Identifying the origin of local flexibility in a carbohydrate polymer

Correlating the structures and properties of a polymer to its monomer sequence is key to understanding how its higher hierarchy structures are formed and how its macroscopic material properties emerge. Carbohydrate polymers, such as cellulose and chitin, are the most abundant materials found in nature whose structures and properties have been characterized only at the submicrometer level. Here, by imaging single-cellulose chains at the nanoscale, we determine the structure and local flexibility of cellulose as a function of its sequence (primary structure) and conformation (secondary structure). Changing the primary structure by chemical substitutions and geometrical variations in the secondary structure allow the chain flexibility to be engineered at the single-linkage level. Tuning local flexibility opens opportunities for the bottom-up design of carbohydrate materials.

Natural polymers adopt a multitude of three-dimensional structures that enable a wide range of functions (1). Polynucleotides store and transfer genetic information; polypeptides function as catalysts and structural materials; and polysaccharides play important roles in cellular structure (2–6), recognition (5), and energy storage (7). The properties of these polymers depend on their structures at various hierarchies: sequence (primary structure), local conformation (secondary structure), and global conformation (tertiary structure).Automated solid-phase techniques provide access to these polymers with full sequence control (8–12). The correlation between the sequence, the higher hierarchy structures, and the resulting properties is relatively well established for polynucleotides (13, 14) and polypeptides (15, 16), while comparatively little is known for polysaccharides (17). Unlike polypeptides and polynucleotides, polysaccharides are based on monosaccharide building blocks that can form multiple linkages with different configurations (e.g., α- or β-linkages) leading to extremely diverse linear or branched polymers. This complexity is exacerbated by the flexibility of polysaccharides that renders structural characterization by ensemble-averaged techniques challenging (17). Imaging single-polysaccharide molecules using atomic force microscopy has revealed the morphology and properties of polysaccharides at mesoscopic, submicrometer scale (18–22). However, imaging at such length scales precludes the observation of individual monosaccharide subunits required to correlate the polysaccharide sequence to its molecular structure and flexibility, the key determinants of its macroscopic functions and properties (23).Imaging polysaccharides at subnanometer resolution by combining scanning tunnelling microscopy (STM) and electrospray ion-beam deposition (ES-IBD) (24, 25) allows for the observation of their monosaccharide subunits to reveal their connectivity (26–28) and conformation space (29). Here, we use this technique to correlate the local flexibility of an oligosaccharide chain to its sequence and conformation, the lowest two structural hierarchies. By examining the local freedom of the chain as a function of its primary and secondary structures, we address how low-hierarchy structural motifs affect local oligosaccharide flexibility—an insight critical to the bottom-up design of carbohydrate materials (30).We elucidate the origin of local flexibility in cellulose, the most abundant polymer in nature, composed of glucose (Glc) units linked by β-1,4–linkages (31–33). Unveiling what affects the flexibility of cellulose chains is important because it gives rise to amorphous domains in cellulose materials (34–37) that change the mechanical performance and the enzyme digestibility of cellulose (38). Cellohexaose, a Glc hexasaccharide (Fig. 1A), was used as a model for a single-cellulose chain as it has been shown to resemble the cellulose polymer behavior (12). Modified analogs prepared by Automated Glycan Assembly (AGA) (11, 12) were designed to manipulate particular intramolecular interactions responsible for cellulose flexibility. Cellohexaose, ionized as a singly deprotonated ion in the gas phase ([M-H]⁻¹) was deposited on a Cu(100) surface held at 120 K by ES-IBD (24) (Materials and Methods). The ions were landed with 5-eV energy, well suited to access diverse conformation states of the molecule without inducing any chemical change in the molecule (29). The resulting cellohexaose observed in various conformation states allowed its mechanical flexibility (defined by the variance in the geometrical bending between two residues) to be quantified for every intermonomer linkage. The observed dependence of local flexibility on the oligosaccharide sequence and conformation thus exemplifies how primary and secondary structures tune the local mechanical flexibility of a carbohydrate polymer.Open in a separate windowFig. 