Male Sprague-Dawley rats were divided into four groups: control group fed ad libitum with standard rat diet, NASH group fed ad libitum with high-fat diet to induce NASH and NASH + Gen8 group and NASH + Gen16 group fed with high-fat diet plus intragastric administration of 8 or 16 mg/kg genistein once daily. After 6 weeks, liver samples were collected to determine MDA, TNF-α, PPARγ, and histopathology. The findings were that levels of hepatic MDA and TNF-α increased in NASH group, but 16 mg/kg genistein reduced these levels significantly. Downregulation of hepatic PPARγ was observed in NASH group, but genistein significantly upregulated the expression of PPARγ in both NASH + Gen groups. The histological appearance of liver in NASH group presented pathological features of steatohepatitis which were diminished in both NASH + Gen groups. The results suggest that genistein attenuates the liver histopathology of NASH with upregulation of hepatic PPARγ, reduction of oxidative stress, and inhibition of inflammatory cytokine.Nonalcoholic steatohepatitis (NASH) has become a global chronic liver disease, but no effective medicine has been proven to cure it. This study investigated the protective effects of genistein, a phytoestrogen, on NASH and examined whether it has any effect on hepatic PPAR Nonalcoholic steatohepatitis (NASH) is a chronic liver inflammation caused by fat accumulation in hepatocyte. It is a subset of nonalcoholic fatty liver disease (NAFLD) that covers a spectrum ranging from benign hepatic steatosis to aggressive NASH which can progress to cirrhosis and hepatocellular carcinoma and eventually liver-related death . NASH isγ). PPARγ is a ligand-dependent transcription factor that regulates fat metabolism, inflammation, cell differentiation, and apoptosis [γ was observed in mice with hepatic steatosis induced by high-fat diet (HFD) [γ may play an important role in the progression of NASH.However, nowadays many drugs have been tried in the treatment of NASH and one of the most frequently used therapeutic drugs is insulin-sensitizing drug, most of which are acting on peroxisome proliferators-activated receptor gamma (PPARpoptosis . Increaset (HFD) . On the et (HFD) . Accordiγ expression in NASH model. Consequently, this study determined whether genistein could diminish pathological features of NASH induced by HFD and also affect PPARγ expression in the liver.Genistein , a phytoestrogen, is a main isoflavone found in soy. It is known as a tyrosine kinase inhibitor  and alsoMale Sprague-Dawley rats weighing 180–220 grams were purchased from the National Laboratory Animal Center, Mahidol University, Nakhon Pathom, Thailand. The animals were housed in a controlled temperature room at 25 ± 1°C with 12 h light/dark cycle and were given free access to food and water. The experimental procedure carried out on the animals was approved by the Ethics Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand .n = 8): rats were fed ad libitum with standard rat chow diet (containing 7% fat and 26% protein) purchased from Perfect Companion Group Co., Ltd.  and they were given 0.1% dimethyl sulfoxide (DMSO) once daily by gavage tube for 6 weeks. Group 2 : rats were fed ad libitum with lard-based HFD containing 81% fat and 4% protein for 6 weeks to induce NASH and also administered 0.1% DMSO orally as described in group 1. Group 3 : rats received ad libitum HFD plus intragastric administration of 8 mg/kg genistein  dissolved in DMSO once daily for 6 weeks. Group 4 : rats were given ad libitum HFD and 16 mg/kg of genistein in 0.1% DMSO once daily by intragastric tube for 6 weeks. The amounts of genistein administered were based on the safe doses ranging from 1 to 16 mg/kg body weight according to the pharmacokinetic study of isoflavones by Busby and colleagues [All rats were randomly divided into four groups. Group 1 . The remainder of liver was fixed in 40 g/L formaldehyde to determine histopathology and PPARγ protein expression.At the end of the experimental period, all rats were anesthetized with thiopental . The abdominal wall was opened, and the whole liver was rapidly removed and subsequently washed with cold normal saline. Two small pieces of the liver were frozen in liquid nitrogen and then stored at −80°C for measurements of malondialdehyde (MDA) and tumor necrosis factor-alpha  to examine thiobarbituric acid-reactive substances (TBARS). Briefly, frozen liver tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor on ice. Then the homogenates were centrifuged at 1,600 ×g for 10 minutes at 4°C and the supernatants were collected. After performing in accordance with the manufacturer's protocol, the absorbent of supernatants were measured at 540 nm and MDA concentrations were calculated from a standard curve. Moreover, the total protein contents were also measured to correct the MDA levels which were expressed in nmol/mg protein.α were measured by sandwich ELISA using a colorimetric commercial kit from R&D Systems  according to the manufacturer's protocols. The total protein concentration of each sample was also required to correct the TNF-α level which was represented in terms of pg/mg protein.Portions of liver tissues were homogenized in RIPA buffer. Then liver homogenates were centrifuged at 12,000 ×g for 10 minutes at 4°C and the supernatants were collected. The amounts of TNF-μm. Next, tissue sections were deparaffinized and then retrieved the antigen in microwave. Afterward, the slides were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity and then washed with phosphate buffer saline (PBS). Subsequently, the sections were incubated with mouse monoclonal antibody against PPARγ  at 1 : 50 dilution at 4°C overnight and washed again with PBS. After the tissues were incubated with the secondary antibody for 30 minutes at room temperature, the immunoreactivities were visualized by incubating the slides with diaminobenzidine (DAB) for 5 minutes. Finally, the sections were counterstained with hematoxylin. Under light microscopy, PPARγ immunoreactive cells were identified as those with dark brown in their nuclei and digital images were taken in high magnification field from each sample. A thousand cells were counted manually for each rat and the numbers of positive stained cells were calculated and expressed as the percentage of immunoreactive cells. Quantification of immunostaining intensity was performed by measuring densitometry using ImageJ program .After the liver samples were fixed in formaldehyde, they were embedded in paraffin and cut at 3 Paraffin-embedded liver tissues were sectioned and stained with hematoxylin and eosin. An experienced pathologist who was blinded to the experiment evaluated all samples. All histopathological changes were observed under light microscope. All fields in each section were examined for grading of steatosis, inflammation, and ballooning degeneration according to the criteria described by Brunt et al. .The severity of steatosis was graded as the percentage of parenchymal cells containing fat as follows: 0 = less than 5% of hepatocytes containing fat, 1 = less than 33% of hepatocytes containing fat, 2 = 33–66% of hepatocytes containing fat, and 3 = more than 66% of hepatocytes containing fat.Hepatic inflammation was scored from 0 to 3: 0 = no inflammation, 1 = mild focal zone 3 hepatocyte inflammation, 2 = moderate zone 3 hepatocyte inflammation, and 3 = severe zone 3 hepatocyte inflammation.The presence of ballooning degeneration which is the key character used to distinguish the developed NASH from the less progressive forms of NAFLD was graded from 0 to 2: 0 = no ballooning cell, 1 = few balloon hepatocytes, and 2 = many balloon hepatocytes.p < 0.05.Statistical analysis was performed by the Statistics Package for the Social Sciences (SPSS) software version 18.0 for Windows. Most data were presented as mean ± SD except for histopathological scores which were presented as frequency. Mean comparison among groups of animals was carried out with one-way analysis of variance (one-way ANOVA) followed by LSD post hoc test. Differences were considered statistically significant at p = 0.031). Conversely, genistein reduced the levels of MDA in NASH + Gen8 and NASH + Gen16 groups significantly .As shown in α level was elevated significantly in NASH group compared with the control . On the other hand, the level of TNF-α in liver was significantly lower in NASH + Gen16 group than those in NASH group . Although hepatic TNF-α level in NASH + Gen8 group also tended to be lower than those in NASH group, the difference was not statistically significant.As shown in γ protein expression in the liver was studied by using immunohistochemical technique and PPARγ positive stained cell was identified by dark brown nuclei . Nevertheless, PPARγ expressions in the liver were enhanced by genistein in both NASH + Gen8 and NASH + Gen16 groups with the significant increases of PPARγ immunoreactive cells . Moreover, densitometry analysis also showed the results similar to those of the percentages of PPARγ positive stained cells .PPARn nuclei . As showIn control group, the liver sections represented normal histology as shown in At the present, it is recognized that NASH is one of the most common chronic liver conditions in general population. Even though the pathogenesis of NASH is not yet fully understood, the two-hit hypothesis  is the mγ in NASH model has not yet been revealed. Moreover, the role of PPARγ in NASH has remained contradictory. Therefore, we demonstrated the preventive effects of genistein against NASH and its effect on hepatic PPARγ.Although there is no definitive treatment for NASH, the benefits of genistein, a soy-derived isoflavone with antioxidant and anti-inflammatory activities, in the prevention of NASH have been studied in some research. Nonetheless, the effect of its on hepatic PPAROne of the processes of second hits that activates the transition of simple steatosis to NASH is oxidative stress. MDA, a marker for oxidative stress, is derived from lipid peroxidation which frequently arises in response to oxidative damage . In factα [α plays a role in the evolution of NASH because the expressions of TNF-α and its receptor increase in patients with NASH compared to patients without NASH [α level in NASH rats but administration of 16 mg/kg genistein can reduce this increase significantly. The anti-inflammatory effect of genistein on NASH may be involved in suppression of c-Jun N-terminal kinase (JNK) and nuclear factor- (NF-) κB pathways that leads to the inhibition of TNF-α synthesis [Oxidative stress and lipid peroxidation produce many reactive oxygen species which can trigger production of various inflammatory cytokines, including TNF-α . There iout NASH . Correspynthesis .γ is important to the prevention of NASH, although its role in NASH progression has rarely been described and some results are still unclear and contrary [γ protein expression was significantly dropped in HFD-induced NASH rats when compared with control rats; this result corresponds to the previous studies [γ expression in the liver of NASH group may be owing to an augmentation of TNF-α because TNF-α can inhibit PPARγ activity at both pre- and posttranslational levels [It is known that PPARcontrary , 7. Our  studies , 26. Thil levels .γ expressions in both NASH + Gen groups in this study. Likewise, another study also found an upregulation of PPARγ in the liver of metabolic syndrome rats given genistein aglycone [γ ligand property of genistein which can activate PPARγ in a ligand-dependent manner [γ can attenuate NASH in mice by alleviation of biochemical and histopathological abnormalities. This PPARγ effect could be due to downregulation of proinflammatory cytokines and upregulation of antioxidant as well as redistribution of fatty acid from liver to adipose tissue resulting in the reduction of hepatic steatosis [γ by genistein in this study may contribute to the improvement of steatosis and necroinflammation in the liver sections of NASH + Gen rats.Interestingly, genistein significantly increased hepatic PPARaglycone . This mat manner , 30. Addteatosis , 32. Theγ, on NASH induced by high-fat diet and found that hepatic PPARγ expression was decreased in NASH rats but genistein upregulated its expression as well as diminished liver oxidative stress and inflammation. These appeared to have contributed to the improvement of liver histopathology of NASH. Further research is required to clarify the molecular mechanism behind the effect of genistein on hepatic PPARγ in NASH model, such as the activation of JNK and NF-κB signalling pathways, and the expression of PPARγ target genes in the liver.Overall, the study revealed that feeding HFD for 6 weeks can cause NASH in Sprague-Dawley rats with the same histopathology of NASH as in human in the form of liver steatosis and necroinflammation. Meanwhile, we investigated the protective effects of genistein, which can activate PPARγ expression in the liver. From the findings, it is suggested that genistein effectively attenuates the emergence of NASH, and this may be useful for further studies and applications for NASH protection.In summary, our results show an anti-NASH effect of genistein which is probably related to the increase of PPAR


