FOXO transcriptional activity is associated with response to chemoradiation in EAC.

In this study we aimed to investigate signaling pathways that drive therapy resistance in esophageal adenocarcinoma (EAC). Paraffin-embedded material was analyzed in two patient cohorts: (i) 236 EAC patients with a primary tumor biopsy and corresponding post neoadjuvant chemoradiotherapy (nCRT) resection; (ii) 66 EAC patients with resection and corresponding recurrence. Activity of six key cancer-related signaling pathways was inferred using the Bayesian inference method. When assessing pre- and post-nCRT samples, lower FOXO transcriptional activity was observed in poor nCRT responders compared to good nCRT responders (p = 0.0017). This poor responder profile was preserved in recurrences compared to matched resections (p = 0.0007). PI3K pathway activity, inversely linked with FOXO activity, was higher in CRT poor responder cell lines compared to CRT good responders. Poor CRT responder cell lines could be sensitized to CRT using PI3K inhibitors. To conclude, by using a novel method to measure signaling pathway activity on clinically available material, we identified an association of low FOXO transcriptional activity with poor response to nCRT. Targeting this pathway sensitized cells for nCRT, underlining its feasibility to select appropriate targeted therapies.

Clinical value of ctDNA in upper-GI cancers: A systematic review and meta-analysis.

BACKGROUND: The recent expanding technical possibilities to detect tumor derived mutations in blood, so-called circulating tumor DNA (ctDNA), has rapidly increased the interest in liquid biopsies. This review and meta-analysis explores the clinical value of ctDNA in malignancies of the upper gastro-intestinal tract. METHODS: PubMed, Cochrane and Embase databases were searched to identify studies reporting the diagnostic, prognostic or predictive value of ctDNA in patients with esophageal, gastric and pancreatic cancer, until January 2017. The diagnostic accuracy and, using random-effect pair-wise meta-analyses, the prognostic value of ctDNA was assessed. RESULTS: A total of 34 studies met the inclusion criteria. For esophageal and gastric cancer, amplification of oncogenes in blood, such as HER2 and MYC, can be relevant for diagnostic purposes, and to predict treatment response in certain patient subpopulations. Given the limited number of studies assessing the role of ctDNA in esophageal and gastric cancer, the meta-analysis estimated the diagnostic accuracy and predictive value of ctDNA in pancreatic cancer only (n=10). The pooled sensitivity and specificity of ctDNA as a diagnostic tool in pancreatic cancer were 28% and 95%, respectively. Patients with pancreatic cancer and detectable ctDNA demonstrated a worse overall survival compared to patients with undetectable ctDNA (HR 1.92, 95% confidence interval (CI) 1.15-3.22, p=0.01). CONCLUSION: The presence of ctDNA is significantly associated with a poor prognosis in patients with pancreatic cancer. The use of ctDNA in clinical practice is promising, although standardization of sequencing techniques and further development of high-sensitive detection methods is needed.

Discordance in HER2 Status in Gastro-esophageal Adenocarcinomas: A Systematic Review and Meta-analysis.

Trastuzumab combined with chemotherapy is standard of care for HER2 positive advanced gastro-esophageal cancers. The reported prevalence of HER2 discordance between primary tumors and corresponding metastases varies, hampering uniform patient selection for HER2 targeted therapy. This meta-analysis explores the influence of HER2 assessment methods on this discordance and investigates the prevalence of HER2 discordance in gastro-esophageal adenocarcinomas. PubMed, Embase and Cochrane databases were searched until January 2016. Differences in discordance rate between strict and broad(er) definitions of HER2 status were assessed using random-effect pair-wise meta-analysis. Random-effect single-arm meta-analyses were performed to assess HER2 discordance and the prevalence of positive and negative conversion. A significantly lower discordance rate in HER2 status between primary tumors and corresponding metastases was observed using a strict vs. broad definition of HER2 status (RR = 0.58, 95%CI 0.41-0.82), with a pooled discordance rate of 6.2% and 12.2%, respectively. Using the strict definition of HER2 assessment pooled overall discordance was 7% (95%CI 5-10%). The lowest discordance rates between primary tumors and corresponding metastasis are observed when using a strict method of HER2 positivity. Treatment outcomes of different studies will be better comparable if selection of eligible patients for HER2 targeted therapy is based on this strict definition.

Bioefficacy of beta-carotene dissolved in oil studied in children in Indonesia.

