The Friends of the Indians and Their Foes: A Reassessment of the Dawes Act Debate

In the centennial of the passage of the Northwest ordinance, 1887, the U.S. Congress passed the Dawes Act, continuing the program set up by the earlier document. The Northwest Ordinance had sought to incorporate lands previously uninhabited by whites into the American realm by imposing a Euro-American sense of order onto areas viewed as wild and savage The document created a program by which the western development of the continent would proceed in a rational manner, and by which lands would be attributed worth and meaning in relation to the developed areas to the east. The Dawes Act proceeded in the tradition of the Northwest Ordinance by incorporating Indian reservations of the West into the economic and political sphere of late nineteenth century America. It proposed to break up the communal lands of Indian tribes and nations into quarter sections for individual farmers. In the process, Indian tribes were dispossessed, and white settlers and corporations were granted the lands that had been guaranteed to the tribes forever. The Dawes Act was one of the last measures in the expansionist heritage of the United states by which the remaining enclaves of Indian lands were subjected to the enveloping grid. During the 1880s, the Indian Question became more ominous and more urgent in the eyes of concerned Easterners who called themselves the Friends of the Indians. These reformers created the Dawes Act and encouraged its passage. They were supported by mainstream society in an endeavor to incorporate desired tribal lands into the American realm. It was easy for earlier generations to relegate Indian groups to peripheral and undesirable lands in the infinity of the American West, but by the 1880s, land was becoming scarce and those distant lands once known as the Great American Desert were desired by an ever-expanding nation. The Plains had become an integral part of the American landscape and economy, and its Indian reservations were frustrating the drive to link the continent, from East to West, with white American culture. The boundless continent had become finite. No longer could white America soothe its sense of guilt by compensating Indians with lands in the distant West. With the absence of alternative lands, white guilt was replaced with new and broader justifications for the dispossession of the Indians- justifications that could support the possibility of Indian cultural extinction

Small-scale platform for rapid on-demand manufacturing of recombinant proteins

The current timeline for manufacturing high-quality recombinant proteins used for therapeutics and vaccines, and delivering them to patients, typically requires many months. There are many circumstances, however, where the rapid production, release, and delivery of these biologics could address unmet medical needs, including shortages, emergency situations, and pandemics. This talk will present a new platform developed to produce biologic medicines on demand called Integrated and Scalable Cyto-Technology (InSCyT). This platform comprises an integrated, subliter-scale portable system for the (semi)continuous operation of fermentation, filtration of cell debris from secreted product, innovative affinity-based purification, polishing, and finishing. This program also emphasizes integrated on-line PAT and process control for QbD production and product qualification for release. Examples of the underlying technologies enabling this platform will be presented, along with examples of its integrated operation to produce multiple high-quality recombinant proteins on the same platform, each in less than 48 hours from initial seed to dose. Beyond the applications for rapid manufacturing of biologics at point-of-care, the speed of production enabled by automation and integration highlights other opportunities for vaccine development and conventional manufacturing. These include modeling complete manufacturing processes during process development, rapidly creating materials for preclinical and early-stage clinical testing, and manufacturing therapies for orphan diseases, among others

Integrated manufacturing with microbial hosts for fast process development and production

Integrated and continuously operating processes for recombinant protein drugs that rely on non-mammalian hosts offer potential advantages compared to CHO-based platforms for the rapid production of high-quality biopharmaceuticals. This talk will describe insights and technical advances related to the realization of microbial-based production of proteins gained over the past four years in developing a small prototype system for on-demand production of biologics (Figure). Key learnings discussed will include: 1) how deep ‘-omic level’ understanding of a minimal yeast genome can inform both strain engineering and defined media formulation for enhanced production; 2) new platformable approaches for rapid in silico identification of downstream processes that provide highly orthogonal host-related impurity selectivity between individual chromatographic steps; and 3) examples of integration of these elements to enable hands-free production over days with only minor system changes required to accommodate multiple products. In total, holistic design principles and integration of knowledge together facilitate rapid process development and speed to production at scales relevant for a variety of use cases. These examples highlight the opportunities for employing well-defined alternative hosts with tractable genomes in efficient integrated processes for agile manufacturing. Such strategies should complement on-going and existing efforts to convert CHO-based platforms into continuous operations. Please click Additional Files below to see the full abstract

The Friends of the Indians and Their Foes: A Reassessment of the Dawes Act Debate