Phe residues in Δ-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103 + 47 μM until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of Δ-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of 19F ligand-observe and complimentary 15N,1H-HSQC titrations to monitor interactions from the protein’s perspective. 19F ligand-observe NMR was ratified as mirroring 15N protein-observe but highlighted the advantage that 19F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity.Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (K Protein disulphide isomerase (PDI) is an abundant protein found within the lumen of the ER of all eukaryotes at concentrations estimated to be ~0.8% of total cellular proteina and a’, are redox active and contain conserved cysteine residues in an active site motif of Cys-Gly-His-Cys. The other domains, b and b’, lack these conserved residues and are non-catalytic. Instead, the b’ domain provides the principal binding site for ligand interactionb domain ensures alignment of the functional sites and may confer structural stability to the protein. There is also a short 19 amino acid x-linker region between the b’ and a’ domains allowing inter-domain flexibility, and a C-terminal acidic tail (c) with the ER retention signal KDEL. The complete domain architecture is abb’xa’c.Human Protein Disulfide Isomerase (hPDI) is a 57 kDa protein consisting of four thioredoxin-like domainsb’x has been mapped by NMR45b’ domain is capable of binding small peptides (10–15 amino acid residues) via hydrophobic interactions, independent of disulfide bonds or cysteine residues2b’ domain, along with binding contributions from the redox active domains a and a’, is also essential for the binding of larger peptides and non-native proteins89d values, allows hPDI to bind a wide range of protein folding intermediates and then release the correctly folded protein once the correct conformation is achievedThe ligand binding site on hPDI Humicola insolens) b’xa’ PDI bound to a α-synuclein peptide (αSN) that identified this PDI as being able to capture a hydrophobic ligand segment with key contacts from valine and leucine αSN residuesHumicola insolensb’ is located toward the N-terminal region of b’ whereas the binding site in hPDI b’ appears much larger and registers across the beta-sheet and residues in the C-terminal half of the protein including the x-linker56Obtaining detailed binding information is of considerable importance; ligand specificity is still poorly understood and as yet no structural models exist for hPDI bound to a target ligand. By unravelling the molecular nature of the hPDI-ligand interaction we hope to expand our understanding of the structure-function based specificity of hPDI. Furthermore, medical interest in hPDI and related family members has significantly increased as this protein family have been associated with several diseasesd)1619F NMR has re-emerged as an important and valuable method for studying proteins and biomolecules as illustrated by several recent publications151819202122232415N/1H and 19F NMR chemical shift perturbations to study the molecular detail and specificity of the ligand binding mechanism of the hPDI fragment b’x with a known peptide ligand, Δ-somatostatin 215N enriched protein, selective peptide fluorination and Ala-substitutions in the peptide sequence has enabled a powerful NMR approach to monitoring ligand binding from both molecular perspectives (protein and ligand) that also highlights the potential importance of the phenylalanine over tryptophan for Δ-som recognition by hPDI.A well-documented method of measuring ligand binding by NMR is via chemical shift perturbation to calculate dissociation constants  demonstrated that the peptide binds with an average Kd of 48 ± 35 μM, which whilst suggesting marginally tighter binding than the non-fluorinated peptide, is still within the error range. It was expected that fluorinated peptide would exhibit a lower Kd due to the increased hydrophobicity that fluorination contributes, strengthening hydrophobic interactions. The calculated Kd suggests a modestly tighter binding event with fluorinated peptide, but otherwise fluorinated Δ-som behaves similarly to the non-fluorinated peptide. It may be surprising that fluorinated aromatic residues exhibit increased hydrophobicity when fluorine is the most electronegative element but the phenomenon of increased hydrobicity for fluorinated amino acids has been reported before28Titration of the triple-fluorinated F1,2,3 Δ-som into 0.25 mM Furthermore, fluorine is known to have a lipophilic effect when substituted into benzene compounds and log P values for fluorinated benzenes are higher than non-fluorinated benzenes.Using single and triple fluorinated peptides provided a valuable insight into ligand binding from the peptide viewpoint. All three phenylalanine residues in Δ-som  were fluorinated as it was anticipated they would be integral to the peptide’s binding interaction to hPDI.19F NMR spectra (at a constant peptide concentration of 0.15 mM) and 15N,1H-HSQC spectra (at a constant protein concentration of 0.25 mM). This provided binding perspectives from both peptide and protein. 19F NMR data provided precise curve fits of equation (1) with Kd values in agreement with those obtained by 15N NMR .NMR titrations were carried out by collecting  15N NMR . The 19F19F resonance in the triple-fluorinated peptide correlate well with those obtained from each single fluorinated peptide  in both single and triple fluorinated peptides provides the lowest Kd, with F1 (Phe6) and F3 (Phe11) consistently higher, suggesting that from this data the F2 (Phe7) position may be pivotal to the Δ-som interaction with b’x.Dissociation constants from each  compare  and 2. Ob’x, alanine substitutions were introduced at all phenylalanine positions. As a result three single Phe → Ala mutants, three double Phe → Ala mutants and one triple Phe → Ala peptide were synthesized and monitored for binding using 15N,1H-HSQC experiments that detected the protein. Results for these peptides are shown in d by a single Ala mutant. This does suggest that fluorination influences the affinity, most likely due to the increased hydrophobicity it imparts on the peptide. Observing only single Phe → Ala changes suggests F3 (Phe11) substitution creates the largest increase in Kd and this trend was also observed in the double Ala mutants.With the aim of revealing the importance of the Phe residues in Δ-som for the binding affinity to d that suggests the most significant disruption on the protein’s ability to recognise the peptide. The Phe6,7 → Ala substitution is less dramatic and provides a dissociation constant closest to single mutant peptide Kd values, whereas double substitutions involving Phe11 displayed weaker binding than Phe6,7 → Ala. The effect of Phe11 → Ala within both single and double mutations is is best demonstrated graphically in b’x. Phe11 was also shown to be highly variable in the 19F study; recall single and triple fluorinated peptides differences  to high micromolar Kd using a titration; high quality curve-fits require titration to approaching occupancy and plateau of the curve. In reality such fits difficult to achieve with high micromolar Kd systems, such as protein-folding chaperones like hPDI, because it requires a high concentration of ligand that is beyond the solubility limits of the system. High concentrations of biological molecules also provide additional sources of error such as aggregation and/or increases in viscosity. Although reducing the constant protein concentration can mitigate these issues, there is still a limit to detection to create sufficient signal to noise with adequate resolution to measure changes in chemical shift. The compromise was used here to provide the presented trends in increasing Kd with acceptable signal to noise, resolution and time efficiency of experiments. However, preliminary experiments provided comparable dissociation constants to those reported and provides confidence when inferring Kd trends discussed upon fluorination and alanine substitution.The double Ala mutants displayed weaker binding affinities than the single mutants and substitution of Phe11 to Ala has the largest increase in Knces see  and 2. Tb’x.The Phe6,7,11 → Ala Δ-som peptide did not provide any significant resonance shifts throughout the titration series and was classified as a non-binding peptide. More specifically, any shifts identified at the end point of the titration were extremely small (<0.01 ppm) and did not map to the ligand binding site . These eb’x using a combination of 15N/1H and 19F NMR spectroscopy. Mapping from both the peptide and the protein provides complementary information when measuring dissociation constants as well as the opportunity to interrogate residue specificity. A comparison of the b’x backbone chemical shift perturbations seen on binding of the fluorinated and non-fluorinated peptide confirms that the location of the binding site is unchanged and that the direction and extent of chemical shift perturbation is virtually identical in both cases suggesting a similar overall mode of interaction. Furthermore, chemical shift changes measured from fluorinated and non-fluorinated peptide-ligands are in very close agreement to previous studies and we have previously demonstrated that fluorination of hPDI b’x maintains the binding mode with Δ-somatostatin19F NMR increased the precision of measuring Kd values when compared to 15N,1H backbone amide measurements from HSQC spectra and Kd values measured using fluorinated peptides were marginally smaller than the non-fluorinated counterpart which reflects a higher binding affinity. This observation of fluorination promoting hydrophobic interactions is in agreement with our previous work studying Δ-som binding to hPDI b’x using fluorinated protein that produced a Kd of 23 ± 4 μM at 25 °CThis study presents a detailed analysis of peptide ligand binding to hPDI b’x. Removal of one or two of the phenylalanine residues did not abolish binding, although it did considerably weaken the affinity. However, removal of all three Phe residues from Δ-som prevented the peptide from interacting with b’x and our data suggests that the third phenylalanine residue, Phe11 (F3), makes the largest contribution towards binding affinity. This work highlights the importance of large, exposed, aromatic amino acids in binding to hPDI, much like other molecular chaperonesb’ from thermophilic fungusHumicola insolens PDI has a distinctive mechanism of substrate recognition compared to human PDI. However, protein-folding chaperones are fluid and dynamic protein systems and it is possible that recognition is attributable to thermodynamics in addition to amino acids. All peptides used in this study were individually analysed by 1H NMR under the same buffer and temperature conditions used for the binding studies (data not shown). Peptide 1H NMR spectra were extremely similar and every spectrum displayed the same chemical shift limits with no dispersion. This is consistent with peptides in solution that have no apparent secondary structure or conformational differences. Therefore, fluorination or alanine substitution does not appear to facilitate any significant entropic changes and that the differences in dissociation constants are a result of direct interactions between ligand and target protein. This further supports our conclusions that Phe residues are important for recognition of Δ-som by hPDI and that changes are not due to induced thermodynamic difference by fluorination or alanine substitution.The alanine mutant Δ-som peptides demonstrate that all three phenylalanine residues contribute to the recognition of Δ-som by hPDI 15N and 19F labelling to probe ligand binding that also validates the role of 19F NMR to follow peptide binding from the ligand’s perspective. This approach enabled a hypothesis driven approach to demonstrating the importance of phenylalanine in Δ-somatostatin for substrate recognition by human PDI. This method can be applied to peptide-protein interaction studies where structure-function and molecular mechanistic