerating the callus or somatic embryos was recorded after 2–4 weeks of culturing. The frequency of this regeneration was calculated as the average percentage of buds or calli which formed morphogenic structures. The number of microshoots per explant was determined on a monthly basis. All of the obtained data were processed in the Statistica for Windows program v10.0 . The data of amount of regenerated microshoots per explant, length of explant, and number of internodes were analyzed, statistically averaging analysis of the variance (ANOVA) and standard deviation to find the variability between the different treatments. To see which means differed from each other and to indicate the significant difference during the analysis of percentage and number of investigated explants, Duncan’s multiple-range test was used  in the combination with a fixed IBA concentration (0.09 μM). Through this experimental work, firstly, we were able to induce direct somatic embryogenesis in all 13 studied clematis cultivars, but a genotypic influence was discernable: at 30 days since the buds were placed on media for culturing, the number of somatic embryos formed was different for each cultivar ultivars . In the ultivars . After 2ultivars . In thisultivars , but theultivars . The aveThe frequency of secondary embryogenesis varied across the studied cultivars. A high embryogenesis rate, upward of 65%–100%, was observed in the cultivars ‘Nevesta,’ ‘Crimson Star,’ ‘Serenada Kryma,’ ‘Crystal Fountain,’ and ‘Ay-Nor’ when cultured on the medium with 2.20 μM of BAP and 0.09 μM of IBA . The thrSecondary embryos most often formed along the edges of the somatic embryoids’ cotyledons. Nonetheless, embryo formation directly from the apical zone of embryoids was also noted. Initially, the formation of transparent round structures was observed, but then they became white and increased in size . Since tin vitro are of great scientific and practical interest. Firstly, this is because using mature plant tissue as a primary explant allows one to obtain plant material with economically valuable features. Yet working with mature plant tissues is hampered by the fact that as the plant ages, the processes that result in growth inhibition and reduced regenerative ability also occur in the plant tissues and organs. Microshoots obtained via regeneration are thus characterized by slow growth and weak rooting ability.The widespread use of biotechnological research in the introduction, breeding, and propagation of plants has enabled us to significantly advance our knowledge of plant biology and actively use them in studying morphogenesis via somatic embryogenesis and organogenesis. It is well known that exogenous factors significantly affect the realized morphogenetic capacity of plant tissues and organs; hence, only by combining the results from multiple experiments can an efficient system of somatic embryogenesis and organogenesis for a specific crop be developed that would work in most plant species and cultivars. Most of the clematis plants that housed the NBG-NSC open-field collection are old-aged (more than 170 years old). Studies of the morphogenetic capacity of tissues and organs in mature plants Despite this obstacle, plant scientists all over the world continue to work on this problem, pursuing their research programs and developing more and more biotechnologies applicable to specific crop . For ourIt is known that plant growth regulators play an essential role in the interaction of plant cells, tissues, and organs. Low concentrations of these substances are necessary for the induction and regulation of key physiological and morphogenic processes. For each plant species and cultivar, the optimal concentrations and combinations of plant growth regulators in the culture medium could be experimentally deduced. Thus, cytokinins, in addition to enhancing cell division and growth processes, stimulate cell differentiation, histogenesis, and shoot formation; they also affect callus differentiation and induce the development of axillary buds, the growth of lateral shoots, adventitious bud formation, and subsequent regeneration of plants . In our Theoretical studies in the field of somatic embryogenesis have shown that the whole embryogenic cell mass is determined by the process of embryoid formation, but this is not entirely true. Only some cells are capable of forming a somatic embryo. Observations of clover and pistachios support the hypothesis that growth regulators initiate asymmetric division and lead to a change in cell polarity . It is lPinus halepensis Miller  was able to induce somatic embryogenesis, both primary and secondary. This zone of somatic embryogenesis induction (“inducer”) in culture vessels could be one or a group of somatic embryos. Perhaps, this driver is the center of ractions . A similc embryo . On a suc embryo . It is kc embryo . In our in vitro plant regeneration and the need to change culture media at various stages of morphogenesis . The presented system of somatic embryogenesis allows not only for the propagation of