CAT residue at the enzyme substrate sensing loop, which opens a new paradigm in this widely studied family of enzymes.TsNirK is the first three-copper three-domain nitrite reductase harbouring Gln-coordinated type 1 copper centres. All the structural properties of TsNirK point to an enzyme that, despite having several of the essential catalytic features present in other NirKs, shows two distinctive and unique characteristics: firstly, the putative T1CuC → T1CuN → T2Cu electron-transfer pathway along the same subunit; and secondly, more importantly, is the presence of the Ser 2O and N2). The reaction is catalysed either by a Cu-containing nitrite reductase (NirK) or by a cytochrome cd1 nitrite reductase (NirS), as the simultaneous presence of the two enzymes has never been detected in the same microorganism. The thermophilic bacterium Thermus scotoductus SA-01 is an exception to this rule, harbouring both genes within a denitrification cluster, which encodes for an atypical NirK. The crystal structure of TsNirK has been determined at 1.63 Å resolution. TsNirK is a homotrimer with subunits of 451 residues that contain three copper atoms each. The N-terminal region possesses a type 2 Cu (T2Cu) and a type 1 Cu (T1CuN) while the C-terminus contains an extra type 1 Cu (T1CuC) bound within a cupredoxin motif. T1CuN shows an unusual Cu atom coordination (His2–Cys–Gln) compared with T1Cu observed in NirKs reported so far (His2–Cys–Met). T1CuC is buried at ∼5 Å from the molecular surface and located ∼14.1 Å away from T1CuN; T1CuN and T2Cu are ∼12.6 Å apart. All these distances are compatible with an electron-transfer process T1CuC → T1CuN → T2Cu. T1CuN and T2Cu are connected by a typical Cys–His bridge and an unexpected sensing loop which harbours a SerCAT residue close to T2Cu, suggesting an alternative nitrite-reduction mechanism in these enzymes. Biophysicochemical and functional features of TsNirK are discussed on the basis of X-ray crystallography, electron paramagnetic resonance, resonance Raman and kinetic experiments.Dissimilatory nitrite reductases are key enzymes in the denitrification pathway, reducing nitrite and leading to the production of gaseous products (NO, N Bacteria convert inorganic nitrogen into organic nitrogen sources by assimilatory pathways during the interconversion of NH3, NO3− and NO2−. Dissimilatory denitrification produces dinitrogen by the reduction of NO3− and NO2−, with NO and N2O as intermediaries involving several enzymes in the process.The global nitrogen cycle maintained by some bacteria impacts all forms of life worldwide , catalysed by nitrite reductase (Nir), is the key reaction that initiates the dissimilatory denitrification process in denitrifiers  is composed of two domains  shows all the structural features of the two-domain NirK enzymes with an additional N-terminally fused cupredoxin domain containing a T1Cu centre , which contains a C-terminal cytochrome c domain fused to the two-domain NirK core, is an effective self-electron-transfer system where the donor and acceptor proteins are naturally fused ; this enzyme is a naturally fused type of Nir tethering a cytochrome c at the C-terminus fold as a unique trimeric domain-swapped structure  at a resolution of 1.63 Å together with its biochemical and spectroscopic characterization. This enzyme is a three-domain NirK that shows the T1Cu centre of the two-domain core with a coordination never observed before, a third cupredoxin motif in close proximity to the T1Cu site of the two-domain core and a sensing loop that does not contain the essential AspCAT. We discuss the structural properties of TsNirK in comparison with the best characterized NirKs and the implications on the catalytic mechanism of this novel enzyme.Here we describe the crystal structure of the NirK from 2.2.1.BLAST . The evolutionary history was inferred by using the maximum-likelihood method based on the Whelan and Goldman model ]. The rate variation model allowed for some sites to be evolutionarily invariable . The analysis involved 37 amino-acid sequences. All positions with less than 95% site coverage were eliminated. Fewer than 5% alignment gaps, missing data and ambiguous bases were allowed at any position. There were a total of 256 positions in the final data set. Evolutionary analyses were conducted in MEGA7  plasmid containing the Tsc_c17620 gene  (New England Biolabs Inc.). The recombinant strain was grown aerobically at 37°C overnight with agitation at 200 rev min−1 in lysogeny broth with the addition of 100 µg ml−1 ampicillin as starter culture. Expression was performed using 400 ml (1/100 starter culture) of auto-induction media (ZYP5052) . CuSO4 (200 µM) was added to the high-density culture and maintained in the same condition for 1 h. Finally, the copper-fed culture was induced with 250 µM isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 20°C and 50 rev min−1 for 3 h. Cells were harvested through centrifugation and re-suspended in 20 mM Tris–HCl (pH 8) buffer. Expression levels were evaluated using SDS–PAGE analysis  was disrupted by sonication. The crude extract was recovered by centrifugation at 25 000g for 1 h and dialyzed overnight against 20 mM Tris–HCl buffer (pH 8) supplemented with 100 µM CuSO4 and again centrifuged at 25 000g for 1 h. TsNirK from the crude extract was purified in three chromatographic steps. The crude extract was applied to an anion-exchange column  equilibrated in 20 mM Tris–HCl buffer, (pH 8) and eluted with 600 ml of a 0–500 mM linear gradient of NaCl in equilibration buffer. Deep-blue fractions containing TsNirK were pooled and dialyzed against 20 mM Tris–HCl buffer plus 100 µM CuSO4. The dialyzed pool was loaded onto a Source 15Q matrix column  equilibrated with 20 mM Tris–HCl (pH 8). Bound proteins were eluted with a linear gradient in equilibration buffer . Finally, fractions with TsNirK were concentrated by an Amicon Ultra 30 K nominal molecular weight limit device and loaded onto a Superdex S200 column . Fractions (500 µl) were loaded and eluted with 20 mM Tris–HCl buffer (pH 8) containing 200 mM NaCl. The highly pure turquoise TsNirK fractions were pooled and concentrated to approximately 20 mg ml−1 in 20 mM Tris–HCl (pH 8) and stored at −80°C. Protein purity was evaluated by SDS–PAGE and followed by UV–Vis spectroscopy through the purification procedure.A cell suspension , aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa) and carbonic anhydrase (29 kDa), all from GE Healthcare. The molecular mass of the subunits was estimated by SDS–PAGE according to the method of Laemmli et al.  were added of 250 µl biquinoline solution  and 225 µl of 20 mM ascorbic acid in phosphate buffer pH 6.0 in sequential order. The reaction mixture was maintained at room temperature for 10 min and the absorbance at 546 nm was measured. Standard calibration curve was obtained performing the procedure on 275 µl standard solutions (0–50 µM CuSO4). The copper content in the samples was determined in triplicate.The copper content was determined by performing the biquinoline colorimetric method  , 50 µMSmPaz, and 15 nMTsNirK in a total volume of 1 ml was maintained in a septa-sealed cuvette under argon flux. To exclude dioxygen, all the solutions were flushed with argon for 30 min. The mixture SmPaz–TsNirK was reduced with sodium dithionite (2.5 µl of 200 mM dithionite solution). The reaction was started by the injection of sodium nitrite (10 µl of 100 mM solution). The reoxidation of the electron donor was followed at 597 nm. The reoxidation of SmPaz by the action of NirK from S. meliloti 2011 (SmNir)  measurements were performed at the X-band on a Bruker EMXplus spectrometer at 120 K. EPR spectra were simulated with the EasySpin toolbox based on MATLAB  by an Amicon concentrator. Then 5 µl of 1 M degassed stock solutions of sodium dithionite and sodium nitrite were withdrawn by gastight syringe from the vessels containing the respective solutions and loaded into argon-flushed EPR tubes containing samples of TsNirK (∼200 µl) followed by gentle mixing. The EPR tubes were frozen with liquid nitrogen and kept under these conditions until use. The experimental conditions used were: microwave frequency, 9.45 GHz; microwave power, 2 mW.UV–Vis electronic absorption spectra were recorded on a Perkin–Elmer Lambda 20 UV–Vis spectrophotometer at 298 K. Resonance Raman spectra were acquired at 77 K in a Dilor XY-800 microspectrometer equipped with a Linkam THMS600 freezing microscope stage. Frozen samples were irradiated with 5 mW of a 631.9 nm diode laser (TopMode-633) and the scattered light was collected in backscattering geometry during 2 min at a resolution of 0.4 cm2.7.TsNirK crystals in several conditions within 2 weeks at 16°C. Single crystals grew in 2 µl drops consisting of equal volumes of 8 mg ml−1TsNirK and reservoir solution . Crystals were soaked in reservoir solution containing 30%(v/v) glycerol prior to cryocooling. X-ray diffraction data were collected at Diamond Synchrotron (UK) on beamline I04-1 (0.9282 Å) at 93 K. Data were processed using autoPROC  as the search model. Refinement was carried out through iterative cycles of manual model building in COOT  calculations was used to compute the structure and the Raman spectra of the T1Cu3.3.1.BLAST and protein-sequence-alignment analysis show an N-terminal region typical of two-domain NirKs, and also a C-terminal extension [Fig. 1a)]. This extra domain belongs to the cupredoxin superfamily and shares ∼30% identity with several monomeric cupredoxins (see Table S1 in the Supporting information) involved in electron-transfer processes ]. The bootstrap consensus tree  groups TsNirK within a cluster with uncharacterized putative NirKs, all with extended C-terminal sequences. These sequences not only come from closely related microorganisms of the Thermus genus that share ca 80% sequence identity, e.g. T. brockianus and T. oshimai JL-2, but also from unrelated microorganisms that share only ca 50% sequence identity, e.g. Fraserbacteria sp.  and the methane oxidizer Crenothrix polyspora . The latter allow us to infer that TsNirK belongs to a new subgroup within class III NirKs , as determined by size exclusion chromatography and SDS–PAGE . The copper content obtained was 3.2 ± 0.4 mol Cu per mol monomer. To investigate the overall structure and the interaction of the C-terminal domain fused to the two-domain core [Fig. 1a)], as well as the unique features observed within the sequence alignment [Fig. 1b)], TsNirK was crystallized and its structure solved. Various precipitants from our initial screening yielded characteristic blue single crystals with good diffraction (<1.5 Å) within less than a week. Unfortunately, most of these crystals displayed substantial merohedral twinning, resulting in the trigonal space group H3 incorrectly being indexed as H32. All of these crystals contained a single protomer in the asymmetric unit cell (ASU), with the homotrimeric structure obtained through the threefold crystallographic symmetry axis (data not shown). However, a single crystal was obtained that indexed to C121, containing the entire homotrimer in the ASU. The structure was determined at 1.63 Å through molecular replacement using GtNir (PDB entry 3x1e), which shares 36% identity (57% homology) to the N-terminal region of TsNirK, with the extended C-terminus built in the resulting observed density. Data collection and refinement statistics are shown in Table 1TsNirK’s biological unit are nearly identical, apart from small surface side-chain rotamers, and are related via a threefold non-crystallographic symmetry axis . Main-chain differences and weak density (high B factors) were also observed within the linker region between domain II and III, suggesting a high degree of flexibility.Expression and purification to electrophoretic purity of the product of the optimized TsNirK is composed of three monomers . Three distinct domains can be distinguished within each monomer : domain I , domain II (Leu154–Ala282) and domain III . Domains I and III are located at the periphery of the trimer, while domain II, which is positioned in the core of the homotrimer structure, constitutes the inter-subunits interaction domain . A linker loop connects domain I with domain II (Pro138–Asp153), whereas a second longer linker region (Lys283–Lys308) extends