knowledge is required. There are many different fluorinated amino acid options available within custom peptide synthesis that can be used as explicit probes that will subsequently inform a peptide mutagenesis strategy to test interaction hypotheses. This approach has the potential to be more specific and effective than alanine scanning, particularly with larger peptides. Furthermore, the 19F approach does not require uniform protein labelling, spectral assignment of the protein or costly isotopic peptide synthesis and such peptides are easily synthesized and cost little more than non-fluorinated peptides. In the example reported, both the peptide and protein inform on the binding event in a similar manner and fluorination of peptide residues did not significantly affect the peptide’s binding mode or affinity.As a final point regarding this study, we have also demonstrated a significant use for combined b’x was expressed in E. coli and purified as described previouslyhPDI 6-DMSO and then diluted 20-fold into the NMR sample. To test that DMSO did not influence the protein, 15N,1H-HSQC spectra were obtained with and without 5% DMSO and no changes were observed (data not shown).Unlabelled Δ-som peptide (AGSKNFFWKTFTSS), 4-fluorophenylalanine (4-F) peptides: F1 (AGSKN(4-F)FWKTFTSS), F2 (AGSKNF(4-F)WKTFTSS), F3 (AGSKNFFWKT(4-F)TSS), F1,2,3 (AGSKN(4-F)(4-F)WKT(4-F)TSS), and Phenylalanine → Alanine (Phe → Ala) mutants: Phe6 → Ala (AGSKNAFWKTFTSS), Phe7 → Ala (AGSKNFAWKTFTSS), Phe11 → Ala (AGSKNFFWKTATSS), Phe6,7 → Ala (AGSKNAAWKTFTSS), Phe6,11 → Ala (AGSKNAFWKTATSS), Phe7,11 → Ala (AGSKNFAWKTATSS), Phe6,7,11 → Ala (AGSKNAAWKTATSS) and helix-28 from human serum albumin (RERQIKKQTALVELV) were generated by peptide synthesis and purified >95% by reverse-phase HPLC . To solubilise the hydrophobic peptides, the lyophilised material was first dissolved in 100% D1H) NMR spectrometer equipped with a 5 mm QCI-F cryoprobe. 15N,1H-HSQC spectra were acquired over 45 min on samples containing 0.25 mM b’x and varying peptide concentrations. Each 19F 1D spectrum was acquired over 60 min on samples of 0.15 mM Δ-som with varying protein concentration. Ratios of the variable:static components in each experiment were 1:4, 1:2, 1:1, 1.5:1, 2:1 and 2.5:1. All samples were run at 310 K in 20 mM sodium phosphate buffer (pH 7.0) containing 50 mM NaCl, 5% D6-DMSO, 5% 2H2O and 0.05% sodium azide. 1D NMR data were processed using Bruker Topspin software and referenced using the position of trifluoroacetic acid (−76.55 ppm). 2D data were processed using NMRPipeAll spectra were collected using a 4-channel, 5-amplifier Bruker Avance III 14.1 T (600 MHz 1H)2 + (Δ15N)2]. Dissociation constants (Kd) for each shifting peak were calculated by plotting the chemical shift perturbation (Hz) against ligand concentration (mM) and fitting the curve to the equation:Amide chemical shifts were calculated in Hz as √[. Standard errors from the Levenberg-Marquardt fitting routine were taken as the uncertainties. We determined Kd from 15N,1H HSQC NMR data using two approaches: First, by simultaneous fitting all shifts which are averaged to create a single plot, which is considered an accurate method of Kd determinationd values determined. This hPDI system creates Kd values with shallow binding curve where errors where average chemical shifts reduce fitting accuracy and individual fitting produces higher resoltuion. Therefore, we used Kd values from the averaged individual fit method within this paper to report smaller errors. We believe this method works well for determining high Kd values because it encourages data to be triaged via inspection of individual curve fits and does not register discrepancies across the chemical shift or frequency fluctuation of binding.where Δb’x construct at 25 °C was previously deposited in the BioMagResBank (accession code 15998)15N,1H-HSQCs and HNCA and HN(CO)CA triple resonance experiments were carried out on 15N/13C labelled protein to assign the b’x backbone to 82% at 37 °C .


Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 106 g.mol−1) compared to the native (Mw ~ 1.2 × 106 g.mol−1). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 106 g.mol−1), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein . This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from  Haemophilus influenzae is a small non-motile Gram negative bacterium1H. influenzae strains  in the upper respiratory tract5H. influenzae is most likely to have been an important secondary invader to the influenza virus in the 1890 pandemic and many subsequent influenza epidemics. Nonetheless it is still considered responsible for a wide range of clinical diseases such as meningitis and pneumonia3H. influenzae seem to occur in humans only and are primarily due to type b Haemophilus influenzae or “Hib” (>95% of cases) where it remains a major cause of acute bacterial meningitis. Until the availability of the Hib vaccine, the type b H. influenzae was the main cause of meningitis in children between 6 months and 5 years old, although older children, adolescents and adults can also be infectedInvasive diseases caused by Haemophilus influenzae possess a polysaccharide capsule and that there are 6 capsular serotypes (a–f )Haemophilus influenzae type b, was composed of polyribose-ribitol chains with (3→5) phosphate diester linkages between the ribose moieties. Further work111213Hib is a linear copolymer composed of repeated units of→3)-β-D-Ribf-(1→1)-D-ribitol-(5-OPO3→[(C10H19O12P)n] with a defined molecular size2Since 1931 it has been known that some strains of e.g Streptococcus pneumoniae and Neisseria meningitidis) the type b polysaccharide capsule of H. influenzae is attractive as a vaccine antigen since invasive disease is almost exclusively restricted to only one serotype, which in turn has rendered its capsular polysaccharide a prime candidate for vaccine studies. The first generation of Hib vaccine was based on the purified type b capsular polysaccharide and its ribose-ribitol phosphate repeating units21819Hib conjugate vaccine in 1987 after the pioneering work by Schneerson and co-workersHib-Tetanus toxoid (TT) conjugate Hiberix® and a novel Hib-MenCY-TT (MenHibrix®) by GSK Vaccines. MenHibrix® vaccine administered in accordance to the current Hib vaccine schedule (USA) would have the potential to induce protective antibodies against Hib and meningococcal-CY disease in childrenUnlike other bacteria (3 to >108 g.mol−1), particularly appropriate for large glycoconjugates, many of which are beyond the exclusion limit of SEC-MALSHaemophilus influenzae type b (referred to as PRP native and PRP-ADH respectively) as well as the final PRP- TT conjugate (with a polysaccharide: protein ratio of ca. 0.4) and to establish whether it is the protein component or carbohydrate component which principally influences the physical or hydrodynamic properties in solution This study has also been designed to demonstrate the usefulness of analytical ultracentrifuge based procedures – all not requiring a separation column or matrix - in the characterisation of large glycoconjugate vaccines.Important to their function as a vaccine is the structure and stability of the polysaccharide and conjugates thereof. Molecular weight (molar mass) and molar mass distribution have been documented as being the most important physical parameters affecting the immunogenicity of capsular polysaccharides242627s) versus s were seen in the case of both native Hib polysaccharide  indicative of non-ideality and the absence of significant reversible associative effects  eluting in the void volume of the SEC.Molar mass values estimated from sedimentation equilibrium using the Hib PRP-TT conjugate was then transformed into a corresponding distribution of molar mass using the Extended Fujita Approach of Harding et al.The sedimentation coefficient distribution – which itself gives a good measure of sample heterogeneity - for The transformation is as follows:withandb is a conformation parameter that has already been estimated for a number of polysaccharidess can be found from equation M (e.g. Mw from sedimentation equilibrium) is known for one value of s . The distributions so obtained for two plausible values of b are shown in so20,w, and the concentration dependence coefficient ks). Three different and complementary extrapolation methods values to zero concentration  were used for all 3 samples – all gave good agreement and are shown in For macromolecules of known molar mass the intrinsic viscosity [η] can provide an important measure of the conformational flexibility  volumef/fo = 1 are due to either asymmetry  and ∼200–300 nm for a very stiff rod shaped macromolecule (such as xanthan or schizophyllan)Lp using either intrinsic viscosity3738For a more quantitative estimate of chain flexibility we can use the persistence length 23 mol−1) and A0 and B0 are tabulated coefficients, and the Yamakawa–Fujii equation40where2 can be considered as –ln(d/2Lp) and A3 = 0.1382 if the Lp is much higher than the chain diameter, d. Difficulties arise if the mass per unit length is not known, although both relations have recently been built into an algorithm Multi-HYDFITLp and ML based on minimization of a target function Δ. An estimate for the chain diameter d is also required but extensive simulations have shown that the results returned for Lp are relatively insensitive to the value chosen for d which was fixed at an average of ∼0.8 nm41ML and Lp were treated as variables and the minimum value of the target function Δ was estimated on a 2D contour plot for each sample , but which retained the high flexibility of the native and activated polysaccharide, similar to what we found earlier for capsular polysaccharides from Streptococcus pneumoniae1H-NMR relaxation studies of Berti and coworkersHib conjugated to the non-toxic mutant of diphtheria toxin CRM197: this work also provided evidence of reduced hydration of the protein as a result of the conjugation. Using circular dichroism and fluorescence spectroscopy and 1H-NMR, the protein component of Hib and MenC glycoconjugate vaccines was nonetheless found to be important for pH and thermal stability47All four conformation approaches  show that conjugation with the tetanus protein yielded large polydisperse structures of Hib manufacturing process consists of the following steps: after initial solid preculture steps, the bacteria are incubated in shake flasks. Once the appropriate cell density is reached, the inoculum is transferred to a fermentor for further amplification. The virulent strain of Hib is grown in a medium supplemented with hematin and nicotinamide adenine dinucleotide (NAD). During the growth phase, the pH is regulated with concentrated NaOH solution and the dissolved oxygen is regulated by the stirring speed and air flow rate. At the onset of the drift phase (7–8 hours after inoculation), the pH and dissolved oxygen regulations are switched off and the parameters are left to drift. The fermentation is terminated 10 hours after the inoculation. The broth containing the polyribosyl (PRP) polysaccharide of interest is inactivated by heat and centrifuged to remove cell debris. Purification is assured by Hib precipitation with Cetavlon in the presence of Celite. The complex PRP-CTAB is an insoluble complex retained on the Celite. The PRP polysaccharide is detached from the Cetavlon by using a high salt concentration (0.5 M NaCl). The product is ultrafiltered to eliminate residual nucleic acids, then precipitated with ethanol, dried and kept at −20 °C until conjugation. The residuals (DNA (<0.2%), proteins (<0.1%), LPS(<0.03%)) were very low in the final bulk of polysaccharide and met all the required specifications of the pharmacopeia (WHO/EMEA).The bulk Hib PRP-polysaccharide activation (using adipic acid dihydrazide as linker) prior to its conjugation was using the cyanogen bromide method505153Hib PRPTT conjugate was then purified on a Sephacryl S500HR column using 0.2 M NaCl as the elution solution. The conjugate was then sterile filtered before a final ultrafiltration step, which led to the product meeting the required specifications of the pharmacopeia, with <15% of unconjugated polysaccharide and <5% unconjugated protein. All samples were dissolved in phosphate buffered saline pH ~6.8, I = 0.1 M−1) of each sample and a matching amount of the corresponding reference buffer  in appropriate channels. Samples were run at a rotor speed of 45000 rpm  at a temperature of 20.0 °C. Concentration profiles and the movement of the sedimenting boundary in the analytical ultracentrifuge cell were recorded using the Rayleigh interference optical system and converted to concentration  versus