Critical Evaluation of Strategies for the Production of Blood Coagulation Factors in Plant-Based Systems

The use of plants as production platforms for pharmaceutical proteins has been on the rise for the past two decades. The first marketed plant-made pharmaceutical, taliglucerase alfa against Gaucher’s disease produced in carrot cells by Pfizer/Protalix Biotherapeutics, was approved by the US Food and Drug Administration (FDA) in 2012. The advantages of plant systems are low cost and highly scalable biomass production compared to the fermentation systems, safety compared with other expression systems, as plant-based systems do not produce endotoxins, and the ability to perform complex eukaryotic post-translational modifications, e.g., N-glycosylation that can be further engineered to achieve humanized N-glycan structures. Although bleeding disorders affect only a small portion of the world population, costs of clotting factor concentrates impose a high financial burden on patients and healthcare systems. The majority of patients, ∼75% in the case of hemophilia, have no access to an adequate treatment. The necessity of large-scale and less expensive production of human blood coagulation factors, particularly factors associated with rare bleeding disorders, may be an important area for plant-based systems, as coagulation factors do not fit into the industry-favored production models. In this review, we explore previous studies on recombinant production of coagulation Factor II, VIII, IX, and XIII in different plant species. Production of bioactive FII and FIX in plants was not achieved yet due to complex post-translational modifications, including vitamin K-dependent γ-carboxylation and propeptide removal. Although plant-made FVIII and FXIII showed specific activities, there are no follow-up studies like pre-clinical/clinical trials. Significant progress has been achieved in oral delivery of bioencapsulated FVIII and FIX to induce immune tolerance in murine models of hemophilia A and B, resp. Potential strategies to overcome bottlenecks in the production systems are also addressed in this review

Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples

http://ijs.sgmjournals.org/A taxonomic study was conducted in order to clarify the relationships of two bacterial populations belonging to the genus Weissella. A total of 39 strains originating mainly from Malaysian foods (22 strains) and clinical samples from humans (9 strains) and animals (6 strains) were analysed using a polyphasic taxonomic approach. The methods included classical phenotyping, whole-cell protein electrophoresis, 16S and 23S rDNA RFLP (ribotyping), the determination of 16S rDNA sequence homologies, and DNA-DNA reassociation levels. Based on the results, the strains were considered to represent two different species, Weissella confusa and a novel Weissella species, for which we propose the name Weissella cibaria sp. nov. W. confusa possessed the highest 16S rDNA sequence similarity to W. cibaria sp. nov. but the DNA-DNA reassociation experiment showed hybridisation levels below 49% between the strains studied. The numerical analyses of W. confusa and W. cibaria sp. nov. strains did not reveal any specific clustering with respect to the origin of the strains. Based on whole cell protein electrophoresis, ClaI and HindIII ribotyping patterns, food and clinical isolates were randomly located in the two species-specific clusters obtained

Characterization of Leuconostoc gasicomitatum sp. nov., Associated with Spoiled Raw Tomato-Marinated Broiler Meat Strips Packaged under Modified-Atmosphere Conditions

http://aem.asm.org/Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomatomarinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called “protein swell.” This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species

Characterization of Leuconostoc gasicomitatum sp. nov., Associated with Spoiled Raw Tomato-Marinated Broiler Meat Strips Packaged under Modified-Atmosphere Conditions

http://aem.asm.org/Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomatomarinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called “protein swell.” This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species

Evaluating the analytical performance of four new coagulation assays for the measurement of fibrinogen, D-dimer and thrombin time

L

The endogenous thrombin potential in patients with left ventricular assist device or heart transplant

BackgroundThe Heartmate 3 (HM 3) is a left ventricular assist device featuring less shear stress, milder acquired von Willebrand syndrome, and fewer bleeding incidences than its predecessor the Heartmate II (HM II). The novel surface coating of the HM 3 suggests less contact activation of plasmatic coagulation. We hypothesized that patients with HM 3 exhibit fewer aberrations in their thrombin potential than patients with HM II. We compared these results with the thrombin potential of patients with heart transplantation (HTX).MethodsThrombin generation in plasma samples of patients with HM II (n = 16), HM 3 (n = 20), and HTX (n = 13) was analyzed 3 days after implantation/transplantation and after long-term support (3–24 months) with HM II (n = 16) or HM 3 (n = 12) using calibrated automated thrombography. Heparin in postoperative samples was antagonized with polybrene.ResultsThree days postoperatively HM II patients exhibited a lower endogenous thrombin potential (ETP) than HM 3 and HTX patients (HM II: 947 ± 291 nM*min; HM 3: 1231 ± 176 nM*min; HTX: 1376 ± 162 nM*min, p < 0.001) and a lower velocity index of thrombin generation (HM II: 18.74 ± 10.90 nM/min; HM 3: 32.41 ± 9.51 nM/min; HTX: 37.65 ± 9.41 nM/min, p < 0.01). Subtle differences in the thrombin generation profiles remained in HM II and HM 3 patients under long-term support (Velocity Index: HM II: 38.70 ± 28.46 nM/min; HM 3: 73.32 ± 32.83 nM/min, p < 0.05). Prothrombin fragments 1 + 2 were higher in HM II than in HM 3 patients (HM II: 377.7 ± 208.4 pM; HM 3: 202.1 ± 87.7 pM, p < 0.05) and correlated inversely with the ETP (r = −0.584, p < 0.05).ConclusionWe observed a more aberrant thrombin generation in HM II than in HM 3 despite comparable anticoagulation and routine parameters. A trend toward lower values was still observable in HM 3 compared to HTX patients. Calibrated automated thrombography may be a good tool to monitor the coagulation state of these patients and guide anticoagulation in the future

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements