The metallic edge states are revealed by scanning probe microscopy, scanning Kelvin probe force microscopy and first-principle calculations. Further analyses demonstrate that the edge-state dominated nonlinear transport shows a universal power-law scaling relationship with both temperature and bias voltage, which can be well explained by the 1D Luttinger liquid theory. These findings demonstrate the Luttinger liquid behavior in 2D materials and offer important insights into designing 2D electronics.In atomically-thin two-dimensional (2D) semiconductors, the nonuniformity in current flow due to its edge states may alter and even dictate the charge transport properties of the entire device. However, the influence of the edge states on electrical transport in 2D materials has not been sufficiently explored to date. Here, we systematically quantify the edge state contribution to electrical transport in monolayer MoS 2/WSe2 field-effect transistors, revealing a dominated non-linear edge transport at low temperature, indicating possible Luttinger liquid behavior.Though metallic edge states in monolayer transition metal dichalcogenide have been observed before, how these states contribute to charge transport remains unclear. Here, the authors quantify the edge state contribution to electrical transport in monolayer MoS An in-depth understanding of the surface states has enabled scientists and engineers to optimize the performance of integrated circuits during the past a few decades. Analogous to the surface states of bulk crystals, the edge termination and reconstruction in monolayer two-dimensional (2D) materials  and graphene) can lead to the edge electronic states5, which bring remarkable properties to these 2D materials and spark considerable intriguing applications6. The metallic electronic states at the edges of 2D transition metal dichalcogenides (TMD) have been predicted by first-principle calculations8. However, experimental investigations of the 2D edge states are scarce and little is known about the effects as well as the fundamental mechanism of edge states on electronic transport and performance of monolayer TMD-based electronic devices, which are critical for evaluating their performance for future nanoelectronic devices.Surface states of bulk three-dimensional (3D) semiconductors are critical factors to be considered in designing and fabricating electronic devices, as they can result in trapping and scattering, or acting as recombination centers for free carriers2 and WSe2 field-effect transistors (FETs) and discovery of edge state-induced nonlinear electronic transport and underlying mechanism. Scanning probe microscopy (SPM), scanning Kelvin probe force microscopy (SKPFM) and first-principle calculations reveal that the edge states dominate the charge transport and are responsible for this nonlinear behavior. We further find that the current–voltage characteristics of charge transport show a power-law scaling with respect to both temperature and bias voltage, which is consistent with Luttinger liquid theory and suggests that the edge states are one-dimensional metal with dominant electron–electron interactions.Herein we report a systematic investigation of the edge contribution to charge transport property in monolayer MoS2 flake  curves of the devices with different edge lengths and bulk area  increase with decreasing temperature, and become larger than 50% at the temperature below 50 K  FET with hole carrier is studied. It demonstrates a similar trend, with the edge conductance dominating the overall transport at lower temperature regime  conductive substrate, which is good for scanning tunneling microscope and spectroscopy (STM/S) measurements. Figure 2 island. The apparent height of the island is illustrated by the yellow profile line and is nearly 0.70 nm, which is consistent with the single-layer MoS2 feature. It is notable that no obvious height differences can be found between the edge and the bulk of monolayer MoS2 crystal in the AFM image, which is similar to former studies of MoS2 on HOPG surface13. Figure 2 island obtained in an ultrahigh-vacuum chamber (base pressure < 3 × 10−10 mbar). The profile line of the MoS2 island shows its apparent height of 0.72 nm, also confirming the monolayer nature of the MoS2 sample. Notably, the STM image reveals a brim with high brightness contrast around the edge of MoS2 island  measurements. Figure I/dV spectra taken across the MoS2 edge from HOPG to the bulk MoS2 (data-recording positions are shown in the inset). The tunneling spectrum (#3) acquired at the MoS2 edge shows clearly electronic states (marked by pink) around Fermi level compared to spectra (#4 & #5) from MoS2 bulk, revealing its metallic feature. The edge states stay consistently at different positions along the MoS2 edge, as shown in the Supplementary Fig. 2 (#4 & #5) show semiconductor feature.To get more detailed structural and electronic information about the edges, we zoomed in the area near the edge of the MoSpectrum # acquired17. The calculated band structure indicates that the appearance of additional bands within bandgap  and non-equilibrium Green’s function (NEGF) by using the Perdew-Burke-Ernzerhof (PBE) version of the generalized gradient approximation (GGA)2 domain by Atomistix Tool Kit (ATK)17 by constructing a two-probe model comprised of a semiconducting MoS2 sample with source and drain ends, in which the source and drain are defined by the highly doped (1014 e/cm2) n-type MoS2 to reduce extra calculation time . As the back-gate voltage increases from 0 to 0.5 V, a few more transmission eigenstates start to locate at the bulk of MoS2 channel . Two typical potential curves  are shown in Fig. 2 sample under the given bias voltage. Furthermore, the potential cross the MoS2 channel is simulated by the finite-element method as shown in Fig. 20.To directly reveal the role of the 1D metallic edge state in monolayer MoS2 FET device. Figure IDS–V12) curves of the device at large temperature range from 350 to 6.3 K, where the channel current decreases rapidly with decreasing temperature T. The linear (IDS–V12) characteristics are retained above 50 K, indicating an ohmic behavior. With further decreasing temperature, the characteristic (IDS–V12) curves show clear nonlinear behavior above the threshold voltages (VT)21  characteristics is due to Coulomb blockade (CB)22. However, this theory requires isolated conductive dots separated by a very thin tunneling layer, and the threshold voltage VT extracted in CB model depends linearly on temperature23. These two characteristics of CB model do not agree with our results, suggesting a new mechanism of nonlinear transport in our devices 21 Fig. . A possi26, which results in a power–law dependence of conductance G with temperature by formula of (G = I/V ∝ Tα) and the current with voltage by I ∝ Vβ, in which α and β are the tunneling exponents. In our experimental measurements, the conductance G = IDS/V12 shows a power–law behavior with temperature as G ∼ Tα and gives α = 2.2 at V12 = 0.1 V  curves at different temperatures can be obtained when IDS/T1+α is plotted against eV12/kBT as29:x) is the gamma function, kB is the Boltzmann constant, α and β are the tunneling exponents as mentioned above, I0 is the fitting parameter and γ−1 denotes the number of tunnel barriers in the transport path of Luttinger liquid.An additional feature of the Luttinger liquid theory is that the universal scaling curve of all  curves scale well onto a universal curve over the temperature range from 50 to 6.3 K. This universal curve can be well fitted to Eq. . Under these different gate voltages, the fitting parameter γ−1 based on Eq.  at low temperature shows nonlinear behavior and the corresponding curves can also be scaled to a universal curve with α = 2.15, β = 3, and γ−1 = 100 according to the Eq. . In these devices, the cross points obtained by fitting output data as IDS/Tα1+ against eV12/kBT are analyzed, and the linear dependence of γ−1 as function of edge length is again obtained in the Supplementary Fig. γ−1) increase with increasing channel lengths (edge lengths).This parameter α values extracted from MoS2 and WSe2 FETs, the electron interaction (MoS2) or hole interaction (WSe2) parameter g can be calculated to be 0.102 and 0.106, respectively  interactions along 1D charge transport path in edge-state-dominated MoS2 (WSe2) crystals and this result can be generally extended to all semiconducting TMD crystals.Based on the different 2 transistors. We note that the agreement with Eq.  extracted in our experiment from edge states, indicating that stronger electron–electron (or hole–hole) repulsive interactions along the edges of TMD than the one at the grain boundaries of TMD. In addition, a more general argument could be strengthened that metallic line defects (edges or grain boundaries) in 2D semiconductors all show Luttinger liquid behavior and are ideal 1D systems.We have witnessed two previous works demonstrate that the metallic mirror-twin boundaries in MoS35, in which the periodic lattice deformation, resulting in a sudden increase in sample resistance at the transition temperature. However, all the temperature-dependency resistances of our samples in the Supplementary Fig. 34. Thus, we believed that the likelihood of Peierls transition is low in metallic edge states of TMD  were exfoliated from the commercial MoS2 (WSe2) crystals (SPI Supplies) by scotch-tape method on Si/SiO2 substrate. The source and drain contacts (10 nm Ti/70 nm Au for MoS2 and 70 nm Au for WSe2) were formed through electron beam lithography (EBL), electron beam evaporation and then a lift-off process. The rectangular MoS2 (WSe2) channels were patterned by a second EBL step and subsequently etched using O2/SF6 plasma.Monolayer MoSV1 ~ V6) sense voltage drop. A bias voltage (VDS) is applied across the drain and source terminals. The channel current IDS is measured at the end of drain terminal. The voltage drops measured in this configuration can exclude any effects related to contacts, in which the voltage difference  is measured between the two inner terminals.Electrical measurements were conducted by using a Keithley 4200 semiconductor parameter analyzer and a Lakeshore probe station. The multi-probe measurement configuration is also shown in Fig. −10 mbar. Before STM measurements, the sample was annealed at 150 °C for over 12 h to remove possible adsorbates. A chemically etched tungsten tip was used for imaging. The STM images were recorded in constant current