Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing.

OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.

Prospective evaluation of unmet needs of rural and aboriginal cancer survivors in Northern British Columbia.

BACKGROUND: The unmet needs of cancer survivors in rural, remote, and aboriginal communities are largely unexplored. We explored potential differences between rural survivors (rss) in 4 general population (gp) and 4 First Nations (fn) communities. METHODS: We approached 4 gp and 4 fn rs communities to participate in a mixed-methods project. Participants completed the Hospital Anxiety and Depression Scale (hads) and the Survivor Unmet Needs Survey (suns) and provided demographic information. Each question on the suns can be scored from 0 to 4, with 0 representing "no unmet need" and 4 representing "very high unmet need." A directed approach to content analysis of focus group and interview data was used to triangulate the hads and suns results. RESULTS: We prospectively accrued 23 fn rss and 56 gp rss for this study. More fn rss had borderline or abnormal anxiety (5% vs. 21%, p = 0.02). Compared with gp rss, fn rss had higher unmet needs scores in all categories: Information (2.29 vs. 0.8, p < 0.001), Work and Financial (1.66 vs. 0.5, p < 0.001), Access and Continuity of Health Care (1.83 vs. 0.44, p < 0.001), Coping and Sharing (2.22 vs. 0.62, p < 0.001), and Emotional (2.12 vs. 0.63, p < 0.001). The qualitative findings provided examples and insight into the unmet needs experienced by rss. CONCLUSIONS: First Nations rss had significantly higher anxiety and unmet needs compared with their gp rs counterparts. In addition, different qualitative themes were identified in the groups. Our findings support the development of tailored approaches to survivorship for these populations.

mRNA poly(A) tail, a 3' enhancer of translational initiation.

To evaluate the hypothesis that the 3' poly(A) tract of mRNA plays a role in translational initiation, we constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs were translated, degraded, recruited into polysomes, and assembled into messenger ribonucleoproteins or intermediates in the translational initiation pathway. Relative to mRNAs which were polyadenylated, we found that nonpolyadenylated [poly(A)-]mRNAs had a reduced translational capacity which was not due to an increase in their decay rates, but was attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affected a late step in translational initiation, was distinct from the phenotype associated with cap-deficient mRNAs, and resulted in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibited translation from capped polyadenylated mRNAs but stimulated translation from capped poly(A)- mRNAs. We suggest that the presence of a 3' poly(A) tail may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5' end.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced erythroleukemia: relationship of inactivation to dominant transforming alleles.

The Friend erythroleukemia virus complex contains no cell-derived oncogene. Transformation by this virus may therefore involve mutations affecting cellular gene expression. We provide evidence that inactivating mutations of the cellular p53 gene are a common feature in Friend virus-induced malignancy, consistent with an antioncogene role for p53 in this disease. We have shown that frequent rearrangements of the p53 gene cause loss of expression or synthesis of truncated proteins, whereas overexpression of p53 protein is seen in other Friend cell lines. We now demonstrate that p53 expression in the latter cells is also abnormal, as a result of missense mutations in regions encoding highly conserved amino acids. Three of these aberrant alleles obtained from cells from different mice were cloned and found to function as dominant oncogenes in gene transfer assays, supporting the view that certain naturally occurring missense mutations in p53 confer a dominant negative phenotype on the encoded protein.

Deletion of 5'-coding sequences of the cellular p53 gene in mouse erythroleukemia: a novel mechanism of oncogene regulation.

The p53 gene is rearranged in an erythroleukemic cell line (DP15-2) transformed by Friend retrovirus. Here, we characterize the mutation and identify a deletion of approximately equal to 3.0 kilobases that removes exon 2 coding sequences. The gene is expressed in DP15-2 cells and results in synthesis of a 44,000-dalton protein that is missing the N-terminal amino acid residues of p53. The truncated protein is unusually stable and accumulates to high levels intracellularly. Moreover, it appears to have undergone a change in conformation as revealed by epitope mapping studies. This study represents the first description of an altered p53 gene product arising by mutation during neoplastic progression and identifies a region in the p53 protein molecule that plays a role in determining p53 stability in vivo.

Relationship between incorporation of 9-beta-D-arabinofuranosyladenine in L 1210 DNA and cytotoxicity.