radial position, rls-g*(s) model incorporated into the SEDFIT analytical algorithm of Dam & Schuckg*(s) versus s, where s is the sedimentation coefficient (in Svedberg units S = 10−13 sec). The * indicates the profiles are not corrected for diffusion broadening . This data analysis was followed by the correction to standard solvent conditions - namely the density and viscosity of water at 20.0 °C - to yield s20,W using the algorithm SEDNTERP−1. To account for hydrodynamic non-ideality (co-exclusion and backflow effects), the apparent sedimentation coefficients  were calculated at a series of different cell loading concentration and extrapolated to infinite dilution using the Gralén relationA general description of the ultracentrifugal methods and how they can be used for glycoconjugate and polysaccharide characterisation is given in two recent references29ks is the Gralén or concentration dependence coefficient.where i.e. 4000 rpm (~1200 g), 9000 rpm (~5900 g) and 2000 rpm (~300 g) for PRP native, PRP-ADH and the PRP-TT solution, respectively, at a temperature of 20.0 °C. Scans were taken every one hour and equilibrium was reached after approximately 48–72 hours. Optical records (Rayleigh interference profiles) of the relative concentration distribution of the solute at equilibrium were analysed to give the weight (mass) average apparent molar mass Mw,app using the SEDFIT-MSTAR algorithmM* function of Creeth and Hardingc(r) = the initial loading concentration, c−1). At such low concentrations, non-ideality effects (which tend to lead to underestimates of the molar mass) may be relatively small and we make the approximation that the apparent weight average molar mass Mw,app is equal to the true weight average molar mass MwMz,app using the MFIT algorithm of Ang and RoweSedimentation equilibrium experiments were also performed using the Beckman  Optima XL-I analytical ultracentrifuge again using the Rayleigh interference optics and an automatic on-line data capture system to record equilibrium concentration distribution profiles. The modified long (20 mm) optical path length double-sector titanium cells with sapphire windows were selected and loaded with 0.070 mL of solution  and a matching amount of reference buffer dialysate in the appropriate channels. Samples were centrifuged at rotor speeds selected to give a sufficient fringe increment from meniscus to baseHib native, Hib-ADH and Hib-TT, were carried out using the automated micro-viscometer Anton Parr AMVn  at a concentration series  from 0.1–2.0 mg mL−1 based on the rolling ball viscosity method in which the apparatus measures the time of a (silanized) steel ball needed to roll in a 1.6 mm diameter silanized glass capillary containing the sample. The experiment was performed at different reclining angles of 70° n = 4 times), 60° (n = 4 times) and 50° (n = 6 times) under precise temperature control (20.00 ± 0.01 °C). HugginsDynamic viscosity measurements for c is the concentration, where How to cite this article: Abdelhameed, A. S. et al. A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate. Sci. Rep.6, 22208; doi: 10.1038/srep22208 (2016).


For typical complexes, an assembly funnel occurs in a narrow window of conditions whose location is highly complex specific. However, by redesigning the components this window can be drastically broadened, so that complexes can form quickly across many conditions. The generality of this approach suggests assembly funnel design as a foundational strategy for robust biomolecular complex synthesis.Like protein folding and crystallization, the self-assembly of complexes is a fundamental form of biomolecular organization. While the number of methods for creating synthetic complexes is growing rapidly, most require empirical tuning of assembly conditions and/or produce low yields. We use coarse-grained simulations of the assembly kinetics of complexes to identify generic limitations on yields that arise because of the many simultaneous interactions allowed between the components and intermediates of a complex. Efficient assembly occurs when nucleation is fast and growth pathways are few,  Within cells, bottom-up phenomena organize biomolecules into structures with sizes ranging from angstroms to microns. Precise control over structure at the angstrom and nanometer scales is important for optimizing catalysis in vivo formation of structure across these length scales. Stable nanometer- or angstrom-scale structures generally form as the result of folding a protein or RNA chain with a particular sequence Escherichia coli, for example, more than 20% of known polypeptides become reported members of protein complexes Different physical processes are responsible for the While the development of strategies for the design of synthetic self-assembling complexes have long lagged behind the design of folding processes, recently, a wealth of designed complexes assembled from proteins Here, we test the assumption that self-assembly processes for biomolecular complexes generally reach a high-yield equilibrium state by simulating the kinetics of a variety of generic, idealized assembly reactions. We find that for typical biomolecular complex self-assembly reaction rates and component concentrations To characterize the kinetics of complex assembly, we use a simple model of assembly in which rigid components of a generic complex bind to one another via orientation-specific pairs of complementary interfaces . We assui.e., concentrations typical for large  DNA nanostructures e.g., from entropic or allosteric effects α that determines how the free energy of interaction scales with the number of bonds formed , geometry and reaction parameters  see  and S9. or ) see , S22. Fother to  . These ri.e., high values of To understand why grid and cube complexes assembled so slowly, we investigated the composition of the simulated solution after the completion of the reaction , which provides another, perhaps even larger, challenge in understanding complex assembly.One major assumption in this work is perfectly formed components: we do not address the challenge to form the components in the first place. In successfully forming biomolecular complexes, components must first be properly synthesized and folded or fabricated before they can associate to form a complex. Components that misfold or degrade can alter the assembly landscape by allowing the possibility of nonspecific interactions  Valid species are components, full complexes and multiple component configurations, or intermediates, where all components comprising an intermediate have at least one bond (shared edge) with another component. A black box represents an occupied site whereas a white box represents unoccupied sites on the lattice. (b) Invalid intermediate assemblies are denoted by red “X”s and are lattice configurations that are not connected (do not share an edge) and are not included in our model.(TIF)Click here for additional data file.Figure S2Valid and invalid reactions for the 2×2 grid complex. Examples of valid reactions in a 2×2 grid complex in which (a) one bond, or (b) two bonds are formed. The reverse reaction rate (indicated roughly as arrow length) will change with reaction conditions and bond coupling. Reactions such as in (c) and (d) are not included in our model. In (c), the components do not interact at any edges and would not produce a valid species as a product, and in (d) the reactants share components in the same position, which would in practice block that reaction from happening.(TIF)Click here for additional data file.Figure S3Yields of 1×3 to 1×9 line complexes at various isothermal conditions. Dashed lines indicate thermodynamic equilibrium. Inset diagrams depict the complexes. Here, as in the main text, (TIF)Click here for additional data file.Figure S4Yield of 1×9 line complex at various reaction times, (TIF)Click here for additional data file.Figure S5Assembly size distribution at different isothermal assembly conditions after (TIF)Click here for additional data file.Figure S6e.g.,) but remain far from equilibrium for positive bond coupling.Yields of 2×2, 3×3 and 4×4 square grid complexes at different isothermal assembly conditions and bond coupling constants ((TIF)Click here for additional data file.Figure S7Yields of 2×2, 3×3 and 4×4 square grid complexes as a function of bond coupling constant, (TIF)Click here for additional data file.Figure S8Yield of 3×3 square grid complex for many isothermal conditions, from (TIF)Click here for additional data file.Figure S9Reducing the number of components in the simulation does not significantly affect yield predictions. Yield of 2×2, 3×3 and 4×4 square grid complexes at various isothermal conditions starting with 1000 (instead of 10000) of each component, with the simulated volume adjusted so that (TIF)Click here for additional data file.Figure S10Yield for 2×2 and 3×3 square grid complexes at various isothermal conditions, including yield predictions after long reaction times, (TIF)Click here for additional data file.Figure S11Yields of 2×2, 3×3, 4×4 and 5×5 square grid complexes at various reaction times, (TIF)Click here for additional data file.Figure S12Yield of 2×2, 3×3 and 4×4 spiral complexes at various reaction times, (TIF)Click here for additional data file.Figure S13.Assembly size distributions (in # of components) for 2×2, 3×3 and 4×4 square grid complexes at various isothermal conditions and bond coupling constants. Inset diagrams depict the complexes. All plots are shown after (TIF)Click here for additional data file.Figure S14Timescales of nucleation and rearrangement together determine the rate of complex formation. Both of these timescales are functions of complex size and geometry. The fraction of material in various species as a function of reaction time for 3×3 square grid assembly under different assembly regimes: nucleation-limited at (TIF)Click here for additional data file.Figure S15Size distribution of intermediates for various 2D complexes. Mean size of intermediates (in number of components) after (TIF)Click here for additional data file.Figure S16During an anneal, most complexes are produced during the phase of the anneal that passes through the assembly funnel. Yield of 2×2 and 3×3 square grid complexes during the course of an anneal for various bond coupling constants. Inset diagrams depict the complexes being assembled. The anneal begins from left to right, with the total time of the anneal given as the value of mbly see .(TIF)Click here for additional data file.Figure S17During very long anneals, component depletion can increase the amount of time that the system effectively stays within the assembly funnel regime. Effective reaction propensity is given by (TIF)Click here for additional data file.Figure S18. Yield of 3×3 square grid complex as a function of reaction time for an isothermal assembly Click here for additional data file.Figure S19Deterministic and stochastic solutions are almost identical. To test the similarity of the stochastic solution to the deterministic solution, we simulated the ODEs for the respective complexes using MATLAB’s ode23s solver. Deterministic solution (solid lines) and overlaid stochastically sampled values (dots) of yield for 2×2 and 3×3 square grid and 2×2 spiral complexes at various isothermal conditions. Inset diagrams depict complexes.(TIF)Click here for additional data file.Figure S20Yields of 2×2x2 cube complexes as a function of bond coupling constants (TIF)Click here for additional data file.Figure S21Yield of 2×2x2 cube complexes as a function of bond coupling constant, (TIF)Click here for additional data file.Figure S22Yield of 2×2x2 cube complex at various reaction times, (TIF)Click here for additional data file.Figure S23Assembly size distributions for 2×2x2 cube complex at various isothermal conditions and bond coupling constants. All plots are shown after (TIF)Click here for additional data file.Table S1Complex specifics and parameter space explored in this work.(DOCX)Click here for additional data file.Table S2Criteria for labeling assembly regimes.(DOCX)Click here for additional data file.Text S1Supporting Methods.(DOCX)Click here for additional data file.Text S2Computational Specifics.(DOCX)Click here for additional data file.Text S3Assembly Regime Criteria.(DOCX)Click here for additional data file.Text S4Assembly Distribution Selection.(DOCX)Click here for additional data file.Text S5Further Explanation of Bond Coupling Effect on Yield from .(DOCX)Click here for additional data file.Text S6Computing Thermodynamic Equilibrium of Large Complexes.(DOCX)Click here for additional data file.Text S7Supporting References.(DOCX)Click here for additional data file.


