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Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 16 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in CD46 may reduce the burden of BVDV-associated diseases in cattle, and is consistent with our stepwise,in vitroandex vivoexperiments with cell lines and matched fetal clones.

Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9–mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system. Knockdown of ODF2 and IFT88 resulted in a 50% reduction in the number of cells with primary cilia and significant shortening of the remaining cilia. The expression of GLI1, a downstream target of Hh signaling, was significantly reduced when IFT88 but not ODF2, was downregulated. When morphogenesis was examined using tubule formation in vitro and a novel testicular organoid system, loss of cilia after knockdown of both targets affected cellular assembly and organization. While the Hh pathway was found to be active during morphogenesis in vitro, addition of the Hh antagonist cyclopamine did not affect morphogenesis in either in vitro system. These results indicate that primary cilia are important for morphogenesis in vitro but Hh signaling is not the cilia-mediated pathway responsible for orchestrating morphogenic organization.

Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46, however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Bovine viral diarrhea virus (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genesPTPN12,GRID2, andRABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss ofPTPN12,GRID2, orRABGAP1Lgene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Ribonucleoprotein (RNP) granules are biomolecular condensates—liquid–liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation. Dysregulation of ribonucleoprotein complex granules, previously implicated in neuromuscular disease, can drive pathogenesis in a genetic form of dilated cardiomyopathy, as shown in gene-edited pigs and patient-derived cardiomyocytes.

Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids forGeneTagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates thatin vivotargeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand breakin vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

This paper describes how a group of men, who are invisible to the state because they possess no formal legal documentation, such as ID books or passports, and do not take part in the formal economy, live in a public park in Gezina, Pretoria. It explores how they negotiate some of the ambiguities of street life and community through acts of sharing and illegal public gambling. While the moral and economic principles organising life in the park are plural, forms of everyday communism predominate. I argue that this predominance allows for a form of community that allows the men living there to navigate their daily uncertainties more easily, and explore the ways in which public gambling functions to maintain the conditions for constructive relations amongst them.

The rapid introduction of newer technology and better approaches to management have led to unprecedented increases in productivity, but at its heart, the objective of manufacturing is still to provide the right product in the right quantity at the right time with the right quality at the right price to its customers. This chapter shows how manufacturing organizations strive to meet this objective. Forecasting provides the manufacturer with a basis for anticipating customer demand so as to have adequate product on hand when it is demanded. Aggregate planning approaches establish overall production requirements, and materials requirements planning techniques provide a methodology for ensuring that adequate inventory is available to complete the work required on products needed to meet forecasted customer demands. Job-sequencing methodologies are used to develop shop schedules for production processes to reduce the time for manufacturing products. Finally, Japanese manufacturing philosophy and supply chain management are discussed. Keywords: aggregate planning approaches; forecasting methods; inventory models; Japanese manufacturing philosophy; job-sequencing methodologies; materials requirements planning techniques; production planning; shop schedules; supply chain management

Metal-forming processes use a remarkable property of metals and their ability to flow plastically in the solid state without concurrent deterioration of properties. Metal-forming processes are classified into two categories: hot-working processes and cold-working processes. Some of the hot-working processes that are of major importance in modern manufacturing are: rolling, forging, extrusion and upsetting, pipe welding and piercing. The major cold-working operations can be classified basically under the headings of squeezing, bending, shearing, and drawing. Metal casting provides versatility and flexibility that have maintained casting position as a primary production method for machine elements. Some of the most common molding processes discussed are: injection molding, coinjection molding, expandable-bead molding, and blow molding. In powder metallurgy (PM), fine metal powders are pressed into a desired shape, usually in a metal die and under high pressure, and the compacted powder is then heated (sintered), with a protective atmosphere. Keywords: cold-working operations; hot-working processes; metal casting; metal-forming processes; molding processes; powder metallurgy (PM)

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

FMS layout design is a complex process, involving a large number of alternatives, which contributes generously to the usual two-year FMS planning lead time. The need for a comprehensive, yet easy-to-use methodology or tool which can reduce this large number of alternatives to a more reasonable figure has motivated this research. A survey of existing FMSs was undertaken with the intention of identifying some of the more common (dominant) types of layouts, and to develop, if possible, a methodology for the selection of such layouts. However, almost every FMS was found to have a unique layout. For a given FMS under consideration, the capabilities with respect to the parts which can be manufactured is essentially given. The determining features of the resultant design of the FMS, therefore, are primarily based upon the design of the material handling system which integrates the FMS. However, as the number of machines in a FMS increase, the number of part sequences and alternate configurations increase at a dr...