mode with tunneling current in the range 100–800 pA. The MoS2/HOPG sample was transferred to the ultrahigh-vacuum chamber and degassed at 150 °C for 12 h before STM analyses. STS measurements were using standard lock-in techniques with a voltage modulation of 10 mV and frequency of 973 Hz. Before real-sample STS measurements, the tip was calibrated on a clean Au(111).STM measurements were carried out in a custom-built multi-chamber ultra-high vacuum system. The base pressure was better than 3 × 1016. The local density approximation (LDA) and the wave function is expanded by the single-zeta plus polarization basis for all atoms17. The MoS2 device configuration and a cut-plane representation of the transmission eigenstates throughout the device central region under different gate voltages are shown in Fig. A, B, and C directions, respectively. The real space grid techniques are used with the energy cutoff of 150 Ry in numerical integrations. The quantum mechanical transmission probability of electrons T can be given as17:E, V are the energy and applied voltage, respectively, ΓL/R is the contact broadening functions related with the left/right electrode and GR/A is the retarded/advanced Green’s function of the central region.The electronic transport properties are carried out by ATK toolkit on the basis of density-functional theory in combination with the nonequilibrium Green’s functionFirst-principles calculations for edge states based on density functional theory (DFT) in Part S5 of Supplementary Information were conducted using the Perdew-Burke-Ernzerhof (PBE) version of the generalized gradient approximation (GGA).Supplementary Information


The marker SSR11-1, which is close to the locus, was tested on 78 different cucumber accessions and found to have an accuracy of 94% in resistant and moderately resistant lines but only 67% in susceptible lines. The mapped QTL was delimited within a region of 1,624.0 kb, and nine genes related to disease resistance were identified. Cloning and alignment of the genomic sequences of these nine genes between ‘65G’ and ‘02245’ revealed that Csa6M133680 had four single-base substitutions within the coding sequences (CDSs) and two single-base substitutions in its 3’-untranslated region, and the other eight genes showed 100% nucleotide sequence identity in their exons. Expression pattern analyses of Csa6M133680 in ‘65G’ and ‘02245’ revealed that the expression levels of Csa6M133680 significantly differed between ‘65G’ and ‘02245’ at 80 h after inoculation with CMV and that the expression in ‘02245’ was 4.4 times greater than that in ‘65G’. The above results provide insights into the fine mapping and marker-assisted selection in cucumber breeding for CMV resistance.The commercial yield of cucurbit crops infected with Cucumber mosaic virus (CMV) severely decreases. Chemical treatments against CMV are not effective; therefore, genetic resistance is considered the primary line of defense. Here, we studied resistance to CMV in cucumber inbred line ‘02245’ using a recombinant inbred line (RIL) population generated from a cross between ‘65G’ and ‘02245’ as susceptible and resistant parents, respectively. Genetic analysis revealed that CMV resistance in cucumber is quantitatively inherited. Analysis of the RIL population revealed that a quantitative trait locus (QTL) was found on chromosome 6; named  Cucumovirus in the family Bromoviridae ×100% .A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was used to determine the virus concentration in the apical leaves at 20 days after inoculation . The CMVThe 140 lines of the RIL population were used to construct a linkage map. Simple sequence repeat (SSR) markers were obtained from the genome of cucumber inbred line ‘9930’ . A total® 4.0, where the band pattern of the maternal parent is denoted as a, the band pattern of the paternal parent is denoted as b, and the heterozygous band as h [Genomic DNA was extracted from young cucumber leaves using the modified cetyl-trimethylammonium bromide (CTAB) method reported by Wang . PCR ampand as h .® Excel 2007. QTL analysis was performed via the interval mapping (IM) and multiple-QTL model (MQM) methods with MapQTL® 4.0 . The detected locus was named in accordance with the following scheme: the abbreviation of CMV, chromosome (Chr.) number, and locus number.Statistical calculations were performed by MicrosoftThe authenticity of markers that were closely linked to the CMV resistance gene was tested by the inheritance data of 78 different cucumber inbred lines and hybrids.http://cucurbitgenomics.org/). Gene function was predicted by BLAST based on sequence alignment with sequences in the SwissProt and Gene Ontology (GO) databases. Specific primers were designed to amplify the candidate gene sequences between ‘65G’ and ‘02245’. PCR amplification was performed in a total volume of 25 μL with KOD FX (Toyobo), and the PCR products were sequenced by Sangon Biotech (Beijing).The candidate genes for CMV resistance were identified by comparison with the reference genome of cucumber ‘9930’ (TM Reagent Kit with gDNA Eraser (TaKaRa). qRT-PCR was conducted using SYBR Premix Ex TaqTM II (TaKaRa), and PCR amplification was performed using a qPCR instrument. Actin1 was used as a reference gene for normalizing gene expression values. The analysis of candidate gene relative expression data was performed using the 2−ΔΔCt method.Young developed cucumber leaves  were collected from both parents and were flash frozen in liquid nitrogen; three biological replications of each sample were included. The total RNA was extracted using an RNeasy Plant Mini Kit (TaKaRa 9769) and a PrimeScript1 hybrids had a DI of 35.80 in 2016 and a DI of 34.72 in 2017 and exhibited symptoms that were more similar to the susceptible parent (‘65G’) than to the resistant parent (‘02245’)  . For the‘02245’) . The res‘02245’) .Of the total 1,228 SSR primers used to screen for polymorphisms within the parental lines, 296 (22.98%) were found to be polymorphic. Primers that were evenly distributed across the seven chromosomes were selected for constructing the linkage groups. Ninety-seven primers were selected for the RIL population; these markers were mapped onto seven linkage groups that covered 730.0 cM. The average distance between the markers was 7.5 cM, and each linkage group was distributed across approximately 8–32 markers . The sev® 4.0 identified a QTL for CMV on chromosome 6 (linkage group 6) in both years, which was named cmv6.1. The QTL for CMV was located within 32.7 cM on chromosome 6 and was delimited by two SSR markers, SSR9-56 and SSR11-177. The logarithm of odds (LOD) scores in 2016 and 2017 were 11.58 and 10.09, respectively, and accounted for 31.7% and 28.2% of the phenotypic variation, respectively , the genotype and phenotype were inconsistent. For three lines that were either resistant or moderately resistant , the genotype did not match the phenotype. Thus, marker SSR11-1 is 94% accurate in resistant or moderately resistant lines but 67% accurate in susceptible lines , 151 genes were predicted. Among these genes, nine related to disease resistance were identified. The predicted functions and associated information of these nine resistance genes are presented in The mapped QTL was found to be delimited within a region of 1,624.0 kb on chromosome 6, and after aligning with data from the Cucurbit Genomics Database  was used to examine the temporal expression patterns of Csa6M133680. The results showed that the expression level of Csa6M133680 significantly differed between ‘65G’ and ‘02245’ at 80 h after inoculation with CMV; in addition, the expression of Csa6M133680 in ‘02245’ was enhanced by CMV inoculation and was 4.4 times greater than that in ‘65G’ . TherefoThis study used the RIL population derived from a cross between cucumber inbred lines ‘65G’ (susceptible) and ‘02245’ (resistant) to determine the genetic basis for resistance to CMV. The DIs calculated for this segregating population were continuously distributed, which was appropriate for a quantitative inheritance model. These results are consistent with those of Kooistra and Wang  but diff2 population. These QTLs were named cmv1 through 17 and were located on chromosomes 3, 4, 5, and 6. cmv1 was mapped to chromosome 3, cmv2–cmv7 were mapped to chromosome 4, and cmv15–cmv17 to chromosome 5, the last of which had the largest phenotypic variation. cmv8–cmv14, which were mapped to chromosome 6, were located between markers SSRc68 and SSR20218  [Wang detected seventeen QTLs that control CMV resistance in a cucumber FRCY1, which is related to resistance to CMV in Arabidopsis thaliana [Rx and Rx2 genes, which provide resistance to potato virus X (PVX); the Y-1 gene, which provides resistance to potato virus Y (PVY) in potato; the Tm-22 gene, which provides resistance to Tomato mosaic virus (ToMV); and the Tm-2 gene, which provides resistance to TMV in tomato.Presently, ten virus resistance genes have been cloned from several plant cultivars . These gthaliana , encodesthaliana , 29; thecmv mapping region. Cloning and alignment of these nine genes between ‘65G’ and ‘02245’ revealed that Csa6M133680 had multiple single-base substitutions and that the Csa6M133680 gene product is a bZIP domain-containing transcriptional activator that is homologous to the gene product of RF2b in rice, which inhibits replication by the rice tungro bacilliform virus (RTBV)  .To further determine the possible candidate gene, we conducted qRT-PCR to analyze the temporal expression pattern of Csa6M133680 at different times after inoculation of the two parents. The results showed that the expression level of Csa6M133680 in ‘02245’ significantly increased at 80 h after inoculation with CMV and was 4.4 times greater than that in ‘65G’. Therefore, Csa6M133680 may be involved in CMV resistance regulation in cucumber, and additional fine mapping within the region delimited for CMV resistance will narrow the mapping region for the location of the resistance gene and help elucidate the mechanism of disease resistance.Viruses are the most important diseases of cucumber, and Watermelon mosaic virus (WMV), Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV) and CMV are the four main cucumber viruses. Genes involved in resistance to WMV, PRSV and ZYMV have been mapped to chromosome 6 –32. S2 FS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableCMV02245/CMV02245); b, 02245 allele (cmv02245/cmv02245).Marker genotype designation: a, 65G allele ((DOCX)Click here for additional data file.


Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations  also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product.  •Cell free Hemoglobin Based Oxygen Carriers can deliver oxygen to tissue.•Oxidation of ferrous hemoglobin to ferric and ferryl leads to oxidative toxicity.•Insertion of tyrosine residues enhances reduction of ferryl to ferric hemoglobin.•Some mutants (e.g. βT84Y) also enhance ferric to ferrous reduction by ascorbate.•βT84Y decreases cellular oxidative stress and enhances vascular retention in vivo. Outside the red blood cell, Hb toxicity is in part due to nitric oxide scavenging reactions Maintaining Hb in its functional oxygen carrying ferrous state is key to its function as an HBOC. Most HBOC as delivered contain a fraction (≅5%) of Hb in the ferric state (metHb). The steady state level of methemoglobin is a function of the balance between its formation via the autoxidation of oxyhemoglobin and the reduction of methemoglobin via methemoglobin reductase systems. Inside the red blood cell the enzyme methemoglobin reductase converts methemoglobin to oxyhemoglobin using the reducing power of NADH, although non-enzymatic mechanisms can also contribute Aplysia fasciata), which lacks any tyrosine residues. A variety of phenylalanine to tyrosine mutations were engineered with enhanced electron transfer pathways for ascorbate Aplysia mutations was orders of magnitude greater than that seen following the human mutation. To that end we engineered a range of new tyrosine mutations in the human Hb α and β subunits to see if a similar large increase could be observed. Surprisingly, we found that a subset of these mutations were also able to facilitate the reduction of ferric iron to ferrous iron i.e. return ferryl Hb to a fully functioning ferrous oxy/deoxy oxygen transport molecule. As the ferric form of Hb is the most unstable, triggering heme loss and protein degradation The metHb levels are also important as increasing metHb induces oxidative stress in vivo by reacting with peroxides 22.1E. coli cells carrying the adult or fetal Hb gene were cultivated in a 5 L fermenter . Hb was grown according to methods previously described The cloning, expression and purification of the Hb proteins for in vitro studies was carried out as previously described 2.2CO removal from stored Hb was carried out by illuminating CO hemoglobin samples through a 75 W Xenon to promote photolysis under 100% oxygen flow. CO removal and substitution with oxygen was confirmed by collecting absorption spectra. All concentrations of Hb used were calculated as the tetrameric form, and were commonly 60 µM. Inositol hexabisphosphate (IHP) was added to the Hb solutions to give the ratio of 1.2:1 (tetramer) and deoxygenated under helium flow and gentle shaking for 4 h, at 20 °C. Iminothiolane (IMT) was added to the Hb solutions to give the ratio of 80:1 (IMT:tetrameric Hb) and after 6 min maleimido-PEG added to give the ratio 12:1 (PEG:Hb tetramer). The reactions was stopped with 5.3 M lysine pH 6.5–7.5 after 34 min and 0.9 M cysteine after 46 min. All solutions were prepared using deoxygenated water and kept under nitrogen flow before addition. PEG-Hb solutions were dialyzed in phosphate buffered saline (PBS) using Spectrum Hollow Fibers  to remove unreacted PEG, IMT, lysine, cysteine and IHP. Endotoxin was removed as described previously 2.32 gas mixtures as previously described Upon CO removal Hb solutions left overnight under helium flow in the presence of the Hayashi reducing system before titrations 2.4obs min-1) of the rHb proteins was measured at 37 °C. For each protein, the autoxidation (oxy to met conversion) was measured, and the kinetic traces (577–630 nm) were analysed by single exponential fittings to give kobs (min-1) values. Ferric reduction by ascorbate (met to oxy conversion) was measured at 25 °C in 20 mM NaPi, pH 7.20 and the extent (%) of oxyHb formed calculated with reference to the 100% met and oxyHb spectra of the relevant mutant. Heme loss from metHb (1.7 μM) was measured in 20 mM NaPi, pH 7.20, 37 °C by monitoring the high to low spin state change when heme binds to 2 µM of the high affinity heme scavenger hemopexin . Spectra were taken between 370 and 650 nm and the 417–599 nm difference fitted to a single exponential decay. Ferryl hemoglobin was made by adding a 3:1 ratio of H2O2:metHb and waiting 10–15 min until the spectral change was complete . Trace catalase (1–5 nM) was then added to remove unreacted H2O2. Ferryl reduction (ferryl to met) by the reductant CP20  was analysed by double exponential fits of the time courses (545–630 nm), assigning the faster phase to the α subunit and the slow phase to the β subunit as described previously max /Km was approximated from the respective SEM assuming the variables were independent and using a first order Taylor expansion. Var(x/y) = x2/y2 * ( Var(x)/x2 + Var(y)/y2).Spectra were recorded on a Varian Cary 5E spectrophotometer. The autoxidation , with or without 50 μM ascorbate to the media to a total volume of 250 μL. Cells were placed a plate-reader (Tecan Infinite M200Pro) at 37 °C, 5% CO2 and absorbance spectra 500–650 nm collected every 10 min for 18 h. Kinetic traces were produced by a three-point drop correction across the 577 nm oxy peak using 562 and 592 nm as reference wavelengths. Cellular damage was assessed by measuring levels of lactate dehydrogenase (LDH) released into cellular media after 18 h of exposure to the experimental treatment. The LDH assay  was performed according to manufacturer's instructions. The absorbance at two wavelengths (450 and 650 nm) was obtained using a Tecan Infinite M200Pro plate reader and both values were used to calculate levels of LDH.The metHb proteins were prepared as previously stated 2.63 in an aliquot of 0.8 mL. Next, 1 mg Cy5 was dissolved in 200 μL dimethyl sulfoxide . Subsequently, 1000 μL of PEG-Hb solution and 200 μL Cy5 solution was stirred and mixed at 400 rpm for 5 h at room temperature. The resultant reaction mixture was purified to remove unreacted Cy5 using Size Exclusion Chromatography (SEC) in a NAP 10 column  for cleaning in two steps  using the eluent physiological saline and was collected as in a volume of 1200 μL. For further purification details and characterization see Following endotoxin removal, the PEG-Hb was lablelled with a Cyanine5 dye NHS ester  red fluorescent dye . The pH of the PEG-Hb solution was adjusted to 8.5 with 400 μL of 0.1 M NaHCOAnimals were obtained from Charles River GmbH, Germany. NU(NCr)-Foxn1nu/nu nude mice  were allowed free access to food and water and maintained under temperature, humidity, and light-controlled conditions. All procedures were conducted in accordance with the ARRIVE guidelines and the guidelines set forth by the European Communities Council Directive (86/609 EEC) and approved by the Animal Care and Use Committee of the IEM and the Semmelweis University (XIV-I-001/29–7/2012). In the fluorescent whole-body imaging experiments, the animal was initially anesthetized by Medetomidine (1 mg/kgbw) + Ketamine (100 mg/kgbdw). Anesthesia was maintained using continuous inhalation of 2% isofluorane in medical grade oxygen gas. While being scanned, the animal was placed on heating mat to maintain the rectal temperature at 37.5 °C.The whole-body biodistribution of Cy5-labeled PEG-Hb was measured in vivo at pre-injection and at different post-injection time points . Anesthetised mice were infused by top-loading via the tail vein at a rate of 0.2 mL/min 2). After a calibration measurement of the sample holder's autofluorescence, the same holder with the thin blood film was put into the FOBI optical imaging system and imaged with integration time of 1000 ms and gain level 1. This procedure allowed for repetitive measurements of blood levels of Lumiprobe Cy5 dye either in its free form (control) or bound to PEG-Hb throughout a period of 24 h. Lumiprobe Cy5 NIR fluorescence intensity was normalized to the pre-injection level (i.e. autofluorescence). Following the last imaging time point (24 h) whole-body saline perfusion was applied for 6 min in order to clear blood from the organ tissues. Subsequently, organs were removed and their ex vivo fluorescent images taken with 1000 ms exposure and gain level 1. Mice were terminally anesthetised by the dedicated veterinary use euthanasia drug Euthasol® (ad us. vet. injection).The blood level of labeled PEG-Hb and free Cy5 was measured by smearing one drop of 100 μL blood freshly drawn the from the tail vein onto the white plastic surface of a standardized balance plastic sample holder . Interestingly the addition of a second tyrosine near the α heme (αL91Y) had only minimal or negative effects, suggesting a limitation for heme reduction that is independent of the pathway of electrons arriving at the physiological tyrosine electron donor (αY42). In contrast, inserting a tyrosine residue close to the β heme  increases both the Vmax and lowers the Km for ferryl heme reduction in this subunit. This results in a ten-fold increase in Vmax /Km (from 0.07 in WT to 0.6–1.2 ×10-3 μM-1 min-1 depending on the mutant). The precise position of the tyrosine seems relatively unimportant; “unphysiological” positions are at least as effective as inserting the tyrosine at the homologous site to that in the α subunit (βF41Y). Unsurprisingly, the insertion of a tyrosine near the α heme (αL91Y) did not create a high affinity route for electron transfer to the β heme. One mutant (βK66Y) showed apparently much larger rates of ferryl to met conversion in both the α and β subunits. However, on closer inspection the final spectrum formed was significantly different to the original met spectrum confirming degradative events other than simple reduction were occurring in this mutant Aplysia Mb mutants A comparison was then made of ferryl heme reduction using physiological levels of the plasma reductant, ascorbate . After oAlthough metHb is less oxidatively damaging than ferryl Hb, it is not functional in oxygen transport and can be readily converted back to ferryl by peroxides, a process that additionally forms reactive protein-bound free radicals. It would therefore be beneficial for an extracellular HBOC to be further reduced from ferric metHb to ferrous oxyHb. In the plasma only ascorbate has the reducing power to effect this conversion. The reaction is biphasic with the faster reaction rate generally attributed to reduction of the β subunit NADH and glutathione showed similar direct nonenzymatic rates of metHb reduction in WT compared to ascorbate at the same (30 μM) concentration after 60 min incubation. However, there was no increase in oxyHb formation in the βT84Y mutant .A closer examination of the rate of oxyHb formation at different ascorbate concentrations , revealsCombining mutations in the α and β subunits that enhance ascorbate reducibility did not have an additional beneficial effect . CrucialThe stability and oxidative reactivity of the ferrous form βT84Y was compared to WT following addition to HEK cells. Phenol red free DMEM media was used during the Hb stability and Hb-induced cell damage studies as phenol has the ability to reduce higher oxidation states of Hb and interferes with spectrophotometric assays. Hb was added to cells in both the oxy and met forms and in the presence and absence of ascorbate . FollowiThe effect of the βT84Y mutation on in vivo vascular retention time of a HBOC was