clematis species and cultivars but also to save plant material in the form of slow-growing in vitro collections which could be used to replenish collections of other botanical gardens.In this comprehensive study, both organogenesis and somatic embryogenesis were induced from the base of clematis vegetative buds and microshoots. An increase in regenerative capacity by adventitious shoot formation for 13 cultivars due to TDZ in the culture medium was demonstrated. The main factors affecting the process of somatic embryo regeneration were established: these were mother plant genotype, plant growth regulators (BAP and IBA), a temperature of 26°C, and a light intensity of 37.5 μmol mThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.in vitro experiments. TK prepared the permanent slides for microscopy, and TK and IM described and analyzed them. IM and OM checked and corrected the manuscript. IM planned this research and its experiments and wrote the manuscript. All coauthors agreed with the content of the manuscript and support its conclusions.NZ provided the clematis cultivars for the experiments and assigned them to groups. NI and OM were responsible for the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The improvement of the mechanical properties of Ni-based superalloys is achieved in most cases by modifying the chemical composition. Besides that, the processing can be modified to optimize the as-cast microstructure with regard to the mechanical properties. In this context, the present study highlights the solidification mechanism of several Ni-based superalloys by conducting experiments using a modified, laboratory-scale Bridgman-Stockbarger furnace. In that context, the single-crystal rods are partially melted, directionally solidified and quenched sequentially. Several characterization methods are applied to further analyze the influence of the alloying elements and the variation of the withdrawal rate on the as-cast microstructure. Four stages of solidification are distinguished whereby the morphology observed in the different stages mainly depends on the cooling rate and the local concentration of the carbide forming elements. The effect of carbide precipitation and the effect on the as-cast microstructure is investigated by employing energy dispersive X-ray spectrometry (EDX) and electron backscatter diffraction (EBSD) analysis techniques. A local polycrystalline structure is observed in the single-crystal system as consequence of the influence of the carbon content and the cooling rate. The present work aims to develop strategies to suppress the formation of the polycrystalline structure to maintain the single-crystal microstructure. Ni-based superalloys, based on the superior thermo-mechanical material properties, are widely used in technical high temperature applications like aviation, chemical industry or energy generation industry. Since the requirement of materials with exceptional mechanical properties increases, similarly the development of materials which can withstand these harsh conditions is advanced. The earliest development began with changing the structure of Ni-based superalloys from polycrystalline to columnar and then single crystal. The intention is to completely eliminate grain boundaries so that failure by grain boundary sliding as well as excessive diffusion and the resulting segregation are avoided. In addition, chemical composition alteration by adding, removing or replacing several alloying elements such as carbon and refractory metals are carried out as well ,2,3,4. TBy optimizing the solidification process, the performance of the Ni-based superalloys can be further improved. The solidification process is complex due to the multicomponent system with up to 14 alloying elements . Therefoγ/γ’ eutectic. Other prior investigations involved three-dimensional (3-D) reconstructions from a series of two-dimensional (2-D) planar transverse sections to define the phase formation sequence in Ni-based superalloys [Several studies investigated the solidification process under equilibrium conditions for various Ni-based superalloys using the Calphad method ,17. Thiseralloys ,16. Besieralloys ,18. Howeeralloys .Nowadays, numerical approaches such as the phase field modelling are widely accepted based on various mathematical models to simulate and analyze solidification behavior under settled critical conditions . The simγ matrix as well as the γ/γ’ eutectic is one of the main objectives of the present study. Considering the abovementioned evidences, the quenching method is implemented in the present study. Further, an additional thermocouple wire is mounted in each test substance so that the identification of the phase formation temperatures becomes more precise. Based on various deficiencies of the mostly applied analyzing techniques, the formation sequence of multiple phases during solidification of Ni-based superalloys has not yet been sufficiently explored. In the present paper, much attention is drawn to precisely reveal the real-time microstructure evolution of several Ni-based superalloys, more specifically for CMSX-6, CMSX-6+0.03C and MAR-M247. Since the processing has a strong influence on the solidification process, the effect of withdrawing process on the final microstructure is closer investigated by applying systematically varied withdrawal velocities. Furthermore, the focus of the present work is on the evaluation of the effect of carbon addition on the microstructure and the resulting material properties of the superalloys. As reported in literature for the MAR247 alloy, the formation of carbides is beneficial in view of the high-temperature material properties since grain boundary sliding is impeded by pinning effects ,21. It cThe materials investigated in this study were CMSX-6, CMSX-6+0.03C and MAR-M247, whose compositions are listed in V, i.e., 1 and 4 G of 8 V and G, the corresponding cooling rates of 0.13 Before the procedure began, the furnace was flushed with high-purity argon gas for 80 min to prevent oxidation of the graphite heater and holder, as well as to protect the liquid metal from contamination. During the experiment, the charge was placed on a sample holder and the furnace, which had been preheated to 1773 K (1500 °C), was lowered such that the part of the charge to be melted was encircled by the graphite heater. The furnace was held in this position for 10 min to ascertain uniform melting and homogenize the melt. The furnace was shifted up 50 mm with two alternatives of velocity, hereinafter referred to as withdrawal rate Based on preliminary analysis, which unveiled the exact portion of mushy zone and single crystal regions, the rods were sectioned transversally (perpendicular to the growth direction) and longitudinally . Four samples were generated from each rod, indicating transversal cuts of mushy zone and single crystal regions (TC-MZ and TC-SC) as well as longitudinal cuts of these regions (LC-MZ and LC-SC). The samples were subsequently hot-embedded and metallographically prepared for further analyses. Additionally, a vibratory polisher delivering a scratch-free surface was utilized right before metallographic examinations. If necessary, waterless Kalling’s reagent containing 80 mL of ethanol, 40 mL of HCl and 2 g of Copper (II)Chloride was used as the etching solution to accentuate the structure and intended phases. During observation of each LC-MZ sample under Zeiss Axio optical microscope  at 200× magnification, the earliest visible forms of phases were marked to indicate the phase transformation points and later calculate the formation temperatures.γ/γ’ eutectic island and the precipitated γ’ phase was captured using Zeiss Ultra-55 scanning electron microscope  in Aachen/Germany. ImageJ was also utilized to support the measurement of volume fraction of γ’ precipitates. Chemical characterization of the formed carbides was determined by energy dispersive X-ray spectrometry (EDX) analysis technique in either Zeiss Ultra-55 SEM or Zeiss 1540 XB Crossbeam SEM (Carl Zeiss Microscopy GmbH) in Aachen/Germany. The crystallographic orientation relationship between γ/γ’ eutectic and other structures, i.e., γ matrix and carbides, in LC-SC samples of CMSX-6+0.03C were examined by electron backscatter diffraction (EBSD) technique using Zeiss 1540 XB Crossbeam SEM.Some images of unetched samples were taken, in which the amount of porosity and carbide fraction were analyzed quantitatively employing an image processing software ImageJ . The microstructure development of the γ matrix, carbides, γ/γ’ eutectic and γ’ precipitates. The solidification sequence is elaborated as follows:All six rods possess similar-looking mushy zones with a typical dendrite array, i.e., dendrites growing concurrently in one specific direction. γ matrix: Crystallization of Solidification of fine equiaxed carbides in the interdendritic area: γ/γ’ eutectic: Formation of γ’ phase is indicated by dark-brown patches formed around the dendrite lobes as shown in Solid-state phase transformation: γ matrix compared to prior studies might be caused primarily by the different analysis methods used such as DSC and Thermo-Calc calculation [The lower crystallization temperature of culation ,15,18,26n et al. . On thatn et al. ,27,28. Tγ dendrites continuously grew such that the interdendritic region was consumed by the growing dendritic region, at some point the dendrites would come into contact with the already nucleated carbides in the interdendritic region. Keeping on growing transversely, the dendrites encompassed most of the carbides and the remaining carbides were only partially enveloped [γ/γ’ eutectic was small compared to that of the formed eutectic islands. Thus, the eutectic formation could not have involved eutectic reaction, since an adequate amount of liquid melt as well as space for the corresponding size of eutectic islands to nucleate and grow were not provided by the interdendritic region. Prior studies confirm that the eutectic formation includes a combination of eutectic and peritectic reactions, through which γ’ nucleates from primary γ phase at