ETV6‐RUNX1 gene fusion is one of the most common chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). It is associated with favorable prognosis. The identification of the genomic sequence of the breakpoint flanking regions of the ETV6‐RUNX1 translocation should be the best strategy to monitor minimal residual disease (MRD) in patients with ETV6‐RUNX1‐positive ALL. In this study, the ETV6‐RUNX1 translocation was sequenced by next‐generation sequencing (NGS) in 26 patients with ETV6‐RUNX1‐positive ALL and re‐sequenced by using the Sanger method. Interestingly, the three‐way translocation, including ETV6‐RUNX1, was detected in five patients. Four of them relapsed during or after therapy, while 21 patients without the three‐way translocation were still in remission (P < 0.0001). The three‐way translocation pattern was identical between the diagnosis and relapse samples in three patients, excluding one patient (SCMC‐001245). The relapse samples retained the translocation of ETV6‐RUNX1 relative to the three‐way translocation t at diagnosis, suggesting that the three‐way translocation might be an important risk factor for relapse in patients with ETV6‐RUNX1‐positive ALL and should be further studied.The t  Childhood acute lymphoblastic leukemia (ALL) is a heterogeneous disease comprising different immunophenotypes and various genetic subtypes, including chromosomal translocations, single nucleotide variants, insertion/deletion (indel) of driver genes, and inappropriate expression of oncogenes ETV6‐RUNX1 fusion protein is expressed in 25% of childhood B‐lineage ALL cases and is associated with favorable prognosis following conventional therapeutic strategies The ETV6‐RUNX1 translocation results from the break and rearrangement of chromosomes 12 and 21, two common translocation breakpoints have been described. The major breaking pattern involves a break within ETV6 intron 5 and RUNX1 intron 1, generating ETV6 intron 5‐RUNX1 intron 1 rearrangement. The minor breaking patterns are found in about 10% of ETV6‐RUNX1‐positive ALL and breaks within ETV6 intron 5 and RUNX1 intron 2 or 3, generating ETV6 intron 5‐RUNX1 intron 2/3 rearrangement Bone marrow samples from newly diagnosed patients with childhood ALL were collected from the Department of Hematology and Oncology, Shanghai Children's Medical Center (SCMC). All samples were ETV6‐RUNX1 positive as determined by reverse transcription PCR or by fluorescence in situ hybridization (FISH) from October 2008 to February 2014. The research was approved by the Ethics Committee at SCMC, Shanghai Jiao Tong University School of Medicine.μg of high‐quality genomic DNA (OD260/280 ratio ranging from 1.8 to 2.0) was diluted with 1× low TE buffer in a 1.5 mL LoBind tube to a total volume of 50 μL and fragmented by sonication on the CovarisS2 . Fragmented DNA was repaired, ligated with Illumina adapters, and size selected, aiming for a 350‐ to 400‐base pair (bp) product. The size‐selected product was then amplified (each sample was tagged with a unique index during this PCR procedure) and validated by using the Agilent 2100 Bioanalyzer .DNA extraction was carried out using QIAamp DNA Blood Mini Kit  according to the manufacturer's instruction. Each DNA sample was quantified by agarose gel electrophoresis and Qubit dsDNA BR Assay kit . Briefly, 1 μL 1× binding buffer three times and resuspended in 80 μL of 1× binding buffer. Sixty‐four microliters of 2× binding buffer were added to the hybrid mix and transferred to the tube containing 80 μL of MyOne beads. The mix was rotated for 1 h on a rotator. The beads were then washed with WB1 buffer at room temperature for 15 min once and WB3 buffer at 65°C for 15 min three times. The bound DNA was then eluted with buffer elute. The eluted DNA was finally amplified for 15 cycles using the following program: 98°C for 30 sec (1 cycle); 98°C for 25 sec, 65°C for 30 sec, 72°C for 30 sec (15 cycles); 72°C for 5 min (1 cycle). The PCR product was purified using SPRI beads  according to the manufacturer's protocol. The enrichment library was sequenced on Illumina HiSeq2000 sequencer for paired read 100 bp The amplified DNA was captured with biotinylated oligoprobes , which were designed to tile along the fusion regions, including exon and intron regions of ETV6 and RUNX1. The capture experiment was conducted according to the manufacturer's protocol. Briefly, the DNA library was mixed with Buffer BL and GenCap gene panel probes , heated at 95°C for 7 min and 65°C for 2 min on a PCR machine. Twenty‐three microliters of the 65°C prewarmed Buffer HY (MyGenostics) was then added to the mix and held at 65°C with PCR lid heat on for 22 h for hybridization. Fifty microliters of MyOne beads  were washed in 500 http://www.phrap.org/). SAMtools (http://samtools.sourceforge.net/) was used to extract all mapped reads within 500–1000 bp of each predicted breakpoint. Unmapped reads with mates mapping to the SV region were also included. In addition, a read could not have ≥5 bp of unaligned bases on its ends. Reads supported the SV if the read crossed the breakpoint with at least two extra bases and the read did not align to the reference sequence. Positive breakpoints were re‐sequenced by using the Sanger Method.The sequence variant (SV) detection was completed by using paired‐end sequence data. Insert size distributions were obtained from the mapping results with unimodal insert size distributions. The read pairs should present a mapping quality >30 with a separation distance exceeding four folds of the standard deviation or be in an unexpected orientation. The de novo assembly for all predicted deletions, insertions, inversions, and translocations were performed by using Phrap (μL) was applied on drained slides and developed in the dark for 10 min. Slides were observed with the use of appropriate filters and FISH software. This probe set contains a 350‐kbp probe for the 5′‐end of ETV6 (exons 1–4) and a 500‐kbp probe covering the entire RUNX1 gene.Air‐dried bone marrow slides from archival samples were used for ETV6‐RUNX1 fusion gene detection using a commercial LSI ETV6/RUNX1 extra signal (ES) Dual Color Translocation Probe , according to the manufacturer's instructions. Briefly, slides were washed in 2× saline/sodium citrate buffer for 2 min at room temperature followed by dehydration in an ethanol series , each for 2 min. Ten microliters of the probe mixture for ETV6‐RUNX1 was spotted on the cell sample. A cover slip was then placed on the probe area and the edges were carefully sealed with rubber solution glue. The 4′,6‐diamidino‐2‐phenylindole (DAPI) antifade  and the other two relapsed after treatment . All cases were registered in the pediatric oncology network database (POND). The clinical characteristics and outcomes are presented in Table Homo sapiens, hg19, GRCh37, February 2009) were genomic breakpoint capturing regions of ETV6 and RUNX1, respectively. The target region size was 181 kbp and GenCap probes covered 93.41% of the regions. Target regions were captured and sequenced in all 26 cases of ETV6‐RUNX1‐positive childhood ALL samples. Reads mapping to different chromosomes  were confirmed by capillary electrophoresis  forward chr12 chain fuses with forward chr21 chain (FF) named type a; (2) reverse chr12 chain fuses with reverse chr21 chain (RR) named type b; 3) forward chr12 chain fuses with reverse chr21 chain (FR) named type c; and (4) reverse chr12 chain fuses with forward chr21 chain (RF) named type d  presented the translocation of chromosomes 5, 12, and 21 ), including the classic ETV6‐RUNX1 type c translocation, and the other kind of translocation that the remaining part of ETV6 and RUNX1 fused with chromosome 5. Patient 000966 presented with the tq14;p13;q22) translocation, including the classic ETV6‐RUNX1 type c translocation, and the other kind of translocation that the remaining part of ETV6 and RUNX1 fused with chromosome 6. Patient 001245 presented with the t three‐way translocation, including the classic ETV6‐RUNX1 type c translocation, and the other kind of translocation that the remaining part of ETV6 and RUNX1 fused with chromosome 8. Patient 001579 presented with the t three‐way translocation, including the classic ETV6‐RUNX1 type c translocation, and the other kind of translocation that the remaining part of ETV6 and RUNX1 fused with chromosome 15 .More interestingly, four of the five patients with three‐way translocation had already relapsed, while the other 19 patients without three‐way translocations were all in remission. Furthermore, we found that the four patients with three‐way translocation, who relapsed, did relapse after more than 1 year from diagnosis, while the patients with three‐way translocation who did not relapse just received treatment for 6 months. Although the sample size is limited, the Kaplan–Meier analysis also suggested that there may be a significant difference in terms of prognosis between patients with or without three‐way translocations  and two red  Fig. A. For EThttp://www.phrap.org/), and SAMtools, patient‐specific ETV6‐RUNX1‐positive breakpoints and flanking sequences were detected in each sample. Positive breakpoints were then verified by using the Sanger method. Using NGS combined with the abovementioned analysis tools, we established a method that allowed us to identify the breakpoints and flanking sequences at the genomic level through the design and synthesis of capturing probes across the genomic regions of the ETV6‐RUNX1 translocation breakpoints. Compared to other methods currently used, this method presents advantages such as the lower amount of genomic DNA used, a relatively lower workload for each sample and the ease of use when analyzing multiple samples simultaneously. Moreover, it provides more meaningful information. Briefly, 1 μg of genomic DNA is sufficient for this method. Furthermore, it is a simple and fast method when compared to inverse PCR or long‐distance PCR, which need tedious molecular biological operations. In addition, many samples can be conveniently handled simultaneously in a single flow cell by using high throughput sequencing and analysis. Furthermore, genomic DNA from other types of samples such as bone marrow smear and dried blood spot can also be used. Finally, detailed breakpoints and flanking sequences can be obtained, which are