tested in a mouse top-load model. First the control (αV1M βV1M) and test (αV1M βV1M βT84Y) proteins were PEGylated in the deoxy form using the EURO-PEG-Hb method to prevent rapid renal clearance post mortem direct NIR imaging of the organs cleared from blood are also demonstrated  tyrosine redox centre in the α subunit (αL91Y) did not seem to greatly enhance reduction at this site, although there was a modest increase in the overall rate, possibly via the creation of a new pathway to the β heme.Aplysia Mb model system Aplysia sites  would have to be multiplied by a low electron occupancy of the relevant reactive reduced species to generate the effective rate, the deprotonated tyrosine radical and the protonated ferryl being the relevant reactive species. It is possible that the faster rate in Aplysia is therefore due to a higher intrinsic pK of the protonated ferryl, resulting in a larger population of the reactive species. A limitation due to the intrinsic nature of the ferryl heme in different species/proteins would also explain why changing the position of the redox active tyrosine in Hb has only a small effect on the electron transfer rate. However, as even this relatively modest increase is accompanied by a decrease in lipid peroxidation in cell free assays If the reduced tyrosine radical is formed at 100% occupancy by the addition of saturating levels of reductant the V4.2Given the high oxidation potential -1 s-1 and enhanced by the addition of organic phosphates -3 and 10-2 M-1 s-1– this paper and The ability of ascorbate to reduce directly human met to oxyHb was first shown in the 1940s A more detailed examination of the ascorbate metHb reduction rate showed that the reaction proceeded in two stages, with the reduction of the β being significantly faster than that of the α subunit The results in this paper significantly enhance our knowledge of the mechanism of ascorbate reduction of metHb. -2 M-1 s-1) is consistent with that previously reported for metMb and therefore likely relates to the direct second order reduction of the heme by ascorbate. A more positive heme redox potential was postulated to underpin an enhanced reducibility of the ferric heme in Lumbricus Hb Although the ferric reduction kinetics by ascorbate are similar to the ferryl reduction kinetics in having a specific high affinity saturable component and a non specific low affinity component, the underpinning mechanisms are different. This is why there is no correlation between those mutants that enhance ferryl reducibility and those that enhance ferric reducibility . Table 4The faster rates at low ascorbate concentrations are likely caused by the specific ascorbate binding site on the β subunit. The addition of tyrosine residues that decrease this rate  might act via minor structural changes to the protein perturbing the ascorbate binding site. Mutations that increase this rate  might also act on the binding site, but it is equally possible that they enable an additional electron transfer pathway from the ascorbate binding site to the β or even the α heme. This seems especially likely for the α subunit mutation (αL91Y) and the β mutation that has the fastest rate of electron transfer (βT84Y).4.3Lumbricus) has also been promoted as a benefit of HBOC We have previously demonstrated that engineered human HBOC with an increased reducibility of ferryl Hb by physiological plasma reductants decrease Hb-induced oxidative stress PEG-Hb labeling studies showed the PEGylated proteins had a plasma half time of several days  and 10, The βT84Y product was more PEGylated  than WT 5de novo, HBOC cause increased oxidative stress Ascorbate has been shown to be the key plasma antioxidant for HBOC, maintaining it in the ferrous state and reducing oxidative species such as ferryl and protein free radicals Biotechnology and Biological Sciences Research Council, United Kingdom for financial support (BB/L004232/1).We would like to thank the CEC, BJR and GGAS have patents granted and pending relating to modification of hemoglobin amino acids designed to render a blood substitute less toxic and are shareholders in a related company (CymBlood).


Ovarian cancer is a common gynaecological cancer with a poor prognosis that poses a serious threat to human life and health. It is essential to explore the possible prognostic biomarkers of ovarian cancer. As an important tumour suppressor gene, BCL7A actively participates in the growth of tumours. We aimed to study the prognostic role of BCL7A in ovarian cancer.Through data mining of RNAseq data from the Cancer Genome Atlas database (TCGA), we explored the clinical relevance of BCL7A mRNA expression. As a result, we found that BCL7A is expressed at low levels in ovarian cancer tissues and is correlated with survival status. Survival analysis showed that, compared with those who had higher levels of BCL7A expression, patients with ovarian cancer and low levels of BCL7A generally had shorter overall/relapse-free survival times. Cox regression models showed that low BCL7A expression could be used as an independent prognostication factor for ovarian cancer patients.Low BCL7A expression is an independent risk factor for poor prognosis in ovarian cancer patients. Ovarian cancer is a common gynaecological cancer; its mortality rate ranks first among gynaecological tumours and it poses a serious threat to human life and health , 2. In mAs a key molecule of chromatin remodelling, BCL7A is involved in the process of carcinogenesis. BCL7A is expressed in the ovary, brain, lymph nodes and other tissues. The gene is located on chromosome 12q24.31, and 1 natural variant has been found so far. BCL7A plays an important role in human cancer. It has been found that Myc and IgH are directly involved in the three-way gene translocation of Burkitt lymphoma cell lines, which may contribute to the progression of Burkitt lymphoma , 7. In aTo further evaluate the clinical significance of BCL7A in the prognosis of ovarian cancer patients, we examined the differential expression of BCL7A mRNA in ovarian cancer by exploring the Cancer Genome Atlas database (TCGA). The chi-square test and Fisher’s exact test were used to evaluate the clinical correlation. Survival analysis and a Cox regression model were employed to identify the correlation between BCL7A and ovarian cancer patients’ survival rate.BCL7A expression data and clinical features from the TCGA database, including age, lymphatic invasion, stage, histologic grade, subdivision, new type, longest dimension, sample type, vital status and BCL7A expression, are shown in Table Boxplots revealed that lower BCL7A expression was found in ovarian cancer tissues compared with normal ovarian tissues Fig. . In addiWe used the median of BCL7A expression as a cut-off to divide patients into a BCL7A high-expression group and a low-expression group, and there was no difference in the clinicopathological parameters between the two groups Table .Table 2TP = 0.034), which was consistent with the results of subgroup analysis, especially for advanced stage (P = 0.012), G3 and G4 (p = 0.0097), young patients (p = 0.038) and older patients (p = 0.049).As shown in Fig. P = 0.005, Table A univariate Cox model showed that age and BCL7A expression were two survival related variables. A multivariate Cox model suggested that low BCL7A expression was an independent risk factor for ovarian cancer patients’ overall survival , which was consistent with the results of subgroup analysis, especially in advanced stage (P = 0.014), G3 and G4 (p = 0.0014), and young patients (p = 0.003).As shown in Fig. P = 0.011, Table A univariate Cox model showed that BCL7A expression was the only relapse related variable. A multivariate Cox model suggested that low BCL7A expression was an independent risk factor for ovarian cancer patients’ relapse-free survival  and GTEx , were utilized. Level 3 data were downloaded from the databases. This dataset shows the gene-level transcription estimates, as in log2(x + 1) transformed RSEM normalized counts. There is no existing ethical conflict because all data we used in this study are permitted to be used in research.We have obtained currently available clinical and RNAseq data from both normal and cancerous ovarian tissues. Databases, including TCGA (R (version 3.5.1)  was used


Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved. Changes in S-isotope ratios over time provide clues to understanding the co-evolution of Earth and its biosphere. Here the authors determine the isotope effect of the first reductive enzyme in the sulfate respiration pathway and reinterpret sedimentary S-isotope records based on this biochemical constraint. The sulfide produced by microbial sulfate reduction (MSR) readily reacts with iron to form iron sulfide and in turn pyrite, a redox sink for sulfur and iron4. Thereby, this metabolism provides a linkage between the global sulfur cycle with those of carbon, oxygen, and iron. Given the presence of four stable sulfur isotopes , MSR discriminates against heavy isotopes, resulting in isotope signatures which can be recognized in modern aquatic environments and are also preserved in sedimentary sulfate and sulfide phases over geologic time5. Sulfur isotope fractionation has thus been used both as a diagnostic marker for sulfate reduction and as a tool to reconstruct the early evolution of sulfur metabolisms and ocean redox chemistry10. In particular, small 34S/32S fractionations between sulfate and sulfide before the great oxidation event (GOE) have been considered to reflect low-sulfate concentrations in the Archean oceans12. Such interpretations are grounded in the results of extensive culture experiments over the last half-century, which have revealed a set of relationships between microbial respiration rates, sulfate concentrations, and the kinetic and equilibrium isotope effects controlling net fractionation24. A complete mechanistic understanding of the variables that control sulfur isotope fractionation has yet to be achieved, however, in part due to a lack of information on the individual intracellular processes which cumulatively give rise to net sulfur isotope fractionation between sulfate and sulfide.Sulfate is quantitatively the most abundant water-soluble electron acceptor for biological respiration on the planet26, and fluxes through potential branch-points27. The enzymatic isotope effect is typically a reflection of the transition state formed during the reaction, and flux through a pathway and its branch-points is a complex function of physiological state and environmental conditions. To date, a few experimental constraints on metabolic sulfur fluxes have been imposed either by using the oxygen isotope exchange between sulfite and water24 or by measuring the sulfur isotope compositions of metabolic intermediates in the MSR pathway28. Those studies illustrated the principles underlying physiological variations in sulfur isotope fractionation, but did not give a quantitative measure of the degree of fractionation: it is the enzyme-specific isotope effects which represent the end-members of the kinetic isotope fractionation range. Knowledge of these values will allow for a quantitative interpretation of sulfur isotope fractionation arising from sulfate reduction and a linkage of isotope fractionation to the physiological state of the cell.Kinetic isotope fractionation between sulfate and sulfide is, in theory, dependent