Constitutive activation of the NF-κB signaling cascade is associated with tumourigenesis and poor prognosis in many human cancers including RCC. YM155, a small molecule inhibitor of survivin, was previously shown to potently inhibit the viability of immortalized and patient derived renal cell carcinoma (RCC) cell lines. Here we investigated the role of NF-κB signaling to the anti-cancer properties of YM155 in RCC786.0 cells. YM155 diminished nuclear levels of p65 and phosphorylated p65 and attenuated the transcriptional competencies of the p65/p50 heterodimers. Accordingly, we found that YM155 diminished the transcription of NF-κB target gene survivin. Events that led to the interception of the nuclear translocation of p65/p50 were the activation of the deubiquinating enzyme CYLD by YM155, which led to the inhibition of IKKβ, stabilization of IκBα and retention of NF-κB heterodimers in the cytosol. Importantly, the suppressive effects of YM155 were time-dependent and observed at the 24 h time point, and not earlier. TNF-α, a stimulator of NF-κB signaling did not affect its inhibitory properties. The ability of YM155 to intercept a major transcriptional pathway like NF-κB, would have important ramifications on the pharmacodynamics effects elicited by this unusual molecule. The clear cell histological variant is the most common manifestation of renal cell carcinoma and has a pathology that is unique among other RCC subtypes in its association with the mutated von Hippel Lindau (VHL) tumour suppressor gene.Renal cell carcinoma (RCC) is the most lethal urologic malignancy. Approximately one in three patients with RCC exhibit visceral metastasis6  revealed prominent clustering within the p53 connection pathway. We followed up on three genes that were relevant to RCC pathogenesis and have not been implicated as targets of YM155, namely CYLD , FOXO1 (a tumour suppressor that mediates cell cycle arrest and promotes apoptosis) and ID1 13. Collateral evidence from both mRNA and protein analyses revealed that YM155 downregulated ID1 expression but stimulated CYLD and FOXO1. The activation of CLYD by YM155 was of particular interest because YM155 has been reported to intercept the NF-κB pathway in a non-small cell lung cancer cell line14. The NF-κB transcriptional network plays a pivotal role in regulating cell proliferation, invasion and apoptosis. Briefly, NF-κB is sequestered in an inactive state within the cytosol by inhibitory IκBs which mask the DNA binding ability and hence transcriptional competency of NF-κB. In the presence of activating stimuli, the IκBs are phosphorylated by IκB kinases (IKKs) and directed to the proteasome for degradation. This allows the liberated NF-κB (mainly p65/p50 heterodimers) to be phosphorylated and transported to the nuclei where they recruit chromatin remodelling factors on the NF-κB promoters for activation of target genes.While YM155 suppresses survivin levels in cancer cells, as confirmed in several investigations17. While the effects of YM155 on cell cycle progression and cell death, which are outcomes of p53 regulation, are well established, less is known of its interactions with the NF-κB signaling pathway. These knowledge gaps restrict the rational deployment of YM155 singly or in combination with other agents for cancer chemotherapy. Here we demonstrate that YM155 caused a time dependent attenuation of NF-κB signaling in RCC786.0 cells, primarily by intercepting the nuclear translocation of the p65 subunit and hence curtailing its transcriptional activity.The reciprocity between the p53 and NF-κB signaling pathways which are known to modulate each other’s activities, is widely recognizedVHL gene but they possess wild type p53. We reconfirmed our earlier findings that YM155 potently inhibited the proliferation of RCC7860.0 cells at nanomolar concentrations (growth inhibitory IC50 40 nM)  in the absence of TNF-α Fig. . IntriguWe see here that YM155 exerts a biphasic, time dependent effect on the binding of p65 to its consensus sequence. An early increase in binding affinity (6 h) was followed by a decline at the longer 24 h time point. Both effects were concentration dependent and more pronounced in cells that were not stimulated by TNF-α.19. As shown in Fig. As the binding of NF-κB dimers to their consensus sequences would activate transcription of NF-κB controlled genes, we anticipated YM155 to effect an initial increase in transcriptional activity followed by a subsequent decrease, in line with its biphasic effects on DNA binding. To investigate, RCC786.0 cells were transiently transfected with a luciferase reporter gene whose expression was regulated by NF-κB. These cells were then exposed to YM155 (40 nM) for 6 h and 24 h, in the absence or presence of TNF-α. Similar experiments were carried out with 9-aminoacridine , a known inhibitor of NF-κB dependent transcriptionNext, we asked if the suppression of NF-κB transcriptional activity in YM155-treated cells could be due to diminished levels of p65/p50 heterodimers, in particular p65 which carries the transactivation domain (TAD) required for transcription. To this end, we monitored the levels of p65 and phosphorylated p65 in the cytosolic and nuclear fractions of treated cells at the 6 h and 24 h time points. These experiments were carried out with or without TNF-α  for 24 h. The experiments were repeated in the presence of TNF-α. As shown in Fig. We also probed the levels of CYLD, IKK, phospho-IKK, IκBα and phospho-IkBα at the 6 h time point where there was no suppression of the pathway by YM155. As shown in Fig. BIRC5 which encodes survivin was selected for investigation and the effects of YM155 on the mRNA and protein levels of survivin was examined in Fig. To further validate our hypothesis that YM155 suppressed NF-κB signaling, we proceeded to demonstrate that suppression extended to genes that are regulated by NF-κB. The gene et al. reported upregulated survivin expression in rat dendritic cells stimulated with TNF-α20, while others opined that TNF-α had either no effect on survivin mRNA/protein levels or decreased survivin protein levels23. Notwithstanding these contrarian views, our findings point to the attenuation of survivin expression by YM155 at a late time point, in the absence of TNF-α.A closer inspection of the survivin immunoblots from control cells exposed to TNF-α showed unusual losses in band intensities at both the 6 h and 24 h time points Fig. . These lBIRC5 promoter region can be quantified by qRT-PCR.In the preceding sections, we have shown YM155 diminished the binding of p65 to its consensus sequences, attenuated the transcription of NF-κB related genes in treated luciferase transfected RCC786.0 cells, delayed the translocation of p65 to the nucleus and suppressed levels of phosphorylated p65 within that compartment. These events culminated in the YM155-induced attenuation of survivin expression. To provide further collateral evidence to support our hypothesis, we investigated the recruitment of p65 to NF-κB-dependent promoters by the chromatin immunoprecipitation (ChIP) assay. Briefly, this technique is a means of examining the specific association of transcription factors with DNA in the context of living cells. It provides a snapshot of the interaction between the NF-κB heterodimers (p65/p50) and their DNA binding sites on the target gene within the natural chromatin context of the cell. Primers designed to flank the NF-κB DNA binding region were used to amplify the chromatin fragments immunoprecipitated by the p65 antibody. In this manner, the extent to which p65 binds to the BIRC5 promoter region  in the binding of p65 to two separate sites of the ion Fig. . These o24, and mitochondria as well as processes of which autophagy and the ER stress response have been implicated11. We have shown here that YM155 suppressed the NF-κB pathway through a sequence of events that culminated in the interception of the nuclear translocation of p65/p50. We proposed that the activation of CYLD by YM155 provided the initial trigger which led to the inhibition of IKKβ, thereby stabilizing IκBα in its inhibitory state and the retention of NF-κB dimers in the cytosol. Consequently, the transcriptional competency of these heterodimers was suppressed and we have assembled a body of collateral evidence to support this claim. Accordingly, we demonstrated that YM155 diminished the binding of p65 to DNA consensus sequences, decreased NF-κB transcriptional activity of luciferase transfected cells and suppressed levels of p65 in the nuclear compartment, and through the ChIP assays, provided evidence of diminished binding between p65 and the promoter regions of survivin in YM155 treated cells.YM155 has been a highly controversial anticancer agent since it was reported nearly a decade ago. Proclaimed as the first small molecule to suppress survivin, it soon became apparent that YM155 had pleiotropic effects, affecting diverse targets such as DNA13. It is conceivable that had we monitored the effects of YM155 for a longer time period (>24 h), we would have obtained different results. In fact, we speculate that time dependency in the effects of YM155 may explain the contrarian results of a related study on the interaction of YM155 with the NF-κB pathway. In that report, results were gathered at the 48 h time point from YM155-treated non-small cell lung cancer H1299 cells14. Although the authors concluded that YM155 also suppressed the NF-κB pathway, its effects were directed against the phosphorylation of p50 rather than the nuclear translocation of NF-κB dimers. That the H1299 cells possess mutated p53, unlike RCC786.0 cells which carry wild type p53, may yet be another confounding factor.It should be highlighted that the suppressive effects of YM155 on the NF-κB pathway was only evident at 24 h and not at the earlier 6 h time point, where YM155 had an anomalous stimulatory effect on the pathway. Tellingly, we have earlier shown that in YM155 treated RCC786.0 cells, CYLD mRNA levels were upregulated at the 24 h time point but returned to basal levels after 48 hUnlike the suppressive effects of YM155, evidence to support its early stimulatory activity on the NF-κB pathway was not compelling. Notably, increases in the binding affinity of p65 to its DNA consensus sequences and elevated levels of p65 in the nucleus did not translate to greater transcriptional activity. We speculate that YM155 may have impeded post translational processes or adversely affected the levels of co-activators/ co-repressors that are required for the transcription process.BIRC5 which encodes survivin was selected for investigation and we showed that YM155 downregulated BIRC5 expression, reduced survivin levels and intercepted the interaction between p65 and the DNA binding sites on the promoter regions of survivin. Tellingly, the reductions in the BIRC5 mRNA and protein levels of survivin in YM155 treated cells were only evident at 24 h, at which point, the suppressive effects of YM155 on nuclear p65 were also observed. If the NF-κB modulatory effects of YM155 were related to indirect inhibition of survivin, changes in survivin levels would precede those involved in the NF-κB pathway, which were not observed here. These misaligned time-related trends, together with the stimulatory effects of YM155 on CYLD, led us to propose NF-κB involvement in YM155-induced suppression of survivin, although we cannot completely discount the contributions of other signaling pathways like p53 at this stage of our investigations.To further validate our hypothesis that YM155 suppressed NF-κB signaling, we proceeded to demonstrate that suppression extended to genes that are regulated by NF-κB. The gene Another point that warrants mention is the influence of TNF-α on the effects of YM155. Broadly similar results were obtained regardless of whether cells were treated with TNF-α or not, although some effects were more marked in the presence of TNF-α. Taken together, we propose that the stimulatory effects of TNF-α on the NF-κB pathway were not affected by YM155.25 with reported promising therapeutic effects27. The suppressive effects of YM155 on NF-κB signaling provide a wider perspective on the pharmacodynamic effects elicited by this unusual molecule and potentially, its use in therapeutic combinations to enhance the clinical efficacy in the treatment of RCC and other cancers.In conclusion, we have shown the time dependent (24 h) suppression of NF-κB signaling by YM155 in RCC786.0 cells which occurred independently of TNF-α stimulation. The suppression may be due to diminished ubiquitination induced by greater CYLD activity. Given that NF-κB dysfunction is central to