We have used cesium sulfate density gradient centrifugation to monitor the incorporation of 9-beta-D-arabinofuranosyladenine (ara-A) into L1210 cellular nucleic acids. The results demonstrate the specific incorporation of ara-A in L1210 DNA. We have also found a highly significant relationship between the formation of ara-A incorporated into DNA and loss of clonogenic survival. This relationship was maintained when using ara-A in the presence of the adenosine deaminase inhibitor deoxycoformycin. Furthermore, treatment with increasing concentrations of ara-A resulted in a greater proportion of ara-A residues at the 3'-terminus, consistent with this agent providing a poor primer terminus for elongating DNA strands. These findings are similar to those obtained previously with 1-beta-D-arabinofuranosylcytosine and suggest that the incorporation of arabinofuranosyl derivatives in DNA is one mechanism responsible for cell lethality.

Loss of a highly conserved domain on p53 as a result of gene deletion during Friend virus-induced erythroleukemia.

We have investigated a mutation in the p53 gene leading to expression of a truncated 46,000-dalton protein in a Friend virus-induced erythroleukemia cell line. cDNA sequence analysis revealed a deletion of nucleotide sequences in exon 7 and part of exon 8; 17 additional nucleotides, derived from intron 6, were present in the cDNA and served to maintain the reading frame of the encoded protein. Comparison with p53 protein from other species indicated that the region of the molecule missing in p46 included a highly conserved region. In addition, p46 failed to bind SV40 large T antigen in vitro under conditions which promoted binding of p53 to large T. It seems likely, therefore, that an important functional property of p53 may be affected by the mutation.

Turning down the heat: new routes to inhibition of inflammatory signaling by prostaglandin H2 synthases.

Many nonsteroidal anti-inflammatory drugs act by inhibiting the cyclooxygenase activity of prostaglandin H2 synthase (PGHS), a key enzyme in the biosynthesis of prostaglandins. Gastric toxicity remains a serious problem with the current drugs, however. Recent advances in the understanding of PGHS now suggest two possible approaches to producing drugs with fewer side effects.

Molecular characterization of a chromosome translocation breakpoint t(11;14)(p13;q11) from the cell line KOPT-K1.

Recurrent chromosome translocations involving 11p13 and 14q11 are found in 5-10% of cases of T-ALL. The gene involved in the translocation on chromosome 14 is the T cell antigen receptor alpha or delta. The putative oncogene on chromosome 11 is rhombotin 2 (RBTN2)/translocated in T cell gene 2 (ttg-2), a member of the LIM family of proteins. In this paper we characterize a cell line KOPT-K1 that has a t(11;14)(p13;q11). The breakpoint on chromosome 11 involves an Alu-rich region with the break occurring between two Alu sequences on chromosome 11. In addition, approximately 70 bases from the break on chromosome 11 is a tetranucleotide repeat. Whether either of these structures played a role in the translocation is not known. No heptamer or nonamer sequences, implicated in other rearrangements were found near the breakpoint. The breakpoint on chromosome 11 maps more centromeric than previous translocations of this region. Despite this the RBTN2 gene is highly expressed in KOPT-K1. This cell line will be useful for investigating the role of RBTN2 in leukemogenesis and the mechanism by which the translocation alters the expression of RBTN2.

Induction of differentiation of human myeloid leukemia cells by inhibitors of DNA synthesis.

The HL-60 human leukemic promyelocyte can be induced to mature into terminally differentiated cells using certain nucleosides and chemotherapeutic agents. The mechanisms responsible for this induction of differentiation, however, remain unclear. We have monitored the effects of two specific inhibitors of DNA synthesis to determine whether slowing of DNA polymerization can induce HL-60 differentiation. The results demonstrate that cytosine arabinoside (ara-C) induces nonspecific esterase activity in HL-60 cells and increases surface expression of the monocyte antigen MY-4. The results also demonstrate that aphidicolin, an inhibitor of DNA polymerase which is not incorporated in DNA, induces similar phenotypic changes. The induction of differentiation by both agents was accompanied by loss of clonogenic potential as monitored by colony formation in methylcellulose. These observations suggest that terminal differentiation of HL-60 cells can be induced by drugs known to inhibit DNA synthesis.

Tales of poly(A): a review.

Until recently, evidence to support a translational role for the 3'-poly(A) tract of eukaryotic mRNAs has been mostly indirect, including: a correlation between the adenylation status of individual mRNAs and their translatability in vivo or in vitro, the demonstration that exogenously added poly(A) is a potent competitive inhibitor of the translation of poly(A)+mRNA, but not poly(A)-mRNAs in vitro, and a correlation between the abundance and stability of poly(A)-binding proteins (PABPs) and the rate of translational initiation in vivo. However, more recent studies demonstrate directly that poly(A)+mRNAs can initiate translation more efficiently than poly(A)-mRNAs, and indicate that this effect is: (i) targeted to the formation of 80S initiation complexes, and (ii) likely to be mediated by the cytoplasmic PABP. We suggest that the 3'-poly(A) tail should be considered a translational enhancer which may stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate transcriptional initiation.