1.STM images of cellohexaose (AAAAAA) and its analogs (AXAAXA). Structures and STM images of cellohexaose (A) and its substituted analogs (B–E). Cellohexaose contains six Glcs (labeled as A; colored black) linked via β-1,4–glycosidic bonds. The cellohexaose analogs contain two substituted Glcs, as the second and the fifth residues from the nonreducing end, that have a single methoxy (–OCH₃) at C(3) (labeled as B; colored red), two methoxy groups at C(3) and C(6) (labeled as C; colored green), a single carboxymethoxy (–OCH₂COOH) at C(3) (labeled as D; colored blue), and a single fluorine (–F) at C(3) (labeled as F; colored purple).The effect of the primary structure on the chain flexibility was explored using sequence-defined cellohexaose analogs (Fig. 1). Cellohexaose, AAAAAA (Fig. 1A), was compared with its substituted analogs, ABAABA, ACAACA, ADAADA, and AFAAFA (written from the nonreducing end) (Fig. 1 B–E), where A is Glc, B is Glc methylated at OH(3), C is Glc methylated at OH(3) and OH(6), D is Glc carboxymethylated at OH(3), and F is Glc deoxyfluorinated at C(3). These substitutions are designed to alter the intramolecular hydrogen bonding between the first and the second as well as between the fourth and fifth Glc units (Fig. 1). These functional groups also affect the local steric environment (i.e., the bulky carboxymethyl group) (Fig. 1D) and the local electronic properties (i.e., the electronegative fluorine group) (Fig. 1E). When compared with the unsubstituted parent cellohexaose, these modified cellohexaoses exhibit different aggregation behavior and are more water soluble (12).All cellohexaose derivatives adsorbed on the surface were imaged with STM at 11 K (Fig. 1). The oligosaccharides were deposited as singly deprotonated species and were computed to adsorb on the surface via a single covalent RO–Cu bond, except for ADAADA which was deposited as doubly deprotonated species and was computed to adsorb on the surface via two covalent RCOO–Cu bonds (R = sugar chain). All cellohexaoses appear as chains containing six protrusions corresponding to the six constituent Glcs. The unmodified cellohexaose chains (Fig. 1A) mainly adopt a straight geometry, while the substituted cellohexaoses (Fig. 1 B–E) adopt both straight- and bent-chain geometries. Chemical substitution thus increases the geometrical freedom of the cellulose chain, consistent with the reported macroscopic properties (12).Large-chain bending between neighboring Glc units is observed exclusively for the substituted cellohexaose (Fig. 1). The large, localized bending reveals the substitution site and allows for the nonreducing and the reducing ends of the chain to be identified. These chains are understood to bend along the surface plane via the glycosidic linkage without significant tilting of the pyranose ring that remains parallel to the surface (illustrated in SI Appendix, Fig. S1), as indicated by the ∼2.0-Å height of every Glc (29).The bending angle measured for AA and AX linkages (Fig. 2; Materials and Methods has analysis details) shows that, while both AA and AX prefer the straight, unbent geometry, AX displays a greater variation of bending angles than AA. AX angular distribution is consistently ∼10° wider than that for AA, indicating that AX has a greater conformational freedom than AA. This increased bending flexibility results from the absence of the intramolecular hydrogen bonding between OH(3) and O(5) of the neighboring residue. Methylation of OH(6), in addition to methylation of OH(3), results in similar flexibility (Fig. 2 B and C), suggesting the greater importance of OH(3) in determining the bending flexibility. Steric effects were found to be negligible since AD displayed similar flexibility to other less bulky AX linkages.Open in a separate windowFig. 2.Bending flexibility of AA linkage and substituted AX linkages. Chain bending (Fig. 1) is quantified as an angle formed between two neighboring Glcs (Materials and Methods). The results are given in A for AA, in B for AB, in C for AC, in D for AD, and in E for AF, showing that AX (where X = B, C, D, F) has a higher conformational freedom than AA. The angle distributions (bin size: 10°) are fitted with a Gaussian (solid line) shown with its half-width half-maximum. The computed potential energy curves are shown with the half-width at 0.4 eV and fitted with a parabola to estimate its stiffness (k; in millielectronvolts per degree²).Density functional theory (DFT) calculations support the observations, showing that substitution of OH(3) decreases the linkage stiffness by up to ∼40% (Fig. 2). Replacing OH(3) with other functional groups weakens the interglucose interactions by replacing the OH(3)··O(5) hydrogen bond with weak Van der Waals interactions. The similar flexibility between AB and AC linkages is attributed to the similar strength of the interglucose OH(2)··OH(6) hydrogen bond in AB (Fig. 2B) and the OH(2)··OMe(6) hydrogen bond in AC (Fig. 2C). The negligible steric effect in AD is attributed to the positional and rotational freedom of the bulky moiety that prevents any “steric clashes” and diminishes the contribution of steric repulsion in the potential energy curve. Comparing the potential landscape in the gas phase and on the surface shows that the stiffness of the adsorbed cellohexaoses is primarily dictated by their intramolecular interactions instead of molecule–surface interactions (SI Appendix, Fig. S2). Primary structure alteration by chemical substitution modifies the interglucose hydrogen bonds and enables chain flexibility to be locally engineered at the single-linkage level.We subsequently investigate how molecular conformation (secondary structure) affects the local bending flexibility. We define the local secondary structure as the geometry formed between two Glcs, here exemplified by the local twisting of the chain (Fig. 3). The global secondary structure is defined as the overall geometry formed by all Glcs in the chain, here exemplified by the linear and cyclic topologies of the chain (Fig. 4).Open in a separate windowFig. 