useful for MRD detecting.Genomic DNA from 26 patients with ETV6‐RUNX1‐positive childhood B‐lineage ALL were amplified and captured with biotinylated oligoprobes, and then sequenced on Illumina HiSeq2000 sequencer. According to paired‐end sequence data, mapping results with unimodal insert size distributions, Phrap  or later. Therefore, further follow‐up is needed. Genomic breakpoints on chromosomes 5, 6, 8, and 15 which we tested were all located in introns, gene LMBRD2 on chr5, PHIP on chr6, DPYSL2 on chr8, and AKAP13 on chr15 were involved. All these genes were broke from the middle into two parts and fused with ETV6 and RUNX1, respectively. LMBRD2 encodes LMBR1 domain‐containing protein 2, which has not been reported any association with leukemia. PHIP protein binds insulin receptor substrate‐1, modulates insulin signaling, and infects pancreatic beta cell growth and survival. DPYSL2 encodes a member of the collapsin response mediator protein family, which form homo‐ and heterotetramers and facilitate neuron guidance, growth, and polarity. This gene may play an important role in Alzheimer's disease. AKAP13 encodes a member of structurally diverse proteins called A‐kinase anchor proteins, which are essential in cell signaling pathways. However, none of these four genes have been reported related to leukemia or cancer. Since the chromosomes which involved in three‐way translocations seemed have no relationship with childhood leukemia, it might be just a phenomenon related to leukemia relapse. According to the above results, the three‐way translocation maybe an important risk factor for relapse in patients with ETV6‐RUNX1‐positive ALL and should be further studied.For the four patients who relapsed, especially for two who relapsed after therapy , whether the relapsed clone is identical to that at diagnosis should be determined. When comparing the pattern of three‐way translocation including ETV6‐RUNX1 breakpoint, the relapsed clone was found to be identical to that detected at diagnosis for three patients, suggesting that the relapsed leukemia clone derived from the dominant clone detected at diagnosis. However, for the other patient (SCMC‐001245), the relapsed clone was not identical to that detected at diagnosis. In fact, the three‐way translocation t verified by using the Sanger method at diagnosis was not detected in the relapsed sample, only the translocation t was verified at relapse. The other translocation of chr8 and chr12/chr21 were not detected at relapse. Although the relapse clone of this patient (SCMC‐001245) was partially different, both the sequences of breakpoints and flanking regions of t at relapse were identical to that at diagnosis. Thus, this relapsed clone may be derived from the primary clone detected at diagnosis, while the translocations of t and t were missed during the proliferation and differentiation of leukemia cells.Martineau et al. also detected three‐way translocations (chromosomes 12 and 21) by FISH assays, including FISH using whole chromosome paints (wcps) and Multiplex‐FISH (M‐FISH) in combination with the Spectra Vision 24 color chromosome painting kit. In their opinion, variant translocations are prominent among the few relapse karyotypes reported so far Currently, studies of the recurrent mechanism of ETV6‐RUNX1‐positive B‐lineage ALL concentrate on genetic damages such as CDKN2A/B, CCNC, and other gene deletions. The results show that many deletions are associated with relapse However, the sample size of this study was small, only 26 cases. The sample size needs to be expanded to verify the conclusions of this study and to further our investigation.None declared.


Desulfobulbaceae family can conduct electrons over centimeter-long distances thereby coupling oxygen reduction at the surface of marine sediment to sulfide oxidation in deeper anoxic layers. The ability of these cable bacteria to use alternative electron acceptors is currently unknown. Here we show that these organisms can use also nitrate or nitrite as an electron acceptor thereby coupling the reduction of nitrate to distant oxidation of sulfide. Sulfidic marine sediment was incubated with overlying nitrate-amended anoxic seawater. Within 2 months, electric coupling of spatially segregated nitrate reduction and sulfide oxidation was evident from: (1) the formation of a 4–6-mm-deep zone separating sulfide oxidation from the associated nitrate reduction, and (2) the presence of pH signatures consistent with proton consumption by cathodic nitrate reduction, and proton production by anodic sulfide oxidation. Filamentous Desulfobulbaceae with the longitudinal structures characteristic of cable bacteria were detected in anoxic, nitrate-amended incubations but not in anoxic, nitrate-free controls. Nitrate reduction by cable bacteria using long-distance electron transport to get privileged access to distant electron donors is a hitherto unknown mechanism in nitrogen and sulfur transformations, and the quantitative importance for elements cycling remains to be addressed.Filamentous bacteria of the  On board, the upper 10–12 cm of sediment were discarded to minimize the presence of metal oxides and exclude large burrowing animals. The underlying sulfidic sediment was sealed in airtight bags, brought to the laboratory and stored at 15 °C. Within a few weeks, the bags were opened and the sediment was sieved (sieve mesh size 0.5 mm), homogenized and packed into glass liners or chambers before being incubated. Sediment exposure to air was minimized throughout the handling procedures.3− reduction can sustain distant oxidation of sulfide, the sediment was incubated in a modified version of the flow-through system described by 3− could be controlled  were filled with sulfidic sediment up to ≈3 cm below the upper rim. The chambers were sealed with glass lids to be filled with water without leaving a gas phase, connected to the flow-through system and immersed into an aquarium containing anoxic water to assure that no O2 would diffuse through the sealing. Anoxic, NO3−-amended, artificial seawater  based on MilliQ water  and Red Sea Salts  was pumped from a reservoir into the chambers at a constant rate of 190 μl min−1. Magnetic stir bars driven by an external rotating magnet maintained a homogeneous water column above the sediment in the chambers. The water in the reservoir was maintained at 30 °C and purged with N2 containing 0.04% CO2. Before entering the chambers, the water was cooled down to 13.6 °C to allow a constant exposure of the sediment to gas-undersaturated water, thereby preventing gas bubble formation. Throughout the 64 days of incubation, the NO3− concentration in the inflowing and outflowing water was regularly monitored. The NO3− concentration of the reservoir water remained constant within 266–273 μM.To address whether NOntrolled . Prelimi3−+NO2− (NOx−), O2 and H2S was measured at the end of the experiment using in-house made sensors for NOx−, O2 and H2S. The sediment cores were then sliced in 3 mm sections down to 18 mm depth. Each section was homogenously mixed and sediment samples (approx. vol. 1 ml) were collected in triplicates, transferred into polypropylene centrifuge tubes and frozen at −20 °C for later analysis of the intracellular+porewater NO3− pool. This analysis was performed to address the presence of NO3−-storing organisms  in the sediment.Microprofiles of pH were measured in the sediment after 7, 18, 34, 53 and 64 days of incubation using pH microsensors. The depth distribution of NO3−-free) or anoxic in the presence of 200 μM NO3−. The three parallel incubations lasted for 27, 24 and 28 days, respectively. In the anoxic incubations in presence of NO3− (hereafter referred to as the NO3− treatment), the sediment was packed into glass chambers and incubated in the flow-through system as described above. Additional sediment chambers  were used for the oxic treatment. These chambers were immersed into an aquarium filled with artificial seawater, constantly flushed with air and maintained at 13.6 °C (3−-free) control, the sediment was packed into glass liners  and placed into a sealed aquarium filled with artificial seawater at 13.6 °C. The water was kept stirred by means of an aquarium pump. Anoxic conditions (O2<0.1 μM) were maintained by constantly flushing the water with a gas mixture of N2 (99.96%) and CO2 (0.04%), and monitored throughout the entire incubation with the sensitive STOX O2 sensor . Total hydrogen sulfide (ΣH2S=[H2S]+[HS−]+[S2−]) concentrations were calculated at each depth from the measured H2S and pH values  previously drilled into the glass lid . To prevent O2 diffusion into the glass chamber, the openings were flushed with N2 during measurements. Consumption rates of the measured parameters were estimated by modeling the concentration microprofiles with the algorithm developed by 2, NO3− and HS− were calculated according to Sediment microprofiles of Hg for 10 min. The concentration of pooled intracellular and porewater-dissolved NO3− was determined in the supernatant on a chemiluminescence detector  after being reduced to NO by the VCl3 method  equals the proton consumption by cathodic NO3− reduction minus the proton production due to the non-cathodic consumption of NO3−. This can be expressed as follows:The net proton consumption in the NO3− consumption rate, 3− reduction, nc is the number of moles of protons consumed by one mole of NO3− reduced cathodically and can be either 6 or 10 . 3− flux across the water–sediment interface in the flow-through incubations by means of the following equation:The cathodic NOCo and Ci are the NO3− concentrations in the water at the outlet and inlet of the chamber, respectively; V is the water flow rate and A is the surface area of the sediment core. The current density (Je−) needed to sustain the cathodic NO3− reduction was calculated as follows:where m is the number of moles of electrons consumed by one mole of NO3− reduced cathodically and can be either 5 or 8  and incubated in batch mode in anoxic, NO3−-amended seawater. Anoxic incubations without NO3− served as controls. The O2 concentration in the overlying water  was kept below 15 nM by continuous bubbling with N2/CO2 as described above, except for the short profiling periods, when O2 was up to 2.5 μM. Nitrate concentrations were kept at 80–350 μM (monitored as described above) by regularly adding NO3− to the overlying water. Microprofiles of pH and H2S were recorded after 5, 10 and 12 days to confirm the establishment of electric coupling between NO3− reduction and sulfide oxidation in the NO3−-amended cores. After 12 and 14 days, sediment cores were sectioned in 2 mm intervals down to 10 mm; sections were fixed in 50% ethanol  and stored at −20 °C for fluorescence in situ hybridization (FISH), or snap frozen in liquid nitrogen and stored at −80 °C for DNA analysis. Control cores were sampled after 28 days.To investigate whether cable bacteria can grow by respiring NODNA extraction ), PCR, cloning and sequence analysis were done as previously described (Desulfobulbaceae) and ELF645 (specific for a Desulfobulbaceae lineage confirmed as cable bacteria) were applied to 6 × -diluted subsamples from 4–6 mm depth from NO3−-amended and control sediment of triplicate cores. Filament length density  was determined for probe DSB706-positive filaments using the line-intercept method (Desulfobulbaceae (probe DSB706-positive) filaments in triplicates by checking at least 1000 cells after double hybridizations with both probes, labeled in CY3 or FITC. Analyses were carried out on an Axiovert 200 M epifluorescence microscope (Carl Zeiss).Identification and quantification of cable bacteria by FISH were done as previously described  imaging. Single filaments were picked with a sterile glass hook under microscopic guidance . The areal consumption rate of NOx− was 118 and 104 μmol m−2 h−1 when calculated from microprofile modeling and from 2S oxidation rate derived from microprofile modeling was 11.9 μmol m−2 h−1.Microdistributions of NO3− profile showed concentrations comparable to those measured with the NOx− microsensor in the depth interval 0–3 mm. Below 3 mm depth, the concentration decreased to insignificant values, indicating the absence of NO3−-storing organisms.Pooled intracellular and porewater NO2, ΣH2S, NOx− and pH in three parallel incubations where the sediment was exposed to oxic, anoxic NO3−-amended and anoxic NO3−-free seawater are shown in 3−-free overlying water remained fully sulfidic and the pH decreased along with depth after 24 days of incubation. In the NO3− treatment, ΣH2S was progressively consumed from the surface sediment, and at the end of the incubation (Day 28) the sulfidic front was detected at a depth of 11 mm. Nitrate penetrated 3.8 mm into the sediment and a distinct pH peak indicated proton consumption in the NO3− reduction zone. The estimated current density in the NO3− treatment varied between 6.6 and 8.8 mA m−2 depending on whether N2 or NH4+ was considered as the cathodic end product and whether organic carbon or FeS was the major electron donor for non-cathodic NO3− reduction. The estimated cathodic NO3− reduction represented only 12 and 19% of the total NO3− consumption. In the oxic treatment, ΣH2S was detected at 21 mm depth after 27 days of incubation. Oxygen penetrated 1.8 mm into the sediment resulting in a ≈19-mm-thick suboxic zone. The pH microprofile showed a peak at a depth of 1.8 mm indicating net proton consumption. The cathodic O2 reduction accounted for 34% of the total O2 consumption supporting a current density of 28 mA m−2. Both the pH peak and the sulfide-free zone remained 9 days after the overlying oxic water was replaced with NO3−-amended anoxic water  to 4.7 mm (Day 10) to 7.6 mm (Day 12), and a pH peak appeared at 1.6 mm depth at day 12. The controls remained fully sulfidic and no pH peak developed (data not shown). Filamentous Desulfobulbaceae similar to the previously described cable bacteria (−3 (mean of triplicate cores±s.d.) by the line-intercept method and 85±22 m cm−3 by direct measurement in the NO3−-amended sediment. Filament diameters ranged from 0.4 to 1.0 μm, with an average of 0.6 μm. Only a few and rather short filaments were detected in the controls, with a length density <3 m cm−3. A filament subpopulation hybridized with the more specific probe ELF645  development of a 4–7-mm-wide zone devoid of both NO3− and ΣH2S, consistent with the separation of ΣH2S oxidation from the associated NO3− reduction; (2) consumption of protons in the NO3− reduction zone and proton production at the ΣH2S oxidation depth consistent with the presence of cathodic NO3− reduction and anodic ΣH2S oxidation, respectively. Furthermore, sediment that showed the geochemical traits typical of electric coupling between distant half-cell reactions in the oxic treatment maintained those characteristics after O2 was replaced with NO3− (2S front (up to 5 mm d−1) as a consequence of the interruption of the electric current either by physically disturbing the sediment . Moreover, the activity of Beggiatoa would result in a characteristic pH microprofile with a minimum in the upper sediment and a maximum at the ΣH2S front . Interestingly, only a fraction of all filamentous Desulfobulbaceae (detected by probe DSB706) hybridized with probe ELF645 specifically designed for cable bacteria (Desulfobulbaceae. This conclusion is supported by the 16S rRNA gene sequence analysis that showed a dominance of diverse Desulfobulbaceae sequences outside the previously described cable bacteria cluster (Desulfobulbaceae remains yet to be defined.The presence of filamentous  cluster . The tru3− reduction can sustain the distant oxidation of ΣH2S in marine sediment as previously described for O2, and that cable bacteria are involved. The ability of microorganisms to perform cathodic NO3− reduction has been previously invoked to explain electric current generation in microbial fuel cells (for example, 3− reduction implies electron transfer from a cell to an external electrode or a conductive mineral. For cable bacteria, cathodic NO3− reduction implies the transfer of electrons from distant donors through internal biological structures over millimeter to centimeter distances. The importance of this hitherto unknown mechanism for elemental cycling remains to be addressed.In the present study we have demonstrated that cathodic NO


Rotational spectroscopy and ab initio calculations confirm that both molecules are linear. The geometry of PtC3 was accurately determined by fitting to the experimental moments of inertia of twenty‐six isotopologues. The results are consistent with the proposal of an autogenic isolobal relationship between O, Au+, and Pt atoms.Carbenes of platinum and palladium, PtC Analysis of the rotational spectra reveals that each molecule has a linear geometry and an MCCC connectivity . The results are a successful test of a model proposed by Pyykkö et al.The importance of industrial catalysis by platinum and palladium has prompted extensive studies of their gas‐phase chemistry.3 and PdC3. For PdC3, the range of effective precursors includes C3H4 , C2H2, C2H4, CH4, and C4H4O (furan). For PtC3 the range is narrower, including C3H4 , C2H4, and CH4, all of which were found to be effective. Broadband microwave spectra of the target molecules were recorded between 6.5 and 18.5 GHz  a geometry that is slightly bent at equilibrium but quasilinear in the v=0 state, and 2) an equilibrium (re) geometry that is linear. The ab initio calculations suggest that both molecules are linear at equilibrium. The intensities of PdC3 transitions were found to be highly dependent on the choice of precursor, in the order C3H4>C2H4>CH4. Transition intensities were lower when the population of PdC3 was divided across many isotopic permutations and isotopically enriched allene is prohibitively expensive. These factors prevented measurement of the spectrum of any PdC3 isotopologue that contains both 12C and 13C isotopes. The intensities of PtC3 transitions were insensitive to the choice of precursor and it was possible to generate and record spectra for many isotopic permutations of PtC3  using samples prepared by mixing 12CH4 and commercially supplied 13CH4. It was also found that PtC3 can be generated from a mixture of 12C2H2 and 13CH4 precursors with the result that the spectra of 194Pt12C12C13C, 194Pt12C13C12C, and 194Pt13C12C12C were detected with equal intensities. The observation that the 13C isotope does not preferentially occupy an end position of the C3 subunit strongly implies that the C≡C bond of C2H2 cleaves during the sequence of reactions that generates PtC3 from this set of precursors.Spectra were measured for isotopologues of PtC3 units by rotational spectroscopy. Transition‐metal dicarbides, such as ScC2 and YC2, have been studied previously.3 and PtC3 are calculated at the MP2 level to be 6.1 and 5.6 D, respectively. The lengths of bonds within PtC3 were fitted to experimentally determined rotational constants using Kisiel's STRFIT.3 where the set includes every permutation of C3 that it is possible to generate from 12C and 13C isotopes. The bond lengths thus determined are compared with those in isolated PtC, C3 and OC3 molecules in Table r0 geometry of PtC3 is in good agreement with the re geometry calculated at the CCSD(T) level. The Pt−C bond in PtC3 is longer than found in diatomic PtCr(MC) in PtC3 and in PtCO,3/CO subunits. The r(MC) parameter in PtC3 is shorter than the same quantity in PtCO by 0.031 Å. The first C=C bond (that which is contiguous with the Pt−C bond) of PtC3 is longer than the C=C bond in isolated C3 by 0.022 Å. The set of isotopologues studied is less extensive for PdC3 than for PtC3 and does not permit determination of all bond lengths from the experimental data. If the lengths of C=C bonds within the molecule are fixed as shown in Table r(PdC) is determined to be 1.79898(4) Å. Values of vibrational wavenumbers calculated ab initio are provided in the Supporting Information.The present study is believed to be the first to characterize MC2C3.3 units identified herein and the linear carbon chains that are interceded by Pt/Pd atoms which are a feature of many synthetic coordination polymers.3 and PtC3. It is possible that a fraction of the population of each forms through gas‐phase association of individual metal atoms with intact C3 or other units generated independently of any metal atom.3→MC3 association reactions to yield linear MC3 units are calculated to be −295 kJ mol−1 and −417 kJ mol−1 when M=Pd and M=Pt, respectively . However, it is also possible that the metals themselves initiate the sequence