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Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F

Microplastics are ubiquitous in the environment. While it is well documented that microplastics can be harmful to aquatic and terrestrial life, little is understood about how microplastics affect soil environments. Understanding the processes enhancing or constraining microplastic transport through the environment, including within soils, is essential in order to minimize the negative impacts of microplastic pollution. Soil erosion is thought to be a primary process responsible for translocating microplastics from soils to aquatic environments. This study aims to investigate the processes controlling microplastic movement in response to rainfall and overland flow in laboratory rainfall simulations. Using fluorescent photography, we compared the real- time movement of a natural soil particle, sand, with two types of microplastics of differing densities (linear low-density polyethylene and acrylic) in two size fractions (250-355 μm and 500-600 μm). We quantified the rate and number of microplastic particles transported by overland flow and splash erosion, along with the depth to which the particles migrate into the soil profile. The results from this study represent some of the first empirical data that seek to quantify microplastic surface transport processes. Ultimately, additional research in this area will be required to more accurately estimate the extent to which microplastics are exported from soils and the factors that control this export.

Constructs bearing the cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus (CMV) promoter, or the common carp beta-actin (β-actin) promoter were transferred to channel catfish, Ictalurus punctatus via electroporation. One F

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish (Ictalurus punctatus) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon (Oncorhynchus masou) driven by a carp β-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P

The CRISPR/Cas9 platform holds promise for modifying fish traits of interest as a precise and versatile tool for genome manipulation. To reduce introgression of transgene and control reproduction, catfish species have been studied for upscaled disease resistance and intervening of reproduction to lower the potential environmental risks of introgression of escapees’ as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene from an alligator (Alligator sinensis;As-Cath) into the target luteinizing hormone (LH) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock-in (KI) efficiency (22.38%, 64/286) but low on-target was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). On-target KI ofAs-Cathwas instrumental in establishing theLHknockout (LH−_As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared to the wild-type sibling fish. Furthermore, implanting with HCG and LHRHa can restore the fecundity, spawnability and hatchability of the new transgenic fish line. Overall, we replaced theLHgene with an alligator cathelicidin transgene and then administered hormone therapy to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. This strategy not only effectively improves the consumer-valued traits, but also guards against genetic contamination. This is a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.

CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon (

Long-term storage of oogonia and germ-line stem cells provides an alternative to the limitations associated with cryopreserving eggs of important fish species. These cell types are less vulnerable to the stresses of freezing. Cryopreservation has enormous potential for aquaculture advancement, but protocols must be developed for each species and cell type since its success hinges on various input factors. Blue Catfish Ictalurus furcatus were selected as the test species in this study because of the need to improve fry production of Blue Catfish ♂ × Channel Catfish I. punctatus ♀ hybrids, which can be facilitated by storing oogonia in gene banks. Our objective was to develop a freezing protocol for oogonia of this species. We tested different permeating and nonpermeating cryoprotectants, concentrations of these agents, and freezing rates. We proved that all three factors influenced postthaw recovery of oogonia. Of the permeating cryoprotectants, 1.0 M dimethyl sulfoxide resulted in the most live cells with the highest viability percentages, and adding 0.2 M lactose with 10% egg yolk further improved the results. There were also specific interactions in which the effects of concentration and freezing rate varied among the cryoprotectant treatments. The most effective freezing rate was −1.0°C/min, and cell viability was reduced at −2.5°C/min and −5.0°C/min. From these results, we propose adding 1.0 M dimethyl sulfoxide with 0.2 M lactose and 10% egg yolk to cryomedia and freezing it at a rate of −1.0°C/min. By developing a cryopreservation protocol for a commonly cultured catfish, this work may guide the development of protocols for other species of interest.

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.