at the side of domain I connecting domain II and III, with domain III being on the top of the two-domain core structure . Domain III is closely attached to domain I by surface interactions. The characteristic extra loop (Asn181–Pro191) and the tower loop ] and a calcium atom from the crystallization solution, coordinated by residues of domain I, is found between the monomers. Domain I harbours the characteristic T1Cu and T2Cu centres of two-domain NirKs, whereas domain III has an extra T1Cu centre.The biological unit of 3.3.c). Domain I contains the T1Cu centre at the N-terminal region (T1CuN) and the active T2Cu site [Fig. 2c)]. Domain III contains a second T1Cu centre . Relevant distances and angles of T1Cu centres and their comparison with those observed in others NirKs and blue cupredoxins are shown in Table S2.Coordination around T1Cu and T2Cu atoms is shown in Fig. 2C is an amicyanin-like T1Cu centre , Cys115 Sδ, and a Gln130 O∊1 residue in apical position. This copper site, which shows nearly tetrahedral coordination similar to that observed in stellacyanin . His80 (2.02 ± 0.01 Å), and His114 (2.04 ± 0.03 Å) are provided by domain I, whereas His267 (2.06 ± 0.01 Å) comes from domain II of an adjacent subunit, as usually observed in NirKs.T1CuC centre is buried at ∼5 Å from the molecular surface of TsNirK and is located ∼14.1 Å away from T1CuN; T1CuN  and T1CuC  are ∼12.6 Å and ∼22.3 Å away from T2Cu, respectively [Fig. 2d)]. T1CuC and T1CuN are linked by a chemical path that involves Glu385 and a water molecule [Fig. 2d)]. T1CuN and T2Cu are connected by a typical Cys–His bridge (Cys115–His114) [Figs. 1b) and 2d)].The T1Cu3.4.TsNirK reveals that the loop with an α-helix (His120 to Thr126) located at domain I serves as a scaffold for several inter-domain interactions . The molecular surfaces of both domains involved in the interaction are complementary. Several hydrogen-bond interactions are clearly observed , with a number of water molecules in the contact region also reinforcing the hydrogen-bond network, contributing to the stabilization of inter-domain interactions. At least three interactions were observed in the surrounding area. These interactions take place in the contact region of a β-strand  at the N-terminus of domain III with a β-strand of the domain I : Arg309(O)–Tyr40(N), Val311(O)–Arg42(N) and Val311(N)–Tyr40(O).The unique architecture of 3.5.et al., 1999X4-His substrate sensing loop . The sequence of TsNirK reveals a unique amino-acid composition at the sensing loop that has not been observed before [Fig. 1b)]. The His75-Gly76-Leu77-Ser78CAT-Ile79-His80 substrate sensing loop configures a novel active-site pocket in which the AspCAT(COOH) is replaced by SerCAT(CH2—OH), with SerCAT being in close proximity to the T2Cu centre bound water [Fig. 2d)]. The active-site pocket of TsNirK also harbours the residues Val218 , His216 (HisCAT), Gln239 and Thr240, which have been proposed to be relevant in catalysis in two-domain NirKs. Furthermore, several water molecules connect SerCAT with HisCAT via Gln239 and Thr240 in a hydrogen-bond network [Fig. 2d)].As reported for all NirKs –Ala116–(2 × wat)–Gly91–Asn92].The T2Cu centre can be accessed through an ∼16 Å deep channel  that covers an area of 570 Å3.7.TsNirK was able to reduce nitrite with an apparent turnover of 65 ± 1 s−1, an apparent KM value of 27 ± 2 µM NO2−, and a catalytic efficiency of 2.4 × 106 M −1 s−1 . Furthermore, the enzyme reoxidized a pseudoazurin from Sinorhizobium meliloti 2011 (SmPaz) in the presence of nitrite in an argon-flushed septa-sealed cuvette showing the capacity for interaction with an external cupredoxin-like electron donor .Cys → Cu LMCT band], ∼597 nm [S(π)Cys → Cu LMCT band] and a shoulder within the 700–800 nm region (d–d transitions)  , in line with T1Cu centres in their reduced state. Reoxidation upon addition of nitrite under argon atmosphere partially recovered the as purified protein UV–Vis spectrum showing a slight blue shift of 5 nm of the band at 597 nm.The UV–Vis spectrum is characteristic of a blue-copper-nitrite reductase with absorption bands at ∼447 nm  show partially overlapped nearly axial components, all of them with a solved hyperfine structure at ||g, typical of T1Cu and T2Cu centres in the Cu2+ oxidation state. EPR parameters are given in Table S3. Ferricyanide addition to as-purified TsNirK did not significantly modify either the shape of the line or the intensity, suggesting that the three copper centres are all Cu2+ ions. No EPR signals were observed upon dithionite excess addition. EPR spectra also show the typical behaviour observed in two-domain NirKs upon nitrate addition, i.e. a slight shifting of the ||g feature of the T2Cu EPR signal , which is indicative of T2Cu–nitrite interaction.EPR spectra at 120 K of as-purified 4.TsNirK is the fourth three-domain Nir crystallized so far among the copper nitrite reductases. The overall structure of TsNirK [Fig. 2a)] shows a unique distribution of domains and subunit interactions that differs greatly from those reported for HdNir ]. HdNir, RpNir and PhNir have an extra C-terminal or N-terminal domain harbouring a haem c or a T1Cu cofactor that does not interact with the two-domain core of the same subunit ]. This is not the case with TsNirK, where the extra C-terminal domain interacts directly with the T1Cu–T2Cu complex of the same subunit [Fig. 1c) and the right panel of Fig. 2a)]. The distal T1CuC of TsNirK is located at the C-terminal region, while that of HdNir is at the N-terminal region. Another remarkable difference is that the T1CuC centre of TsNirK is ∼14 Å away from the proximal T1Cu, while the nearest distal T1Cu in HdNir is located at ∼24 Å ] resembles those from blue cupredoxins with intensities and a band distribution similar to those observed in Alcaligenes xylosoxydans Nir (AxNir), Cucumis sativus stellacyanin (CsSte) and amicyanin (see Table S3). TsNirK is intense blue (∊1/∊2 = 0.21) compared with the greenish–blue three-domain HdNir (∊1/∊2 = 0.46) (see Table S3). Addition of an excess of sodium nitrite to dithionite-reduced TsNirK partially recovers the observed as-purified enzyme spectrum, which suggests that the two T1Cu centres are involved in electron transfer. The reoxidation is accompanied by a slight shift to a lower wavelength (5 nm) from the 597 nm band. This type of shift was also observed in the two-domain SmNir when subjected to anaerobic reoxidation in the presence of nitrite . In contrast, for stellacyanins . Hence, this suggests that the TsNirK rR spectrum is the superposition of two distinguishable Cu2+ T1Cu species, a conclusion also predicted by QM/MM calculations .Whereas UV–Vis and et al., 2009et al., 2011et al., 2009Nitrite reduction by NirKs can be divided into three main steps, the interaction between the enzyme and an external physiological electron donor, an internal electron-transfer reaction involving the copper centres, and nitrite-T2Cu interaction to release NO ]. This pocket would allow transient interactions with external physiological electron donors like in other transient complexes ]. The domain III crown seems to act like a compact structure [Fig. 2a)] covering the T1CuN site located at the domain I–II NirK core structure. This constitutes a difference compared with PhNir, in which the extra domain can move apart from NirK core structure allowing the interaction of the external physiological electron donor either with T1Cu or with the tethering cytochrome c  and S3]; in this pathway T1CuC might deliver electrons via a hydrogen-bonded His431 N∊2–wat–Glu385 O∊1–His125 N∊2 chemical path to the T1CuN centre. The water molecule involved in this putative electron-transfer pathway belongs to a hydrogen-bond network that also helps to stabilize domain I–domain III interaction . A similar water-molecule network is also observed in the contact area between the haem c domain and the surface above T1Cu of RpNir . The T1CuN → T2Cu electron-transfer pathway of TsNirK consists of the well characterized Cys–His bridge observed in all NirKs reported so far  residue instead of an AspCAT residue. This fact constitutes novelty from a catalytic perspective, reinforcing the idea that TsNirK belongs to a new group of three-domain NirKs that should be classified separately from those described by Ellis et al. ], with an apparent KM value (27 µM nitrite) only comparable to that of RpNir   (AspCAT–wat–wat–Ala131–Asn90–Asn107)  is not found in TsNirK  ] catalytically equivalent to Glu279 and Thr280 in AfNir ], which is thought to transmit information about the T2Cu status to T1Cu for electron delivery through the Cys–His bridge ]. An occluded water chain connecting SerCAT to HisCAT via a Gln239–Thr340 hydrogen-bond network, which is not observed in most two-domain NirKs, could also be relevant for TsNirK functionality  et al. (1984)et al. (2007)et al. (2004).The following references are cited in the supporting information: Abraham 10.1107/S2052252519000241/lz5022sup1.pdfSupporting tables and figures. DOI: copper-nitrite reductase (NirK) 6hbePDB reference:


The aberrant activation of CDK5 has been implicated in neuronal death in stroke. The goal of this study is to determine whether knocking down CDK5 by a peptide-directed lysosomal degradation approach is therapeutically effective against stroke. We synthesized a membrane-permeable peptide that specifically binds to CDK5 with a chaperone-mediated autophagy targeting motif (Tat-CDK5-CTM) and tested its therapeutic effects on a mouse model of ischemic stroke. Our results showed that Tat-CDK5-CTM blocked the CDK5-NR2B interaction, resulting in the degradation of CDK5, which in turn prevented calcium overload and neuronal death in cultured neurons. Tat-CDK5-CTM also reduced the infarction area and neuronal loss and improved the neurological functions in MCAO  mice. The peptide-directed lysosomal degradation of CDK5 is a promising therapeutic intervention for stroke. Ischemic stroke is a devastating and major cause of morbidity and mortality worldwide. However, due to the narrow time window of thrombolytic therapy, new pharmacological therapeutic approaches are still necessary. Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase that interacts with NR2B and phosphorylates NR2B to promote ischemic neuronal death ,[2]. Tarad libitum. All experiments were carried out according the Institutional Guidelines of the Animal Care and Use Committee (Huazhong University of Science and Technology).Adult (90 ± 5 days old) male C57BL/6J mice were used in all experiments. The mice were housed individually under standard conditions of temperature and humidity and a 12 h light/dark cycle (lights on at 08:00) with food and water Tat-CDK5-CTM (YGRKKRRQRRR-RRPPRSPDHKRYFRDKE-KFERQKILDQRFFE) or Tat-scramble-CDK5 (YGRKKRRQRRR-PHPRSRPRKEDDKRYFR-KFERQKILDQRFFE) peptides with 99.18% purity were synthesized by ChinaPeptides Co., Ltd. . The peptides were numbered, and the investigators did not know which peptide was applied in the experiments.Twenty-four hours after oxygen/glucose deprivation (OGD) and peptide administration, cultured neurons were stained with TUNEL and PI. The number of TUNEL- and PI-labeled cells were expressed as a percentage of the total numbers of DAPI-labeled cells per condition. Seven days after MCAO and peptide administration, the brain sections were stained with fluoro-jade C (FJ) and TUNEL. The number of FJ- and TUNEL-positive cells in each group was counted.Neurological performance was scored daily using modified 7-point neurological scales. A rotarod treadmill was used to measure the motor coordination. The behavioral analysis was always assessed by blinded independent investigators who were unaware of the experimental conditions and treatments.All variable values in the text and figure legends are presented as the means ± SEM. Western blot results, infarct volumes, TUNEL- or PI- or FJ-positive cells, and neurological scores were analyzed using ANOVA with Student-Newman-Keuls (SNK) tests. The behavioral test data were analyzed by two-way repeated-measures ANOVA with Bonferroni comparisons. Significant differences were defined as P <0.05.The cortical neurons were treated with OGD for 60 min and then the neurons were incubated for 2 h with Tat-CDK5-CTM  or the s2+ imaging, we found that Tat-CDK5-CTM significantly reduced calcium influx during the OGD insult  with the vehicle, Tat-CDK5-CTM or Tat-s-CDK5-CTM at a single dose of 0.1, 0.5, or 1 mg/kg body weight  and 20.7±2.6 mm3 (Tat-s-CDK5-CTM) to 9.7±1.2 mm3 (The mice were subjected to MRI at 72 h after the peptide injection. The MRI results showed that the Tat-CDK5-CTM treatment effectively decreased the area of cerebral infarction in all slices . After M±1.2 mm3 .in vivo.Cell death in the mouse brain sections was detected at 7 days after ischemic insult and peptide administration by FJ staining. In this study, the mice were treated with Tat-CDK5-CTM/ Tat-s-CDK5-CTM/vehicle at 6 hours after MCAO operation. We found that Tat-CDK5-CTM reduced the number of FJ-labeled cells in the vulnerable brain regions. The magnitude of FJ labeling reduction was similar to the reduction of the brain infarction . SimilarWe then performed behavioral tests at 4 weeks after MCAO operation and peptide injection . The micin vitro and in vivo. In MCAO animals, the increased expression of CDK5 and p35 was first detected in periinfarcted rat neurons [Here, we demonstrate that the application of the Tat-CDK5-CTM peptide, which can effectively disrupt the binding of CDK5 with NR2B and lead to the lysosomal-mediated degradation of CDK5, is protective against stroke damage both  neurons . CDK5 ha neurons . Tat-CDK neurons . Indeed,www.aginganddisease.org/EN/10.14336/AD.2018.1225The Supplemenantry data can be found online at:


N-glycosidases that depurinate a specific adenine residue  in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. RIPs are grouped into three types according to the number of subunits and the organization of the precursor sequences. RIPs are two-domain proteins, with the active site located in the cleft between the N- and C-terminal domains. It has been found that the basic surface residues of the RIPs promote rapid and specific targeting to the ribosome and a number of RIPs have been shown to interact with the C-terminal regions of the P proteins of the ribosome. At present, the structural basis for the interaction of trichosanthin and ricin-A chain toward P2 peptide is known. This review surveys the structural features of the representative RIPs and discusses how they approach and interact with the ribosome.Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA  N-glycosidases are very potent enzymes, and some of them are among the most potent toxins of plant origin. Ricin, one of the most toxic natural substances discovered in late 19th century by P. H. Stillmark, agglutinates red blood cells [N-glycosidic bond at adenine 4324 (A-4324) in the 28S ribosomal RNA (rRNA) of eukaryotic ribosomes [N-glycosidases are therefore also known as ribosome-inactivating proteins (RIPs). There are also other classes of proteins that inactivate ribosomes. These include phosphodiesterases such as α-sarcin and restrictocin that inactivate ribosome through the hydrolysis of a single phosphodiester bond between G4325 and A4326 in the α-SRL [N-glycosidases and these two terms are used interchangeably here.RNA od cells . It is nibosomes . The looibosomes . In eukaibosomes . rRNA N-N-glycosidase activity, RIPs are also found to possess specific DNA cleavage activities on double-stranded supercoiled DNA and mitochondrial DNA [Under physiological conditions, a single ricin A chain (RTA) molecule depurinates 1000–2000 mammalian ribosomes per minute. Naked rRNA is less susceptible to RIPs, indicating that RIPs need to bind to specific ribosomal proteins before carrying out their catalytic action [rial DNA , superoxrial DNA , phosphorial DNA , or DNA rial DNA , althougrial DNA . With moN-glycosidases. RIPs might play an important role in plant physiology and defense mechanisms because some of them could be induced by development [RIPs are widely distributed in over 100 different plant species and in different organs ,17. Theyelopment , stress elopment , or viraelopment ,23.Based on the number of subunits and the organization of the precursor sequences, RIPs are grouped into three types . Type 1 N-glycosidase catalytic subunit of RIPs are highly conserved. In general, the N-glycosidase subunit of RIPs contains two domains, the large N-terminal domain consists of six α-helices and a six-stranded mixed β-sheet, while the small C-terminal domain consists of an anti-parallel β-sheet and an α-helix with a bend in the middle (α-helix G and H) , structures of more than 20 RIPs are available . AlthougG and H) a. The inG and H) a. At theg of TCS . The ribg of TCS .The structures of RIPs are, in general, well-conserved. For example, the overall root-mean-square deviation (RMSD) of TCS and RTA is 1.1 Å . BetweenN-glycosidase domain (chain A) is linked with a lectin domain (chain B) by a disulfide bond. The chain B binds to β-1,4-linked galactose residues on the cell surface and facilitates delivery of chain A. The overall structure of ricin chain B (RTB) is highly conserved and consists of two homologous domains arisen by gene duplication. Each domain is composed of 12 β-strands and is arranged in a β-trefoil structure of three lobes α, β, and γ [In type 2 RIPs, the β, and γ . The sugβ, and γ .Sequence alignment of all these structure available RIPs indicates the existence of invariant residues, including Y14, R22, Y70, Y111, E160, R163, W192, and S196 as reference to TCS . AlthougIn many RIPs, there are two key glutamate residues in the active site. In TCS, they are E160 and E189. The active site residue E160 serves to stabilize the ribooxocarbenium ion-like transition state intermediate, while E189 acts as back-up for the catalytic glutamate in case the latter is mutated ,38. Howecat value of RTA on naked RNA is 105-fold slower than on intact ribosome [Saccharomyces cerevisiae mutants with P1 or P2 deleted. These mutants are more resistant to the cytotoxicity of RTA [Although the target site of RIPs is a specific adenine residue in the 28S RNA, the presence of ribosomal proteins is essential for efficient catalysis. It was observed that the kribosome . Subsequribosome ,42; RTA ribosome ; and PAPribosome ,44,45. Rribosome  and depuy of RTA . These iThe early RTA and ribosome interaction studies showed that the electrostatic surfaces of RTA and ribosome are essential for the delivery of RTA to the surface of ribosome ,49,50. TRecent structural studies have revealed how eukaryotic stalk protein recruits TCS or RTA to ribosomes. The crystal structure of TCS and P protein C-terminal peptide complex  shows thHowever, this approach of interaction may not be universal. Comparison of primary sequences shows that the residues located at the C-terminal domain, which is responsible for P protein interaction in RIPs, are not conserved . BesidesApart from acting on eukaryotic ribosomes, PAP, dianthin, mirabilis antiviral protein, and Shiga toxin also capable of depurinating prokaryotic ribosomes at A2660 in 23S rRNA, the equivalent adenine in eukaryotic 28S rRNA. The efficiency to prokaryotic ribosomes is about 100–500 times weaker  or compaRIPs are potent toxins. On one hand, they may be used as bioweapons or cause food poisoning. On the other hand, they have good potential applications in pharmacology and biotechnology ,71,72. T


In recent years, an increasing number of studies have shown that elevated expression of cyclin dependent kinase (Cdk5) contributes to the oncogenic initiation and progression of many types of cancers. In this study, we investigated the expression pattern of Cdk5 in colorectal cancer (CRC) cell lines and in a large number of tumor samples in order to evaluate its relevance in this pathogenesis and possible use as a prognostic marker. We found that Cdk5 is highly expressed and activated in CRC cell lines and that silencing of the kinase decreases their migration ability. In tumor tissues, Cdk5 is overexpressed compared to normal tissues due to a copy number gain. In patients with localized disease, we found that high Cdk5 levels correlate with poor prognosis, while in the metastatic setting, this was only the case for patients receiving an oxaliplatin-based treatment. When exploring the Cdk5 levels in the consensus molecular subtypes (CMS), we found the lowest levels in subtype 1, where high Cdk5 again was associated with a poorer prognosis. In conclusion, we confirm that Cdk5 is involved in CRC and disease progression and that it could serve as a prognostic and predictive biomarker in this disease.  CDK5R1) and p39 (CDK5R2), phosphorylation in the T-loop is not required for its activation and most of its substrates are not involved in cell cycle control. The function of this kinase was thought to be restricted to the central nervous system, where it is indispensable for normal brain development during embryogenesis and controls neuronal migration or axonal guidance, among others. Deregulation of Cdk5 is widely described in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s [Cyclin dependent kinase (Cdk5) belongs to the cyclin-dependent kinase family of serine/threonine kinases whose activity depends on a regulatory subunit named cyclin. However, Cdk5 is considered an atypical Cdk because it binds to non-cyclin proteins p35 CDK5R and p39 p39 CDK5R, phosphoEME1) in colorectal cancer (CRC) cell lines [Cdk5 phosphorylates the STAT3 transcription factor on its Ser-727 residue, leading to increased proliferation and promotion of tumor formation in medullar thyroid carcinoma and in prostate cancer cells through the activation of androgen receptor  or resisll lines . Elevatell lines , prostatll lines , and brell lines  tumors all lines . In prosll lines ,12 and ill lines . In CRC,ll lines  and it hll lines .To date, little is known about the possible role of Cdk5 as a cancer biomarker. In the present study, we studied the role of Cdk5 in several CRC cell lines, reproducing results from Zhuang et al.  and mostWe studied protein levels of Cdk5 and p35 by western blotting in a panel of 10 human CRC cell lines and detected both at high levels in all cell lines tested A. In conWe investigated how the transient silencing of Cdk5 affected three important characteristics of tumor cells: proliferation, migration and invasion. Forty-eight hours after siRNA gene silencing, Cdk5 knockdown (siCdk5) efficiency was between 90% and 95% at the protein level in HT29, LoVo, DiFi, and HCT116 cell lines . Under tKRAS (G12D). Cdk5 silencing caused a modest but statistically significant decrease in migration only in the SW48 KRAS G12D cell line and not in the KRAS WT. No differences in invasion ability after Cdk5 silencing were observed in either cell lines  cells. In contrast, silencing of Cdk5 did not decrease the migration and invasion ability of DiFi cells B,C. As Hll lines D,E.p-values 0.198 and 0.046, respectively). Another important characteristic of cancer cells is their ability to undergo an unlimited number of divisions. To study the implications of Cdk5 in their long-term clonogenic capability of CRC cells, we performed colony formation assays in the HT29 and LoVo cell lines . We obseCdk5 mRNA levels in tumor versus normal adjacent colon samples in two different publicly available data sets  (region chr7:151054008–151057848). We observed a statistically significant increase (p = 7.58 × 10−7) in the Cdk5 copy number in primary tumors as compared to the corresponding adjacent normal colon tissues  to Cdk5 expression levels in tumor tissue (p = 4.6 × 10−6) (Broad Institute Cancer Cell Line Encyclopedia)  I.p = 0.049 95%, confidence interval (CI) 0.98–5.88 for cohort B and p = 0.048, 95% CI 0.97–7.43 for cohort C)    = 2.72, 95% CI 1.19–6.23). Similarly, when analyzing the response rates (Chi-square), we only found differences according to Cdk5 levels in the oxaliplatin-treated patients (cohort D) as 87% (13 out of 15 patients) of the low Cdk5 group had complete or partial response, while only 53% (18 out of 34) of the high Cdk5 group responded (p = 0.029). In view of these interesting results in the metastatic setting, we wanted to analyze whether Cdk5 was also predictive for adjuvant oxaliplatin-based therapy response. As we do not have our own cohort with this patient group, we used the TCGA data (cohort F). When splitting this subgroup (n = 49) based on high or low Cdk5, we could not observe any difference in the progression free interval (PFI)  