In the centennial of the passage of the Northwest ordinance, 1887, the U.S. Congress passed the Dawes Act, continuing the program set up by the earlier document. The Northwest Ordinance had sought to incorporate lands previously uninhabited by whites into the American realm by imposing a Euro-American sense of order onto areas viewed as wild and savage The document created a program by which the western development of the continent would proceed in a rational manner, and by which lands would be attributed worth and meaning in relation to the developed areas to the east. The Dawes Act proceeded in the tradition of the Northwest Ordinance by incorporating Indian reservations of the West into the economic and political sphere of late nineteenth century America. It proposed to break up the communal lands of Indian tribes and nations into quarter sections for individual farmers. In the process, Indian tribes were dispossessed, and white settlers and corporations were granted the lands that had been guaranteed to the tribes forever. The Dawes Act was one of the last measures in the expansionist heritage of the United states by which the remaining enclaves of Indian lands were subjected to the enveloping grid. During the 1880s, the Indian Question became more ominous and more urgent in the eyes of concerned Easterners who called themselves the Friends of the Indians. These reformers created the Dawes Act and encouraged its passage. They were supported by mainstream society in an endeavor to incorporate desired tribal lands into the American realm. It was easy for earlier generations to relegate Indian groups to peripheral and undesirable lands in the infinity of the American West, but by the 1880s, land was becoming scarce and those distant lands once known as the Great American Desert were desired by an ever-expanding nation. The Plains had become an integral part of the American landscape and economy, and its Indian reservations were frustrating the drive to link the continent, from East to West, with white American culture. The boundless continent had become finite. No longer could white America soothe its sense of guilt by compensating Indians with lands in the distant West. With the absence of alternative lands, white guilt was replaced with new and broader justifications for the dispossession of the Indians- justifications that could support the possibility of Indian cultural extinction

Closure of Loop-Mediated Isothermal Amplification Chamber on Lab-On-Chip Using Thermal-Responsive Valve

Point-of-care tests for nucleic acids are important for the diagnosis and management of infectious and genetic diseases, biowarfare agents, and for drug research. Recent integration of loop-mediated isothermal amplification (LAMP) onto a lab-on-chip (LOC) platform allows sensitive and specific, detection of target DNA or RNA sequences for point-of-care (POC) applications. However, because of the high risk of contamination to LAMP products, robust and simple methods of hermetically sealing the reaction chamber are essential. In addition, having a method for isolation of nucleic acids at the level of the chip is more effective for POC applications because a laboratory setting is not required to complete the analysis. This report describes an integrated, polymer-based cassette which was designed for detection of DNA/RNA target-sequences by using LAMP and a solid-state nucleic acid purification membrane. The LAMP chamber seals using a self-actuated thermalresponsive valve made from expandable microspheres suspended in Polydimethylsiloxane (PDMS). A flow-through, Flinders Technology Associates membrane (Whatman FT A®) was installed on the cassette with the expectation of providing isolation and purification of nucleic acids. The cassette was designed, fabricated and the efficacy of the valve to seal the LAMP chamber was investigated. The valve underwent expansion and held pressures of233 +/- 5.0 kPa without signs of leakage. A portable, battery-powered, polyimide-based thin film heater, placed outside the cassette, provided thermal control. In addition, a LAMP primer set was designed for the serotonin receptor 5HTRIA promoter gene using Primer Explorer V 4 software (http:/ /primerexplorer.jp/elamp4.0.0/index.html) for LAMP detection of neurotransmitter serotonin. The integrated, portable LAMP cassette will be attractive in global health-care challenges, mainly in resource-poor locations, where the availability of laboratory equipment and/or trained personal is restricted.Digitized from a paper copy provided by the Physiological Sciences Graduate Interdisciplinary Program

