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Leidos, Inc. is a large government science and technology company. When a Leidos employee lost his son to an opioid overdose, he wrote an impassioned email to the CEO, asking him to take action related to the growing societal problem of opioid use disorders. The CEO understood and accepted this important call to action. Leidos’ subsequent efforts included supporting employees and families, as well as community outreach, eventually broadening to include mental health and well-being. This paper outlines the company’s efforts to combat opioid use disorders, understanding that they sit within a larger total worker health umbrella inclusive of mental health and well-being. The hope is that this ongoing journey and its exemplars are helpful for other organizations seeking to make an impact for their employees and society.

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (−0.5, −1.0, −5.0, −10 °C/min until −80 °C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at −1 °C/min, but similar results were obtained using −0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or −1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids due to a lack of elongases and desaturases. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, beta-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92% with 69% homozygosity/bi-allelism, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish simultaneously mutated with the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch. Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and MC4R mutants were 121.6% and 94.1% higher than in controls, respectively. Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA+DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 fish than controls. To our knowledge this is the first example of multiplexed genetic engineering and gene editing of a commercially important aquaculture species for valuable performance traits. With a high mutation rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 channel catfish appears beneficial for application on commercial farms.

CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

Xenogenesis is emerging as an innovative technology for hybrid catfish (channel catfish, Ictalurus punctatus ♀ × blue catfish, I.furcatus ♂) production, where primordial germ cells (PGC), spermatogonial stem cells (SSCs) or oogonial stem cells (OSCs) are transplanted into infertile host species such as triploid fish to produce xenogens. Currently, donor cells are collected from randomly selected immature fish without having prior knowledge regarding the best donor stage, which is critical to increase the efficiency and success of germ cell transplantation in catfish breeding. Therefore, the current study was carried out with the objective of determining the relationships between quantity of stem cells, total length (TL), total weight (TW), and the level of androgens [testosterone (T) and 11-ketotestosterone (11-KT)] in blue catfish (donor fish). Positive quadratic relationships were observed between TL and the number of extracted live SSCs (r2 = 0.51; P

Background Previous data at our facility indicated 37% of patients with Clostridium difficile infection (CDI) were receiving at least one laxative at the time of testing, suggesting the possibility of false-positive results. Nucleic acid amplification testing (NAAT) does not distinguish between colonization and infection with C. difficile. We implemented two interventions to address these issues and evaluated our rates of nosocomial CDI before and after these changes. Methods This was a retrospective study of all positive test results for adult patients with nosocomial C. difficile from October 1, 2017 through March 31, 2019 at Vidant Medical Center, a 911-bed hospital. In June, 2018, we implemented a best practice advisory (BPA) in our electronic health record to recommend against testing for CDI in patients receiving laxatives. We reviewed the number of C. difficile tests ordered before and after initiating the BPA. In December, 2018, we removed NAAT and replaced it with a cell cytotoxicity assay (CCA) for specimens that were enzyme immunoassay (EIA) negative and glutamate dehydrogenase (GDH) positive. Antimicrobial use was measured in days of therapy (DOT) per 10,000 patient-days (PD). Mann–Whitney U test was used for continuous variables. Linear regression was used to monitor antimicrobial use. Results The number of C. difficile tests ordered per month decreased 19.5% after implementing the BPA (P < 0.0001). There was a 44% reduction in the number of EIA+/GDH+ specimens per month after the BPA intervention (P = 0.003). Following substitution of CCA for NAAT for EIA-/GDH+ specimens, there was a 61% reduction in the rate of nosocomial CDI (8.6 cases/10,000 PD to 3.3 cases/10,000 PD; P = 0.005). Total antimicrobial use was unchanged over the course of the study (673 to 677 DOT/10,000 PD). Carbapenem use decreased 56% (P = 0.009); cefepime use increased 85%(p = 0.002); quinolone and clindamycin use were unchanged. Conclusion Laxative use in hospitalized patients is common and likely contributes to a false elevation in the CDI rate by identifying carriers in addition to those who have true infection. Implementing a BPA to reduce inappropriate testing and changing our testing algorithm for Clostridium difficile by substituting CCA for NAAT has resulted in a lower rate of nosocomial CDI. Disclosures All authors: No reported disclosures.