γ-liquid interface as their lattice misfit is small and it later on grows into both remaining liquid and γ dendrite [Since there were not any carbides in the dendrite core, the carbides observed around the dendrite arms nucleate in the interdendritic region . As γ denveloped . Observadendrite ,26,30.Since voids contained within the samples were irregular in both shape and size, their formation was obviously a consequence of solidification shrinkage. In γ and γ’, the microstructural expansion was proposed to decrease the amount of resulting shrinkage porosity. However, it was rather unjustifiable if pores were entirely enveloped by carbides, which would indicate that carbides were nucleated on the pores or they were trapped in the carbides. Carbides are commonly precipitated at temperatures close to the liquidus, while pores are formed at end of solidification where filling by the remaining liquid is not possible. The alongside existence of carbides and pores could be caused by carbides blocking the fluid flow, on the other side of which shrinkage occurs and therefore pores are built up. Partial or complete envelopment of carbides could also indicate that the cores consist of foreign substances such as oxides instead of pores [Contradictory findings revealed that an addition of carbide decreased porosity, which was proven by pores enclosed by carbides or suggested that carbides precipitate in the solid instead of the liquid ,31. Sincof pores ,32. Carbide fraction measurement was carried out on the single crystal part of all samples, with results presented in fication . At highAs explained by Li et al., higher carbon content leads to a greater supersaturation in the later stage of solidification, which induces the growth of well-developed secondary and tertiary dendrite arms . Even thAccording to EDX analysis results, carbides found in this study fall primarily under the MC-type with M dominated by Ti and Ta. Small alumina particles were observed to exist at the cores of a small portion of the faceted carbides, which were reported as well by Fernandez et al. and Zhao et al. ,32. Thesγ dendrite growth, this variation in chemical composition implicates that carbides are formed through several stages [Another portion of carbides without inclusion of alumina particles is also built up by two distinct colors having two different chemical compositions, i.e., an orangish core and a bluish gray overlay, while the rest of the carbides are completely bluish gray in color. On the other hand, the alloy with extremely low amount of carbon contains carbides in bright orange, which indicates that the carbides are rich in Ti whose content may reach approximately 81 wt.%. The bluish gray color represents carbides with high amount of Ta (~39 wt.%) and Mo (~12 wt.%) but not as much Ti (~27 wt.%). In MAR-M247 samples, carbides with another distinct composition, i.e., rich in Ta (~40 wt.%), W (~14 wt.%) and Hf (~10 wt.%) but less in Ti (~16 wt.%) and Mo (nearly zero), were found. The content of carbon in each carbide varies as well, from ~9–11 wt.% in bright orange and orangish carbides to ~18 wt.% in bluish gray carbides. Since carbide nucleation and growth are controlled by the cooling rate and carbide FEs enrichment during l stages ,34.The bright orange carbide is the first carbide to form at low carbide FEs, particularly at limited amounts of carbon as in CMSX-6, and high cooling rate, at which solute diffusion decreases. Accordingly, the carbides formed are exceptionally rich in Ti as the main constituent besides carbon. At lower cooling rate (CMSX-6 (W1)) and/or higher carbon level (CMSX-6+0.03C (W1 and W4)), either more time for solute diffusion or more carbide Fes or even both are available. At the beginning of solidification when elemental segregation is not yet significant, orangish carbides rich in Ti and some Ta are formed. As solidification proceeds, elemental segregation and thus carbide Fes enrichment in the interdendritic region become more pronounced. Correspondingly, the formed orangish carbides grow in the melt with greater amount of carbide Fes such that a different carbide overlay is produced. At the lower part of the mushy zone where more carbides preferentially nucleate, an adequate amount of carbide FEs has substantially segregated into the interdendritic area so that carbides grown therein are entirely bluish gray in color with richer Ta, Mo and C contents. 6C-and/or M23C6-carbides during prolonged heat treatment or exposure [6C-or M23C6-type carbides found in prior investigations [In MAR-M247 samples, another different appearance of carbides was observed that is mainly caused by the chemical composition of the alloy itself. The MAR-M247 alloy contains 10 wt.% of W and a greater amount of Hf and Ta but is less in Ti and Mo. Consequently, the carbides formed are purplish gray in color, loaded with Ta, W and Hf. Some faceted carbides with growing arms in MAR-M247 (W4) sample were found to have a completely different composition, i.e., depleted in Ti and Ta but rich in W (~18–20 wt.%) and Cr ~21–22 wt.