Integrated scalable cyto-technology for recombinant protein bioprocessing

Biological knowledge of infectious diseases and other diseases for which vaccines may provide therapeutic benefits, such as cancer, is growing at an accelerated pace. The implications of this knowledge are improved stratification of diseases, possibilities for personalized treatments, and explicit understanding of protective immune responses to be elicited by vaccines. With this knowledge, it is becoming increasingly feasible to engineer vaccines for specific responses rather than relying on empirical development. Despite this potential, the challenge of routine, low-cost manufacturing of vaccines creates a barrier to transforming health care in both high- and low-resource countries. Vaccines today do not benefit from well-defined, platform-like processes for manufacturing, and concepts such as continuous bioprocessing remain largely within the realm of biopharmaceutical products. The InSCyT platform is an advanced prototype manufacturing system that provides integrated and automated production and purification of multiple protein therapeutics. The system allows end-to-end manufacturing of 100\u27s to 10,000\u27s of doses of recombinant protein drugs in days. It uses a state-of-the-art approach to process design and implementation that takes advantage of a fast-growing, tractable microbial host (Pichia pastoris) and continuous processing for automated, hands-free purification through simple 2- or 3-stage chromatographic processes. The platform design is highly modular, allowing facile process development and process deployment for multiple products. This feature emerges from the predictable behavior of the fermentation and cell culture fluids, and rapid cloning of new molecules, that together facilitate fast development of entirely new processes in weeks. To date, this system has been used to reproducibly manufacture high-quality human growth hormone (hGH), granulocyte-colony stimulating factor (G-CSF), and interferon-α2b (IFN-α2b) in an integrated, automated manner. The speed of production using the InSCyT prototype allows volumetric productivities that compare favorably to those for mammalian-based production. This talk will outline the design and capabilities of the InSCyT system, demonstrate the quality of biologic drugs made to date on the system, and outline opportunities for advancing the platform to provide new capabilities in manufacturing recombinant proteins for use in vaccines. As part of a Gates Foundation-funded Grand Challenge called ULTRA, we have begun to assess the feasibility of manufacturing millions of doses of a trivalent recombinant rotavirus vaccine annually on a small-scale production system like InSCyT. Integrated bioprocessing enabled by systems such as these could offer potential advantages for routine production in local regions with minimal infrastructure, and for democratization of manufacturing capacity for new vaccine concepts and personalized treatments in cancer

Accelerated process development for integrated end-to-end biologics manufacturing

With the exception of monoclonal antibodies, biologics typically require bespoke manufacturing processes that vary widely in the type of and number of unit operations. This constraint leads to custom facility designs and unique strategies for process development for every new molecule. To enable flexible, multi-product manufacturing facilities and to reduce the speed to clinic for new molecules, streamlined manufacturing processes and associated strategies for process development are needed. We have developed a bench-scale, integrated and automated manufacturing platform capable of rapidly producing a variety of recombinant proteins with phase-appropriate quality for early development1. The system comprises three modules for fermentation via perfusion, straight-through chromatographic purification, and formulation. To facilitate the production of multiple products on the same system, we have also developed a holistic strategy for process design to manufacture new products in as few as twelve weeks after obtaining the product sequence. While upstream process development in our host (Pichia pastoris) has been relatively straightforward, there are not many tools currently available for developing fully integrated straight-through chromatographic processes. Therefore, we developed an in silico tool for the prediction of fully integrated purification processes based on a one-time collection of host-related data combined with conventional high-throughput chromatographic screening data for each new target molecule2. We used this tool to develop fully integrated, end-to-end production processes for three molecules (hGH, IFNα-2b, and G-CSF) with at least 45% fewer steps than traditional processes. While our in silico tool allows for rapid resin selection, it may not predict the optimal process for each individual molecule since it is based on conventional high-throughput screening techniques which seek to optimize each chromatographic step independently rather than optimizing a fully integrated, multi-column process. To address this limitation, we have also developed a DoE-like framework for the optimization of fully integrated purification processes once the resins have been selected. First, a series of range finding experiments are carried out on each individual column, similar to conventional screening but with limited analytics. Next, we carry out fully integrated (multi-column) testing of the proposed operational area with more extensive analytics, including host cell protein, DNA, and yield measurements. We use this methodology to develop optimized processes for the end-to-end production of a variety of single domain antibodies with high yield and purity. Further, we present a method for predicting the optimal operating conditions for a new molecule within the same class based only on its biophysical characteristics, reducing the timeline from sequence to early stage, phase-appropriate product to only six weeks. Using these holistic strategies for process development, we have produced over ten different recombinant proteins on our manufacturing platform including enzymes, cytokines, singe domain antibodies, and vaccine subunits. We believe that such integrated strategies for process design could enable the rapid translation from sequence to early stage clinical development of products for a variety of molecules and potentially allow clinical testing of a greater number of high quality molecules for vaccines and biopharmaceuticals. 1. Crowell, L. E. et al. On-demand manufacturing of clinical-quality biopharmaceuticals. Nat. Biotechnol. (2018). doi:10.1038/nbt.4262 2. Timmick, S. M. et al. An impurity characterization based approach for the rapid development of integrated downstream purification processes. Biotechnol. Bioeng. 1–13 (2018). doi:10.1002/bit.2671

Crossword: A Fully Automated Algorithm for the Segmentation and Quality Control of Protein Microarray Images

