ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats.

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.

Creation and Preliminary Characterization of Pregnane X Receptor and Constitutive Androstane Receptor Knockout Rats.

The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s) and phase II metabolizing enzymes and transporter genes in response to stimulation from xenobiotics, including prescription drugs. PXR and CAR knockout and humanized mouse models have proven useful. However, the rat being bigger in size is a preferred model system for studying drug metabolism and pharmacokinetics. Here, we report the creation and preliminary characterization of PXR and CAR knockout rats and PXR/CAR double knockout rats. Whereas the expression of phase I and II enzymes and transporter genes were not upregulated by nuclear receptor-specific agonists pregnenlone-16α-carbonitrile and 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene in the knockout rats, confirming the disruption of respective nuclear receptor(s), our data demonstrate that PXR appears to suppress the basal expression levels of Cyp2b2, Cyp3a23/3a1, Cyp3a2, Cyp3a18, and Ugt2b1 genes, while CAR maintains Cyp2b2 and Ugt2b1 and suppresses Cyp3a9 basal expression levels. In wild-type rats, agonist binding of the nuclear receptors relieves the suppression, and target genes are expressed at levels comparable to knockout rats, with or without drug treatment. Overall, our findings are in good agreement with data obtained from human primary hepatocytes, nuclear receptor knockout cell lines, and mouse knockout models. We believe these models are a useful complement to their mouse counterparts for drug development and as importantly, for functional studies on metabolic pathways involving nuclear receptors.

The Challenge Posed by Geomagnetic Activity to Electric Power Reliability: Evidence From England and Wales.

This paper addresses whether geomagnetic activity challenged the reliability of the electric power system during part of the declining phase of solar cycle 23. Operations by National Grid in England and Wales are examined over the period of 11 March 2003 through 31 March 2005. This paper examines the relationship between measures of geomagnetic activity and a metric of challenged electric power reliability known as the net imbalance volume (NIV). Measured in megawatt hours, NIV represents the sum of all energy deployments initiated by the system operator to balance the electric power system. The relationship between geomagnetic activity and NIV is assessed using a multivariate econometric model. The model was estimated using half-hour settlement data over the period of 11 March 2003 through 31 December 2004. The results indicate that geomagnetic activity had a demonstrable effect on NIV over the sample period. Based on the parameter estimates, out-of-sample predictions of NIV were generated for each half hour over the period of 1 January to 31 March 2005. Consistent with the existence of a causal relationship between geomagnetic activity and the electricity market imbalance, the root-mean-square error of the out-of-sample predictions of NIV is smaller; that is, the predictions are more accurate, when the statistically significant estimated effects of geomagnetic activity are included as drivers in the predictions.

Bone fracture nonunion rate decreases with increasing age: A prospective inception cohort study.

BACKGROUND: Fracture nonunion risk is related to severity of injury and type of treatment, yet fracture healing is not fully explained by these factors alone. We hypothesize that patient demographic factors assessable by the clinician at fracture presentation can predict nonunion. METHODS: A prospective cohort study design was used to examine ~2.5 million Medicare patients nationwide. Patients making fracture claims in the 5% Medicare Standard Analytic Files in 2011 were analyzed; continuous enrollment for 12months after fracture was required to capture the ICD-9-CM nonunion diagnosis code (733.82) or any procedure codes for nonunion repair. A stepwise regression analysis was used which dropped variables from analysis if they did not contribute sufficient explanatory power. In-sample predictive accuracy was assessed using a receiver operating characteristic (ROC) curve approach, and an out-of-sample comparison was drawn from the 2012 Medicare 5% SAF files. RESULTS: Overall, 47,437 Medicare patients had 56,492 fractures and 2.5% of fractures were nonunion. Patients with healed fracture (age 75.0±12.7SD) were older (p<0.0001) than patients with nonunion (age 69.2±13.4SD). The death rate among all Medicare beneficiaries was 4.8% per year, but fracture patients had an age- and sex-adjusted death rate of 11.0% (p<0.0001). Patients with fracture in 14 of 18 bones were significantly more likely to die within one year of fracture (p<0.0001). Stepwise regression yielded a predictive nonunion model with 26 significant explanatory variables (all, p≤0.003). Strength of this model was assessed using an area under the curve (AUC) calculation, and out-of-sample AUC=0.710. CONCLUSIONS: A logistic model predicted nonunion with reasonable accuracy (AUC=0.725). Within the Medicare population, nonunion patients were younger than patients who healed normally. Fracture was associated with increased risk of death within 1year of fracture (p<0.0001) in 14 different bones, confirming that geriatric fracture is a major public health issue. Comorbidities associated with increased risk of nonunion include past or current smoking, alcoholism, obesity or morbid obesity, osteoarthritis, rheumatoid arthritis, type II diabetes, and/or open fracture (all, multivariate p<0.001). Nonunion prediction requires knowledge of 26 patient variables but predictive accuracy is currently comparable to the Framingham cardiovascular risk prediction.

CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos.

The rapid development of CRISPR technology greatly impacts the field of genetic engineering. The simplicity in design and generation of highly efficient CRISPR reagents allows more and more researchers to take on genome editing in different model systems in their own labs, even for those who found it daunting before. An active CRISPR complex contains a protein component (Cas9) and an RNA component (small guide RNA [sgRNA]), which can be delivered into cells in various formats. Cas9 can be introduced as a DNA expression plasmid, in vitro transcripts, or as a recombinant protein bound to the RNA portion in a ribonucleoprotein particle (RNP), whereas the sgRNA can be delivered either expressed as a DNA plasmid or as an in vitro transcript. Here we compared the different delivery methods in cultured cell lines as well as mouse and rat single-cell embryos and view the RNPs as the most convenient and efficient to use. We also report the detection of limited off-targeting in cells and embryos and discuss approaches to lower that chance. We hope that researchers new to CRISPR find our results helpful to their adaptation of the technology for optimal gene editing.

Increased long-term mortality in patients with cardiovascular implantable electronic device infections.

BACKGROUND: Device infection is associated with increased mortality in patients receiving cardiovascular implantable electronic device (CIED) therapy. However, long-term mortality associated with CIED infections has not been systematically analyzed in larger studies. This study sought to determine the long-term mortality associated with CIED infection in a large cohort of Medicare beneficiaries. METHODS: We used a retrospective study design to analyze 3-year mortality in 200,219 Medicare fee-for-service patients admitted for CIED generator implantation, replacement, or revision between January 1, 2007 and December 31, 2007. Multivariate analysis adjusting for age, sex, race, and 28 comorbidities was performed to determine the relative risk (RR) of death in the 12 quarters following CIED infection. RESULTS: Patients with CIED infection, compared to device recipients without infection, had increased mortality that persisted for at least 3 years after the admission quarter for all device types: pacemakers (PMs: 53.8% vs 33%; P < 0.001), implantable cardioverter defibrillator (ICD: 47.7% vs 31.6%; P < 0.001), and cardiac resynchronization therapy-defibrillator (CRT-D: 50.8% vs 36.5%; P < 0.001). After adjusting for patient demographics and comorbidities, significantly increased RR of death following CIED infection persisted for at least 3 years following PM infection, and for at least 2 years with single- and dual-chamber ICD infection. CONCLUSIONS: CIED recipients who develop device infection have increased, device-dependent, long-term mortality even after successful treatment of infection. The etiology of this persistent increased risk of death associated with CIED infection is unknown and merits further investigation.

Comparison of mortality in women versus men with infections involving cardiovascular implantable electronic device.

Device infection is a complication of implantable cardioverter-defibrillator (ICD) therapy that significantly increases mortality. Risk factors associated with death and ICD infection are poorly understood. The purpose of this study was to identify patient characteristics associated with death after cardiovascular implantable electronic device (CIED) infection. This is a retrospective cohort study of 64,903 Medicare fee-for-service patients who received an ICD in 2007, including 1,855 with device infection. Long-term survival was significantly reduced with CIED infection (71.6% vs 85.0%, p <0.001). Regression analysis accounting for age, race, gender, and 28 co-morbidities identified only 2 patient characteristics associated with decreased long-term survival with CIED infection: female gender and human immunodeficiency virus/acquired immunodeficiency syndrome. In patients with CIED infection, women had substantially reduced long-term survival compared with men (67.3% vs 72.9%, p <0.02). The risk-adjusted hazard ratio for long-term mortality with device infection in women compared with that in men increased significantly from 0.86 (95% confidence interval [CI] 0.82 to 0.91) to 1.25 (95% CI 1.02 to 1.53), corresponding to a risk increase of >45%. Importantly, a substantial portion of this excess mortality occurred after the index admission for infection, when the hazard ratio for death in women compared with that in men increased from 0.86 (95% CI 0.82 to 0.91) to 1.20 (95% CI 0.96 to 1.51) with CIED infection, despite little gender difference in admission length of stay, disposition, and cost. In conclusion, women are significantly more likely than men to die with CIED infection. A substantial part of this excess mortality occurs after discharge. It will be important to identify and address the cause(s) of this gender difference in mortality.