The direct functionalization of carbon–hydrogen (C–H) bonds has emerged as a versatile strategy for the synthesis and derivatization of organic molecules. II/PdIV redox cycle are increasingly common. The C–H activation step in most of these catalytic cycles is thought to occur at a PdII centre. However, a number of recent reports have suggested the feasibility of C–H cleavage occurring at PdIV complexes. Importantly, these latter processes often result in complementary reactivity and selectivity relative to analogous transformations at PdII. This mini review highlights proposed examples of C–H activation at PdIV centres. Applications of this transformation in catalysis as well as mechanistic details obtained from stoichiometric model studies are discussed. Furthermore, challenges and future perspectives for the field are reviewed.The direct functionalization of carbon–hydrogen (C–H) bonds has emerged as a versatile strategy for the synthesis and derivatization of organic molecules. Among the methods for C–H bond activation, catalytic processes that utilize a Pd A representative example is the conversion of 2 equiv. of 1 into 2 2 to afford a single isomeric product 4via selective cleavage of C–HA , a 5 : 1 mixture of the isomeric products 6a and 6b is produced (3 at PdII(OAc)2 (>99 : 1 selectivity for activation of HA) and (ii) a subsequent C–H activation of 3 (5 : 1 selectivity for HAversus HB).Several experiments were conducted that suggest that this transformation involves two discrete C–H activation steps that have very different selectivities. For example, the unsymmetrically-substituted substrate  of C–HA . When thrmed 6a, . In conto form 5 , and thiproduced . These rII (step (i)), is followed by oxidation of the resulting palladacycle intermediate A with Oxone to yield PdIV species B (step (ii)). The second C–H activation then occurs at this PdIV intermediate to yield C (step (iii)), which undergoes C–C bond-forming reductive elimination to complete the catalytic cycle (step (iv)).A variety of additional experiments, including cross-over studies and reactivity studies of possible intermediates, implicated the mechanism shown in IV to achieve synthetically useful catalytic transformations. In one elegant example, Michael demonstrated the PdII/IV-catalysed aminoarylation of terminal olefins with NFSI as the oxidant  than both anisole and bromobenzene.d8 showed an intermolecular H/D competition isotope effect of 1.1  in II centres generally show much higher intermolecular competition isotope effects .9A competition between toluene and toluene-anti-aminopalladation to produce alkyl PdII intermediate D (step (i)). D then undergoes oxidation with NFSI to produce PdIV intermediate E (step (ii)) π-coordination of the arene substrate to E to generate F (step (iii)) is followed by C–H cleavage (step (iv)) to afford aryl alkyl PdIV complex G. Finally, C–C bond-forming reductive elimination (step (v)) closes the catalytic cycle.On the basis of these  studies, a full catalytic cycle was proposed. As shown in II/IV-catalysed C–H functionalization reaction involving the oxidative coupling of perfluorobenzamides with simple arenes using NFSI as the oxidant , only F+ oxidants afforded high levels of para-selectivity. This led the authors to propose that the presence of a fluoride ligand on the PdIV center is crucial for achieving para-selective C–H activation.Several studies were conducted to shed further light on the mechanism of this transformation. While substantial yields of oxidative coupled products were obtained with a number of different oxidants (d8 as the arene substrate (kH/kD = 1). This result suggests that the C–H activation at PdIV is not the slow step of the catalytic cycle. Unlike the Michael system, no competition or intramolecular isotope effect studies were reported in this system, so the possible role of π-coordination cannot be assessed from this report.10As shown in via C–H activation at PdIV. For example, in 2008, Inoue and coworkers demonstrated the PdCl2-catalysed C–H arylation of naphthalene with aryl stannanes.12 This reaction was selective for arylation at the α-position of naphthalene 2 and PdCl2, afforded modest yields and selectivities for the C–C coupled products (yields ranging from 12–24% and α : β selectivities from 5 : 1 to 13 : 1). The yield and selectivity could be enhanced dramatically through the use of N–N chelating L type ligands, and the optimal diimine Pd catalyst  afforded low yields and selectivities. On the basis of these investigations, the oxidation of the PdII catalyst by the aryliodonium salt was proposed to be the rate-determining step, and the C–H activation of naphthalene was proposed to occur at the resulting PdIV centre. Additionally, a two-step C–H activation mechanism analogous to that put forth by Michael  followed by subsequent C–H cleavage at the PdIV centre.Rate studies of the Pd-catalyzed naphthalene arylation showed 1 Michael  was prop14 In this system, the site selectivity of furan C–H activation could be modulated based appropriate selection of the terminal oxidant. Using AgCO3 as oxidant, the authors observed poorly selective activation of the furan , an “F+” oxidant that is well known to promote the oxidation of PdII to PdIV,15 afforded >20 : 1 C-5 selectivity in most cases. In the NFTP system, large isotope effect (ratio of products 22-d0/22-d5 = 4.8) was observed when the reaction was run as a competition between benzene and benzene-d6   . The autvia C–H activation at PdIV. These examples show encouraging selectivity trends and demonstrate that valuable synthetic methods can be achieved with C–H activation at PdIV as a likely step. At this stage, most of these transformations have been discovered serendipitously rather than through reaction design. However, moving forward it would be important to rationally design catalytic sequences involving C–H activation at a PdIV centre. A uniting feature of the transformations discussed above is the use of strong oxidants, with “F+” reagents, hypervalent iodine reagents, and inorganic peroxides being particularly common choices. In addition, many these transformations are believed to involve the generation of a PdII–C bond prior to oxidation of PdII to PdIV. This likely serves to accelerate the oxidation event. Finally, most of the catalysts and intermediates in these transformations possess oxidatively stable ligands that are unlikely to participate in competing reductive elimination. All of these features should serve as key design considerations as new reactions are developed.The examples described above summarize the current state of the art in catalytic transformations proposed to proceed IV model complexes were carefully designed to accelerate C–H activation while slowing competing reductive elimination processes from PdIV. These complexes were designed so that the C–H activation would be intramolecular. For example, in complex 24 PdII(2-biphenyl) (CF3) complex 23 with PhICl2 at –30 °C. Warming complex 24 to room temperature resulted in intramolecular activation of the 2-aryl substituent to form the cyclometalated PdIV product 25. To our knowledge, this was the first direct observation of C–H activation at a PdIV centre.Our group has developed organometallic model systems in order to directly observe and study this fundamental reaction. Such studies should ultimately assist in the rational design of new catalytic processes that incorporate this elementary step. The Pdmplex 24 , studied26 (via oxidation of a PdII precursor with PhICl2.18 The tridentate tris(2-pyridyl)-methane ligand (Py3CH) was a key design feature in this study. This strongly coordinating tridentate ligand is well-known to stabilize octahedral PdIV species relative to analogues with bidentate nitrogen donors like bipyridine.19 Thus, it was anticipated that the Py3CH ligand would slow C–H activation and enable more detailed mechanistic investigations of this process. Indeed, the PdIV aryl complex 26 proved stable at room temperature and could be fully characterized by 1D and 2D NMR, HRMS, and X-ray crystallography. Complex 26 did not undergo C–H activation, even upon heating to 90 °C in CDCl3. Instead, C–Cl bond-forming reductive elimination was observed under these conditions. However, when one of the chloride ligands in 26 was exchanged for an acetate, the resulting intermediate 27 underwent clean cyclometalation at room temperature to yield 28. This result suggests that C–H activation at PdIV in this system likely occurs via a concerted metalation-deprotonation mechanism, analogous to C–H activation at PdII.20In a follow up study, the related complex 26  was synt29 . These results in combination with a variety of studies of the dynamic behaviour of complex 26 indicate that C–H cleavage is not the rate determining step in the C–H activation process in this system. The similarity between the KIE observed on this system to those observed in catalytic systems further supports the mechanisms proposed for the catalytic reactions described above.When complex 29  was treaIV complexes. It is also noteworthy that C–H activation at the PdIV centre can be facile at room temperature or below with bidentate ligands or in the presence of acetate ion. Future work on isolated palladium complexes may explore an intermolecular C–H activation event at PdIV.These two examples chronicle C–H activation at discrete and, in one case, isolable Pdvia high valent palladium is a powerful manifold for developing synthetically useful transformations. This mini-review has summarized experiments supporting the viability of C–H bond activation at PdIV and has described mechanistic studies of the C–H bond cleavage event. Synthetically useful catalytic cycles that utilize C–H activation at PdIV remain limited; however, there are great opportunities in this area due to the potential for unique selectivity in these transformations. Although the presently described work is mostly limited to catalytic C–H arylation sequences, as this method is more fully understood, one can anticipate its application to more diverse scaffolds and functionalizations.The catalytic functionalization of C–H bonds