3.Bending flexibility of untwisted and twisted AA linkages. (A) STM image of a cellohexaose containing two types of AA linkages: untwisted (HH and VV) and twisted (HV and VH; from the nonreducing end). The measured bending angles and the computed potential curve are given in B for HH, in C for HV, and in D for VV, showing that the twisted linkage (HV) is more flexible than the untwisted ones (HH and VV). In the molecular structures, interunit hydrogen bonds are given as dotted blue lines, and the pyranose rings are colored red for the horizontal ring (H) and green for vertical (V). The angle distributions (bin size: 10°) are fitted with a Gaussian distribution (solid line) labeled with its peak and half-width half-maximum. The computed potential curves are labeled with its half-width at 0.4 eV and fitted with a parabola to estimate its stiffness (k; in millielectronvolts per degree²).Open in a separate windowFig. 4.Bending flexibility of AA linkage in linear (LIN) and cyclic (CYC) chains. STM image, measured bending angle distribution, and computed potential of AA linkage are given in A for a linear cellohexaose conformer and in B for a cyclic cellohexaose conformer, showing that chain flexibility is reduced in conformations with cyclic topology. The same data are given in C for α-cyclodextrin that is locked in a conformation with cyclic topology. The measured angles (bin size: 10°) are each fitted with a Gaussian distribution (solid line) labeled with its peak and half-width half-maximum. The computed potentials are each labeled with its half-width at 0.4 eV and fitted with a parabola to estimate its stiffness (k; in millielectronvolts per degree²).The effect of local secondary structure on chain flexibility is exemplified by the bending flexibility of twisted and untwisted linkages in a cellohexaose chain (Fig. 3A). The untwisted and twisted linkages are present due to the Glc units observed in two geometries, H or V (Fig. 3), distinguished by their heights (h). H (h ∼ 2.0 Å) is a Glc with its pyranose ring parallel to the surface, while V (h ∼ 2.5 Å) has its ring perpendicular to the surface (29). These lead to HH and VV as untwisted linkages and HV and VH (written from nonreducing end) as twisted linkages.The twisted linkage is more flexible than the untwisted one, as shown by the unimodal bending angles for the untwisted linkage (HH and VV in Fig. 3 B and D, respectively) and the multimodal distribution for the twisted linkage (HV in Fig. 3C). DFT calculations attribute the increased bending flexibility to the reduction of accessible interunit hydrogen bonds from two to one. Linkage twisting increases the distance between the hydrogen-bonded pair, which weakens the interaction between Glc units and increases the flexibility at the twisting point. The increase in local chain flexibility conferred by chain twisting shows how local secondary structures affect chain flexibility.The effect of the global secondary structure on the local chain flexibility was examined by comparing the local bending flexibility of cellohexaose chains possessing different topologies. Cellohexaose can adopt either linear (Figs. 3A and ​and4A)4A) or cyclic topology (Fig. 4B), the latter characterized by the presence of a circular, head-to-tail hydrogen bond network (29). The cyclic conformation of cellohexaose is enabled by the head-to-tail chain folding from the 60° chain bending of the VV linkage. The VV segment in the cyclic chain is stiffer than in the linear chain since the bending angle distribution for the cyclic chain is 6° narrower than that for the linear chain. The observation is corroborated by DFT calculations that show that the VV linkage in the cyclic chain is about three times stiffer than that in the linear chain.To characterize the degree of chain stiffening due to the linear-to-cyclic chain folding, we compare the flexibility of the cyclic cellohexaose and α-cyclodextrin (an α-1,4–linked hexaglucose covalently locked in cyclic conformation). The α-cyclodextrin provides the referential local flexibility for a cyclic oligosaccharide chain. Strikingly, the local flexibility in α-cyclodextrin was found to be identical to that in the cyclic cellohexaose, as evidenced by the similar width of the bending angle distribution and the computed potentials (Fig. 4 B and C). The similar stiffness indicates that the folding-induced stiffening in cellohexaose is a general topological effect unaffected by the type of the interactions that give the cyclic conformation (noncovalent hydrogen bond in cellohexaose vs. covalent bond in α-cyclodextrin). The folding-induced stiffening is the result of the creation of a circular spring network that restricts the motion of Glc units and reduces their conformational freedom. The folding-induced stiffening reported here provides a mechanism by which carbohydrate structures can be made rigid. The dependence of the local chain flexibility on the chain topology shows how global secondary structures modify local flexibility.Using cellulose as an example, we have quantified the local flexibility of a carbohydrate polymer and identified structural factors that determine its flexibility. Modification of the carbohydrate primary structure by chemical substitution alters the mechanical flexibility at the single-linkage level. Changing secondary structure by chain twisting and folding provides additional means to modify the flexibility of each linkage. Control of these structural variables enables tuning of polysaccharide flexibility at every linkage as a basis for designing and engineering carbohydrate materials (30). Our general approach to identify structural factors affecting the flexibility of a specific molecular degrees of freedom in a supramolecular system should aid the design of materials and molecular machines (39) and the understanding of biomolecular dynamics.     