The desirable properties for cutting-tool material include the ability to resist softening at high temperature, which is known as red hardness; a low coefficient of friction; wear resistance; sufficient toughness and shock resistance to avoid fracture; and inertness with respect to workpiece material. Drills are used as the basic method of producing holes in a wide variety of materials. The milling machines use a rotary multitooth cutter that can be designed to mill flat or irregularly shaped surfaces, cut gears, generate helical shapes, drill, bore, or do slotting work. Gears are made by various methods, such as machining, rolling, extrusion, blanking, powder metallurgy, casting, or forging. Three basic methods are used for the manufacturing of threads: cutting, rolling, and casting. Saws have two general areas of applications: contouring and cutting off. Nontraditional, or nonconventional, machining processes are material removal processes that have recently emerged or are new to the user. Keywords: cutting-tool material; drilling machines; gear manufacturing; milling machines; nontraditional machining; thread cutting

A consultant team's evaluation of a system for distributing and controlling medications in a large teaching hospital is described. Through interviews with key personnel from administration, pharmacy, nursing, and the medical staff, an interdisciplinary research group identified problems in the reliability and response times of the hospital's existing medication system. After assessing staff expectations regarding acceptable standards for medication errors and response times and their attitudes toward proposed changes in the medication system, medication-error rates were determined using a pharmacist-observer method. Observations during 34 five-hour periods on four nursing units were conducted over a 17-day period. Medication-error rates were calculated as the frequency of medication errors during the observation period divided by the total opportunities for error (OE), which were defined as doses ordered plus unauthorized doses given. Response times for processing "now," "stat," and routine orders were also determined using work-sampling methods. The total medication-error rate for the nursing units studied was 9% excluding wrong-time errors; more than a third of doses were given more than 30 minutes before or after their scheduled administration times. Response times for "now" and "stat" orders averaged about 23 minutes, in conformance with the desired standard of 30 minutes. However, processing of routine orders required an average of two hours and seven minutes, much of which was attributed to delays in the messenger service. The basic design of the existing unit dose medication system contributed to problems in the reliability and efficiency of the system.

A classification scheme, i.e. taxonomy, is developed for categorizing scheduling research. The purpose of the taxonomy is to provide a means of developing a data base of scheduling research and a method by which the data base can be searched to retrieve journal article and book references. Using the taxonomy a significant number of manuscripts have been classified and a data base has been constructed. The associated program that will allow a user to specify certain attributes, to search the data set for articles and books which deal with shops having those attributes, and then retrieve the referenced bibliographic data has also been developed. This paper describes the classification scheme used, the data base which was developed, the procedures used to specify and retrieve information, and the bibliographic information which can be retrieved. Plans for extending the data base are also discussed.

An optimal approach is applied to even numbered athletic conferences to minimize the total distance traveled by the conference teams when playing conference games. A representative set of constraints is imposed on the problem that is typical of conferences. Only college basketball conferences are actually considered but application could be made to any athletic conference with similar restrictions. Two possible solutions are investigated. The first is a zero-one programming model and the second is an iterative approach leading to the development of a scheduling algorithm. Two examples are provided to show practical application of the algorithm by applying it to the Big Eight and Southeastern basketball conferences. Significant reductions in miles traveled are found for both examples.

Recommendations of consultants for the implementation of a new medication system at a large teaching hospital are described. Based on a previous analysis of the hospital's existing drug distribution and control system that revealed problems in reliability and response time, an interdisciplinary consultant group offered 14 recommendations, which included implementation of a computerized unit dose delivery system and selected clinical pharmacy services. Functions identified for which computerization would produce the greatest benefits included maintenance of patient census data, medication order entry and retrieval, and preparation of a medication administration record for nursing. Recommendations for improving the unit dose system in the hospital consisted of increasing the number of medications packaged in true unit dose form, increasing the frequency of daily deliveries of scheduled medications, sending p.r.n. medications on an on-call basis, decreasing the lead time for preparation of i.v. solutions, and using a pharmacist-manned portable medication cart to reduce workload on the central pharmacy during peak workload periods. Clinical pharmacy services identified as having the greatest cost-benefit ratio were discharge consults, drug therapy monitoring, and drug-use review. Using information from published studies and cost data from the hospital, a net annual savings of over +152,000 was projected with implementation of these services. Improvements in the unit dose system and implementation of clinical pharmacy services were expected to result in substantial cost savings in the study hospital.

This paper deals with a new storage structure layout method called ‘cubic-in-time’, for minimizing the travel time of selected handling equipment in a three-dimensional palletized storage system. Storage-system design algorithms for minimizing travel times are described, the results are analysed, and some general conclusions are drawn.