In patients with high risk stage II and stage III colon cancer (CC), curative surgery followed by adjuvant FOLFOX-4 chemotherapy has become the standard of care. However, for 20 to 30 % of these patients, the current curative treatment strategy of surgical excision followed by adjuvant chemotherapy fails either to clear locoregional spread or to eradicate distant micrometastases, leading to disease recurrence. Preoperative chemotherapy is an attractive concept for these CCs and has the potential to impact upon both of these causes of failure. Optimum systemic therapy at the earliest possible opportunity may be more effective at eradicating distant metastases than the same treatment given after the delay and immunological stress of surgery. Added to this, shrinking the primary tumor before surgery may reduce the risk of incomplete surgical excision, and the risk of tumor cell shedding during surgery.RAS mutated patients). In RAS wild-type patients a third arm testing FOLFOX+ cetuximab has been added prior to colectomy. Patients in the neoadjuvant chemotherapy arms will receive postoperative treatment for 4 months (8 cycles) to complete their therapeutic schedule. The primary endpoint of the study is the histological Tumor Regression Grade (TRG) as defined by Ryan. The secondary endpoints are: treatment strategy safety , disease-free and recurrence free survivals at 3 years, quality of life, carcinologic quality and completeness of the surgery, initial radiological staging and radiological response to neoadjuvant chemotherapy, and the correlation between histopathological and radiological response. Taking into account a 50 % prevalence of CC without RAS mutation, accrual of 165 patients is needed for this Phase II trial.PRODIGE 22 - ECKINOXE is a multicenter randomized phase II trial designed to evaluate efficacy and feasibility of two chemotherapy regimens  in a peri-operative strategy in patients with bulky CCs. Patients with CC deemed as high risk T3, T4 and/or N2 on initial abdominopelvic CT scan are randomized to either colectomy and adjuvant chemotherapy (control arm), or 4 cycles of neoadjuvant chemotherapy with FOLFOX-4  With about 1 million new cases in developed countries annually and 500 000 annual deaths, colon cancer (CC) is the second leading cause of cancer death in Western countries and a significant public health issue  that comThe 20 to 30 % risk of local or distant recurrence observed in patients with stage II/III, non-metastatic CC operated in a curative intent and receiving adjuvant chemotherapy reflects the relative failure of such a strategy to prevent the risk of locoregional spread of tumor cells or eradicate distant micrometastases. Several reasons may explain this failure: the delayed start (up to 4 months) of chemotherapy after the initial diagnosis and the rapid doubling time of colorectal metastases, which may progress during this time interval without chemotherapy . The stiThe potential benefit of such a peri-operative strategy in stage II/III CC is strengthened by: i) recent advances in radiology, which allows a good prediction of tumor stage  prior to surgery , 14; ii)RAS wild type tumors.The Prodige 22 - ECKINOXE trial is a multicenter randomized phase II trial designed to evaluate efficacy (response rate) and feasibility  of two chemotherapy regimens  in a peri-operative setting in patients with stage II/III CC. Control arm includes patients for whom standard treatment comprises surgery followed by adjuvant FOLFOX-4 chemotherapy. This phase II study will assess the feasibility of a peri-operative strategy in these patients and the impact of adding cetuximab in this setting for patients with RAS WTA double central, independent and blinded review of imaging and pathology is planned. All eligible patients will be randomized to either surgery alone or neoadjuvant chemotherapy with simplified FOLFOX-4. Patients randomized to the simplified FOLFOX-4 arm will be randomized to receive Cetuximab or not if they are RAS status of the primary tumor the design will be: Arm A vs. Arm B vs. Arm C for RAS WT patients and Arm A vs. Arm C for those RAS mutated : Colectomy followed by simplified FOLFOX-4 alone every 2 weeks for 12 cycles. Adjuvant chemotherapy will be given to all patients with stage III CC and according to the local practices for stage II cancer.RAS status of the primary colon cancer on biopsies (WT or mutated); (iii) CC classified by abdominal CT scan: high risk T3  - T4 (penetration within adjacent organs) and/ or N2 (more than 3 clustered lymph nodes above 1 cm in shortest diameter); (iv) non metastatic CC ; (v) non complicated primary tumor . Patients with obstructive CC and treated by defunctioning stoma can be included; (vi) absence of synchronous colorectal cancer ; (vii) age ≥ 18 years et ≤ 75 years; (viii) ECOG performance status 0–1 ; (ix) no prior chemotherapy or abdominal or pelvic irradiation; (x) life expectancy of ≥ 5 years; (xi) no history of colorectal cancer; (xii) patients with childbearing potential should use effective contraception during the study and the following 6 months; (xiii) laboratory data including : white blood cell count ≥ 3 × 109/L with neutrophils ≥ 1.5 × 109/L, platelet count ≥ 100 × 109/L, hemoglobin ≥ 9 g/dL , total bilirubin ≤ 1.5 × ULN , ASAT and ALAT ≤2.5 × ULN, Alkaline phosphatase ≤1.5 × ULN, serum creatinine ≤ 1.5 × ULN; (xiv) signed written informed consent obtained prior to any study specific screening procedures.For inclusion in the study, all of the following inclusion criteria must be fulfilled: (i) pathologically confirmed colon adenocarcinoma ; (ii) assessment of Patients are not eligible for this study if any of the following exclusion criteria apply: (i) contra-indication to iodinated contrast medium injection including allergy to iodinated contrast medium and renal insufficiency proscribing iodinated injection; (ii) rectal cancer located within 15 cm from the anal verge by endoscopy or under the peritoneal reflection at surgery or having received radiation therapy prior to surgery; (iii) complicated primary CC ; (iv) synchronous colorectal cancer; (v) metastatic spread at baseline assessment ; (vi) history or current evidence on physical examination of central nervous system disease or; (vii) peripheral neuropathy ≥ grade 1 Common Toxicity Criteria for Adverse Events (CTCAE) v.3.0; (viii) known hypersensitivity reaction to any of the components of study treatments; (ix) presence of inflammatory bowel disease; (x) HNPCC syndrome or polyposis; (xi) major surgical procedure, open biopsy or significant traumatic injury within 28 days prior to study treatment start. Incompletely healed wounds or anticipation of the need for major surgical procedure during the course of the study; (xii) clinically relevant coronary artery disease or history of myocardial infarction in the last 12 months, or high risk of uncontrolled arrhythmia; (xiiii) pregnancy (absence to be confirmed by ß-hCG test) or breast-feeding period; (xiv) previous malignancy in the last 5 years; (xv) medical, geographical, sociological, psychological or legal conditions that would not permit the patient to complete the study or sign informed consent; (xvi) any significant disease which, in the investigator’s opinion, would exclude the patient from the study.RAS WT tumor (FOLFOX-4 vs. FOLFOX-4 – Cetuximab), the present trial will determine which neoadjuvant regimen is the most efficient in terms of response rate in patients with non metastatic locally advanced CC.By evaluating two chemotherapy regimens in patients with The primary objective is to assess the histopathological response to neoadjuvant chemotherapy measured by the Tumor Regression Grade (TRG), as simplified to three categories by Ryan . The TRGSecondary endpoints are the following: (i) postoperative morbidity (occurring within 60 postoperative days) graded 0 to 5 according to the Clavien-Dindo classification . If gradRAS WT patients will be randomised between 3 treatment arms (Arm A vs. Arm B vs. Arm C) and those mutated for RAS between 2 treatement arms (Arm A vs. Arm C). The randomization will be stratified according to the investigator center, T stage (T1/2/3 vs. T4) of the primary tumor on the pretreatment CT scan and N stage (N0-N1 vs. N2) of the primary tumor on the pretreatment CT scan. The screening/baseline assessment will take place within the 3 weeks prior to randomization, randomization will be done between 4 and 8 weeks after surgery and the maximum time between randomization and treatment start will be 15 days.After completion of all the screening evaluations  and signature of the informed consent(s), all eligible patients will be randomly assigned by the randomisation center of the French Federation of Digestive Oncology (FFCD) to one of the treatment arms according to the RAS status: RAS status  of the primary tumor will be performed from biopsies obtained during the diagnostic colonoscopy, by each centers at the “plateformes de génétique somatique des tumours” labelled by the National Institut of Cancer (INCa). Once baseline assessments are completed, check and validation of inclusion and exclusion criteria for the study will be performed at the oncological multidisciplinary meeting of each center.The screening visit must be performed within 3 weeks before the randomisation. During this visit, inclusion and exclusion criteria are checked and validated. The complete pre-therapeutic work-up includes a physical examination, standard laboratory tests with CEA level, complete colonoscopy with biopsies and a CT scan of the thorax, abdomen and pelvis. Clinical tumoural staging (cTNM) will be based on the data obtained from CT scan. The determination of the RAS status of the primary tumor  before chemotherapy administration: documentation of concomitant medication and concurrent procedures; physical examination; vital signs and ECOG performance status; blood sampling for hematology and clinical biochemistry and recording of adverse events since last cycle (including primary tumor related symptoms or complications). The end of treatment visit must be performed 15 days after the 4R (85 mg/m2), with simplified LV5FU2) is administrated intravenously over 48 hours once every two weeks . The FOLFOX-4 regimen will be administrated as follows: Oxaliplatin will be administered as an 85 mg/m2 intravenous infusion over 2 hours (on day 1 only) concomitantly with LV, as a 400 mg/m2 infusion over 2 hours, followed by 5- fluorouracil (5-FU), given as a 400 mg/m2 bolus injection (over 10 minutes), and then as a 2400 mg/m2 continuous infusion over 46 hours. Cycle length is 2 weeks comprising 48 hours of infusion and 12 days of rest. These cycles must be repeated every second week. A total of 12 cycles  will be administrated to all patients in Arm A and B. In Arm C (control Arm), only patients with stage III CC  will receive 12 cycles of adjuvant chemotherapy. For those with stage II CC, the decision of adjuvant chemotherapy in this Arm will be will be done according to the investigator discretion.The simplified FOLFOX-4 . Cetuximab will always be administered first, i.e. the Cetuximab infusion should be completed one hour before chemotherapy begins. The Cetuximab dose must always be based on the body surface area (BSA). There is no restriction for Cetuximab in patient with a BSA > 2.0 m2. As prophylaxis to reduce the risk of an allergic/hypersensitivity reaction, it is mandatory to pretreat the patient as specified below. Cetuximab is administered as follows: the volume is administered over 120 minutes (maximum rate of 5 mL/min). Saline solution (0.9 %) is used to flush the line at the end of the infusion. Vital signs are checked before, during, immediately after and 1 hour after the end of the infusion.- The first infusion is 500 mg/m2. Cetuximab is administered as follows: the volume is administered over 60 minutes (maximum rate of 10 mL/min). Saline solution (0.9 %) is used to flush the line at the end of the infusion.The subsequent weekly doses are 500 mg/mSurgery will be performed 3 to 5 weeks after the last cycle of neoadjuvant chemotherapy (in arms A and B) or within 14 days after the randomisation. The anesthetic evaluation, patient’s information regarding the operative procedure and the organization of the hospitalization will be performed according to the local practices of each investigator center. The colectomy will be performed by laparotomy or laparoscopy (investigator discretion) with respect of the oncological quality criteria of resection. Per and postoperative data will be collected and reported in specific forms.Adjuvant chemotherapy will be administrated for all patients included in arms A (FOLFOX-4) and B (FOLFOX-4 + Cetuximab). Eight additional cycles will be administrated  according the same regimen. In Arm C (control Arm), only patients with stage III CC  will receive 12 cycles of adjuvant chemotherapy. For those with stage II CC, the decision of adjuvant chemotherapy in this arm will be will be done according to the investigator's discretion.For all patients, follow-up assessment will be performed until recurrence and/or death. For the study, the recurrence free survival will be assessed at 3 years. For patients included in the present trial, the follow-up will be systematic and performed thereafter this period according to the good clinical practices. The follow-up schedule and investigations proposed for the present study are those recommended by the FFCD .Every 6 months, the following investigations will be performed: tumor assessment, CEA measurement, survival status, additional cancer therapy. Moreover, the following assessments will be performed during the follow-up: colonoscopy 3 years after surgery then every 3 to 6 years, assessment of neurological toxicity, and assessment of electrolytes  at the second follow up visit (12 months after surgery) if still abnormally decreased at end of treatment assessment.0: an histological response (TRG1-Ryan) rate of 10 % is uninteresting; (ii) H1: an histological response (TRG1-Ryan) higher than 10 % is required to rate pursue investigations in phase III trial. A response rate of 35 % is expected.Patients RAS Wild Type will be randomized between arm A, B and C whereas RAS mutated patients will be only randomized between arm A and arm C. According to Simon (Optimax) 2 steps design, it will be required to include 33 patients/ arm  to test following hypotheses: (i) HAt the first step , after inclusion of the first 13 patients in each arm, if the number of histological response (TRG1-Ryan) is ≤ 1, inclusion in this arm will be stopped and if the number of histological response (TRG1-Ryan) is ≥ 2, inclusion of the next 20 patients will be done. Calculated power at the interim analysis is 97 %.nd step , after inclusion of 33 patients in each arm, if the number of histological response (TRG1-Ryan) is ≤ 6, this arm will be declared uninteresting for further investigations in phase III trial and if the number of histological response (TRG1-Ryan) is ≥ 7, this arm will be declared interesting for further investigations in phase III trial. At the second step calculated power will be 96.3 % and alpha type one error will be 2 %. In control arm no interim analysis is plan for efficacy. Then in this arm, at the second step, calculated power will be 97.2 % and alpha type one error will be 4.2 %. If ≥ 10 % of complications ≥ grade III (Clavien and Dindo classification) within 60 postoperative days are reported, this arm will be declared unsafe.At the 2Interim and final results of this phase II study will be reviewed by an independent data monitoring committee (IDMC). Trial will be pursued in phase III with 2 or 3 arms according to IDMC recommendations. Control arm (no neoadjuvant treatment) will be kept as control arm in phase III whatever the results of phase II study. Required number of patients is 33 × 3 = 99 patients for RAS WT patients and 33 × 2 = 66 for those with RAS mutated. Taking into account prevalence of RAS WT patients of 50 %, it will be necessary to screen 192 patients to obtain 99 patients RAS WT and 66 patients mutated for RAS with a minimal probability of 90 %.Fifty seven French centres will participate in the study: Amiens University Hospital, Caluire et Cuire Hospital in Lyon, University Hospital of Clermont-Ferrand, General Hospital Center of Orléans, European Georges Pompidou University Hospital in Paris, Claude Huriez University Hospital in Lille, North University hospital of Bordeaux, University Hospital of Nîmes, Purpan University Hospital in Toulouse, Paoli Calmettes Cancer Institute in Marseille, Henri Mondor University Hospital in Créteil, North University Hospital in Marseille, Saint-Louis University Hospital in Paris, Pitié Salpêtrière University Hospital in Paris, Ambroise Paré University Hospital in Boulogne-Billancourt, St André University Hospital in Bordeaux, General Hospital Center in Beauvais, Croix-Rousse University Hospital in Lyon, Beaujon University hospital in clichy, Pessac University Hospital of Bordeaux, the University Hospital in Strasbourg, Pontchaillou University Hospital in Rennes, General hospital in Bézier, Robert Debré University Hospital in Reims, Avicenne University Hospital in Bobigny, Cochin University Hospital in Paris, St Anne Clinic in Strasbourg, University Hospital of Nantes, Jean Mermoz Private Hospital in Lyon, University Hospital in Grenoble, La Timone University Hospital in Marseille, Saint Antoine University Hospital in Paris, General Hospital of Pau, Paul Strauss Cancer Institute in Strasbourg, University Hospital of Dijon, Saint Joseph Private Hospital in Paris, University Hospital of Rouen, Rangueil University Hospital in Toulouse, Sainte Barbe Clinic in Strasbourg, Sainte Catherine Institut in Avignon, The Asclepios House Clinic in Avignon, General Hospital of Bayonne, Eugene Marquis Cancer Institut of Rennes, Intercommunal Hospital Center of Creteil, General Hospital of Mont de Marsan, Bichat University Hospital in Paris, Grand Sud Polyclinic of Nîmes, Kenval Polyclinic of Nîmes, Valdegour Clinic Oncogard Center of Nîmes, General Hospital of Boulogne Sur Mer, University Hospital of Poitiers, University Hospital of Brest, Saint Joseph-Saint Luc Hospital in Lyon, Synergia Polyclinic in Carpentras, General Hospital of Avignon, University Hospital of Saint Etienne.This study protocol was approved by the Institutional Review Board, the Ile de France IX ethic committee on November 2011 and the AFSSAPS (Agence Française de Sécurité Sanitaire des Produits de Santé) on September 2011 under the registration number A110936-41. The institutional promoter is the Assistance Publique Hôpitaux de Paris, France. The trial has been registered on ClinicalTrial.gov website under the identification number NCT01675999. This study received a grant from the French National Cancer Institute (INCA) in 2010. The study complies with the Declaration of Helsinki rules and the principles of the Good Clinical Practices guidelines. Informed consent will be obtained from each patient in a written form prior to randomisation. Patient safety and all potential threats to the patients will be monitored every 6 months by an independent data safety monitoring board (DSMB) and, additionally, at the discretion of the DSMB or Promoter. The DSMB also will evaluate the primary endpoint data. Qualified personnel at the sponsor site will also meet every three months to review safety data, including adverse events and serious adverse events. Any information deemed to potentially affect the safety of the trial will be brought to the attention of the DSMB. An Independent committee (IDMC) including at least 1 clinicien, 1 methodologist and pharmacologist will be created before trial initiation. This IDMC should statute on efficacy and safety at the interim analysis regarding decision criteria of Simon design, post operative morbidity.There are several arguments in favor of performing a study to assess the impact on survival of perioperative chemotherapy in the treatment of stage II/III CC. Three prerequisites are however essential. First, systemic chemotherapy must be effective and able to induce a measurable and reproducible response of the primary tumor. Second, current preoperative imaging with abdominopelvic CT scan must allow for an accurate assessment of tumor stage to justify antineoplastic treatment with a limited number of false-positives patients in order not to over-treat patients that do not require such treatments. Finally, the neoadjuvant chemotherapy must be well tolerated and not increase the risk of pre- and post-operative complications.The histopathological changes induced by neoadjuvant chemotherapy and/or radiotherapy have been studied in several digestive cancers, such as cancer of the esophagus, stomach, rectum and liver metastases of colorectal origin , 23–27. Hundt and colleagues reported on the sensitivity of helical CT in imaging colorectal cancer in 37 patients . ArteriaMore recently, Burton and colleagues sought to investigate if the CT scan could elucidate poor prognostic factors for CC in order to select a subset of patients eligible for neoadjuvant treatment . Three pFOLFOX-4 is the standard of care treatment given postoperatively for 6 months in patients with stage III CC –38. In aKRAS (wild-type) metastatic colorectal cancer [The combination of FOLFOX-4 and Cetuximab has also been demonstrated to be effective with regard to response rate (57 to 81 %) and progression-free survival with good tolerance, in patients with non-mutated l cancer –46. Two The aim of our phase II ECKINOXE study is to evaluate the efficacy (response rate) and tolerability of two regimens of neoadjuvant chemotherapy among patients with locally advanced CC. The control arm will consist of patients treated with FOLFOX-4 post-operatively. This study will allow us to investigate the feasibility and effectiveness of this original neo-adjuvant strategy, and to select the best regimen to improve long-term disease-free and overall survivals. A similar study testing another anti-EGFR monoclonal antibody (Panitumumab) is currently ongoing in the United Kingdom . The pilot stage of this randomised controlled trial on the first 150 included has been recently published. Nearly 90 % (85 of 95) of patients in this trial completed preoperative chemotherapy with grade 3–4 gastrointestinal toxicity in 7 %. All 99 tumours in the preoperative group were resected, with no significant differences in postoperative morbidity between the preoperative and control groups. T3 or more advanced tumours was pathologically confirmed in 98 % of patients undergoing immediate surgery and 91 % of patients following preoperative chemotherapy (p = 0 · 10). Preoperative therapy resulted in significant downstaging in terms of TNM stage compared with the postoperative group (p = 0 · 04), including two pathological complete responses.