%). Previous studies reported that the MC-carbides in MAR-M247 may decompose into M–22 wt.%.exposure . Howeverigations ,39. It iSince carbides are repeatedly reported to grow directly from the melt, they become one of the structure-building phases emphasized in the crystallographic analysis. Accordingly, inverse pole figures (IPFs) of TaC and TiC as the dominant carbide constituent are constructed along with that of Ni as the matrix as depicted in γ/γ’ eutectic formation in the LC-MZ sample of CMSX-6 (W4), which were also observed in other alloys. The γ/γ’ growth direction begins with the fine γ/γ’ structure on the γ dendrite lobe as shown in γ/γ’ structure , attached to the surface of γ dendrite arms while also being partially enclosed by the coarse structure (Eγ) and attached to a carbide surface (EMC). The latter type is highly dependent on the carbon level or carbide fraction in the alloy. Increasing carbon content and hence intensifying carbide fraction lead to an increase in the quantity of these eutectic islands.Fine L and EMC types to determine the possibility of homogeneous nucleation of γ/γ’ eutectic and the extent of misorientation caused by carbides, respectively. Moreover, the Eγ type is believed to nucleate on the surface of the γ dendrite since its fine core formed in the early stages is generally attached to the dendrite and, hence, their crystallographic orientation is similar. The crystallographic analysis results of LC-SC samples of CMSX-6+0.03C directionally solidified at different rates are shown in γ/γ’ eutectic, which further signifies that the EL and Eγ types are in fact the same. As suggested by Heckl et al., since neither misorientations nor grain boundaries were present, nucleation and growth of the eutectic occur epitaxially on the primary solidified γ dendrite [γ dendrite. Various 2-D structures might be produced by the same parent γ/γ’ structure depending on the sectioning orientation and distance from the fine eutectic core [EBSD analysis performed was focused on the Edendrite . Accorditic core .MC-type eutectic discloses the expanding influence of misorientation of the carbides as seen in γ/γ’ eutectic nucleates on the surface of MC-carbide as well. As explained by Wang et al., even though there is a greater tendency for γ/γ’ eutectic to nucleate on γ dendrites than MC-carbides due to their complete coherent crystal relationship, nucleation of γ/γ’ eutectic on MC-carbides can also be promoted by elemental segregation [γ/γ’ eutectic to nucleate since Ni, Co and Cr are the γ FEs, whereas Al and Ni are γ’ FEs. The FEs enrichment ahead of MC-carbides merely exceeds the saturation level, which results in a substantial undercooling that eases the γ/γ’ eutectic nucleation. Upon growing, they exchange their respective FEs such that a co-growing mode occurs.On the other hand, the EBSD analysis result on the Eregation . CarbideL-type eutectic islands were found to have a different orientation from the γ matrix [γ/γ’ eutectic nucleation takes place in areas bounded by intermediary surfaces of γ dendrites and MC-carbides. A more reasonable explanation would be that the eutectic islands nucleated on the surfaces of MC-carbides, instead of growing from the melt, but the corresponding carbides were not visible. This could be caused again by the dependence of 2-D structure on the sectioning position and orientation of the whole 3-D structure. Therefore, a further and detailed 3-D analysis on the γ/γ’ eutectic islands is necessary to gain a more assertive assessment of their nucleation mode.A prior investigation reported that some Eγ matrix . These eγ matrix . Aside fγ’ phase precipitated in ideal-cubic morphology with a completely identical size whose particle distribution was uniform such that the channel width was the same everywhere, γ’ volume fraction γ’ area fraction was obtained with the assistance of ImageJ. The calculation results are plotted in γ’ FEs. The latter includes Al, Ti and Ta, which are beneficial for inducing γ’ precipitation, as well as Cr, Mo and W, which are functional for enhancing the γ’ volume fraction [γ’ precipitates could be observed. In that regard the γ’ particles at higher cooling rate are finer and denser, which is supported by calculation results signifying greater values of Assuming that the fraction . The γ’ fraction . A stronus study . The maxγ’ precipitates, as the maximum solid-solid transition rate is seen at larger undercoolings due to deceleration of diffusion with decreasing temperature [γ’ FEs and carbon content. The γ’ FEs affect the overall tendency of γ’ FEs is, however, slightly toned down by the presence of carbon. Carbon bonds Ti, Ta, Mo and W to form carbides in the interdendritic region just below the outset of solidification. The content of these elements in the interdendritic area is depleted to a certain extent throughout the development of γ dendrites alongside the formation of carbides before the γ’ phase is precipitated. Concurrently, γ dendrites grow in the expense of depleted melt in the interdendritic region, which