Single-species PCR assays accurately measure eDNA concentration. Here we test whether multi-species PCR, i.e., metabarcoding, with an internal standard can quantify eDNA of marine bony fish. Replicate amplifications with Riaz 12S gene primers were spiked with known amounts of a non-fish vertebrate DNA standard, indexed separately, and sequenced on an Illumina MiSeq. Fish eDNA copies were calculated by comparing fish and standard reads. Relative reads were directly proportional to relative DNA copies, with average and maximum variance between replicates of about 1.3- and 2.0-fold, respectively. There was an apparent threshold for consistent amplification of about 10 eDNA copies per PCR reaction. The internal DNA standard corrected for distortion of read counts due to non-fish vertebrate DNA. To assess potential amplification bias among species, we compared reads obtained with Riaz 12S primers to those with modified MiFish primers. Our results provide evidence that Riaz 12S gene metabarcoding with an internal DNA standard quantifies marine bony fish eDNA over a range of about 10 to 5,000 copies per reaction, without indication of significant PCR bias among teleost species. In mid-Atlantic coastal samples, eDNA rarity was the main limitation to reproducible detection and quantification, and this was partly overcome by increasing the amount of a DNA sample amplified. Our findings support incorporating a DNA standard in 12S metabarcoding to help quantify eDNA abundance for marine bony fish species.

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.

Objective To evaluate the effects of a best practice advisory (BPA) and a change in the Clostridioides (Clostridium) difficile testing algorithm on nosocomial C. difficile infection (CDI) rates. Methods This was a retrospective analysis at a tertiary care hospital of adult patients who tested positive for CDI between July 1, 2017 and September 30, 2019. In June 2018, we implemented a BPA in our electronic health record recommending 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 replaced nucleic acid amplification testing (NAAT) with a cell cytotoxicity assay (CCA) for stool specimens that were enzyme immunoassay toxin negative and glutamate dehydrogenase positive. Results The number of C. difficile tests ordered per month decreased 14% after implementing the BPA (P = .0001). Following this intervention, the rate of nosocomial CDI (nCDI) decreased by 16.5% (P = .33). Following substitution of CCA for NAAT for enzyme immunoassay toxin−/glutamate dehydrogenase+ specimens, there was a 50% reduction in the rate of nCDI (7.1 cases/10,000 patient days to 3.5 cases/10,000 patient days; P Conclusions Implementing a BPA to reduce inappropriate testing and changing the testing algorithm for C. difficile by substituting CCA for NAAT resulted in a lower rate of diagnosis of nCDI.

One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09-119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97-773, Edwardsiella piscicida E22-10, A. hydrophila ML09-119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL-001 were determined in vitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5 µg of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P < 0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P < 0.05) for cathelicidin-injected groups at 24 and 48 h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long-term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.

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.

The Bible is the most influential book in Western history. As the foundational text of Judaism and Christianity, the Bible has been interpreted and reinterpreted over millennia, utilized to promote a seemingly endless run of theological and political positions. Adherents and detractors alike point to different passages throughout to justify wildly disparate behaviors and beliefs. Translated and retranslated, these texts lead both to unity and intense conflict. Influential books on any topic are typically called “bibles.” What is the Bible? As a text considered sacred by some, its stories and language appear throughout the fine arts and popular culture, from Shakespeare to Saturday Night Live. In Michael Coogan’s eagerly awaited addition to Oxford’s What Everyone Needs to know® series, conflicts and controversies surrounding the world’s bestselling book are addressed in a straightforward Q&A format. This book provides an unbiased look at biblical authority and authorship, the Bible’s influence in Western culture, the disputes over meaning and interpretation, and the state of biblical scholarship today. Brimming with information for the student and the expert alike, The Bible: What Everyone Needs to Know ® is a dependable introduction to a most contentious holy book.

What are interpretive strategies? Interpretive strategies are methodologies of various types used in studying the Bible. Some of these, such as textual criticism and archaeology, we have discussed earlier, and some, such as reception history, we will discuss in subsequent chapters. Many of them overlap,...

Why is it important to study the contexts in which the biblical writers lived? Because the Bible was not written in a vacuum, but in a world—or rather, many worlds—that are different from ours, understanding those worlds helps us understand the Bible. Reading the Bible...

What do I mean by “biblical concepts”? By biblical concepts I mean major ideas and themes that are often grouped under the rubric “biblical theology.” It is a mistake, I think, to speak of “the theology” of the Bible, or of the Hebrew...