Whole-rat conditional gene knockout via genome editing.

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

Genome-wide screen for miRNA targets using the MISSION target ID library.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3Î"TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

Mortality and cost associated with cardiovascular implantable electronic device infections.

BACKGROUND: Cardiovascular implantable electronic device (CIED) therapy can reduce morbidity and mortality, but this benefit can be diminished by CIED infection. Currently, there are limited published data on the mortality and cost associated with CIED infection. METHODS: We analyzed the risk-adjusted total and incremental admission mortality, long-term mortality, admission length of stay (LOS), and admission cost associated with infection in a retrospective cohort of 200 219 Medicare fee-for-service patients admitted for CIED generator implantation, replacement, or revision between January 1, 2007, and December 31, 2007. RESULTS: There were a total of 5817 admissions with infection. Infection was associated with significant increases in adjusted admission mortality (rate ratios, 4.8-7.7; standardized rates, 4.6%-11.3%) and long-term mortality (rate ratios, 1.6-2.1; standardized rates, 26.5%-35.1%), depending on CIED type. Importantly, approximately half of the incremental long-term mortality occurred after discharge. The adjusted LOS was significantly longer with infection (length of stay mean ratios, 2.5-4.0; standardized length of stay, 15.5-24.3 days), depending on CIED type. The standardized adjusted incremental and total admission costs with infection were $14 360 to $16 498 and $28 676 to $53 349, respectively, depending on CIED type. The largest incremental cost with infection was intensive care, which accounted for more than 40% of the difference. Adjusted long-term mortality rate and cost ratios with infection were significantly greater for pacemakers than for implantable cardioverter/defibrillators or cardiac resynchronization therapy/defibrillator devices. CONCLUSIONS: Infection associated with CIED procedures resulted in substantial incremental admission mortality and long-term mortality that varied with the CIED type and occurred, in part, after discharge. Almost half of the incremental admission cost was for intensive care.

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling.

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development.

Comparative academic performance of medical students in rural and urban clinical settings.

OBJECTIVE: To determine whether the academic performance of medical students learning in rural settings differs from those learning in urban settings. DESIGN: Comparison of results of assessment for 2 full cohorts and 1 part cohort of medical students learning in rural and urban settings in 2002 (209 students), 2003 (226 students) and 2004 (220 students), including results for each specialist rotation in the 3rd year and end-of-year examinations in the 2nd and 4th years. SETTING: University of Queensland School of Medicine, Brisbane. Students spent the whole 3rd year (of a 4-year graduate entry programme) conducting 5 specialist 8-week rotations in either the rural clinical division (rural students) or in Brisbane (urban students), all following the same curriculum and taking the same examinations. RESULTS: For the 2002 cohort there were no statistically significant differences in academic performance between rural and urban students. For the 2003 cohort the only significant difference was a higher score for rural students in the end of the 4th-year clinical skills examination (65.7 versus 62.3%, P = 0.025). For the 2004 cohort, rural students scored higher in the 3rd-year mental health rotation (79.3 versus 76.2%, P = 0.038) and lower in the medicine rotation (65.5 versus 68.6%, P = 0.037). CONCLUSION: Academic performance among students studying in rural and urban settings is comparable.

An Arabidopsis Fip1 homolog interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits.