BACKGROUND: More information on the bioefficacy of carotenoids in foods ingested by humans is needed. OBJECTIVE: We aimed to measure the time required for isotopic enrichment of beta-carotene and retinol in serum to reach a plateau, the extent of conversion of beta-carotene dissolved in oil with use of beta-carotene and retinol specifically labeled with 10 (13)C atoms, and the intraindividual variation in response. DESIGN: Indonesian children aged 8--11 y (n = 35) consumed 2 capsules/d, 7 d/wk, for < or =10 wk. Each capsule contained 80 microg [12,13,14,15,20,12',13',14',15',20'-(13)C(10)]beta-carotene and 80 microg [8,9,10,11,12,13,14,15,19,20-(13)C(10)]retinyl palmitate. Three blood samples were drawn per child over a period of < or =10 wk. HPLC coupled with atmospheric pressure chemical ionization liquid chromatography-mass spectrometry was used to measure the isotopic enrichment in serum of retinol with [(13)C(5)]retinol and [(13)C(10)]retinol and of beta-carotene with [(13)C(10)]beta-carotene. The beta-carotene in the capsules used had a cis-trans ratio of 3:1. RESULTS: Plateau isotopic enrichment was reached by day 21. The amount of beta-carotene in oil required to form 1 microg retinol was 2.4 microg (95% CI: 2.1, 2.7). The amount of all-trans-beta-carotene required to form 1 microg retinol may be lower. CONCLUSIONS: The efficiency of conversion of this beta-carotene in oil was 27% better than that estimated previously (1.0 microg retinol from 3.3 microg beta-carotene with an unknown cis-trans ratio). The method described can be extended to measure the bioefficacy of carotenoids in foods with high precision, requiring fewer subjects than other methods.

Photoactivation of rhodopsin: interplay between protein and chromophore.

Data in the literature suggest a finely tuned interaction between ligand (11-cis-retinal) and protein (opsin) in order to allow very efficient photoactivation of the ligand and highly vectorial rhodopsin activation with a huge increase in receptor activity. We have further investigated this interaction using ligand homologues, 13C-ligand labelling or 15N-protein labelling, in combination with Fourier transform infrared (FT-IR) and solid-state magic angle spinning (ss-MAS)-NMR spectroscopy. Using 1D rotational resonance (RR) or double-quantum heteronuclear local field (2Q-HLF) ss-MAS-NMR we report the first structure refinement of the rhodopsin chromophore in situ. These measurements yield a specification of the torsional strain in the for isomerization essential C10-C13 segment of the chromophore. This strain is thought to contribute to the high rate and stereospecificity of the photoisomerization reaction. In agreement with previous data, the C10-C13 segment region reaches a relaxed all-trans configuration at the lumirhodopsin photointermediate. MAS-NMR analysis of [15N]lysine-labelled rhodopsin reveals the presence of a 'soft' counterion, requiring intermediate water molecules for stabilization. FT-IR studies on [2H]tyrosine-labelled rhodopsin demonstrate participation of several tyrosin(at)e residues in receptor activation. One of these, probably Tyr268, is already active at the bathorhodopsin stage. Finally, the effect of ligands with single additional methyl substituents in the C10-C12 region has been investigated. They do not affect the general activation pathway, but perturb the activation kinetics of rhodopsin, suggesting steric interference with protein residues. Possible implications of these results for a structural role of water residues will be discussed, as well.

Medical treatment of a central vein suppurative thrombosis with cerebral metastatic abscesses in a burned child.

A 2-year-old girl admitted with third degree burns (35% TBSA) received 7 weeks poly-antibiotic therapy combined with heparin for a severe Methicillin-resistant Staphylococcus aureus sepsis with multiple metastatic abscesses (lung, skin, brain), from a suppurative thrombophlebitis of the right jugularis interna, extended to the axillary and cava superior veins. Surgical treatment was contraindicated by the local extension. The child was discharged without major neurological sequelae 3 months after admission.

A liquid chromatography-mass spectrometry method for the quantification of bioavailability and bioconversion of beta-carotene to retinol in humans.