causes the dendrites to contain lesser γ’ FEs. This condition ultimately leads to a decrease in Higher cooling rate results in greater undercooling and therefore a finer and denser structure of perature . Howeverγ matrix and γ/γ’ eutectic.The microstructure evolution and solidification sequence for several Ni-based superalloys has been investigated by directional solidification experiments combined with a quenching technique. Aside from that, the influence of carbon and alloying elements and the effect of the withdrawal respective to the cooling rate has been studied. Another focus was directed to the evaluation of the relationship between carbide precipitation and the formation of other phases like the The following conclusions can be drawn:γ, which can be described by γ/γ’ eutectic proceeds via both eutectic γ’, which forms by means of solid-state phase transformation reaction from The solidification sequence of CMSX-6, CMSX-6+0.03C and MAR-M247 begins with the crystallization of An increase in carbon content causes and intensifying porosity for both applied cooling rates. Nevertheless, the porosity amount at the higher cooling rate is rather toned down to a smaller fraction of carbides formed. The presence of carbides in the interdendritic area notably reduces its permeability which hinders the melt flow.EDX analysis indicates that the carbides found in this study mainly correspond to the MC-type, whereby M is dominated by Ti and Ta. At higher carbon concentrations and/or lower cooling rates, the first carbides to precipitate are orangish in color and rich in Ti, which are later on enveloped by a bluish gray overlay loaded with Ta and Mo. In MAR-M247 samples, the carbides observed are rich in Ta, W and Hf, having a purplish gray color. The carbide morphology changes from faceted to Chinese script-type for increasing carbon concentration respective to cooling rate. The size and fraction of the carbides decrease with increasing cooling rate, but the carbide fraction is primarily governed by the carbon content of the alloy. A greater tendency for carbide misorientation and, hence, the presence of local polycrystalline structures can be observed at higher cooling rates. Accordingly, the single crystalline character of the alloy is retained better, applying lower cooling rates.γ/γ’ eutectic begins with the fine γ/γ’ lamellar core as it was found prior to the coarse γ/γ’ structure. The fine eutectic core is crystallized by means of a eutectic reaction, followed by a progressive transition to growth of the coarse structure. The growth continues until it comes into contact with other eutectic islands or a neighboring primary γ dendrite. Crystallographic analysis on CMSX-6+0.03C samples indicates that there is no evidence of homogeneous nucleation of γ/γ’ eutectic. The eutectic islands nucleate on the surface of γ dendrites, and thus the eutectic adopts the crystal structure of the dendrite. A small eutectic island observed to have the same orientation as a carbide but a different orientation from the dendrite confirms that γ/γ’ eutectic does not only nucleate on the dendrite lobe but also on the carbide surface.The growth direction of γ’ precipitates. The γ’ volume fraction is influenced by γ’ FEs, but the effect is slightly depleted by the presence of carbon. Carbon bonds γ’ FEs to form carbides in the interdendritic area so that the γ’ FEs are consumed while γ dendrites are growing. The solidified γ dendrite just before γ’ precipitation becomes more depleted in γ’ FEs as carbon level in the alloy increases and thus the γ’ volume fraction decreases.Increasing cooling rate leads to finer and denser


Evidence for an autoimmune etiology in canine diabetes is inconsistent and could vary based on breed. Previous studies demonstrated that small percentages of diabetic dogs possess autoantibodies to antigens known to be important in human type 1 diabetes, but most efforts involved analysis of a wide variety of breeds. The objective of this study was to evaluate the presence of glutamic acid decarboxylase 65 (GAD65), insulinoma-associated protein 2 (IA-2), and zinc transporter 8 (ZnT8) autoantibodies in diabetic and non-diabetic Australian Terriers and Samoyeds, two breeds with comparatively high prevalence of diabetes, in the United States.p > 0.05). The proportion of IA-2 autoantibody positive samples was significantly higher in diabetic versus non-diabetic Samoyeds (p = 0.003), but substantial overlap was present between diabetic and non-diabetic groups.There was no significant difference in the proportion of samples considered positive for GAD65 or ZnT8 autoantibodies in either breed evaluated, or for IA-2 autoantibodies in Australian Terriers  assays. Future studies using canine specific assays as well as investigation for alternative markers of autoimmunity in these and other canine breeds are warranted. Diabetes mellitus represents one of the most common endocrine diseases in dogs, with certain breeds having a markedly higher risk for developing the disorder. Most dogs with diabetes have a deficiency