oncogenic processes; it has become a target of interest for anti-cancer treatment2 and 95% filtered atmospheric air and maintained at a temperature of 37 oC.RCC786.0 was obtained from American Type Culture Collection . All cells were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) containing L-glutamine, sodium pyruvate, 10% FBS and 100U Penicillin/Streptomycin . All cells were grown in a humidified atmosphere of 5% CORCC786.0 cells were treated with increasing concentrations of YM155 (20–160 nM) or 0.1% DMSO in the presence or absence of TNF-α (20 ng/ml) for 6 h or 24 h. NE-PER Nuclear & Cytoplasmic Extraction Reagents  was used to lyse and extract the nuclear and cytoplasmic protein fraction of cells. The nuclear fractions of the cells were added to a streptavidin-coated 96-well plate with bound NF-κB biotinylated-consensus sequence provided in the NF-κB p65 Transcription Factor Kit  and subsequently processed following manufacturer instructions. The relative NF-κB DNA binding activity of treated cells was determined by comparing with the chemiluminescent signal obtained from control cells.A firefly luciferase reporter vector which contains five copies of NF-κB response elements, pGL4.32[luc2P/NF-κB-RE/Hydro] and a renilla luciferase vector which is a control reporter, pGL4.74[hRluc/TK] were used . RCC786.0 cells were transiently co-transfected with both vectors using Lipofectamine® Transfection Reagent . Twenty-four hours post-transfection, the cells were treated with 40 nM of YM155 or 0.1% DMSO in the absence or presence of TNF-α (20 ng/ml) for 6 h or 24 h. Dual-Glo® Luciferase Assay System  was used to measure the luciferase activities. Transcription activity of NF-κB was calculated as the relative firefly luciferase activity  normalized to the renilla luciferase activity (control reporter). Changes in transcription activity of NF-κB of treated samples were expressed as fold change with respect to the corresponding control samples.To ensure preservation of protein phosphorylation and high total protein yields, cells were lysed directly in M-PER Mammalian Protein Extraction Reagent  supplemented with Halt Protease and Phosphatase Inhibitors . When nuclear and cytoplasmic protein fractions of cells were required, NE-PER Nuclear & Cytoplasmic Extraction Reagents  was used. Quantitation of proteins was made using the bicinchoninic acid (BCA) method . Proteins were electrophoresed by SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were incubated with the indicated primary antibodies  and horse-radish peroxidase conjugated secondary antibodies. All proteins were visualised with Immobilon chemiluminescent detection reagent .st PCR) or 15 cycles (2nd PCR) of 30 seconds at 94 °C, 45 seconds at 55 °C and 60 seconds at 72 °C. A final extension step of 10 minutes was performed at 72 °C. Amplified DNA fragments were resolved by gel electrophoresis, stained with gel red and visualised under UV light.Nested PCR was performed using two sets of primers . Comparative Ct method was used to quantify the expression of the gene of interest.Express SYBR® GreenER™ qPCR SuperMix Universal  and TaqMan® Fast Advanced Master Mix  were used in the detection of PCR products in real time. qRT-PCR was performed using specific primers and DNA probes (Supplementary Tables BIRC5. RCC786.0 cells were treated with 40 nM of YM155 or 0.1% DMSO (control) for 24 h and subjected to cell fixation, chromatin sonication, immunoprecipitation and DNA purification. Rabbit IgG was used as a negative control in the immunoprecipitation experiments. The immunoprecipitated fraction was analysed by PCR and qRT-PCR to determine the abundance of the target DNA sequence(s) relative to the input chromatin.ChIP-IT High Sensitivity kit  was used to determine the association of transcription factor p65 NF-κB with its specific genomic regions on the survivin gene The graphing and statistical analysis software GraphPad Prism version 5.0  and Excel  were used to plot and analyse data. Graphs were plotted to show mean values and error bars which depicted standard error of the mean (SEM). The student’s t-test and analysis of variance (ANOVA) with Bonferroni post–hoc test were used for the comparison of mean values between two and multiple (> 2) groups, respectively. A minimum of 95% level of significance (p < 0.05) was used to define statistical significance. All experiments were repeated at least twice.Supplementary Information


Inadequate regulation of intracellular Cl− interferes with synaptic signalling and is implicated in several neurological diseases. The main inhibitory neurotransmitter of the central nervous system is γ-aminobutyric acid (GABA). GABA hyperpolarises the membrane potential by activating Cl− permeable GABAA receptor channels (GABAAR). This process is reliant on Cl− extruder K+-Cl− cotransporter 2 (KCC2), which generates the neuron's inward, hyperpolarising Cl− gradient. KCC2 is encoded by the fifth member of the solute carrier 12 family (SLC12A5) and has remained a poorly understood component in the development and severity of many neurological diseases for many years. Recent advancements in next-generation sequencing and specific gene targeting, however, have indicated that loss of KCC2 activity is involved in a number of diseases including epilepsy and schizophrenia. It has also been implicated in neuropathic pain following spinal cord injury. Any variant of SLC12A5 that negatively regulates the transporter's expression may, therefore, be implicated in neurological disease. A recent whole exome study has discovered several causative mutations in patients with epilepsy. Here, we discuss the implications of KCC2 in neurological disease and consider the evolving evidence for KCC2's potential as a therapeutic target.Chloride (Cl EGABA lies close to the resting membrane potential (RMP) [EGABA and RMP vary between cell types and compartments. The depolarising or hyperpolarising effect of GABAergic signalling is dependent on the relative RMP and EGABA. When [Cl−]i is high, EGABA is less negative and GABA stimulation results in depolarisation; when it is low, EGABA is more negative and GABA stimulation is hyperpolarising [−]i is usually maintained at a low concentration, enabling inhibitory, hyperpolarising GABAergic signalling i is regulated by the Na+-K+-2Cl− cotransporter 1 (NKCC1) and the K+-Cl− cotransporter 2 (KCC2) i. As maturation proceeds, KCC2 expression increases whilst NKCC1 levels fall  . Using t neurons . During els fall  19, 20], 20−]i iEGABA and is implicated in cortical development systems such as neuro- and synaptogenesis i increase i extrusion in a KCC2-dependent manner in cultured rat hippocampal and cortical neurons i levels and membrane depolarisation at the previously inhibitory synapse i levels than wild-type KCC2 but lower levels compared to the group lacking KCC2 i i . The nee− transport may prove to be an effective therapeutic strategy. High-throughput screening assays have now identified KCC2 activators that reduce [Cl−]i. Gagnon et al. (2013) optimised a first-in-class arylmethylidine family of compounds (CLP257) to lower [Cl−]i i . CLP257 r [Cl−]i . The resr [Cl−]i  but do ractivity . Further1 kinase . Oral tr1 kinase , 107. Si1 kinase . These r− importer GABAAR makes it a potential target for the treatment of several neurological diseases. Currently, phenobarbital (PB), a barbiturate that delays the closing of GABAAR, is the most common first-line drug used for the treatment of seizures [AR agonists [−]i within immature neurons potentially contributes to resistance to pharmacological first-line antiseizure GABAAR agonists in the immature brain i.Activation of the Trk isoform TrkB has been shown to induce phosphorylation of phospholipase Cepilepsy , 119. Caepilepsy . Unlike epilepsy , allowinepilepsy . ANA12 m− homeostasis. A plethora of studies identify KCC2 dysfunction and misregulation as a key component in the development and onset of many neurological diseases. KCC2 is a strong candidate for therapeutic targeting and should be further considered by pharmaceutical investors. It should be noted that the majority of these findings are not made in human neuronal cell lines and are, therefore, limited in their ability to determine the immediate effects of targeting KCC2. Despite this, the data collected from human participants indicates that there is a place for KCC2 pharmaceuticals in the treatment of epilepsy. Continued research in human neuronal cell types may reveal more opportunities for drug development.KCC2 is a key player in the maintenance of neuronal Cl


We hypothesized that liver relaxation times are not only influenced by liver fibrosis, inflammation and fat, but also by air in liver segments adjacent to the lung – especially in MR imaging at 3T. A total of 161 study participants were recruited, while 6 patients had to be excluded due to claustrophobia or technically uninterpretable MR elastography. Resulting study population consisted of 12 healthy volunteers and 143 patients who prospectively underwent multiparametric MR imaging at 3T. Of those 143 patients, 79 had normal liver stiffness in MR elastography  and normal proton density fat fraction , defined as reference population. T1 relaxation times in these patients were significantly shorter in liver segments adjacent to the lung than in those not adjacent to the lung . In liver segments not adjacent to the lung, T1 allowed to differentiate significantly between the reference population and patients with steatosis and/or fibrosis (p ≤ 0.011), while there was no significant difference of T1 between the reference population and healthy volunteers. In conclusion, we propose to measure T1 relaxation times in liver segments not adjacent to the lung. Otherwise, we recommend taking into account slightly shorter T1 values in liver segments adjacent to the lung.Magnetic resonance (MR) T Most conventional liver MR sequences measure relative signal intensities and therefore allow relative comparison between focal disease and adjacent normal hepatic parenchyma3. However, detection and quantification of diffuse liver disease remains challenging4 but is desirable to avoid invasive and expensive biopsies5.Magnetic resonance (MR) imaging of the liver is a powerful tool in diagnosis of focal liver disease and is frequently used in clinical routine6 and MR elastography8. Another emerging technique is the quantification of T1 relaxation time on parametric maps, which is routinely used in cardiac imaging9, for example in diffuse cardiac fibrosis11 or myocarditis12. Recently, the modified Look-Locker inversion recovery sequence (MOLLI) demonstrated great potential for application in diffuse liver disease14.Currently, existing non-invasive imaging biomarkers for diffuse liver disease include proton density fat fraction (PDFF) calculation1 mapping in clinical routine16. Further, it is known from cardiac imaging that T1 values might be influenced if measured in the ventricle close to the lung, why usually measurements in the septum are preferred17. Therefore, specific anatomical conditions of the liver with adjacent air-containing lungs and potential internal confounders, such as hepatic fat and iron composition, should be considered in hepatic T1 mapping2.However, as known from cardiac applications, reference values should be established for different manufacturers, technical parameters and field strengths before using T1 relaxation time is significantly shorter in liver segments adjacent to the lung than in liver segments not adjacent to the lung due to air-induced susceptibility effects at 3T.We hypothesized that T19.Patient characteristics are shown in Table 1 and in 149/155 participants (96%) for T2*  were measurable in 61–63% of patients and the right caudal segments (V and VI) were measurable in 86–88% of patients.Out of 143 patients and 12 volunteers, resulting in 155 participants, there was at least one segment of good diagnostic image quality in 152/155 participants (98%) for T1 values were significantly lower in the cranial slices than in the caudal slices . This did not count for the comparison between cranial segment II and caudal segment III, where no significant difference between both segments was detected . Values between segments were very comparable on each level, except for segment II, which showed values more comparable with the caudal level segments  and 746 ± 81 ms (segments adjacent to the lung) (Table 1 and p = 0.521 for T2*).Normal T1 relaxation time was significantly longer than in the reference population . These results are illustrated in Fig. In positive patients with steatosis as well as in patients with increased liver stiffness, T2* time was a significant confounder of T1 time (p < 10−15), while PDFF, age and sex  were not  for T1 and 0.91 (95%-CI: 0.78–0.97) for T2*.The ICC values were excellent for both T1 relaxation time is significantly shorter in liver segments adjacent to the lung than in liver segments not adjacent to the lung. We calculated a mean T1 value of 780 ± 83 ms at 3T in liver segments not adjacent to the lung , while T1 was around 30 ms shorter in liver segments adjacent to the lung  in a patient population, without focal (based on CT) or diffuse (based on PDFF and MR elastography) liver disease.This study shows that T1 underestimation. Similar effects are known from myocardial T1 mapping21. In addition, there is a T2-dependency of the MOLLI based T1 mapping sequence due to its bSSFP design, therefore influencing the measured T1 value. Another explanation might be partial volume effects at the liver dome since the slice thickness is 10 mm  were drawn carefully to exclude liver vessels and outer 10 mm of the liver border, to prevent this possible bias as much as possible.This difference may be explained by different reasons. One possibility are susceptibility effects from adjacent air in the lungs. Susceptibility differences between liver and lungs cause off-resonance, which may lead to T mm Fig. , which i1 relaxation time difference between segment II and III was smaller. A potential explanation for this different behaviour might be that segment II is in close anatomical neighborhood to the heart and not to the lungs as the cranial segments VII, VIII and IVb are. We therefore propose to measure T1 relaxation time in liver segments V and VI with excellent inter-reader-reliability, when a representative T1 quantification of the liver is intended. These segments are normally targeted for liver biopsy and may be regarded as representative for the whole liver. However, for a more detailed segmental analysis of T1 relaxation times in the liver shorter normal T1 values in liver segments adjacent to the lung should be taken into account. T2* seems to be more prone to respiratory and pulsation artefacts from the beating heart, which might explain lower image quality in segments of the left liver lobe (adjacent to the heart) and in cranial segments, while image quality in caudal left segment V and VI showed the best image quality. Electrocardiography gating might improve image quality, which was not tested in this study.While differences between cranial and caudal segments in the right liver lobe and segment IVa vs. IVb were comparable, the T1 values of the liver at 3T using the MOLLI technique. Katsube et al. presented a mean value of 836 ± 69 ms in 16 patients with normal liver function. The slightly lower values in our study  may be explained by a smaller population in the study of Katsube et al., as well as the by the fact that Katsube et al. defined the normal population based on normal liver function in laboratory tests, which does not exclude patients with steatosis or early liver fibrosis23. Other published normal T1 values were generated with other mapping techniques, but are nevertheless in a similar range, such as 824 ± 61 ms in 26 healthy volunteers using a spiral GRAPPA-based method24, 825 ± 49 ms in 6 healthy volunteers using an inversion recovery method with refocusing pulses25, and 745 ± 65 ms in 8 healthy volunteers using MR fingerprinting26. Slight differences might be explained by different techniques of assessment and small sample sizes used in the cited studies. This underlines the recommendation, that normative values should be established for the particular site and set-up for different manufacturers, technical parameters and field strengths before using T1 mapping in clinical routine16. In accordance with other published studies, increased T1 time correlated with increased liver stiffness in MR elastography as a non-invasive surrogate for liver fibrosis23. Yoon et al. published T1 values in patients with chronic liver disease (863 ± 81 ms) and Child A liver cirrhosis (879 ± 86 ms)27 in the same range as we present here (849 ± 107 ms for early liver fibrosis and 910 ± 98 ms for significant fibrosis). T1 values calculated with commercially available software solutions using the MOLLI mapping technique have been shown to correlate with the severity of NAFLD/NASH and fibrosis28 and may predict clinical outcome in these patients29. We demonstrated an increased T1 time in patients with steatosis, which is in accordance with other published studies30. This is a paradox, since fat has a much shorter T1 time than water. According to recent publications, off-resonance effects in imaging voxels containing a mixture of fat and water signals may explain this finding32.There are very few published data on normal T1 and T2*. One known confounder inducing T1 shortening is liver iron content33, however patients with iron overload have been excluded in this study. Liver blood distribution and oxygenation levels are might influence T1 and T2* times as well. These effects and possible influences on T1 and T2* need further investigation. In multivariate analysis of reference patients without focal or diffuse liver disease and healthy volunteers, we could not demonstrate a significant age- or sex- dependency of the measured T1 values. However, there was a collinearity between T1 and T2* values. Further research should be performed to show whether T1 is T2* dependent  or whether it is the other way round and T2* is influenced by T1 due to a too short TR in the used T2*-mapping sequence. Also using a multiparametric approach combining T1, T2*, MRE and PDFF might deliver further insights.In addition to fat and fibrosis content as well as susceptibility-effects in liver segments adjacent to the lung, there are several other potential confounders of T1 mapping of the liver. For an accurate non-invasive characterization of diffuse liver disease, a combined analysis of different MR parameters such as T1 mapping, T2* mapping, elastography and PDFF should be performed. Further research will show how a multiparametric combination of those MR imaging biomarkers may help differentiating and quantifying diffuse liver disease.Our study highlights the importance of standardized technical parameters and well-defined normal values when performing T34 and steatosis grades35, as the non-invasive gold standard, and we included negative and positive controls based on these imaging techniques. Another limitation is that we did not obtain full 3D coverage of the liver, as we used commercially available Siemens MOLLI sequences with 2D acquisitions on three transverse slices. Nevertheless, we present a segmental comparison of T1 mapping in a reference population, as well as in negative and positive controls. Newer 3D mapping sequences or MR fingerprinting may offer even more applications for future use.Our study has several limitations. First, we focused on a population without known chronic liver disease. Due to the study design and associated ethical considerations, liver biopsy was not possible. Instead, we used MR elastography and PDFF, which have been shown to correlate very well with biopsy-confirmed fibrosis1 maps in the liver at 3T, we propose to measure T1 relaxation times in liver segments not adjacent to the lung. Otherwise, we recommend taking into account slightly shorter T1 normal values in liver segments adjacent to the lung.When analysing T1 and T2* mapping, proton density fat fraction (PDFF) quantification and MR elastography.This prospective cross-sectional study was approved by the institutional review board  and was conducted in accordance with relevant guidelines and regulations after obtaining written patient informed consent. All participants underwent multiparametric MR imaging at 3T in our institution between 03/2016–06/2017, including TA total of 161 study participants were recruited, while 6 patients had to be excluded due an incomplete MR exam due to claustrophobia or technically uninterpretable MR elastography. Resulting study population consisted of twelve healthy volunteers without a history of liver disease (negative controls) and 143 patients Fig. . The incClinical information and laboratory test results were recorded for the included patients. Clinical parameters included age, gender, body mass index (BMI), history of diabetes or hypertension, daily drug intake, tobacco use and alcohol consumption. Biological parameters included dyslipidemia, platelet count, quick value, total bilirubin levels, gamma-glutamyltranspeptidase (GGT), aspartateaminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, albumin, creatinine and hematocrit.1 of 5% (=40 ms) between the upper and lower liver segments with a significance level of 0.05 and a power of 0.8, a sample size of 72 was needed. A mean T1 time of 800 ms with standard deviation of 85 ms was based on a pilot readout for sample size calculations.To detect a mean difference in T1 mapping, we used an axial-acquired, MOLLI single breath-hold sequence (echo time (TE) of 1.01 ms, data acquisition window of 740 ms, inversion time (TI) 225 ms , flip angle (FA) 35°, 8-mm slice thickness, field-of-view (FOV) 384, matrix 154 × 192 pixels, and scan time of 11 s) with a 3-3-5 design . T2* mapping was performed with a multiecho gradient echo (GRE) single breath-hold sequence . T1 and T2* maps were generated on three single slices in the upper, mid and lower liver. PDFF was calculated using the Dixon method with axial T1-weighted axial vibe images  to differentiate patients with and without liver steatosis. For MR elastography, a pneumatic driver (Resoundant) was placed on the right upper quadrant transmitting shear waves by continuous acoustic vibrations with a frequency of 60 Hz. The liver shear stiffness in kPa in the right upper liver lobe was determined with a gradient echo-based elastography sequence  using the 95% confidence map of stiffness. A shear modulus ≥2.8 kPa was considered to represent early liver fibrosis , while a shear modulus ≥3.5 kPa was defined as significant liver fibrosis (corresponding to histology fibrosis grade ≥F3)37.Patients were examined with a 3T-MR system  in a fasting state (>6 h). For T1 and T2* mapping, 8 polygonal regions of interest (ROI) were drawn in liver segments II-VIII by the radiologist (V.O.) who was blinded to the patient’s clinical history. In liver segments without excellent image quality (e.g. due to motion artefacts) or in segments that were not captured on any of the three acquired slices, no ROI was drawn at this location, and thus no value was assigned to the respective segment. The mean ROI size was 685 ± 203 mm2. Large blood vessels, bile ducts and regions with partial volume, including air or perihepatic fat at the liver border, were excluded . For T38 and GraphPad Prism . Clinical characteristics were compared between groups using the Wilcoxon test for continuous variables or Fisher’s exact test for categorical variables. The p-value for significance was <0.05. T1 and T2* mapping parameters with good quality were then compared between segments using a paired Wilcoxon test. To address intersegmental variability and to identify the best area for reference, the median values of the liver segments adjacent to the lung  and those not adjacent to the lung  were calculated for the reference population and compared with the negative and positive controls using the Wilcoxon test.Analysis was performed with the statistical software package R 1 (segments not adjacent to the lung) as the outcome and sex (dummy-coded), age, PDFF and T2* (segments not adjacent to the lung) as variables. Age and sex were chosen as basic demographic characteristics that might bias the measured T1 in the liver while PDFF and T2* were added as known representatives of liver fat and iron content that might influence T1. Pearson correlation was used to compare MR elastography shear modulus with T1 values. For interrater reliability, T1 and T2* relaxation time was measured in segment VI in 20 randomly selected consecutive patients by a second radiologist (A.H.), who was blinded to the patient’s clinical history and had 7 years of experience in hepatic imaging. The two-way consistency intraclass correlation (ICC) was then calculated and classified as follows: ICC 0.4-0.59 defined as fair; 0.6–0.74 defined as good; and 0.75–1.00 defined as excellent39.To assess possible confounders in the reference population and among healthy volunteers (n = 79 + 12), a multivariate regression model was used with TThis prospective cross-sectional study was approved by the institutional review board (IRB number 282-15) and was conducted after obtaining written patient informed consent.