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis Fip1 homolog is characterized. The gene for this protein resides on chromosome V and encodes a 1196-amino acid polypeptide. Yeast two-hybrid and in vitro assays indicate that the N-terminal 137 amino acids of the Arabidopsis Fip1 protein interact with poly(A) polymerase (PAP). This domain also stimulates the activity of the PAP. Interestingly, this part of the Arabidopsis Fip1 interacts with Arabidopsis homologs of CstF77, CPSF30, CFIm-25, and PabN1. The interactions with CstF77, CPSF30, and CFIm-25 are reminiscent in various respects of similar interactions seen in yeast and mammals, although the part of the Arabidopsis Fip1 protein that participates in these interactions has no apparent counterpart in other eukaryotic Fip1 proteins. Interactions between Fip1 and PabN1 have not been reported in other systems; this may represent plant-specific associations. The C-terminal 789 amino acids of the Arabidopsis Fip1 protein were found to contain an RNA-binding domain; this domain correlated with an intact arginine-rich region and had a marked preference for poly(G) among the four homopolymers studied. These results indicate that the Arabidopsis Fip1, like its human counterpart, is an RNA-binding protein. Moreover, they provide conceptual links between PAP and several other Arabidopsis polyadenylation factor subunit homologs.

Novel alternative splicing of mRNAs encoding poly(A) polymerases in Arabidopsis.

The Arabidopsis thaliana genome possesses four genes whose predicted products are similar to eukaryotic poly(A) polymerases from yeasts and animals. These genes are all expressed, as indicated by RT/PCR and Northern blot analysis. The four Arabidopsis PAPs share a conserved N-terminal catalytic core with other eukaryotic enzymes, but differ substantially in their C-termini. Moreover, one of the four Arabidopsis enzymes is significantly shorter than the other three, and is more divergent even within the conserved core of the protein. Nonetheless, the protein encoded by this gene, when produced in and purified from E. coli, possesses nonspecific poly(A) polymerase activity. Genes encoding these Arabidopsis PAPs give rise to a number of alternatively spliced mRNAs. While the specific nature of the alternative splicing varied amongst these three genes, mRNAs from the three "larger" genes could be alternatively spliced in the vicinity of the 5th and 6th introns of each gene. Interestingly, the patterns of alternative splicing vary in different tissues. The ubiquity of alternative splicing in this gene family, as well as the differences in specific mechanisms of alternative processing in the different genes, suggests an important function for alternatively spliced PAP mRNAs in Arabidopsis.

Pharmaceutical "charge compression" under the Medicare outpatient prospective payment system.

Analysis of the actual acquisition costs of a sample of pharmaceuticals demonstrates that payment rates for pharmaceutical therapies under the Medicare hospital outpatient prospective payment system (OPPS) are systematically biased against fully reimbursing high cost pharmaceutical therapies. Under the Centers for Medicare and Medicaid Services' (CMS') methodology, which assumes a constant markup, a bias in the cost estimate occurs when hospitals apply below average markups in establishing their charges for pharmaceutical products with above average costs. We developed a model of the relationship between product costs and charge markups. The logarithmic model shows that an increase in the acquisition cost per episode can be expected to lead to a reduction in the charge markup multiple. When markups for pharmaceuticals decline as acquisition cost increases, a rate-setting methodology that assumes a constant markup results in reimbursement for higher cost products that can be far below acquisition cost. The incentives in the payment system could affect site of care choices and beneficiary access.

Nursing practice and the definition of human death.

This paper aims to highlight the gap in nursing literature of discussion of the definition of human death--to show that nurses should engage in such discussion. For the nursing role in the care of brain dead patients and their relatives may unwittingly promote and foster a definition of human death which is fundamentally flawed. A person can be warm, pink, have an independently beating heart and be breathing, yet still be diagnosed as brainstem dead. Nursing literature which discusses issues surrounding brain death (as opposed to brain death itself), proposes that nurses should suppress any reservations which they may have in accepting that a patient with the characteristics described is dead; and that they should try to allay any reservations which relatives of such dead patients might have. But what if the concept of brainstem death is flawed? Surely, as accountable professionals, nurses should not accept the role just referred to without satisfying themselves that the concept of brainstem death is coherent and robust. This paper tries to show that, on examination, this is not the case. The definition of human death which guides practice in the UK and elsewhere is fundamentally flawed. Instead of suppressing their own intuitions, and the intuitions of patients' relatives in the management of patients diagnosed as brainstem dead, nurses should critically examine the definition of death which currently informs clinical practice. Our conclusion is that this definition is false.