AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i.

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.

The physician assistant: Shifting the Paradigm of European medical practice?

INTRODUCTION: Physician Assistants are medical care providers working under supervision and/or in collaboration with a medical doctor. The Physician Assistant profession has its origin in the United States, but in the last decade has also reached other nations to overcome medical staffing issues. With little summarized literature available, the aim of this study is to portray the Physician Assistant movement in Europe. METHODS: A literature search was conducted in Academic Search Premier, CINAHL, ERIC and MEDLINE databases. In addition, European PA educational programs, professional associations, and local experts on the PA profession were queried. RESULTS: Currently, in Europe there are three countries in which physician assistants are trained and are working. The educational models of physician assistant training in the United Kingdom, Germany and the Netherlands differ, as do the degrees offered by the training institutions. CONCLUSIONS: There is scant literature about physician assistant training and practice in Europe available in the common scientific databases. The paucity of literature makes it difficult for an outsider to observe the developments and to value the impact of a new profession on national health systems. Further high-quality research is needed to adequately characterize physician assistant education and implementation across Europe.

The att locus of Rhodococcus fascians strain D188 is essential for full virulence on tobacco through the production of an autoregulatory compound.

The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified. The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence. To gain insight into the function of the att locus, sequence and expression analyses were performed. The att locus contains nine open reading frames homologous to arginine and beta-lactam biosynthetic genes. att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR-type transcriptional regulator. The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts. Because the induction of the fas locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R. fascians.

The plant pathogen Rhodococcus fascians colonizes the exterior and interior of the aerial parts of plants.

Rhodococcus fascians is a plant-pathogenic bacterium that causes malformations on aerial plant parts, whereby leafy galls occur at axillary meristems. The colonization behavior on Nicotiana tabacum and Arabidopsis thaliana plants was examined. Independent of the infection methods, R. fascians extensively colonized the plant surface where the bacteria were surrounded by a slime layer. R. fascians caused the collapse of epidermal cells and penetrated intercellularly into the plant tissues. The onset of symptom development preceded the extensive colonization of the interior. The meristematic regions induced by pathogenic strain D188 were surrounded by bacteria. The nonpathogenic strain, D188-5, colonized the exterior of the plant equally well, but the linear plasmid (pFiD188) seemed to be involved in the penetration efficiency and colonization of tobacco tissues.

The fas locus of the phytopathogen Rhodococcus fascians affects mitosis of tobacco BY-2 cells.

The effect of Rhodococcus fascians, the causal agent of leafy gall disease, on the mitotic behavior of synchronized tobacco Bright Yellow-2 (BY-2) cells was investigated. Incubation of aphidicolin-synchronized BY-2 cells with R. fascians cells specifically resulted in a broader mitotic index peak, an effect that was linked to an intact and expressed fas virulence locus. The obtained results pointed towards an effect of R. fascians on the prophase of mitosis. The relevance of these results to the virulence of the bacterium is discussed.

Targeting quality improvement activities for depression. Implications of using administrative data.

BACKGROUND: Large health care organizations may use administrative data to target primary care patients with depression for quality improvement (QI) activities. However, little is known about the patients who would be identified by these data or the types of QI activities they might need. We describe the clinical characteristics and outcomes of patients identified through administrative data in 2 family practice clinics. METHODS: Patients with depression aged 18 to 65 years were identified through review of encounter/administrative data during a 16-month period. Patients agreeing to participate (N=103) were interviewed with the Primary Care Evaluation of Mental Disorders questionnaire and completed the Depression Outcomes Modules (with an embedded Medical Outcomes Short Form-36 [SF-36]), Symptom Check List-25 (SCL-25), and Alcohol use Disorders Identification Test. Follow-up assessments were completed by 83 patients at a median of 7 months. RESULTS: A large majority of identified patients (85%) met full criteria for a Diagnostic and Statistical Manual of Mental Disorders depressive disorder; those not meeting criteria usually had high levels of symptoms on the SCL-25. Seventy-seven percent of the patients reported recurrent episodes of depressed mood, and 60% reported chronic depression. Although most improved at follow-up, they continued to have substantial functional deficits on the SF-36, and 60% still had high levels of depressive symptoms. CONCLUSIONS: QI programs that use administrative data to identify primary care patients with depression will select a cohort with relatively severe, recurrent depressive disorders. Most of these patients will receive standard treatments without QI interventions and will continue to be symptomatic. QI programs targeting this population may need to offer intensive alternatives rather than monitor standard care.

Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition.

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4'-phosphopantetheine prosthetic group by holo-ACP synthase. The Km value for NodF is 61 microM, as compared to 2 microM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for beta-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E. coli acpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1-33 of AcpP fused to amino acids 43-93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes).

The effects of insurance coverage on the quality of prenatal care.