As evidenced from detailed characterization and density functional theory (DFT) calculations, Y3Pd2 realizes negatively charged Pd species, a low work function and a high carrier density, which are expected to be beneficial for the efficient Suzuki coupling reaction of activated aryl halides with various coupling partners under mild conditions. The catalytic activity of Y3Pd2 is ten times higher than that of pure Pd and the activation energy is lower by nearly 35%. The Y3Pd2 intermetallic electride catalyst also exhibited extremely good catalytic stability during long-term coupling reactions.Suzuki cross-coupling reactions catalyzed by palladium are powerful tools for the synthesis of functional organic compounds. Excellent catalytic activity and stability require negatively charged Pd species and the avoidance of metal leaching or clustering in a heterogeneous system. Here we report a Pd-based electride material, Y 3Pd2, which serves as an efficient and stable catalyst for Suzuki coupling reactions.In Suzuki coupling reactions, excellent catalytic performance require negatively charged Pd species and the avoidance of metal leaching or clustering. Here the authors implanted Pd sites into an intermetallic electride, Y To overcome the high activation barriers of the substrates, electron-rich active sites have been proposed in most homogeneous palladium catalysts by the introduction of electron-donating ligands5. However, the use of costly and potentially toxic palladium complexes and ligands remains a controversial issue with respect to sustainable chemical industry6. In contrast, heterogeneous catalysts are preferred over homogeneous catalysts due to their facile reusability and compatibility with flow systems11. There have been numerous efforts to develop heterogeneous catalysts for Suzuki cross-coupling reactions, and most of them are based on the design of palladium clusters or nanoparticles with highly electron-donating supports, photogenerated electrons or hot electrons in heterojunctions14. In particular, Pérez-Ramírez et al. recently developed single-atom catalysts, in which isolated Pd atoms anchored into the exfoliated graphitic carbon nitride, attract considerable attention due to its excellent activity and stability towards Suzuki cross-coupling reaction, surpassing the performance of state-of-the-art homogeneous catalysts and other conventional heterogeneous catalysts15. Electron-rich isolated palladium sites with high electron donation ability generally exhibit excellent catalytic activity for Suzuki cross-coupling reactions20. However, the stability issue for the loaded palladium catalyst particles has not yet been resolved; severe particle aggregation and/or leaching of active Pd sites on the catalyst surface during the reaction leads to poor recyclability23. There are few approaches to significantly enhance the activity and stability of heterogeneous palladium-based catalysts because the electron density of the active metal is not easily stabilized.Palladium-catalyzed Suzuki cross-coupling reactions have drawn considerable attention as a powerful tool for carbon-carbon bond formation, and are extensively used in organic synthesis chemistry27. When these alloys are used, the binding energy of the oxygen containing species on Pt can be modified moderately by changing the electronic structure of the active Pt sites31. Our group recently reported that ternary intermetallic compounds (LaCu0.67Si1.33 and LaCoSi) have a unique activation ability for H2 and N2 molecules, and thus exhibit excellent catalytic activity for the chemoselective hydrogenation reaction and ammonia synthesis, respectively33. These intermetallic compounds host solid active sites, high carrier densities, and low work functions (LWFs). The combination of rare earth metal and active transition metal can also induce charge transfer from the rare earth metal to the transition metal, which results in negatively charged active sites. As a consequence, electron transfer from the catalyst to the lowest unoccupied molecular orbitals (LUMOs) of absorbed molecules can be realized, which leads to weakening of the chemical bonds of the absorbed molecules and enables molecular dissociation with lower activation energy (Ea)37. Inspired by these developments, we have attempted to design a Pd-based intermetallic electride that should have both solid Pd sites and strong electron donation ability associated with a high carrier density and a LWF.Recently, Nørskov and colleagues reported active and stable cathode catalysts of Pt alloyed with early transition metals, such as lanthanum, scandium, or yttrium for electrocatalytic oxygen reduction reaction3Pd2, as an efficient catalyst for Suzuki cross-coupling reactions. The work function of Y3Pd2 (ΦWF ≈ 3.4 eV) is comparable to that of metallic yttrium (ΦWF ≈ 3.3 eV); however, Y3Pd2 is stable in air and under the reaction conditions for Suzuki cross-coupling. The measured carrier density is ne ≈ 1 × 1022 cm−3, which is much higher than that for oxide and carbon supports. Together with the negatively charged Pd, associated with different electronegativity between yttrium and palladium, Y3Pd2 exhibits stable and high catalytic activity for Suzuki cross-coupling reactions. The high carrier density and LWF for Y3Pd2 enable the activation of aryl halides with a significantly reduced Ea (about 35% less) compared with that for a pure Pd catalyst, and the turnover frequency (TOF) and recycle number exceed those for other reported Pd-based heterogeneous catalysts under similar reaction conditions. We consider that the electron transfer from the negatively charged Pd to aryl halides could be the key factor for the dissociation of aryl halides and the intrinsic catalytic ability of the active Pd sites involved was significantly enhanced in the Y3Pd2 intermetallic electride material.Herein, we propose a Pd-based electride, Y3Pd2 crystallizes with a trigonal structure 38. Consequently, large volumes of Y4 tetrahedral and Y6 octahedral cages are realized in Y3Pd2, and thus the X1, X2, and X3 sites can be considered as periodically distributed cavities. Bader charge analysis using pseudo atoms of these interstitial sites revealed that 0.87, 1.05, and 1.05 electrons are confined in the X1, X2, and X3 sites to give a formal valence state of [Y3Pd2]+:e‒, which is consistent with the electride concept. Motivated by the presence of interstitial sites, we conducted DFT calculations to clarify the electronic structure. Figure 3Pd2 exhibits a metallic band structure. The electronic bands near the Fermi level are partially associated with empty X1, X2, and X3 sites , the most stable surface, was calculated to be 3.4 eV .As predicted by the DFT calculations, Y eV Fig. , which isee Fig. . Therefo3Pd2 and pure Pd metal catalysts. For both reactions, the Y3Pd2 catalyst exhibited rather higher reaction rates than those of pure Pd metals  based on total Pd was firstly calculated to be 7.6 h−1, which is several times higher than that of pure Pd metal (0.9 h−1) with comparable particle size under the same conditions  by an order of magnitude  values at different reaction temperatures, a set of Arrhenius plots were produced by plotting lnr0 as a function of 1/T. The slope of the Arrhenius plot was used to calculate the apparent Ea for Y3Pd2 to be 48.4 kJ mol−1 for the Suzuki coupling of iodobenzene and phenylboronic acid , which provides evidence that the Suzuki coupling reaction is highly promoted over Y3Pd2. Similarly, Ea for the coupling of bromobenzene  surface. The activation process of bromobenzene molecule proceeds with the calculated energy gain of 0.11 eV on Y3Pd2 (0001), much lower than that of 0.53 eV on Pd (111)  from the Y3Pd2 (0001) surface to the adsorbed aryl halides via the active Pd site  and extended X-ray absorption fine structure (EXAFS) spectroscopy. The results indicated that the absorption edge for the Pd species in intermetallic compounds was located at an even lower energy relative to that for the Pd foil, which implies that Pd is negatively charged in Y3Pd2  patterns  are in the range of 1400–1600 cm−146. In the low-frequency region, the main peaks at around 996, 1014, and 1060 cm−1 can be assigned to trigonal ring breathing, out-of-plane C–H (γC–H) deformation and halogen-sensitive vibration, respectively46. Compared to pure Pd metal, the two frequencies at 1575 and 1473 cm−1 involved with the admixture of C–I deformation on Y3Pd2 exhibited an obvious redshift  for Y3Pd2  spectroscopy has been reported to be a powerful technique for understanding the electronic effects related to adsorbed molecule activation processes on a solid catalyst. Therefore, to elucidate the interaction between the aryl halides and the catalyst, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) spectra were measured for iodobenzene adsorbed on Ysee Fig. . Moreovesee Fig. . Both of3Pd2, based on the experimental results. Here, Y3Pd2 with high electron density of Pd and positively charged Y act as the adsorption sites for the aryl halide (iodobenzene or bromobenzene). Next, the C–X group with an electron withdrawing element X  of the aryl halide molecules can be preferentially adsorbed on Yδ+-Pdδ− sites through strong electrostatic interaction. The negatively charged Pd served as the electron donation sites to weaken the C–X bond, resulting in a significantly suppressed Ea, which is confirmed to be the rate-determining step for the reaction. The subsequent reductive elimination and transmetallation processes are followed by the formation of the final coupling products. Although the detailed mechanism remains undetermined at this stage, it is important to emphasize that electron transfer from the negatively charged Pd to the aryl halide could be the key factor for activation of the aryl halide that results in high catalytic activity for the Suzuki coupling reactions.Figure 49. Thus far, recently studied Pd based heterogeneous catalysts could only be reused less than 10 times under mild alkaline conditions51. In the present work, under the same reaction conditions, Y3Pd2 could be recycled 20 times without loss of activity, which indicates much higher stability than other reported Pd-based heterogeneous catalysts  as compared with Pd (111) (–0.95 eV), indicating a relative lower dissolution potential of the Pd species in Y3Pd2, which accounts for the high stability during the catalytic reaction53. Moreover, the segregation of a Pd atom from the bulk towards the surface requires 1.79‒2.67 eV for Y3Pd2 (0001) , as shown in Supplementary Fig. 3Pd2, in which Pd sites could be stabilized through ionic and metallic bonding within the Y3Pd2 lattice. These results well explain the excellent stability of the Y3Pd2 intermetallic electride catalyst.The recyclability of catalysts is one of the most important properties for practical applications. It is worth noting that bare Pd nanostructures or even supported Pd nanoparticles in conventional Suzuki coupling reactions typically suffer from a significant loss in activity because traditional heterogeneous Pd-based catalysts act by the release of small amounts of soluble Pd with the dissolution and re-deposition process, which often induced an aggregation of the loaded Pd speciessee Fig. . Moreovesee Fig.  and a chsee Fig. , which hure Fig. . Most imble Fig. . Note th3Pd2  or electron-withdrawing (F and CF3) substituents were present  of biphenyl for the coupling of bromobenzene with phenylboronic acid pinacol ester (Supplementary Table −1) were comparable to that of reported Pd single-atom catalyst tested in flow system, while much higher than those of other reported heterogeneous catalysts tested in a batch reactor15. The results confirmed the general applicability of the Y3Pd2 catalyst with high chemoselectivity and functional group tolerance.Aromatic and Aliphatic Alcohols. The reaction protocol was further extended to various aryl halides and boronic acids to verify the scope and activity