Characterization of IXINITY (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslationalmodifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band onCoomassie-stained gels; activity assayswere normal and showed97% -carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX

Characterization of IXINITY (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslationalmodifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band onCoomassie-stained gels; activity assayswere normal and showed97% -carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX

Characterization of IXINITY" (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97% -carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX

Characterization of IXINITY (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslationalmodifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band onCoomassie-stained gels; activity assayswere normal and showed97% -carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX

Multiproxy paleodietary reconstruction using stable isotopes and starch analysis: the case of the archaeological site of Playa del Mango, Granma, Cuba

Paleoethnobotanical and stable isotope studies have demonstrated that the indigenous groups that populated the Antilles, traditionally understood as dependent exclusively on wild resources, cultivated and consumed both C3 and C4 plants even before the arrival of the ceramic-bearing Arawak groups. However, the relative importance of cultigens and the differential use of plants, especially maize, between populations and individuals remains un-known. In this paper we combined the analysis of stable isotopes (delta 15N, delta 13Cco, delta 13Cen, delta 13Cap, delta 34S) of 27 in-dividuals from the archaeological site of Playa del Mango, Cuba with the identification of starch grains in dental calculus. The stable isotope results indicate that the sampled population had a 70:30 C3/C4 diet, where at least 65 % was based on C3 protein. Starches from C3 (e.g., Marantaceae, Ipomoea batatas) and C4 plants (Zea mays) were found in similar proportions (50:50). These results support that the lack or abundance of starch grains cannot be used to infer directly the frequency at which C3 and C4 plants were consumed within a small popu-lation. Statistically significant differences between females and males in the carbon isotope composition of diet, and its energy portion, suggests a differential consumption of plants by sex. Playa del Mango individual diets were statistically different from those of coeval sites, supporting our previous findings that groups with different dietary traditions concurrently inhabited Cuba in precolonial times. The study demonstrates the power of combined use of stable isotope models, and starch analysis, to provide a more nuanced reconstruction of dietary practices in past human populations.Archaeological science

Operation 3D Printers Renishaw AM400 for 3D Printing by Method SLM

BYRTUS, R. Obsluha 3D tiskárny Renishaw AM400 pro 3D tisk metodou SLM: Bakalářská práce. Ostrava: VŠB – Technická univerzita Ostrava, Fakulta strojní, Katedra obrábění, montáže a strojírenské metrologie, 2017 47s. Vedoucí práce: Ing. Marek Pagáč, Ph.D. Hlavní náplň bakalářské práce spočívala v provedení rešerše všech dostupných metod aditivní výroby a vytvoření uceleného postupu procesu stavby metodou SLM na zařízení Renishaw AM400 za použití korozivzdorného práškového kovu AISI 316L. Teoretická část se zabývala teorii 3D tisku, představením jednotlivých metod tisku a systémů. Dále byla představena samotná metoda selektivního laserového tavení kovů, specifikace práškových kovů a 3D tiskárna na kov. Praktická část je věnována obsluze stroje, jeho kalibrací a činnostem potřebným k zahájení a dokončení tisku.BYRTUS, R. Operation 3D Printers Renishaw AM400 for 3D Printing by Method SLM: Bachelor thesis, Ostrava: VŠB – Technical University of Ostrava, Faculty of Mechanical Engineering, Department of Machining, Assembly and Engineering Metrology, 2017 47s. Thesis head: Ing. Marek Pagáč, Ph.D. The main aim of the bachelor thesis was to investigate all available methods of additional production and to create a complex process of the SLM method of the Renishaw AM400 using a stainless steel AISI 316L powder. The theoretical part deals with the theory of 3D printing, introducing the individual printing methods and systems. In addition, the method of selective laser metal melting, the specification of powder metals and the 3D metal printer were introduced. The practical part is devoted to machine operation, calibration and activities required to initiate and complete printing.346 - Katedra obrábění, montáže a strojírenské metrologievýborn

Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators

The human X and Y chromosomes evolved from an ordinary pair of autosomes, but millions of years ago genetic decay ravaged the Y chromosome, and only three per cent of its ancestral genes survived. We reconstructed the evolution of the Y chromosome across eight mammals to identify biases in gene content and the selective pressures that preserved the surviving ancestral genes. Our findings indicate that survival was nonrandom, and in two cases, convergent across placental and marsupial mammals. We conclude that the gene content of the Y chromosome became specialized through selection to maintain the ancestral dosage of homologous X-Y gene pairs that function as broadly expressed regulators of transcription, translation and protein stability. We propose that beyond its roles in testis determination and spermatogenesis, the Y chromosome is essential for male viability, and has unappreciated roles in Turner (tm) s syndrome and in phenotypic differences between the sexes in health and disease

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

A comparison of two strains of the hospital pathogen Enterococcus faecalis suggests that mediators of virulence differ between strains and that virulence does not depend on mobile gene element

Targeted Amplicon Sequencing (TAS): A Scalable Next-Gen Approach to Multilocus, Multitaxa Phylogenetics

Next-gen sequencing technologies have revolutionized data collection in genetic studies and advanced genome biology to novel frontiers. However, to date, next-gen technologies have been used principally for whole genome sequencing and transcriptome sequencing. Yet many questions in population genetics and systematics rely on sequencing specific genes of known function or diversity levels. Here, we describe a targeted amplicon sequencing (TAS) approach capitalizing on next-gen capacity to sequence large numbers of targeted gene regions from a large number of samples. Our TAS approach is easily scalable, simple in execution, neither time-nor labor-intensive, relatively inexpensive, and can be applied to a broad diversity of organisms and/or genes. Our TAS approach includes a bioinformatic application, BarcodeCrucher, to take raw next-gen sequence reads and perform quality control checks and convert the data into FASTA format organized by gene and sample, ready for phylogenetic analyses. We demonstrate our approach by sequencing targeted genes of known phylogenetic utility to estimate a phylogeny for the Pancrustacea. We generated data from 44 taxa using 68 different 10-bp multiplexing identifiers. The overall quality of data produced was robust and was informative for phylogeny estimation. The potential for this method to produce copious amounts of data from a single 454 plate (e.g., 325 taxa for 24 loci) significantly reduces sequencing expenses incurred from traditional Sanger sequencing. We further discuss the advantages and disadvantages of this method, while offering suggestions to enhance the approach