Basement membranes (BMs) are specialized extracellular scaffolds that influence behaviors of cells in epithelial, endothelial, muscle, nervous, and fat tissues. Throughout development and in response to injury or disease, BMs are fine-tuned with specific protein compositions, ultrastructure, and localization. These features are modulated through implements of the BM toolkit that is comprised of collagen IV, laminin, perlecan, and nidogen. Two additional proteins, peroxidasin and Goodpasture antigen-binding protein (GPBP), have recently emerged as potential members of the toolkit. In the present study, we sought to determine whether peroxidasin and GPBP undergo dynamic regulation in the assembly of uterine tissue BMs in early pregnancy as a tractable model for dynamic adult BMs. We explored these proteins in the context of collagen IV and laminin that are known to extensively change for decidualization. Electron microscopic analyses revealed: 1) a smooth continuous layer of BM in between the epithelial and stromal layers of the preimplantation endometrium; and 2) interrupted, uneven, and progressively thickened BM within the pericellular space of the postimplantation decidua. Quantification of mRNA levels by qPCR showed changes in expression levels that were complemented by immunofluorescence localization of peroxidasin, GPBP, collagen IV, and laminin. Novel BM-associated and subcellular spatiotemporal localization patterns of the four components suggest both collective pericellular functions and distinct functions in the uterus during reprogramming for embryo implantation.
Basement membranes (BMs) are specialized extracellular scaffolds that provide architecture and modulate cell behaviors in tissues, such as fat, muscle, endothelium, endometrium, and decidua. Properties of BMs are maintained in homeostasis for most adult tissues. However, BM ultrastructure, composition, and localization are rapidly altered in select uterine tissues that are reprogrammed during pregnancy to enable early maternal-embryo interactions. Here, our data exhibit both static and dynamic BMs that were tracked in mouse uterine tissues during pre-, peri-, and postimplantation periods of pregnancy. The data exhibit spatial-temporal patterns of BM property regulation that coincide with the progression of adapted physiology. Further interpretation and discussion of these data in this article are described in the associated research article titled, “Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming” (C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, 2016) [1].
Orally delivered drugs offer significant benefits in the fight against viral infections, and cost-effective production is critical to their impact on pandemic response in low- and middle-income countries. One example, molnupiravir, a COVID-19 therapy developed by Emory, Ridgeback, and Merck & Co., had potential to benefit from significant cost of goods (COGs) reductions for its active pharmaceutical ingredient (API), including starting materials. A holistic approach to identifying, developing, and evaluating optimized synthetic routes, which includes detailed COGs modeling, provides a rapid means to increase the availability, uptake and application of molnupiravir and other antivirals in global markets. Identification and development of alternate processes for the synthesis of molnupiravir has been conducted by the Medicines for All Institute at Virginia Commonwealth University (M4ALL) and the Green and Turner Labs at the University of Manchester. Both groups developed innovative processes based on synthetic route design and biocatalysis aimed at lowering costs and improving global access. The authors then performed COGs modeling to assess cost saving opportunities. This included a focus on manufacturing environments and facilities amenable to global public health and the identification of key parameters using sensitivity analyses. While all of the evaluated routes provide efficiency benefits, the best options yielded 3-6 fold API COGs reductions leading to treatment COGs as low as <$3/regimen. Additionally, key starting materials and cost drivers were quantified to evaluate the robustness of the savings. Finally, COGs models can continue to inform the focus of future development efforts on the most promising routes for additional cost savings. While the full price of a treatment course includes other factors, these alternative API synthetic approaches have significant potential to help facilitate broader access in low- and middle-income countries. As other promising therapeutics are developed, a similar process could enable rapid cost reductions while enhancing global access.