What languages was the Bible written in? The Bible was written in three languages. The first is Hebrew, which was the primary language of the ancient Israelites for most of the biblical period, until Aramaic gradually replaced it. Hebrew continued to be used by Jews...

What are the Bible’s highest values? When the early first-century CE rabbi Hillel was asked by a prospective convert to explain the whole Torah while the prospective convert was standing on one foot, Hillel reportedly replied: “What is hateful to you, do not do to...

What is reception history? Reception history is a scholarly term for what we could call the study of the afterlives of the Bible: how, since it was being formed, the Bible has been used, from popular culture to high art. Such cultural influence of canonical...

What is the Bible? The Bible is the sacred scripture of Judaism and Christianity. In its pages we encounter some of the most memorable characters in world literature: Adam and Eve, Cain and Abel, Abraham and Sarah, Jacob, Moses, Samson and Delilah, David and Bathsheba,...

Who wrote the Bible? The simple fact is that we don’t know who wrote much of the Bible. Although the names of apparent authors are found in many biblical books and parts of books, scholars have concluded that not all of those individuals wrote the...

Long-term storage of spermatogonial stem cells (SSCs) represents the next step for advancing aquaculture research by providing valuable genetic resources for genetic enhancement programs and xenogenesis technologies. Hybrid catfish, the cross between channel catfish, Ictalurus punctatus ♀ and blue catfish, I. furcatus ♂, are in high demand by the US aquaculture industry, but production can be limited by the availability of blue catfish gametes. Therefore, xenogeneic stem cell transplantation is a powerful method for generating blue catfish gametes within faster growing and maturing channel catfish hosts. These technologies could be facilitated by having cryopreserved SSCs from testicular tissue on-hand in gene banks. Cryopreservation protocols for blue catfish testicular tissues and for other fish species are still being optimized, and currently, data is lacking on the effects antioxidants and antifreeze proteins (AFPs) have on cellular post-thaw viability of SSCs. The objective of this study was to analyze the individual and combined effects of antioxidants (catalase, hypotaurine, and ascorbic acid) and AFPs (I and III) on post-thaw type A spermatogonia cell production and viability from testicular tissue. In this study, there were no improvements with individual antioxidant or AFP treatments. However, when the antioxidants hypotaurine and catalase were tested in combination with different AFPs, three treatments had higher type A spermatogonia cell production than the control, including hypotaurine 7 mM + AFPI 1 μg/mL, hypotaurine 3.5 mM + AFPI 0.1 μg/mL, and hypotaurine 7 mM + AFPIII 0.1 μg/mL. These treatments along with hypotaurine 7 mM + AFPI 0.1 μg/mL and catalase 100 IU + AFPI 0.1 μg/mL had higher viability than the control. From these results, we recommend adding these antioxidant-AFP combinations to future cryopreservation protocols. Overall, this is the first study showing that combining specific antioxidants and AFPs can improve spermatogonia cryopreservation for a fish species.

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

No consensus exists regarding the definition of ventilator-associated pneumonia (VAP). Even within a single institution, inconsistent diagnostic criteria result in conflicting rates of VAP. As a Level 1 trauma center participating in the Trauma Quality Improvement Project (TQIP) and the National Healthcare Safety Network (NHSN), our institution showed inconsistencies in VAP rates depending on which criteria was applied. The purpose of this study was to compare VAP definitions, defined by culture-based criteria, National Trauma Data Bank (NTDB) and NHSN, using incidence in trauma patients.A retrospective chart review of consecutive trauma patients who were diagnosed with VAP and met pre-determined inclusion and exclusion criteria admitted to our rural, 861-bed, Level 1 trauma and tertiary care center between January 2008 and December 2011 was performed. These patients were identified from the National Trauma Registry of the American College of Surgeons (NTRACS) database and an in-house infection control database. Ventilator-associated pneumonia diagnosis criteria defined by the U.S. Center for Disease Control and Prevention (used by the NHSN), the NTDB, and our institutional, culture-based criteria gold standard were compared among patients.Two hundred seventy-nine patients were diagnosed with VAP (25.4% met NHSN criteria, 88.2% met NTDB, and 76.3% met culture-based criteria). Only 58 (20.1%) patients met all three criteria. When NHSN criteria were compared with culture-based criteria, NHSN showed a high specificity (92.5%) and low sensitivity (28.2%). The positive predictive value (PPV) was 84.5%, but the negative predictive value (NPV) was 47.1%. The agreement between the NHSN and the culture-based criteria was poor (κ = 0.18). Conversely, the NTDB showed a lower specificity (57.8%), but greater sensitivity (86.4%) compared with culture-based criteria. The PPV and NPV were both 74% and the two criteria showed fair agreement (κ = 0.41).The lack of standard diagnostic criteria for VAP resulted in variable reporting to different agencies. Emphasis on establishing a consensus VAP definition should be undertaken.