of chemical reactions that leads to dehydrogenation of the precursor. There is extensive evidence from previous studies that both Pt and Pd atoms undergo bond‐insertion and cleavage reactions with hydrocarbons.2 and MCCH2 have both been generated2 are expected to be above the upper frequency limit of the spectrometer and both MCH2 and MCCH2 will have comparatively low dipole moments which significantly decrease the intensity of their rotational transitions relative to those of MC3.The described results confirm that the heavier elements of Group 10 can form linear arrangements similar to that previously identified for Ni3 relative to MC2 or MC4. Calculations of the geometries of CAu2+, CAu3+, Pt2C, Pt2C3, and Au2C2 revealed analogies between the behavior of each of Au+ and Pt and a chalcogen atom such as O.106s0 configuration is analogous to the 2pσ0 hole on oxygen, and the 5dπ orbitals of platinum participate in π‐bonding interactions analogous to those involving the 2pπ orbital of oxygen. The existence of a family of stable molecules was thus predicted. An orbital energy level diagram for PtC3 is presented in Figure S1 in the Supporting Information. There are striking similarities between the geometries of MC3 measured during the present work and that reported earlier for OC3 by Brown et al.2C3 are analogues of the well‐known, stable oxocarbons carbon dioxide, monoxide, and suboxide, respectively, each of which have been known since the 19th century. Similarly, PtSi2C2 would be ethylene dione,3, but not yet PtC2, have been detected. The results of the present work thus support the suggestion that platinum can be regarded an isoelectronic, isolobal counterpart of oxygen. The proposal can be further assessed with reference to previous works.An empirical model proposed by Pyykkö et al.4 and CO on Ptn clustersnOm clusters3C+ cluster ion12O9) is known to be stable, suggesting that Pt9C12 might be generated in an equivalent structural form. An experimental studyn+ and CuCn+ revealed ion intensities in the mass spectra that are significantly stronger where n=3 than for clusters of other sizes.3+ relative to other cluster sizes from these results. As in the present work, the experiment performed by Ticknor et al.3 was generated with a significantly higher abundance2 within the expanding gas sample and this may cause the generation of AuC3+ to be favored over the generation of clusters of other sizes, regardless of the thermodynamic stability of AuC3+. Indeed, during a previous study, signals for NiC3+ and NiC6+ were detected in mass spectra with higher intensity than units containing 1, 2, 4, or 5 carbon atoms,+ ion is not isoelectronic and isolobal with O. However, the reported fragmentation behavior of AuCn+ is also notable. Clusters where n is odd lose only the metal atom on photodissociation whereas those with an even value of n display an additional loss channel corresponding to the loss of an odd number of carbon atoms. The overall result is that chains  containing an odd number of carbon atoms tend to be formed during photofragmentation, consistent with the proposal of Pyykkö et al. The perspective thus emerging from the collected results of spectroscopic experiments is that the proposal+ centers with the O atom is powerful and useful with respect to structural trends in gas‐phase clusters that contain Pt, Pd, Au+, and C centers.Reports of clusters containing multiple carbon and platinum or palladium atoms are scarce. The adsorption of, and reactions of, CHAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. 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Our novel direct synthesis of graphene/TiO2 heterostructure is achieved by C60 deposition on transition Ti metal surface using a molecular beam epitaxy approach and O2 intercalation method, which is compatible with wafer scale growth of heterostructures. As-grown heterostructures exhibit inherent photosensitivity in the visible light spectrum with high photo responsivity. The photo sensitivity is 25 times higher than that of reported graphene photo detectors. The improved responsivity is attributed to optical transitions between O 2p orbitals in the valence band of TiO2 and C 2p orbitals in the conduction band of graphene enabled by Coulomb interactions at the interface. In addition, this heterostructure provides a platform for realization of bottom gated graphene field effect devices with graphene and TiO2 playing the roles of channel and gate dielectric layers, respectively.Heterostructures consisting of two-dimensional materials have shown new physical phenomena, novel electronic and optical properties, and new device concepts not observed in bulk material systems or purely three dimensional heterostructures. These new effects originated mostly from the van der Waals interaction between the different layers. Here we report that a new optical and electronic device platform can be provided by heterostructures of 2D graphene with a metal oxide (TiO In graphene field effect transistors (GFET), graphene provides the active layer where the channel carriers are modulated by gate biasing through the dielectric layer. In graphene optical devices a dielectric layer is needed to induce a built-in potential within the structure so that photo-excited electrons can be extracted from the photosensitive materials into the graphene layer on top. Currently the photosensitive 2D materials such as MoS2 or WS2 are widely investigated to enhance photo-responsibility. Recent studies of graphene/photosensitive 2D heterostructures have revealed new physical phenomena such as Moire patterns582/Gr heterostructure resulted from Van Hove singularities2 has been considered since it is a well-known material that has been widely used for light harvesting2 a very attractive material for the realization of both electronic and photonic devices. The schematic diagram of our growth approach is shown in 2 dielectric layer. We demonstrate this dual functionality by depositing two top metal contacts on the Gr/TiO2 heterostructure to serve as source and drain electrodes. The gating voltage is applied through the underlying TiO2 on n-type SiC in a bottom gated FET configuration. With the same device configuration, the photosensitivity under visible light illumination is measured by the change of photoconductivity with zero gate voltage. We believe that the observed photo response at zero gate voltage is caused by the optical transitions between the valence band maximum, associated O 2p orbitals in TiO2, and the conduction band minima, formed by the C 2p orbitals of the graphene in the Gr/TiO2 heterostructure. The schematic diagrams of device and measurement configurations are shown in 2 heterostructure have been conducted in air.Graphene (Gr) is a very attractive material for diverse optoelectronic and electronic device applications due to its extraordinary transport properties, broad spectral-bandwidth, and fast response time12360 flux dependence were studied. The growth temperature was kept at 1400 °C during these experiments. First, the C60 flux was held at 1.6 × 10−7 Torr while the growth time varied. As growth time increased, the 2D peak position shifted linearly to lower wavenumber. After 30 min. growth, the 2D peak was located at ~2712 ± 5 cm−1. With longer growth time (90min.), it shifted to 2705 ± 1.5 cm−1. The FWHM of the 2D peak showed similar behavior to peak position. The value of the FHWM decreased from 71 ± 9 cm−1 (30 min. growth) to 56.5 ± 2 cm−1 (90 min. growth). The quality of the graphene was estimated from the ratio of the D and G peaks. The growth time dependence of the D/G ratio also exhibited similar behavior to position and FWHM of the 2D peak. Increased growth time resulted in a decrease of the D/G ratio from 0.32 ± 0.07 (30 min. growth) to 0.23 ± 0.01 (90 min. growth). The C60 flux dependence with two different fluxes was also evaluated. The growth was done at 1400 °C for 2 hr. With increasing flux from 0.8 × 10−7 to 1.6 × 10−7 Torr, we observed improvement of graphene quality with a decrease of the D/G ratio and 2D FWHM by 40% and 20%, respectively. The peak position also shifts to lower wavenumber from 2706 cm−1 to 2703 cm−1. Next, the effect of O2 intercalation at 500 °C at various times was studied with Raman analysis. The 2D peak position shifted slightly to lower wavenumber. A 4hr. intercalation led a 2D peak shift of only 5 cm−1, to lower wavenumber. The 2D FWHM showed entirely different behavior. Initially it broadened compared to that of as-grown graphene  and then started to narrow . The graphene quality variation was estimated by comparing the D/G ratio before and after intercalation. We did not observed any variation of the ratio for intercalation up to 60 min. but a significant increase was observed beyond 60 min.To investigate the graphene growth on Ti metal surfaces, the growth time and C2 surfaces. 60 deposition on both TiO2 (red color curve) and Ti (blue color curve). As seen in 3/2 and Ti 2p1/2 peaks for TiO2 are located at 459.1 eV and 464.8 eV respectively. However, our results  shows no O 1s peak corresponding to TiO2. Therefore, we conclude that C60 deposition results in graphene and TiC formation on both Ti and TiO2. The formation of TiC upon C60 deposition could be explained by a carbon thermal reduction reaction2 reacts with carbon to form TiC and releases CO gas above 1300 °C. The high substrate temperature (1400 °C) during C60 deposition could initiate carbon thermal reduction reactions between molecular C60 and the TiO2 surface.Graphene growth was attempted on both Ti and TiO results  show the2 has been obtained from oxidation of TiC in the temperature range from 623 K to 1073 K in air2 was accomplished with an O2 intercalation procedure. Intercalation was conducted under controlled O2 pressure (10−4 Torr) in the XPS chamber (details in Method). 