We investigate effective equations governing the volume expansion of spatially averaged portions of inhomogeneous cosmologies in spacetimes filled with an arbitrary fluid. This work is a follow-up to ...previous studies focused on irrotational dust models (Paper I) and irrotational perfect fluids (Paper II) in flow-orthogonal foliations of spacetime. It complements them by considering arbitrary foliations, arbitrary lapse and shift, and by allowing for a tilted fluid flow with vorticity. As for the first studies, the propagation of the spatial averaging domain is chosen to follow the congruence of the fluid, which avoids unphysical dependencies in the averaged system that is obtained. We present two different averaging schemes and corresponding systems of averaged evolution equations providing generalizations of Papers I and II. The first one retains the averaging operator used in several other generalizations found in the literature. We extensively discuss relations to these formalisms and pinpoint limitations, in particular regarding rest mass conservation on the averaging domain. The alternative averaging scheme that we subsequently introduce follows the spirit of Papers I and II and focuses on the fluid flow and the associated
1
+
3
threading congruence, used jointly with the
3
+
1
foliation that builds the surfaces of averaging. This results in compact averaged equations with a minimal number of cosmological backreaction terms. We highlight that this system becomes especially transparent when applied to a natural class of foliations which have constant fluid proper time slices.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to ...be partly generated by enzymatic cleavage with heparanases, resulting in
-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-
sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.
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In heparin, some 3-
-sulfated sequences do not meet the structural requirements of the ATIII binding pentasaccharide. These "non-conventional" sequences are the object of this study. In a previous ...paper (Mourier P. Heparinase digestion of 3-
-sulfated sequences: selective heparinase II digestion for separation and identification of binding sequences present in ATIII affinity fractions of bovine intestine heparins), we demonstrated that unsaturated 3-
-sulfated disaccharides detected in exhaustive heparin digests were specifically cleaved by heparinase I. Consequently, building blocks analyses of heparins using heparinases I+II+III digestion could be compared with experiments where only heparinase II is used. In these latter conditions of depolymerization, the 3-
-sulfated sequences digested into unsaturated 3-
-sulfated disaccharides with heparinases I+II+III, were heparinase II-resistant on their non-reducing side, resulting in longer new building blocks. These properties were used to study the structural neighborhood of these 3-
-sulfated moieties, which have still-undefined biological functions. In this part, heparinases I+II+III and heparinase II digestions of porcine mucosa, bovine mucosa and bovine lung heparins were compared in six fractions of increasing affinity for ATIII. Tagging of building blocks by reductive amination with sulfanilic acid was used. The distribution of 3-
-sulfated building blocks in the ATIII affinity fractions was used to examine the ATIII binding of these sequences.
Binding to antithrombin-III (ATIII) determines the anticoagulant activity of heparin. The complexes formed between heparin and ATIII result from a specific pentasaccharide sequence containing a 3-
...sulfated glucosamine in medium position. Building block analysis of heparins, following heparinase digestion, is a critical method in quality control that provides a simple structural characterization of a complex product. Hence, in these applications, study of the digestion of 3-
-sulfated moieties merits special attention. With heparinase II, specific inhibition of cleavage of the non-reducing bond of 3-
-sulfated units is observed. This specificity was erroneously generalized to other heparinases when it was observed that in exhaustive digests of heparins with the heparinase mixture, resistant 3-
-sulfated tetrasaccharides were also obtained from the specific ATIII-binding pentasaccharides. In fact, the detection of unsaturated 3-
-sulfated disaccharides in digests of heparin by heparinases I+II+III, resulting from the cleavage of the 3-
sulfated unit by heparinase I in non-conventional sequences, shows that this inhibition has exceptions. Thus, in experiments where heparinase II is selectively applied, these sequences can only be digested into tetra- or hexasaccharides where the 3-
-sulfated glucosamine is shifted on the reducing end. Heparinase I+II+III and heparinase II digests with additional tagging by reductive amination with sulfanilic acid were used to study the structural neighborhood of 3-
-sulfated disaccharides in bovine mucosal heparin fractions with increasing affinity for ATIII. The 3-
-sulfated disaccharides detected in heparinase I+II+III digests turn into numerous specific 3-
-sulfated tetrasaccharides in heparinase II digests. Additionally, ATIII-binding pentasaccharides with an extra 3-
-sulfate at the reducing glucosamine are detected in fractions of highest affinity as heparinase II-resistant hexasaccharides with two consecutive 3-
-sulfated units.