nor OS . It is worth mentioning that when analyzing the entire TCGA cohort , no differences could be observed in PFI and OS  A,B, and ohort C) . To studohort C) . As showI and OS , highligp = 0.026, As we observed a difference in migration and invasion when we silenced Cdk5 in our in vitro studies, we analyzed the link between Cdk5 expression and several gene sets related to EMT in the TCGA cohort. EMT is a well described process whereby cells undergo multiple biochemical changes that enable them to assume a mesenchymal phenotype, characterized by enhanced migratory capacity, invasiveness, and elevated resistance to apoptosis. We found that only for the TGF-β signaling within EMT did high Cdk5 levels correlate with higher pathway activation  receptor [KRAS mutations. Patients from cohort B were split according to KRAS mutational status (WT or mutated) and Cdk5 expression was again categorized as high or low. In the KRAS WT group, patients with high or low Cdk5 levels had similar DFS   B. We didl status . p = 0.036, 95% CI 1.07–8.10) (CRC has recently been classified into four consensus molecular subtypes (CMS) , namely,07–8.10) C, while 07–8.10) . It is wCdk5 has emerged as a possible drug target in several tumors, including CRC. Here, we confirm previous results implicating its role in CRC pathogenesis , but impCdk5 gene found in primary tumors as compared to normal adjacent tissues, a fact that was also pointed out by Robb et al. [Our results indicate that Cdk5 and its activator p35 are broadly expressed in human CRC cell lines; however, Cdk5 was found to be mainly bound to p25, the truncated form of p35. The binding of p25 to Cdk5 is indicative of constitutive activation of Cdk5 . In humab et al. .KRAS (LoVo) or in rapidly accelerated fibrosarcoma B (BRAF) (HT29) genes. Indeed, signaling through the MAPKs is one of the main deregulated pathways in CRC and other authors had reported an interaction between Cdk5 and different effectors of this pathway. Unfortunately, in the only previous study conducted for Cdk5 in the context of CRC, all the cell lines tested had a deregulated MAPK pathway [Cdk5 gene. We observed that in this case, Cdk5 downregulation had no effect on migration or invasion of cells, which is in agreement with previously published data [RAS/BRAF mutant CRC, Cdk5 could phosphorylate ERK5 and that the effects of abrogation of Cdk5 expression were due to the inhibition of ERK signaling [KRAS or BRAF mutations seem to preferentially activate the ERK1/2 pathway and when treated with ERK5 inhibitors, cell proliferation is unaffected [Reduced Cdk5 expression was associated with decreased migration and invasion in vitro but not with cell proliferation. This has been observed previously in cell lines from different tumor origins ,11,12,13hed data . It was affected ,24. KRAS-mutant tumors. These results point to the prognostic value of Cdk5, which seems to be related with a more aggressive phenotype as we and others have reported in cell lines and in vivo models. Thus, those tumors expressing high levels of Cdk5 could have an enhanced capacity to invade and migrate from primary sites to metastatic sites. This is in agreement with what has been reported for other kinds of cancer, for example, breast cancer, where the Cdk5-FAK pathway downstream of TGF-β signaling is necessary for EMT [We could successfully translate our results into a clinical setting as high levels of Cdk5 were associated with a higher risk of relapse in early-stage MSS CRC patients that did not receive any treatment after primary tumor resection, especially in patients with  for EMT . Indeed,We want to remark the homogeneity of these two cohorts of localized CRC patients and the fact that the patients did not receive any treatment, as this underscores the importance of Cdk5 as a biomarker in a priori good prognostic tumors. Whether treatment should be considered for these patients has to be explored in further studies.We also investigated the predictive value of Cdk5 through analysis of the association between tumor protein expression and the outcome of metastatic CRC patients receiving two standard chemotherapy regimens: Oxaliplatin plus 5-fluorouracil and irinotecan plus 5-fluorouracil. Several studies have shown that Cdk5 plays a role in DNA damage repair and resistance, including the one induced by oxaliplatin and irinotecan . HoweverCdk5 gene silencing conferred high sensitivity to this kind of drugs [For the moment, these results suggest that Cdk5 could be a good target to develop drugs against; on the one hand, they could be used in an adjuvant setting to prevent disease relapse and on the other hand, in a metastatic setting in combination with oxaliplatin. Another possibility would be a combination with poly (ADP-ribose) polymerase (PARP) inhibitors, as it was reported that of drugs . UnfortuFinally, we wanted to explore the possible association between Cdk5 and the recently described CMSs  using thKRAS G12D/+ mutated cell lines , were used in the present study. Cell lines were grown as a monolayer in RPMI 1640 , DMEM  and Ham’s F-12 (LoVo) media . DMEM and RPMI media were supplemented with 10% of heat-inactivated fetal calf serum (FCS) , 1% penicillin/streptomycin , 2 mmol/L L-glutamine , and 10 mmol/L HEPES . The Ham’s medium was supplemented with 20% of heat-inactivated FCS and 1% penicillin/streptomycin . All cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO2. Cells were periodically tested for Mycoplasma contamination and authenticated by short tandem repeat profiling with in-house methods.Human tumor-derived colorectal adenocarcinoma cell lines HT29, LoVo, DLD1, LS513, HCT116, DLD1, Caco2, LS174T, SW1417, SW480, HCT15, and DiFi from the American Type Culture Collection, along with SW48 parental and KRAS WT, and BRAF mut. V600E; LoVo: MSI, KRAS mut. G13D, and BRAF WT; HCT116: MSI, KRAS mut. G13D, and BRAF WT; DiFi: MSS, KRAS WT, and BRAF WT; SW48: MSI, KRAS WT, and BRAF WT.The molecular status of cell lines used for experiments: HT29: MSS, Cdk5 was transiently silenced using a mix of three different siRNAs  and lipofectamine RNAiMAX in OptiMem medium according to the manufacturer’s instructions . A negative transcription control (siNTC)  was used in all the experiments. Silencing was validated by western blotting and experiments with a minimum of 70% silencing levels were considered.Cell proliferation was analyzed 24, 48, 72, and 96 hours after siRNA Cdk5 silencing. Cells were permeabilized with 0.3% Triton X-100 and 30 μM propidium iodide  was added and the fluorescence signal corresponding to the cell quantity was measured at 645 nm on the Varioskan reader . HT29 and LoVo siNTC and siCdk5 cells were plated in six-well plates at a density of 300 cells/well. After 12 days in culture, the cells were washed with phosphate-buffered saline (PBS), fixed with a methanol/acetic acid (3:1) solution for 10 min and stained with a solution of crystal violet (0.5%) for 10 min. Colonies were counted manually.Migration and invasion assays were performed using transwell plates . For the invasion assay, the upper chambers were coated with 21 μL of Geltrex™ . For both the assays, the lower chambers were filled with a medium containing 10% FCS and cells were seeded in 2% FCS medium in the upper chamber. Migrated/invasive cells that had reached the lower side of the membrane were fixed with 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet. The ImageJ software was used for quantification.Cells were homogenized in lysis buffer and the supernatant was incubated with a Cdk5 primary antibody : sc-6247) overnight at 4 °C. The mix was incubated 2 hours with protein G-coupled beads  and the beads were washed and boiled for 5 minutes in a loading buffer to detach the protein complex. The protein levels were examined by western blotting. Cells were homogenized in a radioimmunoprecipitation assay (RIPA) plus buffer. Frozen tissue samples were disintegrated manually and were homogenized with RIPA plus buffer using the gentleMACS Dissociator system . Protein concentration was determined using the DC™ Protein Assay  and 50 μg of the protein was loaded and subjected to electrophoresis in 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels  and transferred onto Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membranes . After blocking , membranes were incubated overnight with specific primary antibodies against Cdk5 , p35 , and α-Tubulin . Membranes were incubated with IRDye rabbit and mouse secondary antibodies   and scanned and analyzed on the Odyssey imaging system (LICOR Biosciences). Non-cropped western blots from Six different cohorts (named A to F), with a total of 811 samples from different patients, were used in this study. A tissue microarray (TMA) was built for cohorts D and E as previously described . SubsequCDK5 gene expression was studied by qPCR in cohort C as previously described [escribed ,28. BrieTwo publicly available cohorts were used: Colonomics (NCBI BioProject PRJNA188510) and TCGA. The colonomics cohort (cohort B) consisted of 98 paired adjacent-normal and tumor tissues from stage II microsatellite stable patients and 50 colon mucosae samples from healthy donors . Gene expression data, assessed by Affymetrix Human Genome U219 expression array, had previously been analyzed . n = 448) and 25 samples annotated as stage IV TCGA-rectum adenocarcinoma (READ), 473 samples in total. The classification of TCGA samples into four CMS was in agreement with Guinney et al. [For studies using the TCGA cohort (cohort F), we included all samples annotated as TCGA-colon adenocarcinoma (COAD)  of at least three independent experiments and statistical analysis was performed with Graphpad Prism V.4 software . Comparisons among different experimental conditions were carried out through the T-student or two-way ANOVA test followed by Bonferroni post-test. Values of A survival analysis was performed with the survival and survminer R packages. Kaplan–Meier curves depicting disease-free survival (DFS), time to progression (TTP), progression-free interval (PFI), and overall survival (OS) were accompanied with the log-rank test to verify significance in survival curve differences. The Cox regression hazards models were performed to quantify the effect of gene expression and survival (adjusted for age and stage). Differences in response to treatment were evaluated in cohorts D and E by contingency tables and chi-square or Fisher’s exact tests as appropriate. Cdk5 gene expression comparison between different CMS subtypes was performed using one-way ANOVA. Regarding TCGA data, comparison between primary and normal tumor samples  were performed using paired samples T-tests. Both variables were compared using the Pearson correlation test. The GSVA R-package  was used to compare the gene expression levels of EMT and TGF-β signaling in EMT pathways in Cdk5 low vs. Cdk5 high tumors. A Wilcox’s test was performed to evaluate the statistical significance.We combined in vitro studies with the analysis of a high number of tumor samples from different CRC stages and confirmed that Cdk5 is involved in CRC progression and that it could serve as a prognostic and predictive biomarker in this disease. Our findings also suggest that Cdk5 could help decide which patients should receive adjuvant therapy and whether oxaliplatin or irinotecan should be used in the treatment of metastatic disease.