A growing family of receptor genes for lysophosphatidic acid (LPA) and other lysophospholipids (LPs).

A missing component in the experimental analysis of cell signaling by extracellular lysophospholipids such as lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) has been cloned receptors. Through studies on the developing brain, the first such receptor gene (referred to as vzg-1) was identified, representing a member of the G-protein coupled receptor (GPCR) super family (1). Here we review the neurobiological approach that led to both its cloning and identification as a receptor for LPA, along with related expression data. Summarized sequence and genomic structure analyses indicate that this first, functionally identified receptor is encoded by a member of a growing gene family that divides into at least two subgroups: genes most homologous to the high-affinity LPA receptor encoded by vzg-1, and those more homologous to an orphan receptor gene edg-1 that has recently been identified as a S1P receptor. A provisional nomenclature is proposed, based on published functional ligand actions, amino acid composition and genomic structure whereby the receptors encoded by these genes are referred to as lysophospholipid (LP) receptors, with subgroups distinguished by letter and number subscripts (e.g., LPA1 for Vzg-1, and LPB1 for Edg-1). Presented expression data support the recently published work indicating that members of the LPB1 subgroup are receptors for the structurally-related molecule, S1P. The availability of cloned LP receptors will enhance the analysis of the many documented LP effects, while their prominent expression in the nervous system indicates significant but as yet unknown roles in development, normal function, and neuropathology.

Effect of ara-A on differentiation and proliferation of HL-60 cells.

The HL-60 human leukemic promyelocytic cell line can be induced to mature into terminally differentiated cells using certain chemotherapeutic agents. We have recently demonstrated that two inhibitors of DNA synthesis, cytosine arabinoside (ara-C) and aphidicolin, can induce HL-60 differentiation with the appearance of monocytic markers. These pyrimidine antimetabolites may have affected DNA methylation patterns and resulted in altered gene expression, or the differentiated phenotype may have occurred by inhibition of DNA replication. Consequently, we have extended these studies by using the purine analog, adenine arabinoside (ara-A), which also acts as an inhibitor of DNA synthesis. The results demonstrate that ara-A also induces HL-60 non-specific esterase activity and enhances expression of myeloid cell surface antigens, MY-4 and MO-1. The induction of a differentiated phenotype by ara-A occurs after partial inhibition of DNA synthesis, a finding similar to that observed with ara-C and aphidicolin. These observations indicate that purine, as well as pyrimidine analog inhibitors of DNA polymerization can induce differentiation of HL-60 cells along a monocytic lineage. These findings may be relevant to recent clinical trials that have employed low doses of ara-C in an attempt to induce differentiation of malignant hematopoietic cells.

Novel intracellular signaling function of prostaglandin H synthase-1 in NF-kappa B activation.

Many potent nonsteroidal antiinflammatory drugs (NSAIDs) exert their effects by inhibiting the cyclooxygenase activity of prostaglandin H synthase-1 (PGHS1, thus disrupting prostaglandin biosynthesis. However, these drugs do not block the activation of NF-kappa B, an inducible transcription factor which regulates numerous inflammation-related genes. Here we demonstrate that PGHS1 peroxidase, a NSAID-insensitive activity of PGHS1, mediates NF-kappa B activation through an intracellular reactive oxygen signaling pathway. Overexpression of PGHS1 strongly potentiated NF-kappa B activation by phorbol esters and dramatically elevated the generation of intracellular reactive oxygen species (ROS) in response to low concentrations of t-butyl peroxide. Both functions were dependent on PGHS1 peroxidase activity and could be suppressed by the potent antioxidant pyrrolidine dithiocarbamate. In contrast, elimination of PGHS1 cyclooxygenase activity by NSAIDs or site-directed mutagenesis failed to block ROS production or NF-kappa B activation. Thus, PGHS1 peroxidase serves an intracellular signaling function leading to NF-kappa B activation, separable from its role in prostaglandin synthesis.

Nursing facilities and managed care.