2 intercalation progressed. Besides peaks for as-grown graphene at 284.7eV and TiC at 281.5 eV, three peaks associated with C-O (286.1 eV), C = O (288.1 eV), and the π → π* (291.5 eV) transition were observed in the C 1s spectra (not shown here). Since oxygen-carbon bonding was observed in the as-grown sample as well, the existence of C and O bonding might be due to air exposure during transfer from the growth chamber to the XPS chamber. In as-grown graphene/Ti structure, graphene peak is located at 284.7 eV with ~1.2 eV FWHM. However, the shift of peak position to 284.2 eV and the FHWM narrowing from 1.2 eV to 1 eV were observed as intercalation proceeds. We attribute the shift in binding energy to substrate interactions between the Ti and TiO2. The conversion of TiC into TiO2 during intercalation is shown in 4+ cation doublet located at 458.8 eV and 464.6 eV and the metallic type bonding (titanium carbide) Ti0 peaks located at 454.7 eV and 460.9 eV were observed. These peaks are associated with Ti 2p3/2 and Ti 2p1/2 transitions, respectively. The evolution of chemical composition at different temperatures and intercalation times is also shown in 4+ and Ti3+ along with TiC peaks  and its peak intensity increases with further intercalation. We calculated the activation energy for TiO2 conversion by O2 intercalation and graphene etching by the O2 (detail in Method). The calculated activation energy for TiO2 conversion is about 0.29 eV while activation for graphene etching is 0.41 eV, which indicate that TiO2 conversion is the dominant reaction in our intercalation experiments.Intercalation between epitaxial graphene and its substrate has been widely studied as a method of decoupling the strong interaction from the substrate14151660 deposition on Ti metal and O2 intercalation were also investigated with Raman measurements. 2 exposure. In the as-grown sample, Raman measurements show the typical Raman fingerprint of graphene with the presence of the 2D, G, and D peaks. Importantly, a fit to the 2D peak gave a single Lorentizan at ~2700 cm−1. Typically, C60 deposition on SiC resulted in Bernal stacked few layer graphene−1. This suggests that graphene on Ti metal is not Bernal stacked but is very similar to turbostractic stacking of graphene2 intercalation (2 layer (Eg = 3.2 eV) after O2 intercalation enhances the transmittance of visible incident Raman laser into SiC substrate. It results in Raman activation of SiC substrate and convolution of SiC and graphene Raman spectrum. The formation of TiO2 after intercalation was confirmed by observation of TiO2 vibration modes , an A1g and B1g mode doublet at 516 cm−1, and a B1g mode at 399 cm−12, A1, and E1 modes (black colored annotations), while two distinct TiO2 related Raman peaks (orange color annotations) were observed after O2 intercalation. The peaks at ~143 cm−1 and ~640 cm−1 are attributed to the Eg mode (O-Ti-O bending vibration) of anatase TiO2.The Con modes . TiO2 ha2/n-SiC heterostructures we fabricated bottom gated FET devices. Ti/Au contacts were deposited for source and drain contacts on top of the 1 cm × 1 cm samples.  The channel length was about 6 mm. The schematic diagram of GFET structure is shown in 2. The gate response was measured for a gate voltage range of ±20 V and is shown in g ≈ +3 V, which indicates that the intercalated graphene exhibits hole doping. d ≈ 2.9 mA at Vg = 15 V and Vd = 0.5 V). These results might be due a high capacitance due to the TiO2 dielectric layer.To explore the electrical properties of our Gr/TiO2 has been widely used as photosensitive material. However, its large bandgap (3.2 eV) has restricted its use to ultraviolet applications since the photo response is only activated under ultraviolet radiation. Several approaches have been proposed to extend its photo response to a broader range such as dye sensitization, doping, and coupling with semiconductors23242/n-SiC heterostructures at room temperature (RT). The schematic diagram of measurement is shown in −1 conductance. Upon illumination, the device showed an increased current flow through the source-drain contacts with increased current flow at high bias voltage. The conductance of the graphene improved to 0.06 Ω−1, indicating a visible light photoresponse in our heterostructure. The calculated photo-responsivity of the heterostructure is about 278 mAW−1, which is much higher than that reported for modified graphene photo-detectors such as graphene metal junctions (6.1 mAW−1)−1)−1)g = −20 V, the graphene photoconductivity resulted in a conductance of 0.052 Ω−1 while it increased to 0.06 Ω−1 at 20 V. The difference between negative and positive gate voltage might be related to variations of net current flow through the channel. The response to turning the light on and off was measured under 0.2 V bias between source-drain as shown in −1, which is lower than that of our photo detector. Since the large bandgap (3.2 eV) of TiO2 does not allow for a photo-response to visible light, the photo-response observed here might originate from the Gr/TiO2 heterostructure. A possible mechanism for the enhanced visible light photoresponse seen in our Gr/TiO2 heterostructures may be provided by Geng, Liu and Yao2/graphene composites they calculated the electronic properties of atomic scale TiO2 clusters on graphene films and predicted anchoring of the clusters by charge rearrangement at the interface due to an interaction between the Ti 3d orbitals of the TiO2 and the p orbital of the graphene sheet. This results in extra states in the bandgap of TiO2 which enable absorption of visible light by the composite and the transfer of photoexcited electrons from the TiO2 valence band to the graphene conduction band. The subsequent separation of electrons and holes in graphene and TiO2 leads to long carrier lifetimes. In addition, since the interface between TiO2 and graphene plays a critical role, they also suggested that increasing the interface contact area could improve the photo response. We speculate that a similar process may be taking place in our comparatively large area heterostructures. The absorption of light takes place primarily in the TiO2 closest to the interface where their density of states is modified by the presence of the graphene layer. Electrons then transfer to the graphene layer and increase the electron density. The separation of electrons and holes reduces recombination and the holes in the TiO2 can screen the gating effect under positive bias while inducing more holes in the graphene layer under negative bias, leading to a reduced photoconductivity at negative bias. This model explains our experimental results shown in the gate response characteristics  with a metal carbide interface layer (TiC). After O2 intercalation the byproduct TiC layer was completely converted into TiO2, which resulted in a graphene/oxide heterostructure. We showed that this heterostructure exhibits dual functional properties, showing both electronic field effect phenomena and an enhanced visible light photosensitity. The field effect phenomena and enhanced photosensitivity were demonstrated in bottom gated GFET devices.We demonstrated that C60 molecules were deposited on the Ti layers in a high temperature molecular beam epitaxy system. The growth temperature and carbon flux were 1400 °C and 1.6 × 10−7 Torr. The growth time was 30 min. For the study of graphene growth on TiO2 surfaces, the as-deposited Ti/n-SiC sample was annealed in an O2 environment at 800 °C for 1 hr. The oxidation of the metallic Ti layer was confirmed by XPS analysis. The graphene growth was performed with the same growth conditions as those for Ti/n-SiC. After graphene growth, the sample was exposed to air while being transferred to the XPS chamber for O2 intercalation experiments.Metallic titanium layers (20 nm) were grown on an n-type SiC wafers by e-beam evaporation. CFor the field effect transistor device tests, Au/Ti metal contacts (source and drain) were deposited on the graphene surface. The photo response experiment was performed by illumination with a white LED at room temperature.2 was estimated by high resolution XPS spectra. The rate constant (k) for TiO2 formation due to intercalation was obtained by analyzing the temporal change in the ratio of TiC to TiO2 atomic concentrations at each intercalation temperature. By fitting an Arrhenius plot (Log (k) vs. 1/T), the activation energy (Eg) for oxidation was calculated. The graphene etching effect during O2 intercalation was monitored by the reduction of the graphene C 1s peak (284.7 eV) intensity. The etching activation energy was also calculated by estimating rate constant (k) and fitting the Arrhenius plot (Log (k) vs. 1/T). The temporal variation of C 1s ratio between as-grown and intercalated samples was used to calculate the rate constant (k) at each intercalation temperature.The temporal change in atomic concentration of TiC and TiO−8 Torr the sample was transferred to the analysis chamber. After initial XPS scans were acquired the sample was transferred back into the load-lock chamber for O2 intercalation. For the intercalation experiments research grade O2 was introduced into the chamber with a leak valve. The background pressure in the chamber was maintained at 1.0 × 10−4 Torr. Once the desired pressure was reached the sample was heated from the backside of the sample holder with a 10.6 μm CO2 IR laser. The laser output power was adjusted until the target temperature was reached. The temperature of the sample was measured with a pyrometer. After the intercalation the chamber was pumped down until a background pressure 3.0 × 10−8 Torr was reached. The sample was then transferred back into the analysis chamber for XPS data acquisiton.After graphene growth, the sample was transferred to the load-lock of the XPS system. When the background pressure of the load lock chamber reached 3.0 × 107/2 (84.0 eV) peak with an overall resolution of ~1 eV. Each spectrum was fit with a Shirley background followed by fitting convoluted Gaussian and Lorentzian peak shapes for each chemical state in the region of interest. For the C 1s graphene and Ti 2p metal peaks an additional asymetry parameter was added to the Gaussian-Lorentzian convolution. The fitting procedure consisted of a Levenberg-Marquardt routine that minimizes χ2.The XPS data was acquired with an Scienta Omicron standard Mg Kα (1253.6 eV) X-ray source and Argus hemispherical analyzer. High resolution scans of the C 1s, O 1s, and Ti 2p regions were acquired at 20 eV pass energy with 0.1 eV steps. The binding energy scale was calibrated against the Au 4fHow to cite this article: Park, J. et al. Approach to multifunctional device platform with epitaxial graphene on transition metal oxide. Sci. Rep.5, 14374; doi: 10.1038/srep14374 (2015).