on the isotope effects of the enzymes involved, the relative rates of each enzymatic reaction26. Recently, Leavitt et al.29 conducted the first enzyme-specific sulfur isotope fractionation measurements, using DsrAB, a partial enzyme complex of dissimilatory sulfite reductase, and found the produced thiosulfate and trithionate to be depleted in 34S by 15‰ at the reduced sulfur S0 position of those compounds relative to the reactant sulfite. Although this reaction system lacked the DsrC subunit required to achieve full conversion of sulfite to sulfide30, the measured fractionation was much smaller than the previous theoretical value of ca. 50‰26, revealing possible discrepancies between experimental and indirect fractionation estimates and further motivating the need for the experimental assessment of enzymatic isotope effects. Remaining to be determined is the sulfur isotope fractionation by Apr, the first reductive enzyme in the MSR pathway, as well as for the full DsrABC complex. Fractionation by the Apr enzyme is predicted to exert greater control on the overall fractionation of the whole pathway as the Apr step becomes rate limiting at fast respiration rates31. So far, a normal isotope effect of 25‰ has been inferred for Apr26, but this value has never been measured.MSR to sulfide is carried out by three different enzymes: sulfate adenylyltransferase , adenosine phosphosulfate (APS) reductase Apr; EC 1.8.99.2), and dissimilatory sulfite reductase   were calculated to be 20.3 ± 0.5‰ and 20.1 ± 0.8‰ at 32 and 20 °C, respectively. This is the first experimental constraint on sulfur isotope fractionation by Apr, and is about 5‰ smaller than the previously inferred value of 25‰26 that was based on the equilibrium sulfur isotope fractionation between sulfate and sulfite at a physiologically relevant temperature26. Because the equilibrium isotope effect for reversible reactions equals the difference between kinetic isotope effects for forward and backward reactions33, the assumption of a 25‰ fractionation value is equivalent to assuming no kinetic fractionation in the backwards reaction. Our new measured value of 20‰ for the forward (reductive) direction suggests that Apr likely exhibits a small, inverse isotope effect when operating in the reverse (oxidative) direction. We note that an enzymatic preference toward heavy isotopes, while rare, has been previously reported in other reactions, e.g., for nitrite oxidation36. °C Fig. . As the 34S Fig. , consistres Fig. , indicatres Fig. . From th37. While many enzymatic reactions display weak temperature dependence of the kinetic isotope effect at the actual catalytic step, the steps in the reaction which precede or follow the catalytic step might contribute to the temperature dependence of the observed isotope effect40. The similarity of the measured sulfur isotope effects at 20 and 32 °C despite the nearly quintupled rate therefore indicates that the catalytic bond-breaking step is nearly rate-limiting for APS reduction, rather than substrate binding or product release. In contrast, fractionation of sulfur isotopes in whole cell studies has been found to change with temperature41 and also to be inversely correlated to the cell-specific sulfate reduction rate (csSRR)18. These differences between enzyme- and cellular-level fractionations suggest that the relationships between physiology and sulfur isotope fractionation are not rooted in the kinetics of an individual enzyme, but are instead expressed by the overall sulfate reduction pathway. We explore this next with the aid of a recently established model framework31 and our quantitative constraint on the sulfur isotope effect of Apr.The almost identical fractionations at two different temperatures may have implications for whole cell-derived isotope fractionation, as well as for the mechanism of enzymatic catalysis. APS reduction is thought to be a multistep reaction, which begins with nucleophilic attack on the sulfur of APS by the flavin cofactor FAD, followed by loss of AMP during collapse of the covalent APS-flavin adduct31. The less reversible the reaction, the less isotope effects of the subsequent reactions will be expressed. Reversibility can be recognized as a measure of the relative degree of rate limitation at different steps in a pathway, which is ultimately set by the thermodynamic conditions of the reaction43:b and f denote backward and forward rates, R is the gas constant, T the temperature, and ΔG is the free energy change associated with the reaction. As the value of ΔG strongly depends on the concentrations of involved reactants and products, Wing and Halevy31 proposed a model with intracellular metabolite levels as the central determinant of reversibility and thus sulfur isotope fractionation. We updated their model to include the newly determined value for 34εApr and used it to explore varying redox potentials of the electron donating half-reaction at constant sulfide concentration  greater than −5 kJ mol−1, the resulting reversibility is as small as 0.1 . Although such a small difference between the redox potentials of electron carriers and APS  can contribute to maintaining reversibility in APS reduction and thereby produce larger isotope fractionation, such low energy yields make it difficult to account for the energy needed to generate a proton gradient that is presumed to be coupled to the oxidation of membrane-bound electron carriers56. However, if electron flow from physiological carriers to APS or sulfite were to be directly coupled to proton motive force generation, the reaction energies of these processes would be added to the expected energies of the APS or sulfite reduction. Because ion translocation to the outside of a chemiosmotically charged membrane is endergonic, the reversibility of a directly coupled APS or sulfite reduction step would increase as a result of the additional energy requirement , it may0.1  would likely account for a substantial fraction of pyrite formation, like those in some modern euxinic basins63. More importantly, neither scenario explains why the maximum sulfur isotope fractionation during the Archean would be so similar to the sulfur isotope effect of APS reductase without exceeding it.In contrast to modern environments, sulfur isotope fractionations between sulfate and sulfide rarely exceed 20‰ in rocks older than 2.5 Ga Fig. . Such sm2, play a role in controlling the expression of sulfur isotope fractionation. Previous culture studies have demonstrated qualitatively that decreasing availability and reactivity of electron donors leads to larger sulfur isotope fractionation19, and our experimental determination of the Apr kinetic isotope effect provides quantitative support for these qualitative arguments. Based on the results above, we consider the possibility that the availability of more favorable electron donors was responsible for reduced sulfur isotope fractionation during the Archean. In an anoxic ocean harboring other microorganisms competing for reductants, using sulfate as an electron acceptor would have been a major evolutionary innovation. Sulfate-reducing microbes would likely have outcompeted ancient methanogens and acetogens for common electron donors such as H2, because sulfate reduction provides a greater free energy yield than CO2 reduction66. Considering H2 as an electron donor, at 100 µM concentration of sulfate and 10 nM H2, hydrogen oxidation coupled to sulfate reduction still yields −43.5 kJ mol−1, while methanogenesis provides only −13.7 kJ mol−1; acetogenesis is unfavorable at +20.4 kJ mol−1. In addition to the thermodynamic favorability of sulfate reduction when considering common electron donors, sulfate-reducing microorganisms would be able to utilize higher potential (lower energy yielding) reductants for respiration, again by virtue of delivering electrons onto sulfate, although these low-energy electron donors would likely have been utilized only if high-energy ones were substantially consumed. Competition with iron reducing microorganisms could also be considered. However, limited access to iron-bearing detrital phases yields lower rates of iron reduction in the water column67, and sulfate reduction often precedes iron reduction even in the low-sulfate freshwater sediments68. The absence of sulfur isotope fractionations greater than 20‰ is thus consistent with the hypothesis that privileged access to electron donors would be able to drive the Apr reaction irreversibly. A similar scenario could explain, at least in part, why sulfur isotope fractionation larger than 20‰ became widespread after 2.5 Ga, when the GOE, triggered by the rise of oxygenic photosynthesis, made O2 available as an electron acceptor69. With increasing oxygen concentrations, aerobic microorganisms could outcompete sulfate reducers for access to electron donors, relegating sulfate reducers to lower net energy environments. Adaptation to this thermodynamically less favorable lifestyle may have made the MSR reaction more reversible, leading to larger fractionation.Not only sulfate concentration but also electron donors, such as organic matter or H2, also seems plausible in accounting for the rise in sulfur isotope fractionation values after the GOE. Regardless though, it is now clear that the electron donor is a contributing determinant to isotope fractionation during MSR, with the Apr-catalyzed reaction exerting significant control on the observed fractionation. Consequently, differences in δ34S values of sulfate and pyrite cannot be used as a quantitative constraint on sulfate concentration, e.g., in the Archean oceans12, without additional information about the reductant pool.This hypothesis is yet speculative, and is not exclusive of other effects such as those from low-sulfate concentration. Indeed, simultaneous loss of electron donors and increasing sulfate concentration, both as a result of increasing atmospheric ODesulfovibrio vulgaris Miyazaki as previously described70, flash frozen in 75 mM Tris-HCl buffer (pH 7.5), and stored at −80 °C for later assay. All chemicals, except for APS, were used as received from the manufacturer without further purification. The APS sodium salt was obtained from Sigma-Aldrich (CAS No. 102029-95-8), and given the relatively low-purity grade (85%) it often contained >10% sulfate (mol/mol) as impurity. The stock solution of APS (~40 mM) was prepared using degassed deionized water, split into a series of 250 μl aliquots, and stored at −80 °C until needed. Upon thawing, 50 μl of 0.2 M BaCl2 solution was added to each aliquot, and precipitated BaSO4 was removed by centrifugation. Excess Ba2+ was eliminated from solution by adding 100 μl of 0.1 M Na3PO4 solution, and again, the insoluble Ba2(PO4)3 was removed by centrifugation. After dilution, the final APS concentration in the working stock solution was set to 7.5 mM. A few tens of mM phosphate would be present in this working stock, which was not quantitatively significant because more than ten times larger amount of phosphate was added to the reaction mixture to buffer the pH  equipped with an AS11-HC column , using a gradient elution with KOH as mobile phase28. The concentrations determined by IC are subject to an error of ±5%. For isotope analysis, the eluant fractions corresponding to sulfate and APS were collected, and APS was quantitatively converted to sulfate via hydrolysis at 65 °C for 12 h.APS reductase enzyme was purified