Electronic nose (E-nose), a kind of instrument which combines with the gas sensor and the corresponding pattern recognition algorithm, is used to detect the type and concentration of gases. However, the sensor drift will occur in realistic application scenario of E-nose, which makes a variation of data distribution in feature space and causes a decrease in prediction accuracy. Therefore, studies on the drift compensation algorithms are receiving increasing attention in the field of the E-nose. In this paper, a novel method, namely Wasserstein Distance Learned Feature Representations (WDLFR), is put forward for drift compensation, which is based on the domain invariant feature representation learning. It regards a neural network as a domain discriminator to measure the empirical Wasserstein distance between the source domain (data without drift) and target domain (drift data). The WDLFR minimizes Wasserstein distance by optimizing the feature extractor in an adversarial manner. The Wasserstein distance for domain adaption has good gradient and generalization bound. Finally, the experiments are conducted on a real dataset of E-nose from the University of California, San Diego (UCSD). The experimental results demonstrate that the effectiveness of the proposed method outperforms all compared drift compensation methods, and the WDLFR succeeds in significantly reducing the sensor drift. Electronic nose (E-nose) is known as machine olfaction, consisting of the gas sensor array and corresponding pattern recognition algorithms, and is used to identify gases. Zhang et al.  and WangA number of methods have been applied to cope with the sensor drift of E-nose. From the perspective of signal preprocessing methods ,7, frequThe above methods can suppress the drift to a certain extent, but the effects of these methods are limited due to the weak generalization of drift data. The sensor drift will make a variation of data distributions between the collected samples previously (data without drift) and the collected samples later (drift data). It will cause a great decrease in classification accuracies when the model trained with the data without drift is directly applied in testing samples with the drift. Therefore, in the sensor researches and pattern recognition communities, it is challenging to find a drift compensation algorithm with good robustness and adaptability.In these cases, domain adaption techniques are a proper solution to deal with the problem of inconsistent data distributions between the source and the target domain samples. These techniques also have broad applications in many research fields, including natural language processing, machine vision, etc. ,15,16. IInspired by Wasserstein GAN (WGAN)  and specThe rest of this paper is organized as follows. The basis of the proposed method is presented in In this section, a brief introduction of the Wasserstein distance will be given. It is the basis of the proposed method. x to y, and the common cost function is based on l norm, such as 1-norm x to another random location y, and it satisfies the marginal constraint Wasserstein distance is used to measure the distance between two probability distributions and is defined asThe Kantorovich-Rubinstein theorem shows that the dual form of Wasserstein distance  can be wnce, and  has showIn domain adaption techniques, the source and the target domain are defined by describing “S” and “T”, respectively. We have the training set The sensor drift of the E-nose leads to inconsistent data distributions between the previously collected samples (source domain) and the later collected samples (target domain), which means that the model trained with source domain samples may be highly biased in the target domain. To solve this problem, a new method (WDLFR) is proposed in this paper. The learned feature representations are invariant to the change of domain by minimizing the empirical Wasserstein distance between the source and the target feature representations through an adversarial training manner. d-dimensional representation with the network parameters The adversarial method is composed of two parts, including the feature extractor and the domain discriminator implemented by a neural network. The feature extractor network is used to learn the feature representations of the source and the target domain, and domain discriminator is used to estimate the empirical Wasserstein distance between the feature representations of both domains. First, considering a sample of any domain f, we can train a domain discriminator, as suggested in [For the function ested in , to learAccording to the feature extractor network, the feature representations of the source and the target domain are If the function of the domain discriminator Here, the question of enforcing the Lipschitz constraint is raised. A weight clipping method was presented in , aiming Now, the Wasserstein distance is continuous and differentiable almost everywhere, and an optimal domain discriminator can be trained first. Then, by fixing the optimal network parameters of domain discriminator and minimizing the Wasserstein distance, the feature extractor network can learn the feature representations with the domain discrepancy reduced. Therefore, the feature representations can be estimated by solving the minimax problemFinally, by iteratively learning feature representations with lower Wasserstein distance, the adversarial objective function can learn domain invariant feature representations.The final purpose of this paper is to ensure that the classifier of the source domain will have a good performance in the target domain. Considering the above domain adaption method, it may be impossible to learn the optimal feature representations. Because the WDLRF method can learn domain invariant feature representations and guarantees transferability of the learned feature representations, the source domain classifier is feasible to the target domain. However, the learned domain invariant feature representations are not enough class-distinguished. Therefore, the supervision information of the training set denoted by the source domain will be integrated into the domain invariant feature representation learning as suggested in . The ovek-th dimension value of the distribution Next, the combination of the feature representation learning and the classifier will be introduced. Several layers can be added as the classifier after the feature extractor network. The objective of the classifier Finally, the final objective function is obtained by combining the Equations (8) with (11)The WDLFR algorithm can be implemented using the standard back-propagation with two iterations. In a mini-batch size including labeled source data and unlabeled target data, the domain discriminator can firstly be trained to optimal point by gradient ascent. The mini-batch gradient ascent method divides all samples of two domains into several batches and updates the network parameters of the neural network in each batch way, which can reduce the computational complexity. In other words, the mini-batch gradient ascent method divides the training set into several small training sets. Then, in order to reduce the distribution discrepancy across domains, we simultaneously minimize the estimated empirical Wasserstein distance across domains and the classification loss computed by labeled source samples to update the feature extractor network. Finally, the learned feature representations are domain-invariant and class-distinguished, since the parameter The sensor drift changes the features of the collected data. Furthermore, it also makes data distributions different. Domain adaption techniques can reduce the difference of distributions among domains. Therefore, the proposed WDLRF method can be used to reduce the drift of the E-nose. Algorithm 1 The Proposed WDLFR Method: Asserstein Distance Learned Feature Representations Combining with ClassifierRequire: Labeled source data 1. Initialize feature extractor, domain discrimination, classifier with random weights Repeat: 2. 3.  Sample m instances   Sample m examples Fori = 1, …, kdo4.  5.   6.   7.   Calculate spectral normalization weights 8.   End for9. 10. 11. Until12. In this section, the real sensor drift benchmark dataset of the E-nose from UCSD is used to evaluate the effectiveness of the WDLFR method, and the experimental results of the proposed WDLFR method are compared with that of other drift compensation algorithms in E-nose.The real sensor drift benchmark dataset, consisting of data from three years, was collected by Vergara et al.  at UCSD.To more intuitively observe the data distribution discrepancy of all batches, the two-dimensional principle component scatter points of the original data are plotted in In this paper, all experiments are performed using Tensorflow and the training model is optimized using Adam optimizer. The advantage of the Adam optimizer is that each iteration of the learning rate has a clear range, and it makes the change of parameters very stable. Under the situation of the best experimental results, the constructions of the WDLRF method are as follows. The feature extractor network contains an input layer of 128 neuron nodes and an output layer of 200 neuron nodes. The domain discriminator is designed with an input layer of 200 nodes, one hidden layer of 10 nodes, and an output layer of 1 node. The classifier is composed with an input layer of 200 nodes and an output layer of 6 nodes. All the activation functions adopt the ReLU function, except a Softmax function for the classifier. After normalizing all samples from source and target domains, the samples from the source and target domain are first inputted into the feature extractor network. Then, the extracted source domain features are inputted into the classifier, and the source and target domain features are inputted into the domain discriminator to evaluate the Wasserstein distance. Finally, it updates the feature extractor network by learning the classifier and the domain discriminator at the same time. Therefore, the extracted features from the feature extractor network have domain invariant characteristics, and the distribution consistencies of source and target domain are greatly improved.The classification accuracies are used as a criterion to judge the drift reduction. A goal that the WDLFR method aligns with the distribution of the source and target domain is to improve the performance of the classifier. Therefore, all experiments are conducted under Experimental Setting 1 and Setting 2. In order to better verify the effectiveness of the proposed WDLFR method in this paper, the proposed approach compares with principal component analysis (PCA) , linear Setting 1: Take Batch 1 as the source domain for training model, and test on Batch b, b = 2, 3, …, 10.Setting 2: Take Batch b as the source domain for training model, and test on Batch (b + 1), b = 1, 2, 3, …, 9.Under the setting 1, the first batch with labeled data is used as the source domain, and the b-th  batch with unlabeled data is considered as the target domain. If the process of learning domain invariant feature representations from the both domains is regarded as a task, a total of nine tasks with pair-wise batches (Batch 1 vs. Batch b)  are implemented in this Experimental Setting 1. Inspired by PCA scatter points in In order to represent the effect of the proposed WDLFR method on drift suppression intuitively, the recognition results of all comparison algorithms under Experimental Setting 1 are reported in Under Experimental Setting 2, the b-th batch of dataset is used as source data, and the (b + 1)-th batch of dataset is viewed as target data, b = 1, 2, 3, …, 9, i.e., the classification model is trained on Batch b and tests on Batch (b + 1). The experimental comparison results of recognition accuracy for each algorithm are reported in In order to solve the issue of inconsistent distributions caused by the sensor drift in the E-nose, a novel drift compensation algorithm (WDLFR) is proposed in this paper. The WDLFR can effectively reduce the distribution discrepancy by taking the merit of the good gradient property and generalization bound of Wasserstein distance. Furthermore, the WDLFR can reduce the drift of the E-nose. The characteristics of the WDLFR are as follows: (1) The feature extractor network and the domain discriminator are trained by an adversarial manner, so that the extracted features by the feature extractor network can eventually cheat the domain discriminator to generate domain invariant feature representations; (2) It combines the feature extractor with classifier to make the learned domain invariant feature representations class-distinguished. Finally, in order to verify the effectiveness of the WDLFR, we experiment on the dataset of the E-nose from USCD, and the classification accuracy is better than other comparison algorithms using the proposed WDLFR method.In the future, we will continue to expand the work from the perspective of adaption classifier. It establishes a residual relationship between the source and the target domain classifier, and combines the feature extractor network with the adaption classifier to compensate the drift.