of Suzuki coupling reactions catalyzed by Yd2 Table . All of 3Pd2, was developed for Suzuki cross-coupling. The in situ stable intermetallic electride Y3Pd2 possesses both high carrier density and a LWF, and thus exhibit much higher catalytic activity than Pd metals and other conventional Pd based supported catalysts. Mechanistic studies demonstrated that negatively charged Pd sites induce electron injection into the antibonding orbitals of C–X, significantly promoting the activation of aryl halides, which is the rate-determining step for the investigated Suzuki cross-coupling reactions. Given the heterogeneous nature of the catalyst, Y3Pd2 could be easily separated and recycled for more than twenty cycles without significant loss in activity. Our results show the possibility of tailoring the electronic structure of active transition metals such as Pd by the formation of intermetallic compounds with early transition elements—an approach that should be broadly applicable for the tuning of catalytic properties.Guided by the active sites integrated in the intermetallic electride concept, an intermetallic electride catalyst, Y3Pd2 was directly synthesized by arc-melting yttrium and palladium ingots with the ratio of 3.1: 2 under an argon atmosphere. The ingot obtained was grounded using an agate motor in Ar filled glove box. The obtained powder was then pelletized and sealed into an argon filled stainless tube, and annealed at 900 °C for 90 h. Stainless tubes were sintered in nitrogen atmosphere in order to prevent their oxidation. Y3Pd, Y3Pd4, and YPd3 were prepared by arc-melting method similar with that for the preparation of Y3Pd2. Ball-milling of Y3Pd2 was conducted using heptane and ethanol as solvents under argon atmosphere, and the rotation rate was set to 150 rpm. In order to avoid overheating, Y3Pd2 was milled for 10 min with 10 min of interval, and this sequence was 10 times repeated. Pd/C, Pd/Al2O3, and Pd–Pb/CaCO3 used in this study were purchased from Sigma-Aldrich, and directly used without any pretreatments.Elemental yttrium (ingot) and palladium (ingot and powder) used in this study were purchased from Kojundo Chemical Lab. Co., Ltd. (Japan), and directly used without any pretreatments. YThe electronic transport properties, magnetic susceptibility and heat capacity were measured using a physical property measurement system (PPMS) and a superconducting quantum interference device (SQUID) vibrating sample magnetometer (SVSM) from Quantum Design.2CO3, and Pd  catalyst were mixed in 5 ml of solvent. The autoclave was then flushed three times with Ar and reactions were performed in the autoclave at about 30–70 °C. The products were analyzed using gas chromatography (GC) and product identification was further confirmed from gas chromatography-mass spectrometry (GC-MS) measurements. The TOF was calculated from the reaction rate at a low conversion level derived from the number of Pd atoms exposed to the catalyst surface . Prior to the analysis, the catalyst was treated with flowing Ar (50 ml min−1) at 400 °C for 30 min and then flowing He (50 ml min−1) at 400 °C for 15 min. To calculate the metal dispersion, a stoichiometry of Pd/CO = 1 was assumed. Because each Pd site was isolated by the surrounded Y atoms in the most stable surface Y3Pd2 (0001). The nearest separation between surface isolated Pd sites is ~0.43 nm, sufficiently longer than the bond length of CO molecule (~0.11 nm). In addition, the CO adsorption to Y powder was below the detection limit at the same condition. Therefore, it is reasonably assume each active ensemble of Pd-Y corresponds to one CO molecule. For theoretical prediction, both of the Pd atom numbers in one unit cell of Y3Pd2 crystal and surface area of the corresponding unit cell were calculated. Then the number of the Pd atoms per unit cell’s surface area were extended to the measured specific surface area of the experimentally prepared Y3Pd2 powder.The number of moles of Pd atoms exposed on the surface was estimated by experimental measurement and theoretical prediction, respectively. For experimental measurement, the CO-pulse chemisorption  was measured at 50 °C using a He flow of 30 ml minFor the recycling study, the Suzuki coupling reaction was performed with organohalide and phenylboronic acid, maintaining the same reaction conditions as described above except using recycled catalyst. After each completion of the reaction, the catalyst powder was washed by ethanol with a certain amount of water several times to remove the organic and inorganic impurities from the used catalyst. Finally, the resultant powder was dried in a vacuum at room temperature, weighed, and reused in the next run.λ = 0.15418 nm). Nitrogen sorption measurements  were performed to evaluate the Brunauer–Emmett–Teller surface area of the catalysts. The surface morphology of the catalyst samples was investigated using high-angle annular dark field scanning transmission electron microscopy . XPS  measurements were performed using Mg Kα radiation at <10−6 Pa (8 kV bias voltage applied to the X-ray source). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield Plus 400 instrument (Bruker Corporation) using tetramethylsilane as an internal standard . X-ray absorption fine-structure (XAFS) measurements were performed on the AR-NW10A beamline of the Photon Factory Advanced Ring at the Institute of Materials Structure Science, High Energy Accelerator Research Organization, Tsukuba, Japan. A Si (311) double-crystal monochromator was used to obtain a monochromated X-ray beam, and spectra were obtained in transmission mode. Y3Pd2 with BN (dried at 300 °C) were mixed in an argon-filled glovebox and the mixture was pressed with a hand press apparatus to obtain a pellet sample, which was sealed in a plastic bag for measurement. XAFS spectra were analyzed using the Athena and Artemis software packages43. The FEFF6 code44 was used to calculate the theoretical spectra.The crystal structure was analyzed using an X-ray powder diffractometer  with monochromated Cu Kα radiation  with the Perdew−Burke−Ernzerhof (PBE)−1. An alumina sample cup containing approximately 30 mg of catalyst was introduced into a water-cooled stainless steel heat chamber equipped with KBr windows . The sample was heated at 200 °C under vacuum for 2 h and then cooled to room temperature. After the pretreatment, iodobenzene was supplied into the system. The infrared spectrum of the sample at room temperature prior to the adsorption of substrates was used as the background for the difference spectra obtained by subtraction of the background from the spectra of the catalyst samples with adsorbed substrate.DRIFT spectra were measured using a spectrometer  equipped with a mercury–cadmium–tellurium detector at a resolution of 4 cmSupplementary Information


The first systematic study is presented of π interactions between non-aromatic rings, based on the authors’ own results from an experimental X-ray charge-density analysis assisted by quantum chemical calculations. The study includes quinoid rings, planar organic radicals and metal chelates. The stacking observed covers a wide range of interactions and energies, ranging from weak dispersion to unlocalized two-electron multicentric covalent bonding (‘pancake bonding’). e.g. quinones) than those involving delocalized systems (e.g. aromatics), and (iii) the main component of the interaction is electrostatic/multipolar between closed-shell rings, whereas (iv) interactions between radicals involve a significant covalent contribution (multicentric bonding). Thus, stacking covers a wide range of interactions and energies, ranging from weak dispersion to unlocalized two-electron multicentric covalent bonding (‘pancake bonding’), allowing a face-to-face stacking arrangement in some chemical species (quinone anions). The predominant interaction in a particular stacked system modulates the physical properties and defines a strategy for crystal engineering of functional materials.The first systematic study of π interactions between non-aromatic rings, based on the authors’ own results from an experimental X-ray charge-density analysis assisted by quantum chemical calculations, is presented. The landmark (non-aromatic) examples include quinoid rings, planar radicals and metal-chelate rings. The results can be summarized as: (i) non-aromatic planar polyenic rings can be stacked, (ii) interactions are more pronounced between systems or rings with little or no π-electron delocalization ( They do not meet any of the criteria for these two categories but they can stack, as demonstrated by our examples of molecules belonging to different chemical classes: quinones (including charged ones), a variety of planar organic radicals, including semiquinones and tetracyanoethylene, and metal-chelate rings. Our findings are in agreement with the observation that more favourable stacking interactions can be achieved by exploiting the interactions of non-aromatic polyenes rather than aromatic systems, an idea put forward by Bloom & Wheeler  they are weak.In the present paper we emphasize that the presence of a stacking motif does not depend on the presence of an aromatic system. Different chemical species comprising π systems that are inclined to stacking arrangements can include a plethora of intermolecular interactions. The examples studied include quinoid rings, semiquinone anion radicals and their combinations, and metal-chelate rings  are −0.61 and −0.95, respectively . Unlike aromatics, they have distinguishable single and double C—C bonds. Therefore, the electrostatic potential in the rings is not uniform, but the molecules contain alternating electron-rich and electron-depleted areas .Quinoid rings are not aromatic: the harmonic oscillator model of aromaticity (HOMA) indices of benzoquinone (BQ) and tetrachloroquinone  interplanar separation   by a combination of X-ray charge-density analysis and quantum chemical computation  calculations of isolated clusters and simulation of the crystal lattice by periodic density functional theory (DFT), is about −10 kcal mol−1 . While this is only a fraction of the electrostatic repulsion of negative charges, the stacks are held together by Madelung energy .Since stacking interactions are, to a large extent, determined by charge density, we might expect that quinones would not stack according to the quadrupolar Hunter–Sanders model. 