We introduce a generalization of the 4-dimensional averaging window function of Gasperini et al (2010 J. Cosmol. Astropart. Phys. JCAP02(2010)009) that may prove useful for a number of applications. ...The covariant nature of spatial scalar averaging schemes to address the averaging problem in relativistic cosmology is an important property that is implied by construction, but usually remains implicit. We employ here the approach of Gasperini et al for two reasons. First, the formalism and its generalization presented here are manifestly covariant. Second, the formalism is convenient for disentangling the dependencies on foliation, volume measure, and boundaries in the averaged expressions entering in scalar averaging schemes. These properties will prove handy for simplifying expressions, but also for investigating extremal foliations and for comparing averaged properties of different foliations directly. The proposed generalization of the window function allows for choosing the most appropriate averaging scheme for the physical problem at hand, and for distinguishing between the role of the foliation itself and the role of the volume measure in averaged dynamic equations. We also show that one particular window function obtained from this generalized class results in an averaging scheme corresponding to that of a recent investigation by Buchert et al (2018 Class. Quantum Grav. 35 24LT02) and, as a byproduct, we explicitly show that the general equations for backreaction derived therein are covariant.
Heparin-antithrombin interaction is one of the most documented examples of heparin/protein complexes. The specific heparin sequence responsible for the binding corresponds to a pentasaccharide ...sequence with an internal 3-
O
-sulfated glucosamine residue. Moreover, the position of the pentasaccharide along the chain as well as the structure of the neighbor units affects the affinity to antithrombin. The development of separation and purification techniques, in conjunction with physico-chemical approaches (mostly NMR), allowed to characterize several structural variants of antithrombin-binding oligosaccharides, both in the free state and in complex with antithrombin. The article provides an overview of the studies that lead to the elucidation of the mechanism of interaction as well as acquiring new knowledge in heparin biosynthesis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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•An analytical comparison was performed on enoxaparins from three different sources.•‘State of the art’ methodologies included strong anion exchange (SAX) HPLC and NMR.•Disaccharide ...analysis showed distinctive profiles linked to the enoxaparin source.•Chemometrics data analysis confirmed the statistical significance of these results.•We defined a protocol for the analysis of originator and generic enoxaparins.
Low-molecular-weight heparins (LMWHs) are complex anticoagulant drugs, made from heparin porcine mucosa starting material. Enoxaparin sodium manufactured by Sanofi is one of the most widely prescribed LMWHs and has been used since 1993 in the USA. In 2010, US Food and Drug Administration approval for supplying generic enoxaparin was granted to Sandoz and subsequently to Amphastar. Little is known, however, of the differences in composition of these preparations. In this study, samples from several batches of generic enoxaparins were purchased on the US market and analyzed with state of the art methodologies, including disaccharide building blocks quantification, nuclear magnetic resonance (NMR), and a combination of orthogonal separation techniques. Direct high-performance liquid chromatography analysis of the different enoxaparin batches revealed distinct process fingerprints associated with each manufacturer. Disaccharide building block analysis showed differences in the degree of sulfation, the presence of glycoserine derivatives, as well as in proportions of disaccharides. Results were compared by statistical approaches using multivariate analysis with a partial least squares discriminant analysis methodology. The variations were statistically significant and allowed a clear distinction to be made between the enoxaparin batches according to their manufacturer. These results were further confirmed by orthogonal analytical techniques, including NMR, which revealed compositional differences of oligosaccharides both in low- and high-affinity antithrombin fractions of enoxaparin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor ...Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline β-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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•An analytical comparison of generic Teva and originator Sanofi enoxaparins was performed.•CTA-SAX, AS11 chromatography, and nuclear magnetic resonance were used.•Generic enoxaparins ...have distinct antithrombin binding oligosaccharide compositions.•Manufacturing process modifications affect heparin depolymerization selectivity.•Compositional changes in oligosaccharides may affect the generic enoxaparin pharmacology.
Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of ...factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP U.S. Pharmacopeia heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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