Hybrid catfish, the progeny of channel catfish (Ictalurus punctatus) females × blue catfish (I. furcatus) males, are in high demand by the United States aquaculture industry because they outcompete each of the parent species for pond and raceway culture. Unfortunately, fry production can be a limiting factor due to a lack of natural hybridization between the two species and the necessity to sacrifice males for artificial fertilization. Males are costly to maintain in hatcheries and take longer to mature than channel catfish males. Therefore, it is essential to use the minimum quantity of sperm that allows for the highest fry production. In this study, channel catfish eggs from 9 females were collected from two different locations and fertilized with six different sperm to egg ratios ranging from 1.0 × 103:1 to 1.0 × 105:1. Embryos were then incubated under common environmental conditions until hatch. Overall average hatch success ± SE using sperm to egg ratios between 1 × 103 and 1.0 × 105 ranged from 18.3 ± 4.3 to 48.8 ± 8.3%. Overall, there was a significant effect of sperm to egg ratio (P 1 × 104:1 sperm per egg had no significant improvement on hatch success. Both study locations indicated significant impacts of sperm to egg ratio (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.

Mr. F. Smith was a 63-year-old man admitted to the Veterans Administration hospital with fever, respiratory distress, and a possible recurrent pneumonia. He had entered a community hospital with pneumonia approximately 18 months earlier. His 80 pack-year tobacco history and 10-year emphysema history complicated the clinical course on the first admission, and his status worsened to the point of respiratory failure. He suffered a cardiac arrest while on a ventilator in an intensive care unit. He was asystolic for approximately 5 minutes but was resuscitated.

Lewis lung carcinoma (LLC) clones were used in in vitro models for dissemination to identify mechanisms regulating the stimulation of metastatic LLC-LN7 migration by prostaglandin E2 (PGE2) or forskolin plus 3-isobutyl-I-methylxanthine (IBMX), and the lack of responsiveness to generated cAMP in non-metastatic LLC-C8 cells. The regulatory subunits of protein kinase A (PKA) from LLC-LN7 cells bound more 8-N3-32P-cAMP, even though production of regulatory subunits was equal to that in LLC-C8 cells. Protein kinase C (PKC) differentially regulated PKA activation in the LLC variants. PKC activation inhibited PGE2-stimulated migration by LLC-LN7 cells. Inhibition of PKC with staurosporine stimulated LLC-LN7 cell migration to a level comparable with that induced by PGE2. However, PGE2 did not further stimulate the migration of staurosporine-treated cells. The PGE2 or staurosporine stimulation of LLC-LN7 cell migration was dependent on PKA activation. The effects that modulation of PKA and PKC had on LLC-LN7 cell migration paralleled the effects on endogenous protein phosphorylation. LLC-LN7 cell autophosphorylation was stimulated to a similar degree by PGE2, forskolin plus IMBX, staurosporine, or the combination of staurosporine and forskolin plus IBMX. In contrast, neither migration nor autophosphorylation was stimulated in non-metastatic LLC-C8 cells by cAMP elevation or by PKC inhibition. Autophosphorylation, although not migration, of LLC-C8 cells was stimulated by forskolin plus IBMX when PKC activity was inhibited. These results suggest that the increased PKA response of metastatic LLC-LN7 cells is contributed by an increased binding of cAMP by the PKA regulatory subunits and a reduced level of regulation by PKC.