A method based on high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (APCI LC-MS) was developed for the quantification of the bioavailability of retinyl palmitate and beta-carotene and the bioconversion of beta-carotene to retinol in humans. Following oral administration of [8,9,10,11,12,13,14,15,19,20-13C10]-retinyl palmitate and [12,13,14,15,20,12',13',14',15',20'-13C10]-beta-carotene at physiological doses to children between 8 and 11 years of age, blood samples were drawn and serum was prepared. Retinol and beta-carotene were extracted from 0.2- and 1.0-mL serum samples, respectively, and analyzed using reversed-phase HPLC with a C30 column interfaced to an APCI mass spectrometer. Unlike other LC-MS assays for carotenoids, no additional purification steps were necessary, nor was any derivatization of retinol or beta-carotene required. APCI LC-MS showed a linear detector response for beta-carotene over 4 orders of magnitude. Using selected ion monitoring to record the elution profile of protonated circulating beta-carotene at m/z 537 and [13C10]-beta-carotene at m/z 547, the limit of detection was determined to be 0.5 pmol injected on-column. To assess the ratio of labeled to unlabeled retinol, selected ion monitoring was carried out at m/z 269, 274, and 279. These abundant fragment ions corresponded to the loss of water from the protonated molecule of circulating retinol, [13C5]-retinol (metabolically formed from orally administered [13C10]-beta-carotene), and [13C10]-retinol (formed by hydrolysis of [13C10]-retinyl palmitate). The ratios of labeled to unlabeled retinol and the ratio of labeled to unlabeled beta-carotene were calculated. Combined with standard HPLC measurement of beta-carotene and retinol concentration and a mathematical model, these results showed that this simple LC-MS method can be used to quantify beta-carotene bioavailability and its bioconversion to retinol at physiologically relevant doses.

Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: electronic structure of the retinylidene chromophore in rhodopsin.

11-Z-[8,9,10,11,12,13,14,15,19,20-(13)C10]Retinal prepared by total synthesis is reconstituted with opsin to form rhodopsin in the natural lipid membrane environment. The 13C shifts are assigned with magic angle spinning NMR dipolar correlation spectroscopy in a single experiment and compared with data of singly labeled retinylidene ligands in detergent-solubilized rhodopsin. The use of multispin labeling in combination with 2-D correlation spectroscopy improves the relative accuracy of the shift measurements. We have used the chemical shift data to analyze the electronic structure of the retinylidene ligand at three levels of understanding: (i) by specifying interactions between the 13C-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schiff base in rhodopsin, and (iii) by evaluating the total charge on the carbons of the retinylidene chromophore. In this way it is shown that a conjugation defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative chemical shifts at the odd-numbered carbons (Delta(sigma)odd) of 11-Z-protonated Schiff base models relative to the unprotonated Schiff base can be used to measure the extent of delocalization of positive charge into the polyene. For a series of 11-Z-protonated Schiff base models and rhodopsin, Delta(sigma)odd appears to correlate linearly with the frequency of maximum visible absorption. Since rhodopsin has the largest value of Delta(sigma)odd, the data contribute to existing and converging spectroscopic evidence for a complex counterion stabilizing the protonated Schiff base in the binding pocket.

Chromophore distortions in the bacteriorhodopsin photocycle: evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR.

In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.

Preparation of site-specific isotopically labelled zervamicins, the antibiotic peptaibols produced by Emericellopsis salmosynnemata.

A simple procedure for the preparation of the specifically labelled peptide antibiotic zervamicins IC, IIA and IIB has been developed. The zervamicin molecules are labelled with stable isotopes by culturing the Emericellopsis salmosynnemata on a well-defined synthetic medium containing the highly isotopically enriched amino acid. To obtain the peptide with the specifically and highly enriched amino acid residue, precautions have been taken to prevent any de novo biosynthesis of the particular amino acid from unlabelled precursors. The enrichment of the labelled peptide is determined by mass spectrometric analysis. Following this method we have incorporated [2',4',5',6',7'-2H5]-L-Trp-1, [1'-15N]-L-Trp-1 and [2',3',4',5',6'-2H5]-L-Phl-16 into zervamicins IC, IIA and IIB on the preparative scale and without scrambling of the label. Thus, using the procedures described, isotopically labelled zervamicins can be prepared, allowing them to be studied by solid-state NMR.

Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin.

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [alpha,epsilon-15N2]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [alpha, epsilon-15N2]Lys-rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [alpha, epsilon-15N2]Lys-rhodopsin resonates with an isotropic shift sigmaI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, numax, associated with the pi-pi transition of the polyene chromophore. Experimental modeling of the relation between the numax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center-center distance (deff) between the counterion and the pSB of 0.43 +/- 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.