Our results show that reducing the Fe3Ga4 dimensionality, via nanowire shape, intriguingly modifies its electronic structure. In particular, the bulk system exhibits two transitions, a ferromagnetic (FM) transition temperature at T1 = 50 K and an antiferromagnetic (AFM) one at T2 = 390 K. On the other hand, nanowires shift these transition temperatures, towards higher and lower temperature for T1 and T2, respectively. Moreover, the dimensionality reduction seems to also modify the microscopic nature of the T1 transition. Instead of a FM to AFM transition, as observed in the 3D system, a transition from FM to ferrimagnetic (FERRI) or to coexistence of FM and AFM phases is found for the nanowires. Our results allowed us to propose the magnetic field-temperature phase diagram for Fe3Ga4 in both bulk and nanostructured forms. The interesting microscopic tuning of the magnetic interactions induced by dimensionality in Fe3Ga4 opens a new route to optimize the use of such materials in nanostructured devices.This work reports on the dimensionality effects on the magnetic behavior of Fe Nanowires belong to a new class of quasi-unidimensional materials that have been attracting great interest in the last few years due to their numerous multidisciplinary potential applications, such as functional materials in biomedical sciencesRecently, the novel metallic-flux nanonucleation (MFNN) technique has been successfully developed to nucleate crystalline nanowires inside alumina membrane pores10103Ga4 intermetallic compound synthesized by the MFNN technique in both bulk and nanowire forms. As determined by Philippe et al. Fe3Ga4 presents a complex base-centered monoclinic structure with eighteen Fe atoms per unit cell occupying four non-equivalent sitesT1 = 50 K, while at higher temperatures, antiferromagnetism (AFM) takes place before vanishing at a Néel temperature T2 = 390 K131516173GaAs and GaAs on a GaAs (001) substrate, the Fe3Ga4 compound presents a distinctive photo-enhanced magnetization at room temperature203Ga4 interesting properties. To the best of our knowledge, no such studies about Fe3Ga4 compound have not yet been reported in the literature.In this work we present the magnetic characterization of the Fe3Ga4 magnetic field-temperature phase diagram (H–T), which was constructed for both bulk and nanowire systems using magnetization, specific heat and electrical resistivity measurements. The results are discussed within the framework of the Moriya and Usami’s theory on magnetic phase transitions in itinerant electron systems. As such, this work reveals unambiguous evidence for a dimensionality tuning of the Fe3Ga4 electronic structure that modifies the microscopic exchange parameters considered in Moriya and Usami’s theory. More generally speaking, we strongly believe that the possibility of growing intermetallic compounds in nanowire form will open an interesting branch in the understanding of fundamental properties, as well as permitting to control the nanowire characteristics for desired applications.In this work, we report the dimensionality effects on the Fe3Ga4 nanowires. As shown in μm, giving a length/diameter aspect ratio of around 100. Kα and Ga Lα energies, shown respectively in Scanning electron microscopy (SEM) was first performed to verify the presence, dimensions and composition of the FeKα radiation and θ–2θ scans were recorded in the 25°–55° range. From the study of the X-ray diffraction pattern we can affirm that the Fe3Ga4 phase was formed in the expected C2/m (#12) space group. The obtained crystals present a higher crystalline quality compared to those fabricated via arc melting3, which is commonly observed during Fe3Ga4 growthX-ray diffraction (XRD) data were used to identify the obtained bulk crystalline phase. The measurements were performed using Cu On the other hand, the nanowires structural characterization required synchrotron X-ray diffraction and absorption measurements, both performed at the Brazilian Synchrotron Light Laboratory (LNLS). The main difficulty encountered arises from the alumina membrane present around the nanowires, the latter only forming a small percentage of the measured system.2. As we can see in 23η- and θ-Al2O3 phases η-Al2O3 phase was not clearly observed. Instead, the main features of the observed profile could be modeled by a mixture of two θ-Al2O3 phases with distinct lattice parameters and ~4/1 proportion in the corresponding Bragg peak intensities. The observed lattice parameters, obtained from a Le Bail fit of the observed profile, are a = 11.918(1) Å, b = 2.9535(3) Å, c = 5.6864(4) Å, and γ = 103.92(1)° for the majority phase, and a = 12.052(6) Å, b = 2.998(1) Å, c = 5.771(3) Å, and γ = 103.44(5)° for the minority one. While the refined unit cell volume of the θ-Al2O3 phase of our untreated membrane (V = 187.3(1) Å3) agrees well with the literature value for θ-Al2O3 (V = 187.4–187.9 Å3 253 and V = 202.8(1) Å3, respectively). However, their comparison with unit cell volume values from the literature for isostructural β-Ga2O3  beamline to acquire the XRD spectra of the membrane with embedded nanowires, as well as the untreated empty porous alumina membrane. The data were collected in transmission mode at room temperature and atmospheric pressure with a wavelength of 0.6199 Å. The spot size was 2.0 × 0.2 mm3Ga4 and hematite (α-Fe2O3) were taken as standard samples. The near-edge region of the spectrum (XANES) is shown in 3+ ions, while the strong pre-edge feature at 7112 eV is consistent with the presence of a significant fraction of non-oxidized Fe. The observed spectrum could be fitted by a combination of the spectra from Fe3Ga4 (62(1)%) and α-Fe2O3 (38(1)%), with good agreement both in near-edge region  and the applied magnetic field H (±20 kOe). For the nanowire array characterization, the magnetic field was applied parallel to the nanowires axis.In order to investigate the magnetic properties and build the magnetic phase diagram of the FeT1) and a peak around 390 K (T2). In the case of nanowires, the reduced dimensionality affects T1 and T2 values in opposite ways. As we can see in T1 increases while T2 decreases. Moreover, the relative magnetization drop at the first temperature transition T1 is smaller for the nanowire array compared to the bulk one. This could suggest that we are dealing with a phase transition from a FM to another magnetic phase order with net macroscopic magnetization in the nanowire case (FERRI or coexistence of FM and AFM). As discussed later, this phase coexistence is predictable from Moriya’s theory. This is the first evidence that the nature of this transition differs in the nanowires and in the bulk samples. We also observe that the temperature-dependent magnetization curves exhibit appreciable thermal hysteresis around T1, but only in the Fe3Ga4 nanowires case (not shown here), which decreases when we increase the applied magnetic field. These effects persist for all magnetic field values used in the magnetization measurements, and are characteristic of first order phase transition. In the case of bulk samples, no thermal hysteresis is noticed. Furthermore, we can see that the magnetic signal from Fe3Ga4 phase (in emu/g) is about 500 times smaller that for the bulk crystal, which is consistent with our estimative by XRD that the weight fraction of the Fe3Ga4 phase in the membrane is below 2%.From the magnetization as a function of temperature curves, the two expected magnetic phase transitions are distinguished in terms of a magnetization drop around 50 K  measurements were performed in the 2–100 K and 2–20 kOe ranges of temperature and magnetic field, respectively. As shown in C/T versus T 2 curves at low temperatures give similar Debye temperatures (θB = 230(2) K and θN = 253(15) K for bulk and nanowires, respectively). Since θ is related to the phonon spectrum, which in turn is related to the material crystalline structure, this result demonstrates that the nanowire array exhibits the same crystalline structure than that of Fe3Ga4 bulk phase. Moreover, we observe a reduction in the contribution of the conduction electrons to the specific heat when the system dimensionality is reduced. Remarkably, the electronic coefficient (γ) drops from 25.60 mJ/mol.K2 to almost zero , indicating that the Fe3Ga4 compound tends to become insulating in the nanowire morphology. These data are consistent with the magnetization results, since that a FERRI or FM behavior is most likely to be expected in localized electron magnetism. In addition, field-dependent specific heat measurement performed at 80 K shows a maximum at about 4 kOe for bulk Fe3Ga4 .Bulk electrical resistivity measurements were performed using a standard four-probe technique in the 2–400 K temperature range and under magnetic fields of 0 and 7 kOe. Without applied magnetic field, temperature-dependent electrical resistivity curve exhibits a slope change at approximately 65 K , or a ferromagnetic to ferrimagnetic or coexistence of ferromagnetic and antiferromagnetic transition, if under nanowire form. The specific heat data are also consistent with these conclusions, since a FERRI or FM behavior is most likely to be expected in localized electron magnetism. Finally, at T2, the applied field decreases the temperature of maximum susceptibility similarly for both systems.Experimental magnetic, specific heat and electrical results yield to the built magnetic phase diagrams of the Feionality . Based o3Ga4 bulk and nanowires by the new technique of metallic-flux nanonucleation. All the results suggest that, based on the Moriya’s theory, there is a transition from ferromagnetic to antiferromagnetic state for Fe3Ga4 bulk, while that for Fe3Ga4 nanowire array, there is a transition from ferromagnetic to ferrimagnetic or a coexistence of ferromagnetic and antiferromagnetic state. These changes in the nature and aspects of the magnetic transitions are supported to arise from electronic structure modifications, which can be tuned by controlling the system dimensionality. These results open the possibility for studying fundamental aspects of magnetic behavior of any intermetallic compound that can be fabricated by the metallic flux method, as well as could allow the design of new nanostructures used in nanometric devices with desired physical features.We have successfully grown both Fe3Ga4 compound were synthesized through the innovating method of metallic-flux nanonucleation (MFNN). The MFNN technique is based on the conventional flux-growth techniqueμm thick, with 250 nm diameter pores arranged in a hexagonal array with 270 nm interpore distance. Both Fe3Ga4 compounds in bulk and nanowire shape were simultaneously obtained in the same bath using the Ga self-flux technique. For this procedure, appropriated quantities of high purity (99.995%) Fe and (99.999%) Ga precursors were deposited in an alumina crucible, together with the nanoporous alumina template properly fixed at the crucible bottom and sealed in a quartz tube under vaccum. In order to form Fe3Ga4 crystals via the flux-growth technique, the sealed tube was heated up to 1100 °C for 12 h, cooled down to 700 °C at 3 °C/h and finally cooled down to 400 °C at 8 °C/h. The filled pores embedded in the nanoporous alumina template were subsequently carefully separated from the bulk crystals.Bulk single crystals as well as nanowires specimens of FeSince the MFNN method yields a simultaneous synthesis of crystalline bulk and nanowires, any intermetallic compound that can be prepared by the flux-growth method can also be, in principle, synthesized in nanowire form. This represents an enormous advantage for the MFNN method.How to cite this article: Moura, K. O. et al. Dimensionality tuning of the electronic structure in Fe3Ga4 magnetic materials. Sci. Rep.6, 28364; doi: 10.1038/srep28364 (2016).