Prolonged-release oxycodone/naloxone in opioid-naïve patients – subgroup analysis of a prospective observational study

Objective: Prolonged-release oxycodone/naloxone (OXN PR) showed improved gastrointestinal tolerability and equivalent analgesic efficacy compared to oxycodone alone in patients with non-cancer pain or cancer pain. This is the first dataset to demonstrate its effectiveness and safety compared to other strong opioids in opioid-naïve patients.

Methods: This is a subgroup analysis of a 4- to 6-week multicenter, observational study. A total of 162 opioid-naïve patients with moderate-to-severe pain of varying etiologies received either OXN PR or other strong opioids (control group). Documented parameters include pain relief (numeric rating scale), bowel function (Bowel Function Index [BFI]), pain-related functional impairment (Brief Pain Inventory Short Form), quality of life (QoL; EuroQol EQ-5D-3L) and a global therapy assessment.

Results: OXN group patients experienced a substantial clinically important reduction in mean pain intensity of 51.4%, compared to a 28.6% reduction in control patients. Although the BFI remained in the reference range in both groups, there was a difference between BFI changes during treatment in favor of OXN PR. The superior effectiveness of OXN PR was paralleled by greater improvements of pain interference and QoL and fewer adverse drug reactions compared to other strong opioids.

Conclusion: The favorable outcomes under real-life conditions suggest that OXN PR provides a valuable option for treatment of moderate-to-severe pain without using weak opioids first.     

Placental pathologies on fetal MRI are associated with high impairment rates: a prospective long-term outcome study

Objective: Placental anomalies visualized at midgestation by MRI are shown to be related to pregnancy outcome. We performed a prospective cohort study to investigate the influence of placental pathologies diagnosed with fetal MRI on long-term neurodevelopmental outcome.

Methods: In our hospital-based, cross-sectional study, all fetal MRI examinations of pregnancies with vascular placental pathology (i.e. infarction with/without hemorrhage, subchorionic thrombi/hemorrhages, intervillous thrombi/hemorrhages or retroplacental hematoma) between 2003 and 2007 were included. The extent of the pathology was expressed as the percentage of abnormality related to the whole placental volume. Pathohistological reports were correlated to MRI findings. Infants were prospectively investigated using Bayley developmental scales at the age of 2–3.5 years. Impairment was categorized as a Bayley scale two SDs below normal (<85 points).

Results: There were 31 singletons and 25 offspring of multiple pregnancies included in the analyses. Impairment rates were 32.2% in singletons and 32.0% in multiple births. No correlation between neuro/motordevelopmental outcome at 2–3.5 years and the type, extent or gestational week at the time of diagnoses of placental vascular pathologies was found.

Conclusion: The long-term outcome of children with vascular placental pathologies on fetal MRI was associated with a high impairment rate after 2–3.5 years, both on motor- and neurodevelopmental Bayley scales. Neurological impairment did not correlate with the extent of placental involvement, intrauterine growth restriction, gestational age at birth or multiple state.     

Man vs machine in emergency medicine – a study on the effects of manual and automatic vital sign documentation on data quality and perceived workload,using observational paired sample data and questionnaires

Background

Emergency medicine is characterized by a high patient flow where timely decisions are essential. Clinical decision support systems have the potential to assist in such decisions but will be dependent on the data quality in electronic health records which often is inadequate. This study explores the effect of automated documentation of vital signs on data quality and workload.