in the production of insulin, a hormone produced by the pancreas that reduces blood sugar, due to a loss of the insulin producing cells in the pancreas (β-cells). The reason for this loss of β-cells is unknown, but previous studies have suggested that the body’s immune system may attack the pancreas in a process called autoimmune destruction. It is well known that autoimmune destruction of the β-cells leads to insulin deficiency in humans with Type 1 diabetes and special blood markers of this autoimmune response, termed autoantibodies, can be detected. These autoantibodies have only been infrequently detected in dogs with diabetes at this time. Yet, most studies have not evaluated the presence of these autoantibodies from large numbers of dogs of breeds with high risk for diabetes, and it is possible that some breeds may have an autoimmune cause for diabetes while others do not. In this study, we measured three different autoantibodies common to human type 1 diabetes (glutamic acid decarboxylase 65 (GAD65), insulinoma-associated protein 2 (IA-2), and zinc transporter 8 (ZnT8)) in a large group of diabetic and non-diabetic Samoyeds and Australian Terriers, two breeds at high risk for diabetes in the United States. An assay commonly performed in human diabetic patients was used in this study. We did not observe that diabetic dogs of either breed were more likely to be positive for any of the autoantibodies than non-diabetic dogs, but the use of the human assay does have some limitations. Our results do not provide support for these specific autoantibodies as markers of autoimmune diabetes using the humans assays in these two breeds. However, studying alternative blood markers of autoimmunity, or developing canine specific assays, may lead to additional information as to the role such mechanisms may have in the development of diabetes in dog.Diabetes mellitus is a common endocrine disease in dogs, with the most frequent presentation being that of insulin deficiency with a life-long requirement for exogenous insulin . ContribCanine diabetes is considered to be similar to human type 1 diabetes (T1D) . AutoantThe role of genetics in human T1D is well established, with human leukocyte antigen (HLA) haplotypes as the major genetic risk factor, along with over 60 non-HLA genes having lesser contributions . With reHence, it has become increasingly apparent that canine diabetes is a heterogenous disease with multiple pathogenic factors , 6. We hp = 0.003). There was no significant difference in age between diabetic (11.4 ± 2.6 years) and non-diabetic (10.6 ± 2.2 years) Samoyeds (p = 0.18). There was no significant difference in sex distribution between diabetic and control groups within Australian Terrier (p = 0.10) or Samoyed (p = 0.22) breed groups (Table p = 0.68). Only eight Australian Terriers and six Samoyeds had a duration of disease less than or equal to one year.One hundred and thirty four  Australian Terrier and 73 Samoyed  serum samples were available for analysis. Age at sample collection was available for 61/63 diabetic and 58/71 non-diabetic Australian Terriers, and for 30/31 diabetic and 32/42 non-diabetic Samoyeds. The diabetic Australian Terriers were significantly older (mean ± SD = 10.7 ± 3 years) than the non-diabetic Australian Terriers (9.45 ± 2.2 years) (p = 0.34).IgG was detected in all samples, with a median of 2467 mg/dL . Median total IgG in the diabetic group for both breeds combined  was not different than the non-diabetic group for both breeds combined  (p > 0.99) , while in Samoyeds 19/31 (61%) of diabetic and 12/42 (29%) of non-diabetic samples were above the level of quantification (p = 0.003)   . For Australian Terriers, 4/63 (6%) of diabetic and 4/71 (6%) of non-diabetic samples were above the level of quantification (and therefore considered to be “positive”)  according to the manufacturer’s instructions to confirm that IgG was present. All samples were measured in duplicate. Autoantibodies for GAD65, IA-2, and ZnT8 were measured using commercial antibody kits from Kronus Inc.  that have been validated for use in human T1D autoantibody screening . This asData were tested for normality using the D’Agostino & Pearson test, and parametric or non-parametric tests were used as indicated. Age was compared for diabetic and non-diabetic groups within each breed using an unpaired t-test with Welch’s correction. Duration of diabetes was compared between breed groups with a Mann-Whitney U test. Sex distribution within each breed group was compared using a Chi squared test. IgG concentrations were compared between diabetic and control groups using a Mann Whitney U test. Given that the autoantibody concentration cutoffs for definitive positivity are unknown for dogs, the proportion of autoantibody ELISA results above the lower limit of quantification (“positive” samples) were compared between diabetic and non-diabetic groups for each autoantibody within each breed using a Chi squared or Fisher’s exact test. Statistical analysis was performed using GraphPad Prism software v8.0 .