T1 mapping is widely used today in CMR, however, it underestimates true T1 values and its measurement error is influenced by several acquisition parameters. The purpose of this study was the extraction of accurate T1 data through the utilization of comprehensive, parallel Simulations for QUAntifying RElaxation Magnetic Resonance constants (SQUAREMR) of the MOLLI pulse sequence on a large population of spins with physiologically relevant tissue relaxation constants.A CMR protocol consisting of different MOLLI schemes was performed on phantoms and healthy human volunteers. For every MOLLI experiment, the identical pulse sequence was simulated for a large range of physiological combinations of relaxation constants, resulting in a database of all possible outcomes. The unknown relaxation constants were then determined by finding the simulated signals in the database that produced the least squared difference to the measured signal intensities.SQUAREMR demonstrated improvement of accuracy in phantom studies and consistent mean T1 values and consistent variance across the different MOLLI schemes in humans. This was true even for tissues with long T1s and MOLLI schemes with no pause between modified-Look-Locker experiments.SQUAREMR enables quantification of T1 data obtained by existing clinical pulse sequences. SQUAREMR allows for correction of quantitative CMR data that have already been acquired whereas it is expected that SQUAREMR may improve data consistency and advance quantitative MR across imaging centers, vendors and experimental configurations. While this study is focused on a MOLLI-based T1-mapping technique, it could however be extended in other types of quantitative MRI throughout the body. In the field of cardiovascular magnetic resonance (CMR) T1 mapping, quantitative measures of myocardial and blood T1 enabled the calculation of important myocardial biomarkers, such as extracellular volume (ECV) fraction and native T1 in the myocardium . Recent While T1 mapping has the potential to improve patient diagnosis, the challenge remains in acquiring reliable data in terms of accuracy and precision. Different T1 mapping methods and parameter sets should yield similar results for a specific tissue type. However, the complex nature of the underlying physics involved and the multitude of parameters that affect image acquisition and post-processing do not allow for consistent reference T1 values of normal myocardium and blood across all methods. Examples of this inconsistency include recent studies on cardiac T1 mapping, which have reported different ranges of T1 values for normal myocardium and blood depending on the methods being used , 11–13.More recently, a single vendor, multicenter clinical study  demonstrThe development of new CMR techniques for obtaining quantitative information usually involves a two-step process: acquisition of images from the MRI scanner with a custom designed pulse sequence and post-processing of the acquired images through a data fitting procedure with closed form equations. Recently, a novel approach to collect and process images for extracting quantitative data from MRI experiments was proposed . This neIn the past, CMR simulations have been used within a limited scope. Simulations have been used to identify the effect of various acquisition parameters on measurement accuracy and precision , 16 and In this study, we propose Simulations for QUAntifying RElaxation Magnetic Resonance constants (SQUAREMR), a new method for extracting quantitative tissue MR data from clinical pulse sequences with the aid of comprehensive, parallel MRI simulations of the Bloch equations. The specific aim was to utilize realistic simulations of MOLLI on a large population of spins so as to compute all possible outcomes of this pulse sequence for a range of physiologically relevant tissue relaxation constants. We hypothesized that quantitative CMR acquired with MOLLI can be improved by comparing the signals acquired from the MRI scanner to the entire pool of possible outcomes that are produced by these simulations for different tissue types. While this study is focused on a MOLLI-based T1 mapping technique, it could however be extended in other types of quantitative MRI throughout the body.Figure In MOLLI T1 mapping, the bSSFP readouts that follow the IR pulse perturb the relaxation process itself and do not allow for an ideal exponential recovery that is based solely on the equilibrium magnetization (M0) and the relaxation constant T1. As a result, the true recovery follows an apparent relaxation constant T1* which is always shorter than the true relaxation constant T1. MOLLI reconstruction compensates for this apparent relaxation by means of a 3-parameter exponential signal model and the “Look-Locker” correction factor . HoweverThe basic concept of this study is based on the premise that the signals obtained from the simulation of a clinical pulse sequence on simulated tissue with specific relaxation constants  would be identical to the measured signals acquired from the MRI scanner by applying the same pulse sequence on real tissue with the same relaxation constants . Therefore, for the same pulse sequence, obtaining identical simulated and measured signals would ideally indicate that the tissue constants were identical in both the simulation and the MRI scanner. Using a closed form expression of the clinical pulse sequence was not necessary but instead the identical pulse sequence was simulated and was applied on a computer model of spins. The solutions of the Bloch equations provided the temporal evolution of each spin magnetization vector under the influence of the RF pulses and magnetic field gradients of the pulse sequence.A basic block diagram of SQUAREMR is shown in Fig. The acquisition parameters described the MOLLI pulse sequence. These included the following parameters: number of inversion-recovery-prepared modified-Look-Locker experiments, number of ECG-triggered single-shot acquisitions per modified-Look-Locker experiment, number of pausing heart cycles between modified-Look-Locker experiments, IR RF pulse duration and shape, TR and TE durations, bSSFP readout RF pulse duration and shape, slice thickness, acquisition scheme, Field-Of-View (FOV), matrix size, SENSE factor, receiver bandwidth (rBW) and number of startup TRs in the linear flip angle ramp.In MOLLI, the TIs for a number of the single-shot images depend on the actual heart rate and cannot be known before the pulse sequence execution. Therefore, the simulations were performed after image acquisition.A comprehensive MR physics simulator, MRISIMUL, was used , 20. ComFor each MOLLI experiment, the identical pulse sequence was simulated on a population of spins for a large range of physiological combinations of T1 and T2. T1 and T2 values of 200–1900 msec and 20–400 msec respectively were simulated with a step of 1 msec. Simulations were not performed for combinations where T2 > T1. To explore faster SQUAREMR execution times, a step of 5 msec was also used. The ranges of T1 and T2 values were chosen based on physiological myocardial and blood values found in the literature , 14 withSimulations were performed on a single-node system consisting of a server style computer of 2 hexa-core  processors, 32 GB RAM and four Tesla C2075 GPU cards. Each Tesla C2075 graphics card utilized a total dedicated memory of 6 GB GDDR5 and a total of 448 stream processors.To investigate SQUAREMR performance, two sets of pre-Gd MOLLI experiments were performed. For a given experiment, the total SQUAREMR execution time could be reduced by decreasing either the total number of spins within the spin model or the total number of pulse sequence time steps or the T1 map size or a combination of them.The first set of MOLLI experiments was performed with a simulation temporal step of 10 μsec and an acquisition matrix size of 128x128 whereas the second set of experiments was performed with a simulation temporal step of 5 μsec and an acquisition matrix size of 320x320. To further investigate how spin model size relates to SQUAREMR performance, two different test cases were considered for each set of experiments. For the first test case, a short T1 and T2 sampling step of 1 msec was combined with a large number of 101 spins along slice thickness resulting in a total of approximately 533,000 database entries. For the second test case, a longer T1 and T2 sampling step of 5 msec was combined with a small number of 21 spins along slice thickness resulting in a total of approximately 21,500 database entries. For both test cases, T1 and T2 values of 600–2000 msec and 20–400 msec respectively were simulated.4 and Agar , even in cases of “tissues” with long T1s and MOLLI schemes that do not allow for full relaxation of long T1s prior to the next inversion. Conventional MOLLI post-processing in these cases presented a bias higher than 100 msec for the phantoms with high T1 values.Results from the first phantom are shown in Fig. p < 0.05; 4(1p)3(1p)2 scheme bias of 11.4 ± 16.1 msec vs. 90.2 ± 89.3 msec, p < 0.05; 5(0p)3 scheme bias of 16.9 ± 27.2 msec vs. 101.3 ± 94.3 msec, p < 0.05; N = 6], 3 scheme bias of 12.1 ± 20.5 msec vs. 54.7 ± 45.7 msec, N = 10) against their reference T1 and reference T2 values. SQUAREMR demonstrated small error  in estimating T1 for all T1 and T2 combinations. The error was also relatively small in estimating T2  for phantoms with high T1 and low T2 values. For other T1 and T2 combinations, SQUAREMR yielded larger errors  in estimating T2.Figure p < 0.001, N = 12). The average myocardial T1 value for segment 9  was 1053 ± 33 msec for conventional MOLLI post-processing and 1148 ± 38 msec for SQUAREMR . The average blood T1 values were 1570 ± 52.8 msec and 1634 ± 50 msec respectively . As other studies have already shown [Figure p < 0.001 for myocardial 5(3p)3 vs. 5(0p)3]. Table Figure To illustrate the performance of SQUAREMR, two different sets of pre-Gd MOLLI experiments were examined. The simulation execution times and the database search times for different MOLLI experiment sizes were recorded and are shown in Table A new method for improving measurements from clinical pulse sequences in CMR relaxometry was presented. The use of parallel simulations was shown to improve the T1 estimates in MOLLI by comparing the MRI signals acquired from the MRI scanner to the entire pool of physiological simulated signals that were produced by parallel simulations of the identical pulse sequence on a population of spins. While the current study explored the feasibility of obtaining T1 properties from MOLLI images, in principle it could be extended in other areas of quantitative MR.MOLLI T1 mapping is widely used today; however the correction of the T1 recovery relies on the a FLASH closed form expression  only becIn the current work, a CMR protocol consisting of three different MOLLI pulse sequences was implemented: 5(3p)3, 4(1p)3(1p)2 and 5(0p)3. For every MOLLI experiment, the identical MOLLI pulse sequence was simulated for T1 and T2 ranges of 200 to 1900 msec and of 20 to 400 msec respectively taking into account realistic aspects of the MR experiment, such as realistic excitation slice profiles and heart rate variation in in-vivo studies. For that purpose, MRISIMUL, a GPU-based, MR physics simulator [The CMR T1 mapping protocol was applied on three phantom setups and on twelve healthy volunteers. All twelve volunteers and phantom setups were examined with the clinical 5(3p)3 scheme. The actual TI timings were extracted from the scanner data and used for the SQUAREMR simulations allowing for more realistic simulations. Four out of twelve volunteers and two out of three phantom setups were imaged additionally with 4(1p)3(1p)2 and 5(0p)3. Results of the CMR protocol on the phantom setups demonstrated improvement of accuracy and a substantially reduced T1 variability across the different MOLLI schemes. Compared to conventional MOLLI post-processing, SQUAREMR showed improved accuracy even for long T1s with no pause between modified-Look-Locker experiments. The latter demonstrates that the T1 estimates are not dependent on the duration of the delay between successive modified Look-Locker experiments, which in patient scans may change due to heartrate variations.In order to investigate whether SQUAREMR could also provide T2 estimates from data obtained with a MOLLI pulse sequence, the methodology was applied on phantoms with a range of T1 and T2 values. SQUAREMR showed reasonable T2 values in phantoms with long T1s and short T2s. Larger errors in estimating T2 were observed for the remaining T1 and T2 combinations; however, this was expected for a T1 mapping pulse sequence, such as MOLLI. Previous studies have shown that the MOLLI pulse sequence introduces some T2 modulation on the signal, which is more pronounced for long T1 and short T2 values , 34; theN = 4. SQUAREMR showed substantially elevated T1 values for both myocardium and blood pool when compared to conventional MOLLI post-processing. Conventional MOLLI T1 values in this work were similar to values previously reported in the literature. In particular, the conventional MOLLI scheme 5(3p)3 yielded an average blood T1 value of 1570 ± 52 msec (N = 12). Previous studies have reported 1534 msec with MOLLI [N = 12) was similar to that of previous studies with MOLLI (1034 ± 56 msec [N = 12) was close to that previously reported with SASHA (1639 ± 97 msec [N = 12) which was close to that previously reported with rapid cardiac gated single-shot IR-FSE sequence . On the scheme 5p3 yielde 64 msec ) and two 64 msec ).The application of a parallel realistic simulator in order to correct measured data from the scanner is a new concept. In the past, MR simulations have been used in a limited scope , 16, 17 In this work, some limitations apply. Simulation of magnetization transfer (MT) was not studied although previous simulation studies , 37 suggIn conclusion, SQUAREMR is a new method that allows for quantification of CMR data with already available clinical pulse sequences and with the aid of comprehensive, parallel MRI simulations. In this study, a MOLLI-based T1 mapping example was investigated demonstrating improvement of accuracy in phantom studies and consistent mean T1 values and consistent variance across the different MOLLI schemes in humans. This was true even for a wide range of T1 values with MOLLI schemes with no pause between modified-Look-Locker experiments, indicating potential value for myocardial tissue characterization within just 8 heart beats.The methods presented in this study represent a different approach in quantitative CMR with existing clinical pulse sequences. SQUAREMR allows for correction of quantitative CMR data (e.g. MOLLI T1 maps) that have already been acquired by simulating the clinical pulse sequence that was used for the data acquisition. Last, it is expected that in the future SQUAREMR may improve data consistency and advance quantitative MR to become more robust across imaging centers, vendors and experimental configurations and the technique may be extended in other areas of quantitative MR imaging.The study was approved by the local ethics committee and all subjects provided written consent for publication of this study and accompanying images .