The catastrophic collapse of the pelagic ecosystem at this time was probably the dominant factor responsible for the end-middle Norian conodont extinction.Extinctions within major pelagic groups  occurred in a stepwise fashion during the last 15 Myr of the Triassic. Although a marked decline in the diversity of pelagic faunas began at the end of the middle Norian, the cause of the middle Norian extinction is uncertain. Here we show a possible link between the end-middle Norian radiolarian extinction and a bolide impact. Two palaeoenvironmental events occurred during the initial phase of the radiolarian extinction interval: (1) a post-impact shutdown of primary and biogenic silica production within a time span of 10 The biodiversity crisis at the Triassic–Jurassic boundary (TJB) has traditionally been identified as one of the “big five” mass extinctions of the Phanerozoic245−1, an entry angle of 45° and a crystalline target (density = 2750 kg m−3). The size range of such a crater is consistent with the size of the Manicouagan crater 17Here, we show that the remarkable turnover of siliceous plankton (radiolarians) in the end-middle Norian was triggered by a large impact event. Such an event has been inferred from anomalous concentrations of platinum group elements (PGEs)710712We analysed a large dataset of Upper Triassic radiolarian occurrences in the Inuyama area72021Capnodoce and most Capnuchosphaera species. An initial phase of diversification (phase D1) started in the upper sublayer of the claystone and in the overlying chert bed. A second major wave of diversification (phase D2) is observed ~33 cm above the ejecta horizon. Given an average sedimentation rate of 1.1 mm kyr−1 in the middle Norian chert succession  from the study section at Sakahogi , but that a negative δ13Corg excursion is present in the upper claystone sublayer . Above the claystone interval, δ13Corg values vary from −27.09‰ to −24.57‰ .To evaluate changes in primary productivity, we examined stratigraphic variations in organic carbon isotopic values  with meteoritic Os (characterized by low 187Os/188Os ratios); the meteoric Os was vaporized at the time of the impact and was subsequently dissolved into seawater. The 187Os/188Os ratio, which is lowest in the lower sublayer, gradually increases towards the upper sublayer, and reaches pre-impact levels in chert samples overlying the claystone layer. No discrete extraterrestrial particles were observed in the upper sublayer claystone, which indicates that the 187Os/188Os ratio of this sublayer records the Os isotope composition of ancient seawater187Os/188Os values in the upper sublayer claystone after the impact event may reflect post-impact removal of excess dissolved meteoritic Os from seawater, which occurred over a period of 104–105 yr, as Os residence times range from 10 to 60 kyr  shifts in δ13Corg values across the ejecta layer. The global implications of the magnitude and short duration of the negative δ13Corg excursion reported here remain to be verified at other middle–late Norian boundary intervals worldwide.The negative δkyr refs  and 30. −1, and a constant dissolution rate of biogenic silica during that time, the mass accumulation rates (MAR) of radiolarian silica in the pre-impact chert beds is estimated at ~0.1 g cm−2 kyr−1, which is the same as the biogenic silica flux near the equator in the modern Pacific Ocean (0.1–0.3 g cm−2 kyr−1)−2 kyr−1 in the upper sublayer claystone, and then remained low during the deposition of sponge spicule-rich cherts. On the other hand, the MAR of the siliceous sponge silica increased markedly across the claystone layer of the ejecta deposit, from ~0.01 to ~0.08 g cm−2 kyr−1, and subsequently decreased to ~0.01 g cm−2 kyr−1 after the E2 event. Our data and previous data22Following the resurgence in primary productivity after the E1 event, the biogenic silica content had recovered to pre-impact values by the first chert bed overlying the claystone layer. However, our analysis reveals that major biotic components of the bedded chert changed temporarily from radiolarians to siliceous sponges, for ~0.3 Myr after the impact. Assuming a constant sedimentation rate in the middle–upper Norian chert succession of 1.1–1.6 mm kyrCapnodoce and Capnuchosphaera species. These middle Norian radiolarians are very rare above the E1 interval, whereas a small spumellarian species is abundant within the E2 interval; this spumellarian species is reported as Spumellaria gen. et sp. indet. A, and its occurrence can be used to identify the stratigraphic position of the ejecta layer in other Triassic chert sections within the Jurassic accretionary complexes in Japan2124Decreases in the sinking flux of radiolarian silica during the E1 and E2 events may reflect a decline in radiolarian production in middle Norian taxa, including in Did the middle–late Norian extinction event occur uniformly on a global scale, or does it represent a regional phenomenon in the Panthalassa Ocean? The record of radiolarian faunal change across the middle–upper Norian boundary has been established at the species level in several regions37Epigondolella bidentata conodont zone and the base of the Gnomohalorites cordilleranus ammonoid zone in western North America39Parvigondolella species survived across the ejecta layer, but that an important middle Norian Epigondolella species became extinct just below the impact horizon  ammonoid extinctions that occurred in the middle–upper Norian boundary3940 horizon . The preThis study has revealed that late middle Norian open-ocean ecosystems experienced profound disruptions after a large impact event (chondritic impactor of 3.3–7.8 km in diameter), and that the event was possibly related to the 90-km-diameter Manicouagan crater in Canada. Although many marine sections over the past 540 Myr have been examined, no catastrophic collapse in marine ecosystems caused by an extraterrestrial impact has yet been described, with the outstanding exception of the Cretaceous–Paleogene boundary (KPB) crisisTo extract radiolarians for biostratigraphic analyses, 53 chert samples were soaked in a dilute HF solution (5%) for 24 h, and were then passed through a 32-μm mesh sieve. This treatment was repeated 2–5 times to obtain sufficient residue for analysis. After drying the residue, radiolarians were handpicked under a binocular microscope and were observed using a scanning electron microscope. Conodonts were handpicked from the same residues used for the radiolarian biostratigraphy.13Corg) determinations was ± 0.5‰. The analyses showed good reproducibility of values, with 3% or less fluctuation . All isotopic results are reported in conventional delta (δ) notation, defined as per mill (‰) deviations from the Vienna Peedee Belemnite (VPDB) standard value.Samples for whole-rock geochemical analysis were collected from 45 chert and claystone beds across the ejecta layer. Veins and strongly recrystallized/weathered parts of the samples were avoided to minimize the effects of diagenetic and metamorphic overprinting on the sediment geochemistry. Samples were crushed and fragments were carefully hand-picked to avoid contamination by altered and weathered materials. These hand-picked fragments were then pulverized in an agate mortar for carbon isotope analysis. All glassware used in the analyses was baked for 3 h at 450 °C, and all tools were cleaned with methanol between uses to avoid organic carbon contamination. Approximately 0.8 g of each sample was treated with an excess of 6 M hydrochloric acid for 48 h at 60 °C. Samples were then rinsed five times with ultrapure water (>18 MΩ) and dried. Carbon isotope ratios and carbon and nitrogen contents were measured using an elemental analyser (FlashEA 1112) coupled to a Thermo-Finnigan Delta Plus Advantage isotope ratio mass spectrometer at the Centre for Advanced Marine Core Research, Kochi University, Japan. Isotopic measurements were repeated two times for 19 samples across the claystone (ejecta) layer to check the reproducibility of the results. The carbon isotope ratios were calculated using standard Alanine; the precision of the organic carbon ratio (δ2B4O7) flux at ~1190 °C for 7 min in a Pt crucible. The analytical results were generally within 3% (relative percent difference) of accepted values of reference material JB-2, issued by the Geological Survey of JapanMajor element  contents of the bulk-sediment samples from the cherts and claystones in the Sakahogi section were measured using a Rigaku ZSX Primus II X-ray fluorescence (XRF) spectrometer at the University of Tokyo. After drying the powdered samples at 110 °C for ~12 h, loss on ignition (LOI) was calculated from the weight loss during ignition at 950 °C for over 6 h. Fused glass beads for XRF analysis were made from a mixture of 0.4 g of ignited sample powder and 4 g of lithium tetraborate  have similar values. Consequently, only the per-taxon rate with singletons is discussed.where NThe Triassic bedded cherts in the Inuyama area comprise biogenic silica (radiolarian tests and siliceous sponge spicules) and terrestrial components derived from the continental crust (aeolian dust)22rad and Sisponge, respectively) were then calculated as follows:where PAAS is a shale standard based on the composition of post-Archean average Australian shalesrad and Vsponge are the volumetric compositions of the radiolarians and siliceous sponges based on thin section analysis, respectively. Then, the mass accumulation rate (g cm−2 kyr−1) of radiolarian and sponge silica  was calculated as follows:where Vρ is the bulk density of chert (2.46 g cm−3) or claystone (1.69 g cm−3) and a is the sedimentation rate for chert (0.11–0.16 cm kyr−1) or claystone (0.09 cm kyr−1). The sedimentation rate of the claystone layer was calculated from the measured thickness (3.2 cm), and the time interval of its deposition was estimated using a mean oceanic residence time of Os (~3.5 kyr)where How to cite this article: Onoue, T. et al. Bolide impact triggered the Late Triassic extinction event in equatorial Panthalassa. Sci. Rep.6, 29609; doi: 10.1038/srep29609 (2016).