from 74. Samples were introduced to plasma via an ESI PFA-50 nebulizer and Cetac Aridus II desolvator. Sulfur isotope ratios of the sample and working standard were measured in 50 alternating cycles, and instrumental blank was estimated after each sample block. The mean blank value was subtracted from the measured signal for each mass. The measured 34S/32S ratio was calibrated using a linear interpolation between the two bracketing standard values. Sulfur isotope ratios are reported here using the conventional delta notation:34Rsample and 34RVCDT are the 34S/32S ratios of sample and Vienna-Cañon Diablo Troilite (VCDT), respectively. Our working standard was calibrated against the IAEA S-1 reference material (δ34SVCDT = −0.3‰) and has a δ34S value of −1.55‰ ± 0.16 (2σ) on the VCDT scale. Analytic reproducibility for δ34S has been previously evaluated as ±0.2‰ (2σ)74. Measured concentrations and isotopic compositions of APS and sulfate were summarized in Supplementary Table δ34S(x) is the sulfur isotopic composition of the total, APS, or sulfate sulfur pools, and [APS] and [SO4] are their molar concentrations. Calculated isotopic compositions of total sulfur varied within 10% of that of the beginning, which is a threshold value commonly used in isotope studies75. Closure of isotope mass balance indicates that assumption of a closed system is valid, and no unknown sulfur pool was present in our experiments. As shown in Supplementary Table δ34S value of accumulated sulfate from that of sulfite produced during the assay. Given that no additional sulfate evolved in the control experiments, the contribution from an initial sulfate pool was removed via isotope mass balance:δ34S(SO3) is the isotopic composition of produced sulfite, δ34S(SO4) and δ34S(SO4)0 are sulfur isotope compositions of the accumulated and initial sulfate, and [SO4] and [SO4]0 are sulfate concentrations at the time of sampling and at time zero. Errors from isotope analysis via ICP-MS (0.2‰) and concentration measurement via IC (±5%) were propagated via first-order Taylor series expansion, resulting in an uncertainty estimate for δ34S(SO3) values of better than 0.5‰. Sulfur isotope fractionation factor (34εApr) was calculated using an approximate solution to the Rayleigh distillation equations76:f is the fraction of the remaining APS, δ34S(APS)0 and δ34S(APS) are sulfur isotope compositions of the initial and remaining APS, respectively, and δ34S(SO3) is isotopic composition of produced sulfite. Using linear regression analysis, values of 34εApr were obtained from the slope of -lnf versus δ34S(APS) and (f·lnf)/(1 − f) versus δ34S(SO3). All analytical errors were propagated via Monte Carlo simulation (n = 5000).The samples containing dissolved sulfate were dried on a hot plate and diluted in 5% nitric acid to a sulfate concentration of 20 µM to match the in-house working standard. Isotopic analysis was conducted on a Thermo Fisher Scientific Neptune Plus multi-collector inductively coupled plasma mass spectrometer (MC-ICP-MS), operated in medium resolution following the method previously described31, we ran the model with a constant sulfide concentration but varied redox potential for the electron carriers coupled to the MSR pathway  in the Archean rocks. Since the proliferation of Archean 33S studies has also yielded a record of the ratio between the more abundant isotopes, 34S and 32S, we supplement the existing compilation with the Archean δ34S data collected during the last decade (Supplementary Data Sulfur isotope data from modern environments, shown as a histogram in Fig. Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Caenorhabditis elegans, demonstrating for the first time the presence of blocking in this animal, and dissociating it from context conditioning. We present an initial genetic dissection of these phenomena in a model benzaldehyde/NH4Cl aversive learning system, and suggest that blocking may involve an alteration of memory retrieval rather than storage. These findings offer a fundamentally different explanation for blocking than traditional explanations, and position C. elegans as a powerful model organism for the study of higher order conditioning.Higher-order conditioning phenomena, including context conditioning and blocking, occur when conditioning to one set of stimuli interacts with conditioning to a second set of stimuli to modulate the strength of the resultant memories. Here we analyze higher-order conditioning in the nematode worm  Learned associations about the external world are a valuable source of information for animals to exploit in guiding their behavioral choices, but constraints must exist on what associations are learned if they are to remain predictively useful. In rodents, context conditioning and blocking have both been characterized as mechanisms by which memories interact to attenuate or enhance their effects, providing mechanisms by which knowledge about one association can inform the value of others. These “higher-order conditioning” phenomena provide a powerful means where by simple, one-to-one associations about the world can combine in predictively useful ways.1, and to a more limited degree in invertebrates4. Context conditioning has been demonstrated in Caenorhabditis elegans for both associative5 and non-associative6 memories.In context conditioning, context cues present during training become associated with a memory such that retrieval of the memory is enhanced in the presence of the context cues, and hindered in alternate contexts. The role of context in associative learning has been extensively studied in rodents and humans  and a complex conditioned stimulus (CS1 + CS2) is prevented, or “blocked”, if one of the components of the conditioned stimulus is already associated with the US. Blocking is often explained as a failure to attend to the non-associated component of the conditioned stimulus, because the prior association between the associated component and the unconditioned stimulus is sufficient to fully predict the US. In this view, it is necessary that the US be “surprising” to the subject to form new associative memories9, which proposed that conditioned stimuli become associated with an unconditioned stimulus based on the scale of the mismatch between the strength of pre-existing associations with the US and the strength of the present association.Blocking offers some of the strongest evidence that learning depends not on mere temporal contiguity, but on contingency, and is now viewed as an essential phenomenon for any viable theory of learning to explain. The insight that a cue must be “surprising” to be learned about was later expanded and formalized in the influential Rescorla-Wagner model of classical conditioning10, and researchers have advocated for the adoption of simpler model systems to further our understanding of it11.Despite blocking having been the subject of extensive investigation in mammals, and having been enormously influential on theories of learning and memory, the neural and molecular mechanisms underlying it remain poorly understood. Studies of blocking in rodents have been plagued by problems of replicabilityApis mellifera), intramodal blocking has been demonstrated12 for training to some odorants. The sea slugs Hermissenda crassicornis and Aplysia californica both appear to exhibit blocking13, as does the terrestrial slug Limax maximus14. Conversely, learning in the fruit fly Drosophila melanogaster does not appear to exhibit blocking16. Blocking in C. elegans has not hitherto been shown.Efforts to study blocking in invertebrates have met with mixed success. In honeybees  that would produce a separation between naïve approach and trained aversion similar in extent to that seen in benzaldehyde learning. We found that training to 100 mM NH4Cl, followed by testing on a gradient produced by a 5 μL point of 2.5 M NH4Cl opposite a 5 μL point of 20 mM NaAc, produced the greatest alteration of post-training chemotaxis relative to untrained controls, and that which was most similar  to benzaldehyde/food-deprivation conditioning , before pairing food-deprivation (US) with CS1 and benzaldehyde (CS2) simultaneously for a second hour  was similar to that in the CS1-only control condition, learning to benzaldehyde (CS2) was significantly reduced  compared to the CS2-only control condition  in this paradigm and in the control, CS1-alone training paradigm, while a significant block of NH4Cl (CS2) learning was observed, suggesting that direction of blocking was solely dependent on stimulus order, and not on stimulus identity  blocked learning to benzaldehyde (CS2) in subsequent experiments.In blocking, it is the order of the conditioned stimuli that determines which stimulus is blocked rather than the identities of the stimuliity Fig. . This de4Cl (CS1) context they were trained in. We reasoned that if the decreased learning of the CS2/US pairing was the result of context conditioning, matching the testing context to the training context would restore learning, while if it was result of blocking it would not  in the NHnot Fig. . We obsehen-1(tm501). hen-1 encodes a secreted protein expressed in ASE neurons whose activity is required for the integration of sensory stimuli and behavioral plasticity21. While it has previously been reported that hen-1(tm501) mutations result in a partial loss of NaCl/food-deprivation associative learning, we find that under our training conditions hen-1(tm501) fully eliminates learning to NH4Cl, while preserving salt sensation. We predicted that hen-1(tm501), being unable to pair NH4Cl (CS1) to the US, will be able to fully pair benzaldehyde (CS2) to the US, and thus exhibit no blocking of benzaldehyde learning. When tested to benzaldehyde in the blocking condition, however, hen-1(tm501) surprisingly showed a partial block of benzaldehyde learning similar to that of N2  – an independent loss of function hen-1 allele21 – showed a similar set of phenotypes  worms homozygous for a putative null allele of CREB1 exhibited wild type learning and blocking in a NH4Cl blocking benzaldehyde paradigm  evolution of blocking in C. elegans than in mammalian brains.Previous work has reported several paradigms under which context conditioning occurs in 18. We find that nuclear localization of EGL-4 in AWC occurs even when the behavioral output of benzaldehyde/food-deprivation learning has been partially blocked by prior NH4Cl learning. This is consistent with blocking of benzaldehyde learning occurring downstream or parallel to EGL-4 nuclear localization in AWC, perhaps in a pathway involved in memory retrieval. Since benzaldehyde is sensed by the AWC neurons24, while NH4Cl is primarily sensed in an AWC-independent fashion25, blocking may be occurring in a downstream interneuron.In principle, blocking could exert its effect either before on (or before) memory formation, or on memory retrieval. Nuclear localization of EGL-4 in AWC has been shown to be both necessary and sufficient for benzaldehyde/food-deprivation associative learninghen-1(tm501) worms, which are incapable of NH4Cl/food-deprivation associative learning, can still undergo blocking, suggests that learning to CS1 is not required for CS1 to block CS2. Since CS1 would not seem to have any predictive value for the unconditioned stimulus in these animals but remains capable of blocking learning to CS2, this finding appears to challenge the Rescorla-Wagner model