Biological assays formatted as microarrays have become a critical tool for the generation of the comprehensive data sets required for systems-level understanding of biological processes. Manual annotation of data extracted from images of microarrays, however, remains a significant bottleneck, particularly for protein microarrays due to the sensitivity of this technology to weak artifact signal. In order to automate the extraction and curation of data from protein microarrays, we describe an algorithm called Crossword that logically combines information from multiple approaches to fully automate microarray segmentation. Automated artifact removal is also accomplished by segregating structured pixels from the background noise using iterative clustering and pixel connectivity. Correlation of the location of structured pixels across image channels is used to identify and remove artifact pixels from the image prior to data extraction. This component improves the accuracy of data sets while reducing the requirement for time-consuming visual inspection of the data. Crossword enables a fully automated protocol that is robust to significant spatial and intensity aberrations. Overall, the average amount of user intervention is reduced by an order of magnitude and the data quality is increased through artifact removal and reduced user variability. The increase in throughput should aid the further implementation of microarray technologies in clinical studies.Camille and Henry Dreyfus Foundation (Camille Dreyfus Teacher-Scholar Award

The effectiveness of faecal removal methods of pasture management to control the cyathostomin burden of donkeys

Background: The level of anthelmintic resistance within some cyathostomin parasite populations has increased to the level where sole reliance on anthelmintic-based control protocols is not possible. Management-based nematode control methods, including removal of faeces from pasture, are widely recommended for use in association with a reduction in anthelmintic use to reduce selection pressure for drug resistance; however, very little work has been performed to quantitatively assess the effectiveness of such methods.<p></p> Methods: We analysed data obtained from 345 donkeys at The Donkey Sanctuary (Devon, UK), managed under three different pasture management techniques, to investigate the effectiveness of faeces removal in strongyle control in equids. The management groups were as follows: no removal of faeces from pasture, manual, twice-weekly removal of faeces from pasture and automatic, twice-weekly removal of faeces from pasture (using a mechanical pasture sweeper). From turn-out onto pasture in May, monthly faecal egg counts were obtained for each donkey and the dataset subjected to an auto regressive moving average model.<p></p> Results: There was little to no difference in faecal egg counts between the two methods of faecal removal; both resulted in significantly improved cyathostomin control compared to the results obtained from the donkeys that grazed pasture from which there was no faecal removal.<p></p> Conclusions: This study represents a valuable and unique assessment of the effectiveness of the removal of equine faeces from pasture, and provides an evidence base from which to advocate twice-weekly removal of faeces from pasture as an adjunct for equid nematode control. Widespread adoption of this practice could substantially reduce anthelmintic usage, and hence reduce selection pressure for nematode resistance to the currently effective anthelmintic products.<p></p&gt

Rational design of expression vectors for high quality biologics

Commercial proteins (e.g. antibodies, enzymes, vaccine components) for applications from biopharmaceuticals to commodity chemicals require low-cost manufacturing of high-quality products. The engineering of recombinant hosts to achieve large quantities of high-quality heterologous proteins is crucial to both minimizing costs and maximizing safety and efficacy (in the case of biopharmaceuticals). High-quality proteins are properly folded and full-length (intact), with native N-, and C-, termini and bear no significant proteolysis or other degradation (oxidation, deamidation, etc…). As most expression hosts rely on recombinant DNA technology for production of the heterologous protein, the transgene cassette provides an early, and inexpensive, opportunity for optimization of quality and protein titer. Commonly, transgene cassettes include a promoter, a heterologous gene of interest, and terminator for expression of the heterologous gene. A targeting sequence for guided recombination and selective marker for isolation of positive clones are also key elements. In engineering the transgene cassette, factors such as the promoter for heterologous gene expression, target site for transgene integration, sequence for translation initiation, and mRNA codon-optimization of the gene of interest are critical design points for a given protein-expressing strain. Here, we demonstrate an approach to transgene cassette optimization in the methylotrophic yeast, Pichia pastoris, informed by functional genomics. Omics-based techniques such as RNA-Seq, ATAC-Seq and ribosomal foot-printing afford greater upfront understanding for subsequent optimized strain engineering on a product-by-product basis. These types of data are cheap and easy to acquire for yeast and can indicate host- or sequence-derived bottlenecks in transgene transcription, translation and expression. Linking these data to product quality attributes can enlighten the design of the expression vector for fast in silico optimization of wide-ranging factors such as gene dosage balance, translation efficiency, and balanced cell kinetics enabling high-quality protein production. Collectively, we show that these tools can enable fast vector design for new heterologous protein-producing strains, including those expressing recombinant vaccines, and robust optimization when engineering higher productivity cell lines