Purpose The aim of this study was to evaluate the level of burnout syndrome and quality of life (QoL) among Polish massage therapists, and determine their relationship with sociodemographic and work-related variables. Methods A group of 43 participants aged 28–63, who were blind or poor-sighted were recruited for the study. They were surveyed with sociodemographic data questionnaire and the Polish versions of the Maslach Burnout Inventory-General Survey and WHOQOL-BREF. Results The overall level of exhaustion was 6.79 ± 4.45, cynicism was estimated at 7.30 ± 3.43, and professional efficacy was 23.3 ± 5.44. Regarding QoL, the psychological domain was the highest (73.6 ± 10.0), while the physical domain was the lowest (61.1 ± 6.94). None of the sociodemographic variables or occupational factors had any statistical relationship with any burnout subscale. Significant correlations were found between the psychological domain of QoL and marital status , years of practice , hours of practice per week  and private practice . Significant relationships were found between the environmental domain of QoL and the place of residence  and between hours of practice per week . A significant positive correlation was noted between professional efficacy and the social relationship domain . Conclusion Job activity plays a crucial function in the psychosocial rehabilitation of massage therapists with visual impairment. This was confirmed by the low risk of burnout, and the psychological domain being the highest of QoL. Burnout was first defined by Freudenberger in 1974 as the decrease in the power and energy of individuals who are faced with a workload . RecentlBurnout is known to be one of the most important reasons for absenteeism or turnover , and hasA poor level of quality of life (QoL) could reduce work performance and it may constitute a source of burnout and early retirement among health practitioners . MultiplIt should be noted that the job professional activity is seen as an important determinant of QoL . The posVisual impairment (VI) is acknowledged as a major health issue. According to last available global estimates 285 million people in general population had problems with sight and 39 million of them were blind . VI has Many of the job categories for blind and partially-sighted people have been determined by The European Blind Union, and in the healthcare category, these include physical therapists, physiotherapists or massage therapists. Educating people with VI as massage therapists has a long tradition. In Japan, it began in the seventeenth century and has since continued as a form of rehabilitation and retraining . WorkingA small number of reports indicate that burnout in massage therapists with VI is low and it has been suggested that an understanding of mental health can allow prevention of burnout in this group , 24. FewThe study group comprised 43 people  aged 28–63 (mean = 46.3 ± 8.28). All participants were active massage therapists working in health units in Poland who were blind  or poor-sighted . Most participants had been visually impaired for more than 5 years . All participants had been diagnosed with VI according to the International Classification of Diseases (ICD)-10 .The study sample was recruited from the National Association for Blind and Partially-Sighted Massage Therapists and Physiotherapists in Poland, which is one of the sections managed by the Polish Blind Union (PBU). Nowadays, the National Association includes 50 active members. Membership requires confirmation of VI, a background in medical educational and documented membership in PBU.All members of the association were invited to participate in the study. Interviews were conducted by one trained interviewer. The questionnaires were completed by participants via telephone and the oral personal agreement was the inclusion criteria. The exclusion criteria comprised the presence of any diagnosed intellectual disability, or any physical disability other than a visual one.The study was approved by the Bioethics Committee of the Medical University of Lodz (no RNN/311/15/KE).The sociodemographic questionnaire was constructed for the needs of this study. Information on age, gender, level of VI (blind/poor-sighted), and the age of VI onset were recorded, as well as level of education, place of residence, marital status, household structure, and whether the respondent’s incomes were the main source of livelihood. Additionally, participants were asked about the working variables such as duration of practice, hours of practice per week and if they had a private practice Table .The WHOQOL-BREF is the one of the best known instruments that has been developed for cross-cultural comparisons of QoL. It has been adopted to many languages, and its simplicity of use was the main reason for its selection in the study . The PolIn the WHOQOL-BREF, the physical domain is connected with activities of daily living, dependence on medicinal substances and medical aids, energy and fatigue, mobility, pain and discomfort, sleep and rest and work capacity. The psychological domain consists of assessment of body image and appearance, negative feelings, positive feelings, self-esteem, spirituality/religion/personal beliefs, thinking, learning, memory and concentration. The social relationship domain incorporates personal relationships, social support and sexual activity. The environmental domain corresponds to financial resources, freedom, physical safety and security, health and social care: accessibility and quality, home environment, opportunities for acquiring new information and skills, participation in and opportunities for recreation/leisure activities, physical environment (pollution/noise/traffic/climate) and transport .Each item of the questionnaire was scored on a response scale from 1 to 5. To estimate the QoL value, all raw domain scores obtained by WHOQOL-BREF were transformed to a score ranging from 4 to 20, and then transformed linearly to a 0–100 scale. All domain scores were scaled in a positive direction, with higher scores denoting higher QoL .The Maslach Burnout Inventory-General Survey (MBI-GS) was used. It consists of 16 items that are scored on Likert scale from 0 (never) to 6 (every day) according to the frequency of occurrence of each symptom. Questions apply to the feelings and attitudes of the respondent. Burnout is understood here as “a crisis in one’s relationships with work”; this does not necessarily mean a crisis in relationships with people but is connected to cynicism about the value of personal work and doubts regarding one’s capability to perform. In contrast, engagement indicates excellent performance of work and self-confidence about effectiveness. In MBI-GS, burnout is measured on the three subscales: EX, CY and PE. The EX was taken to mean fatigue, but not necessarily caused by service recipients. CY is an expression of indifference and distant attitude towards work. PE is connected with the social and non-social aspects of occupational accomplishment, as well as worker’s expectations .Higher scores on the EX and CY subscales and a lower level on the PE subscale indicate a higher level of burnout , 31. The2) were used. A p-value of < 0.05 was regarded as statistically significant. Statistical analysis was conducted using Statistica Software, version 10.The nonparametric Mann–Whitney U-test was used to compare the mean values of two groups of variables and independent gender groups, and the nonparametric Kruskal–Wallis test was used to compare three groups of variables. The relationships between quantitative variables were analysed using the correlation coefficient rank ρ (rho); the significance of this factor was estimated by the Students’ t test. The Chi square test was used to assess differences between subgroup and gender. The effect sizes were reported to illustrate the strength of the statistical relationships and Cohen’s d and epsilon squared . The QoL regarding the health determinant was 3.72 ± 0.59, and this was slightly higher among women than men .Among the whole group, the highest level of QoL was observed in the psychological domain (73.6 ± 10.0), and the lowest level in the physical domain (61.1 ± 6.94). In men, the highest QoL was found in the psychological domain (73.1 ± 10.7), while in women, it was found in the environment domain (75.5 ± 9.43). The physical domain was the lowest, both in men and women . Women reported higher scores than men in the psychological domain (74.5 ± 8.60 and 73.1 ± 10.7), sociological domain (69.7 ± 12.5 and 62.5 ± 13.0) and environmental domain (75.5 ± 9.43 and 71.8 ± 10.5). Men and women demonstrated very similar scores in the somatic domain; the differences between the sexes were not statistically significant  . Additionally psychological domain was positively correlated with years of practice , hours of practice per week  and private practice . A significant relationship was also found between the environmental domain and place of residence  and a significant positive correlation with hours of practice per week .Neither the physical domain nor the social relationship domain had any significant relationship with any sociodemographic variables or occupational factors Table . A signiNo significant correlation was found between the level of EX, CY and any of the domains of QoL and overall and health-related QoL. Only PE was found to have a significant positive correlation with social domain  (Table A low level of burnout was observed in all subscales among massage therapists. These results are consistent with a previous study which indicated a low level of burnout in this group . Our finThe therapists are also more prone to burnout than other health professionals, because they are usually in close interaction with their patients during the rehabilitation process. Physical therapists are seen as a group vulnerable to burnout because of the specific nature of their relationship with chronically ill, aggressive and depressive patients . The risWe assume that our massage therapists with VI were less prone to burnout than other therapists because they have more resources to allow them to work with patients with impairments. Their own success in the psychosocial rehabilitation process, possibly confirmed by their success in being actively employed, may further support and strengthen their process of rehabilitation. It could also provide them with better tools to understand the needs of patients, allowing them to be better able to stimulate patient motivation or cope with aggressive or depressive reactions.The results from our study confirm that male participants achieved a higher level for EX, CY and PE than female participants, but the difference was not significant. There is no strong evidence that the level of burnout is dependent on sex, but some results indicate that women are more prone to burnout than men  which caSome demographic and organizational variables such as gender, being less-educated or working in a hospital have a significant influence on the development of burnout . Our preJob activity has a vital meaning for QoL and unemployment has a substantial, negative effect on QoL in the general population . For peoOur findings indicate that our participants obtained the highest level of QoL in the psychological domain, but the lowest in the somatic domain. In contrast, health-care staff reported their QoL to be highest in the physical domain, but lowest in the environmental domain . It coulOur study found that while women tended to demonstrate slightly lower general QoL, they also recorded slightly higher scores throughout the health-related QoL domain. Generally, women are prone to lower QoL scores than men in the occupationally active population . This haThe main conclusion from our survey is that higher social QoL corresponds to greater personal efficacy. Previous studies suggest that social support and having family and other relationships serve a protective function against burnout . AdditioOur study has some limitations: the study is based on a small number of participants and the lack of similar studies makes it difficult to generalize our results. Nonetheless, our findings do indicate reinforce the need for the development of further studies confirming the positive impact of professional work on the functioning of people with VI. Moreover, the future studies should determine the prevalence and the association between work-related musculoskeletal disorders and quality of life among physical therapist with VI. It should be noted that low vision is associated with high risk of developing musculoskeletal problems, especially in the neck/scapular area . AdditioIt was found that the studied massage therapists were at low risk of burnout. Of the examined domains, the psychological domain demonstrated the greatest QoL and the physical domain the least. Additionally, high QoL was found in the social domain, corresponding to higher personal efficacy. Based on this data, we conclude that job activity among people with VI plays a crucial function in psychosocial rehabilitation. To improve the social functioning of this group, we recommend the implementation of programs based on professional activation, including continuous education aimed at preparation for employment, and professional counselling at all ages. Another crucial task would be to encourage and support employers in creating jobs for people with disabilities.