2,5-Dihydroxyquinones (DHQs) and their anions favour face-to-face stacking with a short  do not form face-to-face stacks, but stack in an offset fashion, with geometries similar to aromatic stacks ]. Quantum chemical calculations indicate that the energy of interaction is at least −6 kcal mol−1  anions form perfectly staggered stacks (the anions are rotated by 30°) while DHQ2− dianions are perfectly eclipsed (rotated by 0°)  Fig. 3.2.2.et al., 2010et al., 2013et al., 2013et al., 2010et al., 2004et al., 2014et al., 2002et al., 2007et al., 2009aetc. Two types of stack are known: (i) Peierls-distorted stacks with alternating short (<3.2 Å) and long (>3.4 Å) interplanar separations [Fig. 5a)] and (ii) stacks of equidistant radicals . Type (i) is more thermodynamically stable, and it is more common. It can be regarded as stacked radical dimers with coupled spins and such structures are diamagnetic and insulating. The rings in a dimer are bent slightly towards each other; in stacked semiquinones their Cremer–Pople puckering parameter τ is in the range 2.0–4.3°  have been observed: tetrathiafulvalene and its derivatives , which have been interpreted as multicentric covalent bonding. The term ‘pancake bonding’ , it is clear that magnetic exchange and spin coupling make significant contributions to the total interaction. However, the nature and energy of these π interactions remained obscure until quite recently. Quantum chemical studies are available for close dimers of radicals  and they indicate a considerable covalent character ] and with equidistant radicals [Fig. 5b)] ] and provided experimental evidence of pancake bonding .Like other planar radicals, semiquinones also form π stacks ] and a cage  critical point  is found in the centre. The integrated electron density obtained by DFT calculation exceeds 1 e, and the corresponding calculated bond order is 0.80 ].Typical pancake-bonded dimers were found in triclinic −1; this value is similar to other pancake bonds studied by computational methods  and the total is repulsive; however, it is compensated by the cations . In the crystal structure, local repulsions are overcome by Madelung energy. It is interesting to note that the covalent contribution  is −9.4 kcal mola)]. Such an arrangement minimizes the electrostatic repulsion between two negative charges.It is also evident that electrostatic interactions play an integral part in pancake bonding: a map of the electrostatic potential shows that the closest contacts are between the most electron-rich (carbonyl oxygens) and the most electron-depleted (carbonyl carbons) parts of contiguous rings [Fig. 7−3) and the calculated intramolecular bond order is a negligible 0.04  Å], there is very low electron density ], and numerous bonding  critical points are found between the rings [Fig. 8b)], with a maximum electron density of 0.77 e Å−3. Also, a  critical point  is found in each of the symmetry-equivalent close contacts between the rings in the trimer [Fig. 8c)], indicating a cage-like electronic structure.X-ray charge-density analysis of a trimer of tetrachlorosemiquinones ] indicated that the HOMO extends between all three rings of the trimer; the covalent contribution to the total interaction between each pair of rings was calculated by DFT [M05-2X/6-311G] to be −6.8 kcal molca 0.04 e Å−3) and its geometry is similar to the stacking of aromatic rings.A long intertrimer contact reveals a low electron density  and fewer bonding  critical points than in the pancake-bonded dimer, but the  local minimum was present [Fig. 9b)]. This indicates a significantly weaker covalent contribution than in the pancake-bonded dimers, but nevertheless considerably stronger interaction than between the dimers or between aromatic rings. However, this electron density is consistent with DFT calculations , which indicated a weak covalent contribution [Fig. 9c)] with a bond order of 0.26. While the calculation was performed for a pair of radicals only, we can be quite certain that the HOMOs extend along the stack, resulting in some kind of metal-like state. Therefore, we can consider the interactions in a stack of equidistant radicals as weak pancake bonding and conclude that the covalent contribution is critical for semiconductivity.Our X-ray charge-density study of the orthorhombic polymorph of the a)] is nearly identical to that in pancake-bonded dimers, and minimizes electrostatic repulsion.The offset along the O=C⋯C=O axis of 2.057 Å  are no exception ] ]. The molecular planes are close to parallel (α = 2°), with an interplanar distance of 3.28 Å and centroid-to-centroid distances ranging between 3.40 and 3.84 Å. The electrostatic potential plotted onto an HS reveals excess negative charge at the bpy and chloranilate entities [Fig. 11b)], while the central Cu atom has a considerable positive charge. This is in a good agreement with the electrostatic potential of the Cu–CA complex [Cu(CA)2(H2O)2]im2 (im = imidazolium) derived from high-resolution X-ray data [Fig. 10a)]  ]: the polymer chains run parallel, with an interplanar separation of 3.26 Å and an offset of ca 1.6 Å normal to the direction of the polymer. Again, the closest contacts are between electron-depleted Cu and electron-rich chloranilate rings [Fig. 12b)], and the contact surfaces of two contiguous chains are relatively flat, comprising a multitude of small facets [Fig. 12c)].Similar interactions exist between 1D coordination polymers [Cu(CA)(MeCN)]n : the Cu⋯O contact of 2.574 (2) Å is much shorter than the sum of the van der Waals radii, but nevertheless significantly longer than the sum of the covalent radii  indicated weak bonding between Cu and O, leading to the conclusion that the Cu coordination is in fact 4+1, making this one of the longest examples of a Cu—O bond. Analysis of the VDP [Fig. 13b)] shows that the face corresponding to the long Cu—O bond is ca 50% smaller than the faces representing the other four Cu—O bonds, but more than twice as large as typical faces representing intermolecular contacts.A curious case of a borderline phenomenon between coordination bonding and π stacking was noted in [Cu(CA)(phen)]3.et al., 2015a−1  to >15 kcal mol−1 .We have presented a stacking model of planar polyenic systems which is primarily based on experimentally determined charge density, previously published by us (Molčanov i.e. aromatics) and the strongest are between rings with little or no electron delocalization (such as quinones).It is interesting to note that, among closed-shell molecules, the weakest interactions are between fully delocalized π systems (One can summarize: (i) in common stacking of aromatic rings, dispersion and electric quadrupoles are dominant; (ii) in stacking of quinones (and other non-aromatic polyenic systems), the prevailing interaction is (multipolar) electrostatic; and (iii) in stacking of metal chelate rings, the prevailing interaction is also electrostatic (multipolar), with the possible contribution of coordination bonding.−3.Planar stacked radicals reveal three possible types of interaction: (i) covalent ‘pancake bonding’, with a considerable electrostatic component ; (ii) electrostatic, with a non-negligible covalent component ; and (iii) dispersion (between pancake-bonded dimers). As our analysis of charge densities indicates, there is no clear-cut border between all these types. In addition, we may establish an AIM criterion for recognizing pancake bonding: a local electron-density minimum [a  critical point] should exist between two radicals, while the electron density in multiple  critical points should exceed 0.7 e Ået al., 2017aStacking interactions exhibit characteristics comparable with those of hydrogen bonding. They both cover a broad range from dispersion  to two-electron/three-centric covalent bonding   are accompanied by stacking interactions and/or hydrogen bonding, and both exhibit a certain degree of covalent bonding – multicentric bonding (Molčanov et al., 2012abet al., 2012bThe predominant interaction in a particular stacked system modulates the physical properties and defines a strategy for crystal engineering of functional materials. The separation distance of stacked quinoid rings correlates with their magnetic characteristics and thus the occurrence of coupled/uncoupled electron spins (Molčanov


Additionally, the TRTS analysis yields an effective bulk carrier lifetime of 4.4 ns, a surface recombination velocity of 6 * 104 cm/s and a doping concentration of ca. 1016 cm−3, thus offering the potential for contactless screen novel optoelectronic materials.The mobilities of electrons and holes determine the applicability of any semiconductor, but their individual measurement remains a major challenge. Here, we show that time-resolved terahertz spectroscopy (TRTS) can distinguish the mobilities of minority and majority charge carriers independently of the doping-type and without electrical contacts. To this end, we combine the well-established determination of the sum of electron and hole mobilities from photo-induced THz absorption spectra with mobility-dependent ambipolar modeling of TRTS transients. The method is demonstrated on a polycrystalline Cu The outstanding benefits of this technique include its non-destructive and contactless nature as well as high time resolutions of <100 fs. Recently TRTS was extended to be used in reflection mode4 and to derive mobilities for thin films on highly conductive substrates5.TRTS is a widely used pump-probe technique to measure the dispersive AC-mobility µe+h is generally derived from the photo-induced THz absorption spectra as shown in Fig. 2ZnSnSe4 thin film sample.A major advantage, and at the same time a potential disadvantage, of TRTS is the measurement sensitivity to all pump-induced charge carriers, i.e. both electrons and holes. This makes both types of carriers accessible – but indistinguishable. Accordingly, the added electron and hole mobility 2ZnSnSe4 has evolved as a promising compound semiconductor material for solar cells. It is structurally closely related to the more established CuSe2 chalcopyrite absorber material, but it has the advantage to contain only earth-abundant elements. As the device efficiency of Cu2ZnSnSe4 solar cells still lags significantly behind record efficiencies of chalcopyrite or CdTe solar cells, fundamental studies of the transport and recombination behavior are expected to identify current performance bottle-necks.During the last years Cu2ZnSnSe4 thin film from TRTS spectra, illustrating the state-of-the-art of TRTS sum mobility measurements. Next, the transient model is developed including dominant first order bulk and surface recombination deduced from wavelength and injection dependent TRTS transients, and ambipolar diffusion verified experimentally by THz emission spectroscopy. Based on these dynamics - ambipolar diffusion, surface, and bulk recombination - TRTS transients are modeled with the continuity equation and the ambipolar diffusion coefficient is determined. Finally, the ambipolar diffusion coefficient and the mobility sum are combined using the Einstein relation to derive the minority and majority carrier mobility, and a transport model for kesterite is proposed. More details on the TRTS technique can be found in the methods section.First, we demonstrate the determination of the sum of electron and hole mobility of the Cu6 to induce surface recombination. In this work we lifted off a 1.55 µm thick coevaporated Cu2ZnSnSe4 film from the molybdenum coated glass substrate which leaves a bare, unpassivated, and possibly damaged surface due to the lifting process.The proposed method works on wafers as well as thin films. The only major requirement for the samples is a relative high surface recombination. If negligible surface recombination is present a sample may be treated by etching, oxidation, or ion bombardmentµe+h as function of THz frequency for the kesterite thin film is shown in Fig. The sum of electron and hole mobility 7. Modeling the mobility by the phenomenological Drude-Smith model for localized transport with equation  of 230 nm and 50 nm for the 1.5 eV and 3.1 eV pump photons, respectively19.Based on the results of the previous sections, we can model TRTS- transient with a joint ambipolar continuity equation for electrons and holes and surface recombination as boundary condition, as described by equations  and 416.Dam(Δn), the effective bulk lifetime τB(Δn) and the front surface recombination velocity S(Δn). Phenomenologically, this simplification is justified when the measured TRTS transients are injection independent as it is found for the highest injection levels in our measurements shown in Fig. Δn above the doping p0 explained by theory in the following sections. Therefore, we will focus on the simpler injection independent modeling of a high injection pair of TRTS-transients excited with 1.5 eV and 3.1 eV photons in Fig. The last simplification is the assumption of the injection independence of the diffusion coefficient Δn shown in Fig. ΔnS(t) which determines the transient TRTS signal via equation  for the effective lifetime at initial times when carriers are distributed over α−1 and in the long time limit t > τDB when carriers have diffused over the sample thickness d to the back surface. Later ones may be taken as start value for the numerical modeling.Third, estimating τFor consistency it was confirmed in Fig. τDB of 7 ns and the carrier distribution at 1.8 ns has not reached the back as shown in Fig. This procedure also excludes back surface recombination if the carriers do not have the time to diffuse to the back. In the presented modeling the 1.8 ns time window is much shorter than the diffusion time to the back surface 20 a surface recombination velocity of 2 × 104 cm/s was derived from TRPL transients of similar kesterite samples based on the analytic approximation (5) of the continuity equation21. However, the time resolution of a TRPL detector can lead to an underestimation of the initial decay and the TRPL maximum can occur significantly after carrier excitation, as shown in Fig. In principle, similar analyses can be performed on time resolved photoluminescence (TRPL) transients. InNow the individual carrier mobilities will be derived from the ambipolar diffusion coefficient and the mobility sum.