of learning, which hypothesizes that blocking of a CS occurs when CS1 alone is sufficient to predict the US.Our finding that 4Cl learning, but not for NH4Cl blocking of benzaldehyde learning, is that HEN-1 is required for recall of the NH4Cl associative memory, but not for its formation, and that the presence of the memory alone  is sufficient to result in blocking of the benzaldehyde associative memory.A possible mechanism by which HEN-1 could be required for NHIf EGL-4 nuclear localization is close to the formation of the benzaldehyde/food-deprivation engram, our finding that this occurs normally in salt blocked wild type worms suggests that salt blocking may not involve hindered storage of the benzaldehyde memory, but rather interference of salt to the retrieval of the benzaldehyde memory. Moreover, this interference must be specific to the mechanisms of blocking and not context conditioning, where salt appears to enhance the retrieval of the context memory. Inhibition of recall of an association during blocking, rather than inhibition of formation of the association, is similarly inconsistent with the predictions of the Rescorla-Wagner model.26. That retrieval-failure is similarly the most parsimonious explanation of blocking in C. elegans both strengthens evidence for it as a broadly cross-species mechanism of higher level learning, and demonstrates the utility of C. elegans as a genetically tractable model in which to infer the mechanisms of higher order mammalian learning.A mechanism for blocking based on retrieval-failure has previously been proposed in ratshen-1(tm501) JC2154, pyIs500 [ofm-1p::GFP + odr-1p::dsRed + odr-3p::GFP::egl-4] JZ500 were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). hen-1(ut236) was a generous gift from Takeshi Ishihara.Wild-type N2 (Bristol strain), Escherichia coli27. Age-synchronized worms were grown at 23 °C until the young adult stage (48 h) before behavioral experiments. All behavioral assays were performed on 100 mm chemotaxis media (CTX) petri plates5 at 20 °C. Behavioral experiments were performed on In assays testing the approach to salts, salt gradients were established by adding 5 μL of the indicated concentration of ammonium chloride (NH4Cl) or sodium acetate (NaAc) approximately 3.5 h before testing. In assays testing benzaldehyde (Bnz) approach, 1 μL 1% benzaldehyde and 1 μL 100% ethanol (EtOH) were added to opposite sides of the CTX plate 2 cm from the edges immediately after worms were placed on the plate during testing. Immediately following, 1 μL of 1 M sodium azide was placed on top of each point of benzaldehyde, ethanol, NH4Cl and NaAc to anaesthetize the worms, preserving their initial choice. Before beginning the assays, worms were washed from the cultivation plates and rinsed twice with sterile water to remove residual bacteria. Milli-Q water was used in every transfer to another plate before worms were dried with Kimwipes. All chemicals were purchased from Sigma-Aldrich, St. Louis, Missouri. All testing phases allowed worms to undergo chemotaxis for 1 h, after which they were counted. Dead or damaged worms were not scored. The Chemotaxis Index (CI) was calculated according to the equation CI =  - (# worms at counterpoint))/(# worms on plate). Worms were scored as being at a point if they were within a 20 mm radius of the point of salt or odorant placement. A score of +1 indicates complete approach to either NH4Cl or benzaldehyde, while a score of −1 indicates complete avoidance. For behavioural assays, all groups in single graphs were run on the same day under the same conditions.Animals were cultivated on nematode growth medium (NGM) seeded with OP50 For experiments where multiple tests were performed, p-values were adjusted using Bonferroni corrections. All assays were run on at least two days, in duplicate or triplicate, with 20–400 young adult worms per plate. Plates having fewer than 20 or greater than 400 worms were excluded from analysis. As this sometimes resulted in uneven sample sizes between conditions, all comparisons for statistical significance were made with Welch’s variant of two-sided t-tests. All Ns represent number of plates, except in Fig. 4Cl was used to establish a salt gradient during testing against an increasing NaAc gradient (2 mM to 2.5 M), as indicated. For all salt chemotaxis experiments, gradients were allowed to develop in the agar for 3–5 hours before testing. For naïve approach assays, worms were placed on CTX and allowed to chemotax for 1 h to the previously established salt gradients. For NH4Cl learning assays, worms were starved for 1 h on 100 mM NH4Cl CTX agar before being transferred to CTX testing plates with prior established salt gradients, and allowed to chemotax for 1 h.5 μL of 2.5 M NH4Cl for 1 h, prior to the addition of 2 μL of 100% benzaldehyde on the lid for a second hour, before being tested to salt (2.5 M NH4Cl vs 20 mM NaAc) or benzaldehyde  separately. In the naïve condition, worms were left to starve for 2 h and tested to NH4Cl or benzaldehyde. In the benzaldehyde trained control, worms were starved for 1 h before being transferred to new plates to which 2 μL 100% benzaldehyde was added to the lid, after which they were conditioned for 1 h before testing. In the NH4Cl trained control, worms were starved for 1 h before being transferred to 100 mM NH4Cl CTX plates and starved for 1 h before testing.In the blocking condition, worms were adapted to 100 mM NH4Cl and benzaldehyde for 1 h. The naïve condition was performed identically to the NH4Cl blocking benzaldehyde paradigm. Worms were starved in the presence of 2 μL 100% benzaldehyde in the benzaldehyde trained control. They were starved for 1 h before being transferred to 100 mM NH4Cl CTX plates for 1 h in the NH4Cl trained control.Worms were adapted to 2 μL of 100% benzaldehyde for 1 h prior to exposure to both NH4Cl blocking Benzaldehyde assay, except worms in the blocking condition were also tested for benzaldehyde approach on 100 mM NH4Cl CTX agar.Performed identically to the NH4Cl blocking benzaldehyde paradigm with naïve, and NH4Cl and benzaldehyde trained controls as described above. Animals were washed from the training plates and placed on an agarose pad on a slide, paralyzed with 1 mM levamisole, and covered with a cover slip. Slides were imaged immediately on an Olympus Fluoview FV1000 confocal microscope using a 60x oil immersion objective and analyzed with ImageJ. Percent GFP nuclear localization was calculated as the fraction of GFP found in the cell nucleus divided by the total present in the cell.Worms were conditioned in the NHSupplementary Figures


To assess whether elevated thyroid-stimulating hormone (TSH) levels before conception can predict poor outcomes of assisted reproductive technology (ART).Prior to July 2018, we searched the PubMed, EMBASE, COCHRANE, Google Scholar, and CNKI databases for studies. Retrospective or prospective reports that compared ART results in patients with subclinical hypothyroidism (SCH) with normal thyroid function were selected. Two reviewers separately reviewed each potential article for qualification, analyzed the quality of the studies according to the Newcastle-Ottawa scale, and extracted the data. The PRISMA guidelines were adopted.P = 0.43), clinical pregnancy rate (CPR) ; P = 0.74), pregnancy rate (PR) ; P = 0.99), and miscarriage rate (MR) ; P = 0.26). Furthermore, when a higher TSH level was used as the cut-off value to diagnose SCH , a significant difference was found in the MR ; P = 0.02) between the two groups of ART-treated women. However, when a broader cut-off value was used to define SCH, no significant differences were observed in the LBR ; P = 0.14), CPR ; P = 0.052), or PR ; P = 0.74) between the two groups of ART-treated women.We selected a total of 18 publications that included 14,846 participants for this meta-analysis. When the TSH cut-off value for SCH was set at 2.5 mIU/L, no significant differences were observed in ART-related outcomes between SCH patients and normal women. The evaluated outcomes included the live birth rate (LBR) , No difference was observed in ART outcomes when a TSH cut-off value of 2.5 mIU/L was used. However, when a broader TSH cut-off value was used, preconception SCH resulted in a higher miscarriage rate than in normal women.The online version of this article (10.1186/s12958-018-0424-0) contains supplementary material, which is available to authorized users. SCH affects 2–5% of all pregnant women in the United States. This disease is characterized by abnormalities in the hypothalamic-pituitary-thyroid axis that are reflected by alter standard serum thyroxine (T4) levels and increased serum TSH levels . OpinionOvert hypothyroidism (OH) is defined as a high level of TSH and a low level of T4 and is related to infertility as well as adverse pregnancy outcomes , includiThe relationship between ART outcomes and thyroid function has been a hot topic and the subject of a great deal of debate in recent years . The reaDuring a pregnancy, variations occur in the normal range of TSH levels, and this has led to the introduction of SCH, a more vaguely defined complication observed in pregnant patients . While tART patients experience many barriers when attempting to achieve conception, and identifying the optimal range of pregestational TSH levels is now specifically recognized as an important parameter. Furthermore, the superovulation caused by ART generates a rapid increase in E2 levels  that incThough obstetricians can correct TSH in patients who are already pregnant, by inspecting data associated with a large cohort of ART patients, in this study, we attempt to determine whether elevated TSH levels in the preconception period predict adverse outcomes in ART patients. We seek to address whether it is appropriate to apply traditional TSH criteria in patients about to undergo ART  and to prompt reproductive medical organizations to develop methods to increase the ability of practitioners to identify and treat ART patients whose TSH status puts them at high risk of adverse results.The included studies were limited to prospective or retrospective studies that compared ART consequences in patients with SCH with normal thyroid function. Publications were excluded if (1) only women with SCH were described without a comparison group consisting of women without SCH, (2) the included women had overt hypothyroidism or hyperthyroidism (3) TSH was not evaluated before the ovarian stimulation cycle we begun, (4) the number of IVF/ICSI cycles was not specified, (5) no specific TSH cut-off value was used to define SCH.These searches were performed in the Medline, PubMed, EMBASE and COCHRANE, Google Scholar, CNKI databases. For instance, Medline was searched using the following search string for articles published from January 1990 to July 2018: (((“insemination”[MeSH Terms] OR “insemination”[All Fields]) OR (“fertilization in vitro”[All Fields] OR “fertilization in vitro”[MeSH Terms] OR (“fertilization”[All Fields] AND “vitro”[All Fields]) OR “fertilization in vitro”[All Fields])) OR (“reproductive techniques”[MeSH Terms] OR (“reproductive”[All Fields] AND “techniques”[All Fields]) OR “reproductive techniques”[All Fields])) AND ((((“hypothyroidism”[MeSH Terms] OR “hypothyroidism”[All Fields]) OR ((“thyroid gland”[MeSH Terms] OR (“thyroid”[All Fields] AND “gland”[All Fields]) OR “thyroid gland”[All Fields] OR “thyroid”[All Fields] OR “thyroid (usp)”[MeSH Terms] OR (“thyroid”[All Fields] AND “(usp)”[All Fields]) OR “thyroid (usp)”[All Fields]) AND (“physiopathology”[Subheading] OR “physiopathology”[All Fields] OR “dysfunction”[All Fields]))) OR (“thyroid diseases”[MeSH Terms] OR (“thyroid”[All Fields] AND “diseases”[All Fields]) OR “thyroid diseases”[All Fields] OR (“thyroid”[All Fields] AND “disorder”[All Fields]) OR “thyroid disorder”[All Fields])) OR (“thyroid diseases”[MeSH Terms] OR (“thyroid”[All Fields] AND “diseases”[All Fields]) OR “thyroid diseases”[All Fields] OR (“thyroid”[All Fields] AND “disease”[All Fields]) OR “thyroid disease”[All Fields])). We included all published retrospective or prospective studies. All relevant publications were retrieved. We also systematically reviewed the reference lists of the identified articles to identify additional reports that could be included in the meta-analysis. We made no attempt to identify unpublished reports.Study selection and Data Collection Processes.Two authors (Zhao T and Chen BM) performed the original screening of the titles and abstracts of all studies, and citations considered irrelevant by both observers were excluded. The PRISMA flow diagram Fig.  providedWe evaluated the quality of the articles according to the Newcastle–Ottawa scale, which is an effective method for scoring observational and non-randomized studies. All articles were evaluated independently by two authors. Discrepancies were solved by consensus. The Newcastle–Ottawa scale employs a score system based on the following three primary criteria: the selection of participants, the comparability of study groups, and outcome or, for case-control studies, an assessment of exposure. While ‘comparability of cohorts’ was scored as 2, 1 or 0; the other two primary criteria were scored based on eight items, each scored as either 0 or 1. Accordingly, the quantitative estimation of the total quality of each individual article ranged from 0 to 9. In the meta-analysis, articles were considered high quality if they obtained seven or more scores, those obtaining four to six scores were believed medium quality, and those obtaining three or less scores were believed low quality.We extracted information related to the research characteristics, the quality of the publications and the test results from each included article. The primary outcome was the LBR per woman, which was defined as the number of childbirths that led to one live born baby. The following secondary outcomes were applied: (i) the CPR per patient, which was defined using conceptions diagnosed by ultrasonography, including cases in which there was one or more gestational sacs in the uterus; (ii) the PR per woman, with pregnancies diagnosed as serum b-hCG levels ≥5 mIU/mL within 14 days following ART; and (iii) the MR per clinical conception.We adopted the Cochrane Collaboration’s tool for assessing risk of bias to evaluate the risk of bias in individual studies. We analyzed the following risk of biases: 1) selection bias  2) performance bias  3) detection bias  4) attrition bias  5) reporting bias .Except for the above-mentioned types of biases, other two types of biases were included 1) sampling bias  2) measurement bias .P value less than 0.05. Random effects and fixed effects models were used in this meta-analysis. To evaluate between-study heterogeneity, both the ×2-based Q statistic test and the I2 statistic were applied. We defined I2 values of 25%, 50%, and 75% as representing low, moderate, and high heterogeneity, respectively. We used a random effects model to pool outcomes when high heterogeneity was observed; whereas we used a fixed effects model to pool outcomes when heterogeneity was not high.We used Stata (version 11) to analyze the data. We calculated a combined odds ratio (OR) with a 95% confidence interval (CI) to assess the strength of the connection between SCH and the risk of adverse pregnancy-related outcomes. The significance of the combined OR  was identified with the Z test. We defined significance as a We evaluated the effect of each article on the overall risk appraisal by sequentially omitting each report to validate the authenticity of the results of the meta-analysis. We utilized Begg’s funnel plots and Egger’s linear regression test to estimate potential publication bias. The present study fulfilled the criteria of Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) , CPR , LBR , and MR  (Table The detailed characteristics of the involved studies are presented in Table P = 0.43). The I2 value (58.2%) calculated for these studies indicated high heterogeneity, and we therefore used a random effects model to evaluate the pooled effect estimate  did not indicate the presence of asymmetry for these studies  than in women with normal thyroid function , ate Fig. . A sensiP = 0.14). The I2 value (67%) indicated a high degree of heterogeneity. We therefore utilized a random effects model to pool the effect estimate  and Begg’s funnel plots (P = 0.92) indicated no asymmetry among these studies ; ate Fig. . A sensi7, 1.11; P = 0.74). The I2 value was 39.9%, indicating low heterogeneity. We therefore chose a fixed effects model to pool the effect estimate  and Begg’s funnel plots (P = 0.92) indicated no asymmetry among these studies . The meta-analysis indicated no association between SCH and the CPR. Comparison of the CPR between women with SCH and euthyroid women indicated no significant difference ; ate Fig. . When eaP = 0.052). The I2 value (18.1%) indicated low heterogeneity. We therefore utilized a fixed effects model to pool the effect estimate  and Begg’s funnel plots (P = 0.39) indicated no asymmetry among the studies ; ate Fig. . A sensiP = 0.99). The I2 value was 0%, indicating an absence of significant heterogeneity  and Begg’s funnel plots (P = 0.27) did not indicate the presence of asymmetry in the involved studies  and PR. We failed to find a meaningful association between the presence of SCH and the PR ; ity Fig. . A sensiP = 0.74). The I2 value (67.2%) indicated high heterogeneity. We therefore utilized a random effects model to pool the effect estimate  and Begg’s funnel plots (P = 0.45) indicated no asymmetry among the studies ; ate Fig. . A sensiP = 0.26). The I2 value was 67.2%, indicating high heterogeneity. We therefore chose a random effects model to pool the effect estimate  and Begg’s funnel plots (P = 0.59) indicated no asymmetry in the studies ; ate Fig. . A sensiP = 0.02). The I2 value (59.8%) indicated high heterogeneity. We therefore utilized a random effects model to pool the effect estimate  and Begg’s funnel plots (P = 0.90) indicated no asymmetry among these studies ; ate Fig. . A sensiThe quality assessment of the studies included in the meta-analysis is shown in Table This meta-analysis was specifically aimed at evaluating the associations between preconception maternal TSH levels and ART outcomes. We found that a stricter TSH cut-off value did not seem to influence ART outcomes including the LBR, PR, CPR, and MR. Furthermore, using a broader TSH cut-off value resulted in a significant difference in poor ART outcomes including the MR.Many previous studies have researched the association between SCH during conception and maternal outcomes, but their conclusions have been inconsistent. In 2016, a meta-analysis focused on thyroid dysfunction indicated that SCH during pregnancy is related to multiple poor neonatal and maternal outcomes, especially abortion . HoweverMoreover, subclinical thyroid function disorder is a focus of studies evaluating patients undergoing ovarian stimulation because it has been suggested that these patients experience a decrease in thyroxin levels and an increase in TSH levels during ovulation induction.Due to the increased subsequent risk of hypothyroidism in participants with a TSH level above 2.0 mIU/L , the NAWhen a broader TSH cut-off value was used, we found a significant difference in the MR between SCH patients and normal women. This outcome is consistent with some random clinical trials (RCTs). These trials demonstrated that treatment of SCH patients who were defined using a broader TSH cut-off value appeared beneficial. An RCT was conducted in patients aged 20–40 years old with SCH  who were undergoing IVF . A totalIn addition, we found that an increasing number of clinicians are uncertain regarding whether to perform preconception thyroid function tests, especially for women seeking ART . RoutineIn contrast to our findings, a recent prospective trial published by Negro et al.  includedThere are several limitations to this meta-analysis. First, the sizes of the populations included in many of the original studies and the total number of patients were small. Second, because some original articles were retrospective, it is probable that other factors may account for the differences observed in outcomes. In retrospective cohort studies, it is feasible that there will be selection bias, problems with the quality of the original publications, publication bias, confounding and heterogeneity. Third, TPO, an important confounding factor, could not be eliminated in several of the original papers because of limitations associated with the studies. In spite of these limitations, the current meta-analysis has some strengths. Our methodology was rigorous, and the outcomes achieved after the results of these trials were pooled were more credible. We also comprehensively compared the CPR, PR and MR as secondary outcomes between normal women and SCH patients to explore the influence of preconception SCH on ART outcomes. Furthermore, we analyzed the data using two different cut-off values for TSH levels to explore the association between SCH and adverse ART outcomes. The results suggested a new diagnosis standard to define SCH in woman seeking ART.Our data indicate no difference in ART outcomes when a TSH cut-off value of 2.5 mIU/L is used. However, using a broader cut-off value of TSH, we found a higher MR in SCH patients seeking ART than in the control group. We suggest that a thyroid function test should be a routine examination in women seeking ART.Additional file 1:Figure S1. PRISMA Checklist. (EPS 538 kb)Additional file 2:Figure S2. a-d Sensitivity analysis of the studies included in the meta- analysis. The figure (a-d) shows the OR obtained by combined analysis of the remaining studies after the successive exclusion of each study individually. The excluded study is listed on the left, and the corresponding horizontal lines indicate the OR and CI obtained by re-calculation after its exclusion. The CI for the overall meta-analysis of the studies is indicated by two vertical lines. (TIF 746 kb)Additional file 3:Figure S3. a-d Begg’s funnel plot for publication bias analysis. Each point represents a separate study. (TIF 347 kb)Additional file 4:Figure S4. a-d Sensitivity analysis of the studies included in the meta- analysis. The figure (a-d) shows the OR obtained by combined analysis of the remaining studies after the successive exclusion of each study individually. The excluded study is listed on the left, and the corresponding horizontal lines indicate the OR and CI obtained by re-calculation after its exclusion. The CI for the overall meta-analysis of the studies is indicated by two vertical lines. (TIF 587 kb)Additional file 5:Figure S5. a-d Begg’s funnel plot for publication bias analysis. Each point represents a separate study. (TIF 316 kb)