OBJECTIVE: To examine the extent to which Illinois nursing facilities have developed relationships with other healthcare providers, particularly managed care organizations (MCOs). STUDY DESIGN: A cross-sectional survey of nursing facilities designed to determine: 1) relationship objectives; 2) obstacles to developing relationships; 3) currently available services; 4) staffing for these services and; 5) nursing facility approaches to networking. The survey was sent to a census sample of 867 nursing facilities serving the elderly in Illinois. Descriptive and multivariate logistic regression analyses of relationships determined to be formal/risk-sharing were performed. STUDY POPULATION: The sample included 523 Illinois nursing facilities. A total response rate of 60% was achieved (523/867). RESULTS: Higher strategic goals, urban location, nonprofit ownership status, higher percentages of private pay and/or Medicare clients (vs Medicaid), and provision of home care and subacute services were all significant predictors of formal or risk-sharing relationships with MCOs. CONCLUSIONS: Facilities with more relationships and higher goals have more formal/risk-sharing relationships with MCOs. Facilities in urban areas have more relationships, likely due to the fact that rural facilities have fewer options and operate in different markets. In addition, nursing facilities rely on Medicare referrals from hospitals, and these Medicare patients, especially those in urban areas, are increasingly controlled by MCOs.

Building a professional environment in long-term care. The role of clinical career development.

1. To be successful, an organizational career development program must include a differentiation of the responsibilities for which the various parties (employer, employee, career counselor) will be held accountable. 2. Project outcomes revealed that the career mobility program was attractive to nursing personnel and facility management personnel alike. However, it was more attractive to nonlicensed than licensed personnel. 3. Of the staff who participated and were promoted, the majority remained in their jobs. The program was most successful in enhancing retention with personnel who received within-level promotions. 4. The process of career development requires collaboration and support from all levels of leadership and staff throughout the organization. A career development program, including a career mobility program with a strong career counseling component, can serve as a catalyst in professionalizing the long-term care work environment.

After-hours call in a primary care nursing practice.

This paper describes the recorded concerns of clients and the management activity of Master's prepared nurse providers in an urban, ambulatory, university affiliated primary care nursing practice during after-hours call. Retrospective record review was used to collect data. Pain was the most common client concern, and the abdomen the most frequently identified pain site. The majority of concerns expressed by clients were minor health problems appropriate for telephone management. The most frequent interventions used were nursing measures such as comfort, supported and reassurance. Finally, the nurse providers in this group practice managed the majority of calls with nursing interventions.

Meeting the challenge of unscheduled outpatient visits.

Cancer patients receive multimodal therapy and treatments on an ongoing basis in free-standing cancer centers, infusion centers, and oncology offices and at home. To serve those who require unscheduled evaluation for treatment effects, one hospital developed a program to receive those visits on an inpatient cancer unit.

A professional practice model: two key components.

Professional practice models provide decentralized approaches to nursing practice. The components of such a model should include a primary nursing delivery system, decentralized decision making, salary compensation, self-scheduling and quality circles. This article describes two of the key components of the model--salaried compensation and self-scheduling--on a unit in one agency.

Effect of single post-ovulatory administration of levonorgestrel on gene expression profile during the receptive period of the human endometrium.

The hypothesis that levonorgestrel (LNG) used as an emergency contraceptive interferes with endometrial receptivity remains unproven. We compared the endometrial gene expression profile during the receptive period after administering a single dose of LNG 1.5 mg or placebo on day 1 of the luteal phase. An endometrial biopsy was done on day LH+7 or LH+8 and samples were taken from seven volunteers, each one contributing with one cycle treated with placebo and another with LNG. The expression of 20 383 genes was determined using cDNA microarrays. Real-time RT-PCR was used 1) to confirm the differences found in DNA microarray analysis and 2) to determine the effect of LNG on transcript levels of C3, C4BPα, COX2, MAOA, S100A4, and SERPINB9, known to be upregulated during receptivity, and on cPLA2α, JAK1, JNK1, CTSL1, and GSTP1, known to respond to mifepristone. Additional endometrial biopsies were done during the pre-receptive (LH+3) and receptive (LH+7) period and samples were taken from eight untreated volunteers in order to determine the changes associated with acquisition of receptivity of 14 genes. Mean levels of PAEP, TGM2, CLU, IGF2, and IL6ST mRNAs increased after administering LNG while those of HGD, SAT1, EVA1, LOC90133, ANXA1, SLC25A29, CYB5A, CRIP1, and SLC39A14 decreased. Except for the level of ANXA1 transcript, all changes remained within the range observed in untreated controls, and none of the transcripts responding to mifepristone changed in response to LNG. Post-ovulatory administration of LNG caused minimal changes in gene expression profiling during the receptive period. Neither the magnitude nor the nature or direction of the changes endorses the hypothesis that LNG interferes with endometrial receptivity.