Here, we show that the domain walls of this system generate an additional contribution to the Hall conductivity tensor and a transverse magnetization. The former is an electric field lying in the same plane with the magnetic field and electric current and therefore a planar Hall effect. We demonstrate that in-plane rotation of spins inside the domain wall would explain both observations and the clockwise or anticlockwise chirality of the walls depends on the history of the field orientation and can be controlled.Magnetic domain walls are topological solitons whose internal structure is set by competing energies which sculpt them. In common ferromagnets, domain walls are known to be of either Bloch or Néel types. Little is established in the case of Mn The structure of domain walls is of interest to the antiferromagnetic spintronics. Here the authors find an additional contribution to the Hall conductivity tensor and a transverse magnetization generated by domain walls in Mn3Sn and report that the sign of this contribution depends on the prior history of the sample. They are either of Bloch type, where the magnetization rotates in a plane parallel to the wall plane, or of Néel type, whose magnetization vector rotates in a plane perpendicular to the wall. Thanks to high-resolution scanning probes of local magnetization, they can be visualized2. Theoretical proposals for other more sophisticated spin textures have recently emerged3. In addition to their fundamental interest, the attention to domain walls is driven by the quest for new spintronic devices4. Much less is known about antiferromagnetic domain walls.A domain wall is the topological defect of a discrete symmetry. In ferromagnetic materials, these are narrow boundaries separating magnetic domains with different polarities. Their width and structure are set by the competition between the exchange energy and the magneto-crystalline anisotropy energy7 in the Mn3X  family of noncollinear antiferromagnets11. The discovery followed theoretical predictions13 and preceded the observation of a variety of other anomalous transverse responses by thermal and optical probes20. These materials constitute new platforms for antiferromagnetic spintronics22. The structure of domain walls have been a subject of theoretical23 and experimental studies24. Evidence and arguments for a nontrivial spin texture in domain walls are available, but no direct image of their magnetic structure, yet.A large anomalous Hall effect (AHE) was recently discovered3Sn. The first observation is that in the narrow magnetic field window of multiple domains, there is a planar Hall effect (PHE), which consists in an electric field oriented parallel (and not perpendicular) to the applied magnetic field. The thermoelectric counterpart of this effect, namely a planar Nernst effect (PNE) was also detected. The second observation is the existence of a transverse magnetic response in the same narrow field window. Employing micron-size Hall sensors in close proximity with the sample26, we monitored the local magnetic field at the surface and found in the same field window a finite off-diagonal magnetization: a finite magnetization oriented perpendicular to the orientation of the applied magnetic field. We will argue below that a satisfactory explanation of both these observations is provided by a specific spin texture inside the domain walls. The third result is that the sign of the emergent electric field  depends on the history of the magnetic field orientation. We will show that this is caused by residual minority domains promoting a specific chirality. This last observation constitutes a new case of memory formation in condensed matter recording a direction27.Here, we report on three distinct experimental observations leading us to identify the in-plane structure of the domain walls in Mn3Sn, which we call PHE and PNE. The experimental configuration is sketched in Fig. z-axis (J//z) and the magnetic field was oriented along the y-axis (H//y). Electric field was measured simultaneously along both x- and y-axes. Ex, which represents an electric field vector perpendicular the magnetic field and the charge current, is the Hall response. As seen in Fig. 24. As the magnetic field is swept, three different regimes succeed each other24. In regime I, the system hosts one single domain. When the applied magnetic field (opposite to the magnetization of the dominant domain) exceeds a threshold, new domains nucleate and regime II starts. At sufficiently large magnetic field, the system becomes single-domain again (regime III). As seen in , there is a nonvanishing Ey (Ex). This is a planar Hall effect, with an electric field, which is parallel and not perpendicular to the magnetic field. Note that this signal only emerges in the presence of domain walls. Its amplitude is comparable to the amplitude of the topological Hall effect (THE)  Fig.  extractez-axis. When the magnetic field is oriented along the y-axis, there is a finite Ex. It represents the anomalous Nernst effect, which also displays a hysteretic jump , the magnitude of PHE (PNE) remain a sizable fraction (≈0.3–0.4) of the total AHE (ANE) and there is no significant evolution with temperature. We will show below how the PHE, the PNE and their odd parity in field, are set by the internal structure of domain walls23 in this system.We carried out an extensive set of temperature-dependent measurements, see Supplementary Fig. Figure ly/lx, where ly(lx) is the length of the sample along the y-axis (x-axis). When ly/lx ≈ 1 [inset (a)], bulk magnetization for the field along two orientations are almost coincident. But when ly/lx ≈ 3, the hysteresis loop is wider when the field is oriented along the longer axis, in agreement with that was reported previously15. As seen in the Fig. As seen in Fig. y- (x-)axis, the new domains nucleate at the xz(yz)-surface and immediately occupy the area (5 × 5 μm2) probed by a Hall sensor. The wide hysteresis loop of the bulk magnetization monitors the gradual enhancement produced by the smooth occupation of the center of the sample.Additional insight is brought by surface magnetization data obtained with Hall sensors. As shown in Fig. We used the Hall sensors to look for an off-diagonal magnetic response, namely a finite magnetic field perpendicular to the applied field. The mutual configuration of the sample, the magnetic field and the Hall sensors for quantifying longitudinal and transverse magnetization (TM) are shown in [inset (e)] and [inset (f)]. The obtained data at room temperature is shown in Fig. 23), which would explain our results, is sketched in Fig. θ = π) is located at the center and another domain with opposite polarity (θ = 0) at the periphery. . In the (more or less thick) wall separating these two domains, spins rotate smoothly and concomitantly in the x–y plane. The texture along x-axis is such that at the center of the domain wall, the adopted configuration has an orientation perpendicular to the two domains. Figure π/2 and −π/2. This would correspond to an either clockwise or anticlockwise rotation of spins depending on the specific domain configuration at the center and the periphery. Note that domain walls of this type, with in-plane rotation of two possible signs, follow directly from the hierarchy of scales discussed in ref. 23, in which the Dzyaloshinskii–Moriya interaction is much stronger than an in-plane twofold anisotropy. The origin of the twofold anisotropy will be discussed in future work.A spin texture for domain walls  would generate a magnetic field perpendicular to and electric field parallel to the orientation of the applied magnetic field. This is the origin of the TM and planar Hall effect. The angle-dependent study of the AHE24 has established that the orientation of the electric field associated with anomalous Hall effect is set by the orientation of spins . Therefore, the π/2 spin configuration in the center of domain wall would naturally gives rise to an electric field perpendicular to those generated by the θ = 0 and θ = π domains.Such a texture would provide a natural explanation for the TM and the planar Hall effect observed in regime II. The in-plane tilt of spins , then the PHE and the TM signals will be even (symmetric) in field. On the other hand, if what remains fixed is the sense of the rotation (clockwise or anticlockwise), then the signals will be odd (or asymmetric) in field, because the spin configuration inside the domain wall will be opposite during the two sweeps.In this picture, the sign of the signals reflects the chirality of the domain wall. Consider a hysteresis loop with the magnetic field swept from a ain Fig. . If durix-axis from 0.5 to −0.5 T and back. This corresponds to switching domains from θ = 0 to θ = π configurations and back to the starting point. The measurement was preceded in the first case by a field rotation from +π/2 to 0 and in the second case, by a rotation from −π/2 to 0. As one can see in Fig. 27. By subtracting the two sets of data or adding them, one can extract the symmetric  hysteretic loop, the sample is practically single-domain with π/2 spin configuration. In principle rotating from π/2 to 0 before the measurement would change the spin configuration of the whole sample from π/2 to 0. However, if residual domains remain stuck in the π/2 configuration, they will set the spin configuration of the domain walls along π/2. If this is the case, then one would expect to see a dependence of the memory effect on the strength of the prior magnetic field. The larger the magnetic field at which the (π/2 to 0) rotation takes place before the measurement, the smaller the fraction of the domains which had stayed in place and the smaller their role in setting the chirality.A plausible answer to this question is provided by the scenario sketched above. When the magnetic field is oriented along B = 0.5, 1, and 2 T. One can see that the magnitude of the PHE and in its symmetric component steadily decreases. This implies that the symmetric component of the PHE set by the chirality of the wall is promoted by the presence of minority domains, whose population decrease with increasing magnetic field. The asymmetric component, on other hand, does not show significant evolution with magnetic field.As seen in Fig. 24, provided that we assume an additional off-plane tilt of spins residing inside the domain walls. Indeed, if the unit vector of magnetization has a finite z dependence 29 and the skyrmionic number will be finite, producing real-space Berry curvature. Such an assumption would not alter the conclusions drawn above. Yet, it is not necessary for explaining the observations reported in the present study.If domain walls with opposite chiralities were evenly distributed in the sample no PHE or TM signal would have been observed. This is not the case. The dominance of a symmetric and history-dependent component in the PHE signal implies that deep inside the sample, minority domains set the chirality of the domain wall. The dominance of the asymmetric and history-independent component in surface TM indicates that wall spin orientation at surface is principally set by the orientation of the magnetic field with only a minor role proposed by the minority domains. We note that the domain wall spin texture proposed here can also generate a