µe and hole µh mobility to the common ambipolar diffusion coefficient Dam(Δn) are dependent on the injection Δn. They are given by the ambipolar Einstein equation (Dam(Δn) following equation  reduces to equation  reduces to equation  mobilities as does the condition for low injection Δn ≪ p0µp/µn.A similar analysis works for low injection transients where the effective mobility can directly be identified as the minority carrier mobility in equation . Howeverp0. Due to the limited space in this article it is given in the Supplementary Fig. The injection dependent transients can also be analyzed to estimate the doping concentration 9.To interpret the TRTS-derived mobilities in polycrystalline thin films the distinction of vertical vs. lateral and intra-grain vs. cross grain boundary transport is important as we have discussed inThe added mobility of electrons and holes derived by the terahertz absorption spectrum in Fig. 23.The ambipolar mobility derived by the terahertz transients is based on vertical ambipolar diffusion into the thin film. As discussed above, the charge carriers do not cross grain boundaries within the time window of the TRTS measurement, allowing to identify the derived mobilities with the intra-grain mobilities, tooHence, the combination of both measurements yields isotropic intra-grain mobilities of electrons as well as of holes.Idevice in operation is vertical and occurs within single grains if these extend throughout the thin film thickness as shown for our sample in Fig. These are relevant mobilities and transport distances for most commercial applications of polycrystalline thin films such as photovoltaics, solar water splitting devices or LEDs. Their charge current 24. Electron mobilities of ca. 100 cm2/Vs have been derived from this method for similar kesterite thin film samples25 comparing well to the TRTS-derived value of 128 cm2/Vs and thus underlining the validity of the current approach.Minority carrier mobilities for vertical transport may also be estimated from a combination of capacitance-voltage, internal quantum efficiency and time resolved photoluminescence measurements on completed solar cells2/Vs30 in the reported majority carrier Hall-mobility in kesterites may therefore be caused by either a variation of the intra-grain transport or by grain boundary barriers depending on the growth conditions.In contrast, Hall effect measurements — as the most common method to derive majority carrier mobility — involve lateral transport which in polycrystalline thin film materials may be dominated by thousands of grain boundaries illustrated in Fig. 9 by the influence of potential fluctuations as illustrated in Fig. The absolute values of the TRTS-derived intra-grain mobilities of electrons and holes are significantly smaller than what is expected from classical (Drude) free carrier transport, were the mobility is directly linked to the effective mass and carrier scattering time. The non-Drude behavior is already obvious from the THz spectra in Fig. 2ZnSnS431. We note that a similar hole mobility of 5 cm2/Vs has been reported for transport in an acceptor band of strongly Mg doped GaAs32.Additionally, the TRTS-derived hole mobility is 20 times smaller than the electron mobility, although the theoretical effective mass is only 2 times smaller. This behavior can be explained by much stronger fluctuations of the valence band compared to the conduction band. Alternatively, the hole transport may be dominated by transport in an acceptor band which has been recently identified to be present in the related material Cu2/V-s ±50%) and majority (128 cm2/V-s ±25%) charge carrier mobility and demonstrated it for an example of a p-type Cu2ZnSnSe4 thin film. The results of this method agree well with electron mobilities derived from carrier collection in Cu2ZnSnSe4 solar cells. The method gives access to the intra-grain and vertical transport in a polycrystalline thin film. The MatLab code for the underlying TRTS transient modeling is freely available on request to the corresponding author and can be applied to any semiconductors with sufficient or, as in this case, induced front surface recombination. This modeling technique additionally determines the surface recombination velocity (5.9 * 104 cm/s), the effective bulk lifetime (4.4 ns) and estimates the doping (ca. 1016 cm−3). Therefore, TRTS is an excellent technique to reveal charge carrier dynamics. In our example, we could explain the partially localized electron transport and the 20 times smaller hole mobility revealed in Cu2ZnSnSe4 by band edge fluctuations and an acceptor band.In summary, we have developed a contact-less method to measure both minority . Scanning this second delay reveals the whole THz pulse transmitted through the sample and therefore the transmission T(t2) and ΔT(t2) in time domain which is then Fourier transformed to T(f) and ΔT(f).The delay ΔT is dominated by free carrier absorption which is proportional to the photo-induced conductivity Δσ = eµΔn consisting of the charge carrier mobility µ and concentration Δn33. Therefore, the measured THz transmission T and its photoinduced change ΔT can be used in equation . However, within the thin film approximation the specific distribution doesn’t affect the THz absorption. Hence, only the integrals of Δσ and Δn over depth are of relevance. Therefore we state equation (ΔnS(t) and the integral conductivity ΔσS.Due to the long wavelengths of the THz radiation (1 THz ↔ 0.3 mm), equation  to calcuT and ΔT(t ≈ 0) is measured shortly after excitation (20 ps) and the initial sheet carrier concentration Δn∫(t = 0) is calculated by equation (φph reduced by their transmission t and reflection r at the sample. The transients were calibrated as shown in the Supplementary Fig. To derive the mobility from equation  the THz equation  with theSupplementary Information


The use of lithium (Li) has dramatically increased during the last two decades due to the proliferation of mobile electronic devices and the diversification of electric-powered vehicles. Lithium is also prescribed as a medication against bipolar disorder. While Li can exert a toxic effect on living organisms, few studies have investigated the impact of anthropogenic inputs on Li levels in the environment. Here we report Li concentrations and Li isotope compositions of river, waste and tap water, and industrial products from the metropolitan city of Seoul. Results show that the large increase in population density in Seoul is accompanied by a large enrichment in aqueous Li. Lithium isotopes evidence a major release from Li-rich materials. Water treatment protocols are also shown to be inefficient for Li. Our study therefore highlights the need for a global Li survey and adequate solutions for minimizing their impact on ecosystems and city dwellers. Lithium use in electronics has increased dramatically, but the environmental impacts are poorly understood. Here the authors show lithium in river and tap water in South Korea is coincident with population density, and that waste water treatment is ineffective at scrubbing this potential toxin. As the secondary Li-ion battery (LIB) is a major and growing industrial channel for the element, approximately 660 million cylindrical Li-ion cells were produced in 2012, of which Korea shared 21% of total LIB manufacturing capacity2. Lithium is also incorporated into alloys and is widely used as a therapeutic drug for treating bipolar disorder since its discovery in 19703. Although future demands will continue to grow and Li recycling may become an integral part of Li business4, there are still few disposal process guidelines for waste LIB. Furthermore, there is a gap in our knowledge concerning the impact of these materials on Li levels in the environment as well as in municipal waters. The biological effect of high Li levels on the diet of several organisms and human beings has however been already reported in several publications10. For aquatic organisms, most of published studies have shown that elevated aqueous Li levels induce toxic effects12. Concerning humans, a growing number of studies have reported an inverse relationship between Li concentrations in drinking water and suicide mortality indices , consistent with its biological role in brain cells15. In contrast, elevated Li concentrations in drinking water may be deleterious and disturb Ca homeostasis during pregnancy16. Interestingly, Li isotopes (the ratio of 7Li/6Li) have been used by Earth scientists and geochemists since, when measured in rivers and soils, they provide key information on soil sustainability and weathering rate on continents, and therefore on the carbon cycle. They are considered as a key isotope proxy of unraveling why and how global climate could be regulated over geological timescale22. Thus, for all these reasons, it becomes increasingly important and urgent to quantify the amount of environmental Li that comes from anthropogenic activities. However, determining the conditions under which Li concentration or Li isotope signature can be impacted by anthropogenic activities remains a challenge.During the last two decades, industrial demands for lithium (Li) resulted in a dramatic increase in Li production and, in 2017, the world production of Li from minerals and brine was 43,000 tHere, to test the effects of anthropogenic activities, we sampled and analyzed different types of water from the Han River (HR) basin. This river is the largest river system in South Korea, in terms of discharge and drainage area, and drains the Seoul Special Metropolitan City (Seoul), the capital and largest metropolis of South Korea. The population of the HR basin is estimated to be 12 million , with δ7Li values all greater than 25‰. This finding is consistent with known mechanisms that fractionate Li isotopes during silicate weathering, such as 6Li-rich clay formation in soils23. The lithium isotope composition of both the BR and NR remains remarkably constant over ~340 km. This finding confirms the negligible impact of lithology on riverine δ7Li values, as generally found in rivers of mixed lithology basins, since the major source of riverine lithium remains weathering, and leaching of silicate rocks and minerals25. Although the topography and runoff are slightly different in both watersheds , and the δ7Li values are high and constant , the downstream part of the HR basin displays a strong and progressive evolution for both parameters  to more than 14 million people at the HR4 site . The effluents correspond to waters treated with various methods to minimize their impact on the environment, and drained back to the river  and may therefore explain the decrease of the δ7Li value displayed by the HR in Seoul  are strongly enriched in Li (up to >1 mM), the second is that their δ7Li values of tap water follow the same evolution from East to West, and are consistent with the values measured in the HR sampled in the same area. This finding strongly suggests that tap water is influenced by the same anthropogenic sources as the river, and confirms that the purification and wastewater treatment processes neither significantly lower Li level nor bias Li isotope