OBJECTIVE: To compare the quality of prenatal care provided to patients with traditional fee-for-service, health maintenance organization, and Medicaid insurance using an evidence-based, community-derived prenatal care guideline. DESIGN: Retrospective cohort study. SETTING: Seven private and hospital-based prenatal care sites in a suburban county in southeast Michigan. PATIENTS: A stratified random sample of 267 patients (93 with Medicaid, 92 with health maintenance organization, and 82 with fee-for-service insurance) receiving prenatal care from community physicians (obstetricians-gynecologists and family practitioners) between January 1, 1991, and December 31, 1992. MAIN OUTCOME MEASURE: Adherence to explicit prenatal care criteria as measured by an evidence-based prenatal care guideline developed by a community panel. "Quality scores" were compared across groups in 4 areas: performance of prenatal screening procedures or tests, visit-based screening, substance use screening, and clinician management of abnormal clinical findings. RESULTS: Patients with Medicaid insurance presented for prenatal care significantly later in pregnancy (14.5 vs 10.5 weeks, P < .01). No significant differences were seen between groups in quality scores for screening tests, clinician management of abnormal clinical findings, visit-based screening, or substance use screening. The overall similarity in quality scores did obscure some significant differences in adherence to individual criteria, particularly in the area of screening tests. Significantly more patients with Medicaid were screened for genital infection (P < .001) and fewer for gestational diabetes (P < .001) or anemia (P < .001) than patients in the other 2 groups. CONCLUSIONS: Although patients with Medicaid presented for prenatal care later in pregnancy and received a different "package" of screening tests than the other 2 groups, there was no overall measurable difference in the quality of prenatal care provided to patients with Medicaid, health maintenance organization, and fee-for-service insurance. Clinicians may have altered screening protocols based on preexisting perceptions of patient risk. Although summary quality measures are a promising tool for comparative research, they provide an incomplete picture of the quality of the prenatal care process and must be interpreted with caution.

Acyl-acyl carrier protein is a donor of fatty acids in the NodA-dependent step in biosynthesis of lipochitin oligosaccharides by rhizobia.

NodA controls transfer of a fatty acid in the biosynthesis of lipochitin oligosaccharides by rhizobia. In an in vitro assay, we used de-N-acetylated chitin oligosaccharides substituted with an O-acetyl moiety as acyl acceptor substrates. We show that acyl-acyl carrier protein is used as a donor in NodA-directed fatty acid transfer.

Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis.

In the biosynthesis of lipochitin oligosaccharides (LCOs) the Rhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence of nodA in the nodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which the nodA, nodB and nodC genes are separately present on different plasmids. Efficient nodulation was only obtained if nodC was present on a low-copy-number vector. Our results confirm the notion that nodA of Rhizobium leguminosarum biovar viciae is essential for nodulation on Vicia. Surprisingly, replacement of R. l. by viciae nodA by that of Bradyrhizobium sp. ANU289 results in a nodulation-minus phenotype on Vicia. Further analysis revealed that the Bradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of the R. l. by, viciae nodFE-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion that nodA is a common nod gene should be revised.

Lipid specificity for membrane mediated partial unfolding of cytochrome c.

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experiments with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.

Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv. viciae is essential for its biological function.

A system for testing the role of the Rhizobium nodF gene in the production of host-specific lipochitin oligosaccharides and in nodulation was developed. We show that a mutant nodF gene, in which the codon for serine residue 45 was changed to that for threonine, still expresses NodF, which, however, is no longer functional.

Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase.

When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034-2040). In Madin-Darby canine kidney (MDCK) cells incubated at 10 degrees C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no C6-NBD-PE could be depleted from the basolateral cell surface by the addition of bovine serum albumin to the medium. Preincubation of the cells with [3H]choline for 2 h at 37 degrees C prior to C6-NBD-PA addition at 10 degrees C labeled non-depletable C6-NBD-PC with a specific activity of > 10 times that of the depletable C6-NBD-PC on the basolateral cell surface, indicating that the latter had not been synthesized by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-PA as substrate for both intracellular and surface C6-NBD-PC synthesis. In addition, C6-NBD-PC synthesis on the cell surface was independent of the location of the C6-NBD-chain on the 1- or 2-position, indicating that the reaction occurred by transfer of phosphorylcholine. Using C6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovered on the basolateral but not on the apical cell surface. The conversion of PC plus ceramide to DG and SM on the basolateral MDCK cell surface suggests that the synthesis of C6-NBD-PC on this surface occurred via the reverse reaction of a SM synthase. Indeed, the surface C6-NBD-PC synthesis was reduced to 40-50% by addition of C6-NBD-ceramide or hydrolysis of cell surface SM by exogenous neutral sphingomyelinase. Since DG activates protein kinase C and ceramide indirectly inhibits this kinase but activates other kinase(s) and phosphatase(s), the phosphocholine transferase at the cell surface may have a regulatory role in signal transduction.