Longitudinal data collection designs are frequently used in the study of developmental variables but typically yield patterns of results that are different from those obtained using cross-sectional designs. Recent research has focused on ways to clarify and reconcile the methodological distinctions between these two designs by utilizing alternative designs that incorporate features of both. This investigation compared the results obtained by the two traditional designs on the Career Maturity Inventory-Attitude Scale with the effects of an alternative design: constructing a composite longitudinal gradient. The two traditional methodologies yielded different results from each other for males, but not for females. However, the alternative design failed to eliminate the obtained differences for males, and introduced differences for females, casting doubt on the efficacy of the “solution.” The implications of these results for developmental research and career maturity were discussed.

The effects of consultant-recommended modifications in a hospital medication system on medication-error rates and response times were evaluated. Fourteen recommendations for improving the medication system in a large teaching hospital were implemented to varying extents over a period of 1.5 years. The response times and medication-error rates of the newly implemented system were then measured using pharmacist-observers as was done in the old medication system. Medication-error rates were then compared on one medical-nursing unit and two surgical-nursing units both before and after implementation of the new system on the medical unit alone. Response times for routine medication orders decreased by 55% compared with the previous system; for "now" and "stat" orders, response times were reduced by 57% for orders filled from the central pharmacy and by 70% for orders filled from medication carts on the nursing units. No significant differences in medication-error rates were found when the old and new medication systems were compared. This finding was attributed primarily to the hospital's failure to implement recommendations related to computer printing and sorting of orders, use of dispensing envelopes for delivering medications in true unit dose form, and packaging of all medications in unit dose form. This study illustrates how the benefits of a unit dose system can be compromised in implementation and the need for close monitoring to ensure that performance standards are maintained.

The approach taken in this paper of isolating seasonal characteristics of products and then using that information to determine when those products should be ordered to minimize the system's purchasing costs appears to be valid. A drawback to using this approach, however, is acquiring appropriate data. The authors used data which they believed to be appropriate for thier market purchasing environment but which are not necessarily valid for other locations and/or environments. The prospective user of this model must find and obtain data suitable for the environment and isolate those products which appear seasonal. Only a small percentage of the items used possess any seasonal properties. There is also the obvious problem of using past data to determine future outcomes. Past price patterns do not necessarily continue into the future, although they should be at least indications, barring unusual farm and/or market situations. As with all decision modeling approaches, however, this model should be followed with caution. While the approach is valid, the procedure is no substitute for knowledgeable management of food service functions. Results from the model should provide additional input to guide food purchasing decisions but should not be used unequivocably. When property used, however, this procedure will allow food buyers to come closer to the goal of optimally purchasing food products from a systems point of view then is currently possible.

A two-stage production line which is both balanced and unbalanced is investigated, where the service times at each station and the interarrival times to the system are independently and normally distributed. Computer simulation is employed in producing interdeparture times of the first station, expected time in the system per arrival and expected queue sizes.

The purpose of this paper is to present a statistically based methodology for selecting the method to be used for generating sample values in a simulation model when alternate methods are available. Choosing which method to use is not a simple task, but very important because it affects the complexity and ultimately the costs associated with the model's development and the model's expected uses. The methodology is presented by means of a case study where it was used in the development of a timber harvesting simulation model. The methodology is primarily based upon analysis of variance and demonstrates one way which has been used in choosing between varying levels of complexity in generating random samples within a simulation model using as a criterion the validity of the alternate methods' results.

When designing a facility layout it is desirable to obtain an optimum design which satisfies certain necessary relationships among departments. Recent research has indicated that applying graph theory to the layout problem can result in the development of improved solutions, but little can be found to indicate how much better the resultant solutions are. The objectives of this paper are to develop a graph theory approach based on research experience and suggestions of Moore, Carrie and Seppanen, to develop a general computer program implementing the procedure and to compare its performance with that of CRAFT, CORELAP and ALDEP. The comparisons are based upon the adjacency relationships satisfied in the resultant layouts. The procedure finds the graph G equivalent to the problem being solved and then generates one of its maximal spanning trees, which after being transformed to its ‘string’ equivalent, is used to extract a maximal planar sub-graph of G. The dual of this sub-graph represents the desired solu...

A technique is presented which can be used to design, or assist in the design of, an integrated materials handling system for a manufacturing facility or a major department of the facility. The developed procedure selects the materials handling equipment to be used to perform a given set of moves in order to miriimize the system's costs associated with the handling, assuming the plant arrangement remains fixed. To demonstrate the selection procedure, an example problem is presented. Since the method is heuristic, no claim of optimality is made.

An application of the Automated Design Layout Program (ALDEP) is presented whereby departmental areas were allocated within a proposed seven story facility for the Birmingham Social Security Payment Center. The criterion used for assignment of departmental facilities to areas was material or folder flow between departments. The final layout proposed was hand adjusted to produce a more aesthetic and practical design.