composition. Altogether Li concentration and Li isotope composition show a binary mixing between a natural end-member (characterized by tributaries – BR and NR − draining rocks and soils upstream) and an anthropogenic end-member, consistent with isotopically light and strongly enriched wastewater  regulations for LIB in Korea31. Therefore, it is likely that the high Li levels measured in waters would come from the release from LIB waste, along with other anthropogenic inputs such as therapeutic drug (Li carbonate), detergent and compost. In order to investigate further this possibility, we collected and analyzed several anthropogenic materials  and display, on average, slightly heavier δ7Li values (15.6‰). Thus, as shown in Fig. 7Li values. Since treated and untreated waters are similarly enriched in Li, and explain the decrease of river δ7Li in Seoul when the population density is high gh Figs.  and 4, L33, such as Zn, Cu, Ni or Hg, for which contaminated zones are clearly identified and monitored, and whose impacts on aquatic organisms and plants have been carefully investigated for many years in ecotoxicology, there is little information on environmental Li and its toxic effect. By illustrating anthropogenic Li inputs in Seoul waters, our study highlights the need to estimate the environmental and health impact of Li-rich materials, particularly in highly populated areas. Understanding the biological and metabolic effects of high Li levels on aquatic ecosystems also remains to be investigated to fill the gap compared to other contaminants. Finally, this study highlights that in urban areas, Li isotopes are more sensitive to anthropogenic inputs rather than local weathering inputs and therefore should be used with caution as a weathering proxy.Compared to other trace metals3. Samples for dissolved anions and total alkalinity (AT) were passed through 0.2 μm filters and collected in I-CHEM LDPE bottles.We collected 27 samples in July 2015 from 22 sites along a 422 km downstream transect between the uppermost reaches and the estuary of the Han River . Analyses of NRCC SLRS-4 and CRM TMDW-A were within ± 5% of certified values. Anion concentrations were measured using a Dionex ICS-1100 ion chromatograph equipped with a DionexTM IonPacTM AS14 anion-exchange column. The total carbonate alkalinity in μeq/L (AT = HCO3 + 2CO3) was measured using a Mettler Toledo T50A titrator with 0.01 M HCl acidimetric titration to an endpoint of pH = 4.5. The percent charge balance error (CBE), as one measure of the data quality, is given by the equation [CBE (%) = (TZ+ − TZ−)/(TZ+ + TZ−) × 100], where TZ+ = 2Ca2+ + 2Mg2+ + K+ + Na+, TZ− = Cl− + 2SO42− + NO3− + AT, and is on average better than ± 2%  mixture of 6 M HNO3 and 100% methanol. Lithium was separated from matrix elements using an AG 50 W−X8 resin (200–400 mesh)26. Then, the sample was dried and re-dissolved in 5% HNO3 (~40 ppb Li). Lithium isotope ratios were measured using a Neptune MC-ICP-MS upgraded with a large dry interface pump at the KBSI and the Korea Institute of Ocean Science & Technology (KIOST). Samples were introduced using a quartz dual cyclonic spray chamber and analyzed with a blank-standard-blank-sample-blank-standard-blank bracketing method. Sample intensities were matched to within 10% of the intensity of the standard. The sensitivity was ~90 V ppm−1 on mass 7 at a typical uptake rate of 100 μL min−1. Prior to isotopic analysis, each sample was checked for the yield and the concentration of matrix elements. The yields were approximately 100%, and the matrix concentration did not exceed 1.5% of the Li concentrations. The lithium isotopic composition is reported in delta notation relative to NIST RM 8545, where δ7Li = [(7Li/6Li)sample/(7Li/6Li)NIST RM 8545 – 1] × 1000. The accuracy and reproducibility of the whole method was validated using the USGS rock reference materials  and seawater standard (IAPSO). BCR-2 yielded +3.6 ± 1.7‰ , BHVO-2 yielded +4.5 ± 0.0‰ , BIR-1 yielded +4.1‰ (n = 1), and IAPSO yielded +31.2 ± 1.5‰ , which were all in good agreement with reported values38.Samples containing ~100 ng Li were dried in Teflon vessels, and the residues were treated with concentrated HNOSupplementary InformationPeer Review File


Present study focuses on rainfall over Western Ghats (WG), a complex topographic region (elevation > 500 m) of India to evaluate and to better understand the satellite behavior in contrast with a flat region (FR) (elevation < 500 m) of central India from 1998 to 2016 using the combinatory data sets of TMPA and IMERG . The categorical Intra Seasonal Oscillations (ISO) of Indian summer monsoon (ISM) namely, Madden Julian Oscillation (MJO) and Quasi Bi-Weekly Oscillation (QBWO) are tested in satellite and India Meteorological Department (IMD) gridded rainfall data sets to find out the satellite performance. As the accurate estimation of rainfall from satellites over higher elevation zones is challenging, here we propose a new perspective to select the rainfall products of satellite for better comparison with ground measurements. Considering the satellite’s best capability in detecting the cold clouds resulting from deep convection and its coupling with higher-level circulation, we show that the rainfall from satellites yield fruitful comparison with ground measurements when moist static stability, tropical easterly jet is above the climatological values. TMPA [Tropical Rainfall Measuring Mission (TRMM) Multi-satellite Precipitation Analysis] and IMERG [Integrated Multi-satellitE Retrievals for Global Precipitation Measurement (GPM)] are the promising multi-satellite precipitation retrieval, which provide opportunities to explore more about weather and monsoon systems over tropical region. Huffman et al.3 provide detailed information on TMPA and IMERG gridded rainfall data including their retrieval algorithm. These data sets are ideal for monsoon and hydrological studies4 although they need calibration at different scales with ground truth obtained from gauge and radar. It is reported that satellite rainfall estimation  shows regional, seasonal and year to year bias11. Gruber and Levizzani12, Kikuchi and Wang13 summarized, satellite retrieval of rainfall from Infrared, microwave and its combination shows serious limitations mainly over land than over ocean. Dinku et al.14 reported that high altitudinal regions pose unique challenges to satellite precipitation retrieval and found that the underestimation of rainfall from the satellite over mountain is due to their incapability in distinguishing the raining and non-raining clouds when IR technique is employed. In cases, where orographic rain is not due to ice aloft, since passive microwave rainfall retrievals are mainly from ice scattering at the upper parts of convective clouds. It also underestimates the surface rain over mountain. Ebert et al.15 also confirmed that satellite generally captures convective precipitation but has difficulty to detect warm rain. Underestimation of rainfall by the satellites over mountainous region has been studied19 in detail and reported that it is also due to the complex nature of the topography over the mountains. Mitra et al.8 made extensive study on rainfall estimation over WG and reported that daily TRMM rainfall underestimate the heavy orographic precipitation. However, a better understanding of satellite rainfall characteristics is important for its effective utilization during the natural hazards such as landslides, floods during active monsoon time. As the spatio-temporal resolution of the satellite data is available on finer scale, study of aforementioned hazards could be possible when the satellite data are used with care.Rain gauge network over land is the most trusted instrument for accurate rainfall measurement. However, in remote areas (e.g. mountainous terrain) sparse rain gauges make the rainfall data erroneous when are averaged over a region. Hence, satellite rainfall retrieval is an alternate option for optimal spatio-temporal analysis of precipitation over such remote areas. In recent times, availability of passive microwave and space borne active precipitation radar along with Infrared technique revolutionized the rainfall estimation and its characteristic study from spaceIn the present investigation, we attempt to study rainfall behavior over a complex topographic region, WG using rainfall data of TMPA and IMERG from TRMM and GPM (a follow-on mission to TRMM) constellation of satellites respectively from 1998 to 2016. The contrast in rainfall behavior during southwest (SW) monsoon season (June to September) over the elevated region of WG with a flat region in central India is presented, using typical Intra Seasonal Oscillation (ISO) indices associated with Indian summer monsoon namely MJO (~30 to 60 days), TEJ (~14 days) and MSS (~14 days) with small periodicity between 2 to 6 days. IMD and satellite data sets were compared over the FR and WG with a perspective to bring them to close agreement in certain conditions where the satellite rainfall data sets with existing algorithm are better compared with IMD rainfall.21. This analysis has been done for understanding the agreement between IMERG and IMD rainfall data sets over India.The spatial correspondence of newly released IMERG V5 and IMD is assessed by considering the different size grid boxes starting from 1° × 1° to 9° × 9° grids Fig.  from 201Prime aim of this study is to characterize rainfall from satellites over the complex topographic region, WG in order to use these data sets for research and application studies more reliably. For this purpose, we have selected a 5° × 5° box  in central India region where the elevation is less than 500 m to examine the changes with respect to an elevated region greater than 500 m over WG . Earlier studies show that TMPA and IMERG are in very good agreement over land49 as IMERG uses similar algorithm of TMPA. The details of the data sets used can be found from Thakur et al.50. India Meteorological Department (IMD), Govt. of India offers gridded rainfall data sets for the landmass of India, developed from the wide range of rain gauges. The data sets being used in the present study comprises with 0.25° × 0.25° spatial resolution and with daily temporal resolution. The basic rainfall data to develop these data sets are taken from the 6955 rain gauge stations over Indian land mass and at least 3000 rain gauges data have been used for any single day. The methodology followed to interpolate the data is Shepard interpolation technique51 and more details on the data sets can be found from52. The density of the rain gauges used in developing these data set along with the topography is shown in [Fig. 55, monsoon studies58 and extreme rainfall events61. For climatology of rainfall, same IMD rainfall product is used for the period 1961 to 1990. IMERG data set is re-sampled using interpolation technique from 0.1° × 0.1° to grid resolution of TMPA and IMD 0.25° × 0.25°.TRMM Multi-satellite Precipitation Analysis (TMPA) 3B42 V7 and IMERG V5 daily 29 were obtained from (http://www.bom.gov.au/climate/mjo/graphics/rmm). Meteorological parameters such as daily zonal (u) and meridional (v) component of wind at 200 mb, specific humidity and temperature profiles at different pressure level with grid resolution 2.5° × 2.5° were taken from NCEP/NCAR reanalysis62. These data sets have been used to estimate the daily tropical easterly Jet (TEJ) and moist static stability (MSS) using the formulae given below.p is specific heat at constant pressure, T is absolute air temperature, L is the latent heat of condensation, and q is specific humidity26. Surface Vertical velocity is derived from ERA- interim reanalysis 0.5° × 0.5° daily data set63. In addition Outgoing Longwave Radiation (OLR) is obtained from Very High Resolution Radiometer (VHRR) on board KALPANA-1 satellite64.Pair of daily Real-time Multivariate MJO series 1 (RMM1) and 2 (RMM2)Supplementary Figures and Tables