Methods

An observational study of 200 vital sign measurements was performed to evaluate the effects of manual vs automatic documentation on data quality. Data collection using questionnaires was performed to compare the workload on wards using manual or automatic documentation.

Results

In the automated documentation time to documentation was reduced by 6.1?min (0.6?min vs 7.7?min, p?<? 0.05) and completeness increased (98% vs 95%, p?<? 0.05). Regarding workflow temporal demands were lower in the automatic documentation workflow compared to the manual group (50 vs 23, p?<? 0.05). The same was true for frustration level (64 vs 33, p?<? 0.05). The experienced reduction in temporal demands was in line with the anticipated, whereas the experienced reduction in frustration was lower than the anticipated (27 vs 54, p?<?0.05).

Discussion

The study shows that automatic documentation will improve the currency and the completeness of vital sign data in the Electronic Health Record while reducing workload regarding temporal demands and experienced frustration. The study also shows that these findings are in line with staff anticipations but indicates that the anticipations on the reduction of frustration may be exaggerated among the staff. The open-ended answers indicate that frustration focus will change from double documentation of vital signs to technical aspects of the automatic documentation system.

     

Incidental findings in multislice computed tomography prior to transcatheter aortic valve implantation: frequency,clinical relevance and outcome

Multislice computed tomography (MSCT) has emerged as the mainstay in patients planned for transcatheter aortic valve implantation (TAVI). Incidental findings (IF) in MSCT are common. However, the exact incidence, clinical relevance and further consequences of IF are unclear and it is controversial whether IF adversely affect patients’ outcome. We analyzed MSCT data of 1050 patients screened for TAVI between January 2011 and December 2014. Median follow-up of patients was 20 months. In total, 3194 IF were identified, which were classified into clinically non-relevant IF (2872, 90%) and clinically relevant IF (322, 10%). In 25% of patients (258/1050) at least one clinically relevant IF was present. Age (80?±?7 vs. 80?±?7 years; p?=?0.198) and EuroSCORE II (3.6% [2.1–5.7] vs. 3.6% [2.1–5.9]; p?=?0.874) was similar between patients with and without a clinically relevant IF. TAVI was performed less frequently in patients with a clinically relevant IF (76% vs. 85%; p?<?0.001), with more patients receiving surgical aortic valve replacement in that group (14% vs. 11%; p?=?0.042), possibly due to the high rate of incidental aneurysms of the ascending aorta (n?=?48). If TAVI was performed mortality did not differ (30-days: 4% vs. 3%; p?=?0.339, 1-year: 11% vs. 14%; p?=?0.226) between patients with and without a clinically relevant IF. Our study is the largest study to analyze prevalence, clinical relevance and therapeutic consequences of IF during screening for TAVI. IF in pre-procedural MSCT are common and clinically relevant in one-quarter of patients. However, these findings had no impact on overall mortality.     

PBPK modeling of irbesartan: incorporation of hepatic uptake

Physiological based pharmacokinetic (PBPK) modeling is now commonly used in drug development to integrate human or animal physiological data in order to predict pharmacokinetic profiles. The aim of this work was to construct and refine a PBPK model of irbesartan taking into account its active uptake via OATP1B1/B3 in order to predict more accurately its pharmacokinetic profile using Simcyp^®. The activity and expression of the human hepatocyte transporters OATP1B1 and OATP1B3 were studied. The relative activity factors (RAFs) for OATP1B1 and OATP1B3 transporters were calculated from intrinsic clearances obtained by concentration dependent uptake experiments in human hepatocytes and HEK overexpressing cells: RAF_1B1 using estrone‐3‐sulfate and pitavastatine clearances, and RAF_1B3 using cholecystokinine octapeptide (CCK‐8) clearances. The relative expression factor (REF) was calculated by comparing immunoblotting of hepatocytes (REF_HH) or tissues (REF_tissue) with those of overexpressing HEK cells for each transporter. These scaling factors were applied in a PBPK model of irbesartan using the Simcyp® simulator. Pharmacokinetic simulation using REF_HH (1.82 for OATP1B1, 8.03 for OATP1B3) as an extrapolation factor was the closest to the human clinical pharmacokinetic profile of irbesartan. These investigations show the importance of integrating the contribution of the active uptake of a drug in the liver to improve PBPK modeling. Copyright © 2015 John Wiley & Sons, Ltd.     

Hepatitis B reactivation during ruxolitinib treatment

Annals of Hematology -