Pharmacodynamics studies have raised questions about how these inhibitors reach SGLT2 in the brush border membrane of the S1 and S2 segments of the renal proximal tubule: are these drugs filtered by the glomerulus and act extracellularly, or do they enter the cell and act intracellularly? To address this question we expressed hSGLT2 in HEK‐293T cells and determined the affinity of a specific hSGLT2 inhibitor, TA‐3404 , from the extra‐ and intracellular side of the plasma membrane. Inhibition of SGLT2 activity (Na+/glucose currents) by TA‐3404 was determined using the whole‐cell patch clamp that allows controlling the composition of both the extracellular and intracellular solutions. We compared the results to those obtained using the nonselective SGLT inhibitor phlorizin, and to the effect of TA‐3404 on hSGLT1. Our results showed that TA‐3404 is a potent extracellular inhibitor of glucose inward SGLT2 transport (IC50 2 nmol/L) but it was ineffective from the intracellular compartment at both low (5 mmol/L) and high (150 mmol/L) intracellular NaCl concentrations. We conclude that TA‐3404 only inhibits SGLT2 from the extracellular side of the plasma membrane, suggesting that it is filtered from the blood through the glomerulus and acts from within the tubule lumen.SGLT2 inhibitors are a new class of drugs that have been recently developed to treat type II diabetes. They lower glucose levels by inhibiting the renal Na An emerging class of new drugs to treat diabetes is the SGLT2 inhibitors which control blood glucose levels by excreting glucose into the urine. Questions have arisen about how these inhibitors reach SGLT2 in the brush border membrane of the S1 and S2 segments of the renal proximal tubule. Using patch‐clamp technique, we characterized the effect of a canagliflozin derivative (TA‐3404) on glucose‐induced currents and we showed that the inhibitor binds to SGLT2 from the extracellular side of the plasma membrane. This provides indirect evidence that SGLT2‐specific drugs act from the tubular lumen and not from the blood through the tubular epithelium. For Na+‐free solution (Choline buffer) NaCl was equimolarly replaced with cholineCl. For whole‐cell patch‐clamp experiments at 37°C, mannitol (100 mmol/L) was added to the extracellular solution to reduce noise and improve stability in the whole‐cell recording mode. The standard intracellular solution (pipette solution) contained, in mmol/L, 145 CsCl, 5 NaCl, 11 EGTA and 10 HEPES/TRIS, pH 7.4. High Na+ intracellular solution contained in mmol/L: 150 NaCl, 11 EGTA and 10 HEPES/TRIS, pH 7.4.The standard extracellular solution  supplemented with 10% fetal calf serum  and 1% penicillin‐streptomycin . Cells were maintained in incubator at 37°C in humidified atmosphere of 5% CO2 and 95% air. Cells were passaged (1:10) every 5 days, and before transfection were seeded in six‐well plates or poly‐L‐lysine‐coated 24‐well plates. Cells were grown till 50–70% confluence and then transfected with 1 μg of the vector containing the hSGLT2 or hSGLT1 coding region (IRES‐hSGLT2 or IRES‐hSGLT1) using the Effectene kit .The protocol that we used to grow and transfect the cells have been already described elsewhere , a specific substrate for SGLTs, and washed in cold Choline buffer three times. The cells were solubilized and a sample was assayed using scintillation counting. Uptake was normalized to total cellular protein. For each tested condition, the sample size was n = 3–4 wells and each experiment was repeated at least twice. Uptakes were measured in the presence and absence of 100 μmol/L phlorizin or the indicated concentration of TA‐3404.Sugar uptake was measured in HEK‐293T cells expressing hSGLT1 or hSGLT2 as described , whereas for hSGLT1, experiments were carried out at 22°C. Currents were filtered at 2 kHz and digitized at 1 kHz.The procedure for whole‐cell patch‐clamp experiments has been explained elsewhere  were obtained by subtracting the baseline current recorded in Na+ buffer to the total current measured in Na+ buffer + glucose: Glucose currents  was determined by measuring the sugar‐dependent steady‐state current or radiotracer uptake as a function of external inhibitor concentration. In these experiments, the glucose concentration was close to the K0.5 of the sugar so the concentration of the inhibitor producing 50% inhibition (IC50) is twice the Ki  were determined by recording the time course of recovery of the D‐glucose‐coupled current after rapid removal of the inhibitor, assuming pseudo‐first‐order binding and dissociation kinetics  related to the off rate: The D‐glucose‐coupled current recovery follows an exponential time course with time constant 1/kon) is calculated from the empirically measured Ki values and koff values: The ON rate . In the graphs, data are shown as mean ± SE.+‐dependent 14C‐α‐methylglucoside (α‐MDG) uptake with an IC50 of 2 nmol/L against SGLT2 and 750 nmol/L against SGLT1 (~350‐fold selectivity for SGLT2 vs. SGLT1). Hereby, we decided to confirm these results investigating the effect of TA‐3404 on hSGLT1 or hSGLT2 activity in HEK‐293T cells. Uptakes of 50 μmol/L α‐MDG into the transfected cells were measured in the presence and absence of inhibitors  generated an inward current of 37 pA that was completely inhibited by the addition of 300 nmol/L TA‐3404  we measured the recovery time of SGLT glucose‐induced current when the inhibitor is rapidly removed from the extracellular solution.The experimental approach to calculate n = 3) seconds). In three experiments, there was no further recovery of the hSGLT2 current over 5 min. This is contrary to the results where phlorizin inhibition was 100% reversible, but the time course for the incomplete reversal was similar to phlorizin  seconds, comparable to the half‐time for phlorizin on hSGLT1  by measuring the effect of increasing concentrations of TA‐3404 on radiotracer uptake (for hSGLT2) or glucose‐induced current (for hSGLT1). We decided to use two different approaches due to the fact that hSGLT2 has low expression, yielding small glucose‐induced currents. For radiotracer uptake experiments, tracer entry in the cell is measured over the period of 30 min leading to a higher sensitivity of the method. For hSGLT2 : for hSGLT1 we computed a kon of 25 × 106 mol/L per sec, whereas for hSGLT2 of 1.4 × 106 mol/L per sec.From these results we were able to calculate the Ki ~2 nmol/L) that is similar to the one measured for phlorizin and drugs such as dapagiflozin   in the glucose‐induced current with the addition of 200 μmol/L phlorizin.We repeated the experiments on hSGLT2 cells, now using a high Naolution 10 mmol/L +/glucose current was ~85 pA. There was no effect when 10 and 200 μmol/L phlorizin or 200 nmol/L TA‐3404 were added to the intracellular solution. + solution (150 mmol/L). As observed for hSGLT2, we did not detect any significant effect on the glucose‐induced current by 10 μmol/L phlorizin or 200 nmol/L TA‐3404, but there was a dramatic decrease in current when 200 μmol/L phlorizin was present in the intracellular solution.Ki 6 nmol/L, but are relatively poor inhibitors of hSGLT1, Ki 390 nmol/L  TA‐3404 binding to hSGLT2 probably involves two components: a reversible component with an Off rate close to that for phlorizin, 0.3 per sec, and a component with an extremely slow dissociation constant. This interesting property of TA‐3404 binding to hSGLT2 warrants further investigation. It should be noted that dapagliflozin binding to hSGLT2 exhibits a very slow Off rate   is due to the selectivity and kinetics of a transporter responsible for reabsorption. The bulk of the drug in circulation is taken up by the liver and excreted as free drug and/or metabolites in the bile.In summary, our results provide indirect evident that SGLT2 inhibitors block the reabsorption of glucose in the early proximal tubule from the glomerular filtrate and that the drugs are reabsorbed further down the tubule. Additional experiments are required to provide direct tests our hypothesis.Xenopus laevis oocytes.We thank Erika Patino for preliminary experiments on the effect of TA‐3404 on hSGLT1 in None declared.