INTRODUCTIONBovine heparins are currently under consideration for the re‐introduction in the US. Protamine sulfate is commonly used to neutralize heparin. Currently, porcine heparin is used for surgical and interventional purposes. The USP Potency of the currently available bovine heparins is lower than that of porcine heparins. This study investigated the differential neutralization of bovine and porcine heparins by protamine sulfate using various laboratory assays.MATERIALS & METHODS8 bovine heparins were obtained from Kin Master, Brazil in powder form with 140–150 U/mg potency. The porcine heparin was obtained from Medefil Inc., U.S.A. in powder form with potency of 190 U/mg. Each of the bovine heparins was supplemented to blood bank plasma at concentrations of 0–10 mg/ml. The effect of protamine sulfate at a fixed concentration of 10 mg/ml was measured using aPTT, thrombin time, amidolytic anti‐Xa and anti‐IIa assays. Saline supplemented heparinized plasmas were used as control. Additionally, the neutralization of the anti‐coagulant effects of a fixed concentration of pooled bovine heparin at 10 mg/ml by protamine sulfate was also studied at a fixed concentration of 10 mg/ml.RESULTSAll 8 bovine heparins produced a concentration dependent prolongation in aPTT and thrombin time assays. Although porcine heparin exhibited stronger anticoagulant effects in these assays. The protamine neutralization profile of both bovine and porcine heparins was similar. Protamine at concentration of 5 mg/ml completely neutralized the equigravimetric amounts of heparins. However, protamine was not effective in neutralizing the higher concentration of these agents. In the amidolytic anti‐Xa assay both heparins produced concentration dependent inhibitory effects, however porcine heparin was stronger. In this assay, protamine differentially neutralized the bovine heparin in comparison to porcine heparin. At 5 mg/ml almost complete neutralization was noted. In the anti‐IIa aminolytic assay, bovine heparins were weaker than porcine heparin. However, protamine sulfate completely neutralized both bovine and porcine heparins. In the whole blood ACT assay at 10 mg/ml, porcine heparin produced stronger anti‐coagulant effect (380 ± 21sec.) in comparison to bovine heparin pool (304 ± 17 sec.). Protamine sulfate at 10 mg/ml, markedly neutralized the anti‐coagulant effects of both heparins. The relative neutralization was comparable between the bovine and porcine heparins.CONCLUSIONSThese studies demonstrate that in the whole blood and plasma based clotting assays the protamine neutralization profile of porcine and bovine heparin is similar when compared at equigravimetric concentrations. However, in the anti‐Xa assays, differences are noted between the two heparins. This may be due to the differential compositions of these heparins at the lower molecular weight component.
IntroductionHeparin is the most widely used anticoagulant drug for medical, surgical and interventional usage. The currently used heparin is primarily obtained from porcine intestinal mucosa. More recently, the reintroduction of bovine heparin is being considered for similar indications. The bovine heparins are mostly obtained from mucosal tissues.AimThe purpose of this study is to cross‐reference the anticoagulant and USP potency of the currently developed bovine mucosal heparins.Materials & MethodsSeveral batches (10–20) of bovine heparins were obtained from two separate manufacturers (A & B) in powdered form. Porcine mucosal heparin was obtained from Medefil Inc. Glendale Heights, IL. USP heparin reference standard (LOT# FOI187) was obtained from US Pharmacopeia Bethesda, MD. Pooled blood bank plasma was purchased from the blood bank at Loyola University Medical Center. Biophen Heparin anti‐Xa and anti‐IIa assays (Aniara, Mason, OH) were used to measure the potency of various heparins against the USP standard. Purified antithrombin supplemented system was used to study the antiprotease activities of these heparins in terms of IC50. For anticoagulant assays, the Triniclot aPTT reagent (Tcoag, Wicklow, Ireland) was used.ResultsIn comparison to the porcine mucosal heparin, the bovine heparins from both sources exhibited considerably lower USP referenced anti‐Xa and anti‐IIa activites. Heparins from manufacturer A exhibited potencies of 145 ± 3 anti‐Xa units/mg and 139 ± 7 anti‐IIa units/mg with an anti‐Xa/anti‐IIa ratio of 1.05. In the case of manufacturer B, heparins exhibited potencies of 141 ± 11 anti‐Xa units/mg and 142 ± 12 anti‐IIa units/mg. Potencies determined by aPTT clotting assay were found to be higher for heparins from both manufacturers (A: 149 ± 4 U/mg; B:(154 ± 13 U/mg) compared to those determined by amidolytic assay. The products from manufacturer B showed wider lot‐to‐lot variation in potency. In the purified antithrombin systems the IC50 of bovine heparins (3.7 ± 0.2 ug/ml for Xa and 0.17 ± 0.1 ug/ml for IIa) were much higher in comparison to the IC50 values of porcine heparin (1.23 ug/ml for Xa and 0.16 ug/ml for IIa).ConclusionsThese studies clearly demonstrate that the currently available USP standard can be used to cross reference the potency of bovine mucosal heparins from various vendors. Furthermore, the bovine heparins exhibit lower potency in comparison to porcine mucosal heparin in both the amidolytic and clot‐based assays. The potency calculated using the plasma‐based clotting assays, such as aPTT, provides higher potency for the bovine heparins in comparison to the amidolytic methods. The marked differences in the IC50 of the Xa inhibition clearly indicates that the bovine heparins differ considerably in the lower molecular weight range in terms of antithrombin binding components, whereas the higher molecular weight components with antithrombin affinity may be similar.
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