topological Hall response as reported previouslyTN = 420 K would presumably erase all history dependence. It would be interesting to compare field-cooled and zero-field-cooled behaviors across the transition temperature in future experiments combining a furnace and a magnet.Heating the sample above 3Sn and found that in this regime, one can observe a planar Hall and planar Nernst effect as well as TM. These observations can be explained by a specific spin texture for domain walls where spins rotate in the pseudo-Kagomé plane. The choice of clockwise or anticlockwise rotation can be controlled by the prior magnetic history of the sample, providing a new platform for memory formation.In summary we put under scrutiny a narrow field window in which there are multiple magnetic domains in Mn3Sn single crystals with a typical size in the range of centimeter were grown by the vertical Bridgman technique24. They were cut to desired dimensions by a wire saw. All transport experiments were performed in a commercial measurement system (Quantum Design PPMS), using the Horizontal Rotator Option. Hall resistivity was measured by a standard four-probe method using a current source and a DC-nanovoltmeter. Two Chromel- Constantan (type E) thermocouples were employed to measure the temperature difference in the case of Nernst measurements.Mnn = 2.5 × 1011 cm−2 (300 K) and it was located 160 nm below the surface. The device was fabricated using electron beam lithography and 250 V argon ions to define the mesa. Supplementary Figure 2 square with a 100 μm interval between two neighboring sensors26. Attaching the device to the surface of the sample, the local magnetic field was determined by measuring the Hall resistivity of the sensor using an AC current source and a lock-in amplifier.Bulk magnetization was measured using a VSM method. For surface magnetization measurements, we employed an array of Hall sensors based on high-mobility AlGaAs/GaAs heterostructure. The density of the 2DEG was Supplementary Information


The functional relationship between flow stress and process parameters  of 21-4N heat-resistant steel during hot deformation was explored, the constitutive equation of peak stress was established, and its accuracy was verified. Based on the dynamic material model, the energy dissipation maps and destabilization maps of 21-4N heat-resistant steel were established at strains of 0.2, 0.4 and 0.6, and processing maps were obtained by their superposition. Within the deformation temperature range of 1060~1120°C and a strain rate range of 0.01–0.1 s−1, there is a stable domain with the peak efficiency of about 0.5. The best hot working parameters (strain rate and deformation temperature) of 21-4N heat-resistant steel are determined by the stable and instable domain in the processing maps, which are in the deformation temperature range of 1120–1180 °C and the strain rate range of 0.01–10 s−1.The hot deformation behavior of 21-4N heat-resistant steel was studied by hot compression test in a deformation temperature range of 1000–1180 °C, a strain rate range of 0.01–10 s It is characterized by high tensile strength and yield strength, good oxidation resistance, excellent chemical stability and good high-temperature mechanical properties, and is widely used in the manufacture of engine valves ,4,5. The50NiL alloy using a Gleeble-3500 thermal simulation test machine, established Arrhenius constitutive equation by using stress-strain data, and used a neural network to compensate the material constant. Hao et al.  was obtained, as shown in n = 5.603.ασ)]-1000/T was obtained, as shown in Q/Rn.α was brought into Equation (11), then the data was subjected to linear regression fitting at different temperatures . The linear regression curve of ln[sinh. Assuming the logarithm of both sides of Equation (6):The value of ασ)] and lnZ were linear, and the data was subjected to linear regression fitting at different temperatures . Then the linear regression curve of lnZ-ln[sinh(ασ)] was obtained, as shown in C was the intercept of the curve. The average intercept was obtained by fitted the curve: lnC = 48.85, C = 1.645 × 1021.From Equation (13), ln[sinh can be rewritten as:Then:C, α, n, Q, into Equation (15) and combining with Equation (12), the peak stress constitutive equation of 21-4N can be obtained:Taking −1; σ—True flow stress, MPa; T—Temperature, °C.In the equation: AARE and the correlation coefficient RR were introduced; the expressions were as shown in Equations (17) and (18). The smaller relative error AARE and the closer correlation coefficient RR were equal to 1, establishing the higher accuracy of constitutive equation as the more accurate prediction.To evaluate the accuracy of the peak stress constitutive equation, the average relative error E—Peak stress experimental value; P—Peak stress prediction value ; In the equation: Z at different temperatures and strain rates. Then, the results were substituted into peak stress constitutive Equation (16), and the corresponding rheological stress was calculated, as shown in Equation (12) was used to calculate The calculated rheological stress was compared with corrected rheological stress  to find AARE = 5.9463%; RR = 0.99343. This indicates that the peak stress constitutive equation established in this paper can well describe the flow stress relationship of 21-4N heat-resistant steel at different temperatures and strain rates.The correlation between experimental stress and predicted stress  of peak stress is shown in Processing maps can reflect the influence of hot working parameters on the microstructure of materials, can simulate intrinsic deformation behavior of materials, and can reveal stable processing domains with good structures and non-stable processing domains with tissue defects. It is a superposition of the power dissipation map and the destabilization map ,39,40.P) of the sample is consumed in two ways: one is power dissipation caused by the plastic deformation of the sample, which is called dissipation (G); the other is power dissipation caused by the structural changes taking place during the plastic deformation process of the sample , which is called dissipation of quantity (J). As shown in Equation (19):At present, the most widely used hot deformation model for metal materials is the Dynamic Material Model (DMM) ,42,43,44G and J are determined by strain rate sensitivity (m), the expression of which is shown as Equation (20):In the equation: Dissipation η) is a function of m, the relationship is shown as Equation (21):The efficiency of power dissipation :The stress under the conditions of strains of 0.2, 0.4 and 0.6 were respectively obtained from the stress-strain curves, as shown in σ-lnm) of each node at different deformation temperatures was obtained. The results are shown in The cubic spline interpolation function was used to interpolate lnη) of each node at different deformation temperatures.Equations (3) and (4) were used to calculate the efficiency of the power dissipation (The cubic spline interpolation function was used to interpolate ln(m/(m + 1)) − lnEnergy dissipation maps and destabilization maps were drawn, and they were overlaid to obtain the processing maps of 21-4N heat-resistant steel.Drawing process of processing maps of 21-4N heat-resistant steel was as follows:η); the power dissipation increases gradually from the upper-left corner to the bottom right; the power dissipation increases with the increase in deformation temperature and the decrease in strain rate; the peak value of power dissipation shows little change with the increase in strain, indicating that the hot deformation process of 21-4N heat-resistant steel basically enters a steady-state deformation stage after the strain variable reaches 0.4, and rheological stress is relatively stable. When the strain is 0.2 (η reaches its peak at a deformation temperature of 1060–1120 °C and a strain rate of 0.01–0.1s−1, which is about 0.50. When the strain is 0.4 (η reaches its peak at a deformation temperature of 1120 –1180 °C and a strain rate of 0.01–0.1 s−1, which is about 0.49. When the strain is 0.6 (η reaches its peak at a deformation temperature of 1120–1180 °C and a strain rate of 0.01–1 s−1, which is about 0.46. Studies have shown that the larger the value of η, the better the machinability of the material, and the deformation domain may be in the best condition for the hot deformation process [n is 0.2 a, η reacn is 0.4 b, η reacn is 0.6 c, η reac process ,46,47.η appear below the right of the safe domain, in which the deformation is 1060–1180 °C, and the strain rate is 0.01–0.1 s−1. According to stress-strain curves, dynamic recovery and dynamic recrystallization occur frequently in this domain, indicating that the materials in this domain have good hot processing performance. The instability domain is concentrated in the upper part, in which the strain rate is 1–10 s−1 and η is at its minimum, and is not suitable for hot processing.−1. Optimum hot working parameters: deformation temperature of 1120–1180 °C, strain rate of 0.01–0.1 s−1. In this domain, η can reach more than 40% and 21-4N has good processing performance. When the deformation temperature is 1000–1120 °C and strain rate is 1–10 s−1, η is relatively small, and the 21-4N has poor machining performance. Therefore, it is necessary to keep away from this domain and the corresponding processing parameters during practical application.Based on the processing maps, the hot working process of 21-4N heat-resistant steel can be obtained: the domain suitable for deformation has a temperature of 1000–1180 °C and a strain rate of 0.01–0.1 sε = 0.6, −1, the microstructure of 21-4N after deformation at the indicated temperatures is shown in In the processing maps of 21-4N heat-resistant steel, the microstructure under different deformation conditions is different. To fully understand the deformation process of 21-4N heat-resistant steel, it is necessary to study the microstructure of different deformation regions. Take the processing map with a strain of 0.6 as an example to analyze the microstructure evolution law of the sample. When During hot deformation of 21-4N heat-resistant steel, rheological stresses decrease with increasing temperature and decreasing strain rate; rheological stress curves show obvious dynamic recrystallization at high temperature and low strain rate; with decreasing temperature and strain rate, rheological stress curves present obvious dynamic recovery; due to work hardening, stress quickly reaches peak, and then gradually drops to a steady rheological stress under the action of dynamic recrystallization, and enters the stable deformation stage.−1. Based on the hyperbolic sine equation, the relationship among the peak stress, deformation temperature and strain rate can be described by the constitutive equation as follows:The hot deformation activation energy of 21-4N heat-resistant steel is calculated to be 583.742 KJ·mol−1.The favorable hot deformation conditions are located in one domain of the processing maps, with a peak efficiency of more than 0.4 occurring at a temperature of about 1120–1180 °C and a strain rate of 0.01–0.1 sThe hot deformation characteristics of 21-4N were evaluated by high-temperature compression test in this study. The following conclusions were drawn from the main results: