Reducing short-acting beta-agonist use in asthma: Impact of national incentives on prescribing practices in England and the findings from SENTINEL Plus early adopter sites.

Short-acting beta-agonist (SABA) over-use in asthma is harmful for patients and the environment. The Investment and Impact Fund (IIF) 2022/2023 financially rewarded English primary care networks that achieved specific targets, including reducing SABA over-use (RESP-02) and lowering the mean carbon footprint per salbutamol inhaler prescribed (ES-02). SENTINEL Plus is a co-designed quality improvement package that aims to improve asthma outcomes and reduce asthma's environmental impact by addressing SABA over-use. We investigated the impact of (i) the IIF incentives and (ii) SENTINEL Plus implementation on asthma prescribing. Using Openprescribing.net data, we demonstrate that IIF 2022-2023 had no significant impact on the total number of SABA prescribed in England (25,927,252 during 12-months pre- and 25,885,213 12-months post-IIF; 0.16% decrease; p=NS), but lower carbon footprint SABA inhaler use increased (Salamol™ prescribing increased from 5.1% to 19% of SABA prescriptions, p < 0.01). In contrast, SENTINEL Plus sites significantly reduced SABA prescribing post-implementation (5.43% decrease, p < 0.05).

Molecular analysis of Peptostreptococcus micros isolates from patients with periodontitis.

BACKGROUND: Recent studies provide strong evidence implicating Peptostreptococcus micros in the pathogenesis of various oral infections, including oropharyngeal abscesses and periodontal disease. To date, very little is known regarding the role of P. micros in periodontal disease. Therefore, a genetic analysis was initiated to differentiate among strains of P. micros infecting periodontal patients. METHODS: Sixty DNA samples of P. micros isolated from 15 patients with periodontal disease were evaluated. Arbitrarily primed polymerase chain reactions (AP-PCR) were performed using primer 3 (AGTCAGCCAC) and primer 13 (CAGCACCCAC). The PCR products were analyzed by gel electrophoresis. RESULTS: The primers produced several unique patterns among the strains tested. Primer 3 resulted in 30 different patterns, whereas primer 13 resulted in 31 different patterns, which were distinct from those seen with primer 3. In 8 of 15 patients, the PCR profile was identical for all isolates cultured from that patient, indicating a clonal infection. In 4 of 15 patients, 2 different genotypes were identified. In the remaining 3 patients, all isolates cultured from these patients exhibited a unique genotype. CONCLUSIONS: While P. micros appears to be heterogeneous throughout a population of periodontal patients, each patient is, for the most part, infected with a limited number of genotypes. These results demonstrate the genetic diversity of P. micros and the usefulness of AP-PCR for future epidemiological studies in understanding the role P. micros plays in periodontal disease pathogenesis.

An apparent case of human T-cell lymphotropic virus type II (HTLV-II)-associated neurological disease: a clinical, molecular, and phylogenetic characterisation.

Several studies have reported an association between HTLV-II and a neurological condition which has come to be called HTLV-II-associated myelopathy and is similar, in some cases, to HTLV-I-associated myelopathy. To further explore the establishment of an etiological link between this virus and neurological disease, we determined the HTLV status of three individuals, one of which presented with symptoms of progressive ataxia. Since the patient with neurological disease and her husband were HTLV-II positive, we had the potential to study one of few cases of an HTLV-II-associated neurological disorder, and the first case in Canada. However, although the individual with the neurological disease was HTLV-II positive, we discovered that her brother, who displays the same clinical symptoms, was not positive for either HTLV-II or HTLV-I. Thus, disease association with HTLV-II became unsupportable. We present here, nevertheless, the first sequence and phylogenetic analysis of an HTLV-II isolate in Canada. This study suggests that cases of HTLV-II and neurological disease must be carefully investigated before any etiological conclusions can be made.

Structural organization of the human TOP2A and TOP2B genes.

Eucaryotic topoisomerase II is an essential nuclear enzyme involved in processes such as chromosome condensation, chromatid separation, and in the relief of torsional stress that occurs during DNA transcription and replication. In cells from vertebrate species, there are two forms of the enzyme, designated alpha and beta. Human topoisomerase IIalpha (TOP2A) is encoded by the TOP2A gene on chromosome 17q21-22, and human topoisomerase IIbeta (TOP2B) is encoded by the TOP2B gene on chromosome 3p24. The protein products of these two genes are important cellular targets of several drugs widely used in the treatment of many human cancers, and a variety of mutations in TOP2A have been associated with the development of drug resistance. In the present study, we have defined the intron-exon structures of TOP2A and TOP2B. TOP2A is approx. 30kb whereas TOP2B is at least 49kb. TOP2A and TOP2B contain 35 and 36 exons, respectively, and both genes contain a high proportion of class 0 introns. Alignment of the amino-acid sequences of the two proteins indicates that the intron-exon organization of the two genes is highly conserved, except for the regions encoding the extreme NH2 and COOH termini of the proteins. These findings suggest strongly that the vertebrate isoforms evolved by duplication of an ancestral gene. Mutations in TOP2A associated with drug resistance show clustering in exons 12, 13, 19-21 and 34-35. Knowledge of the genomic organization of TOP2A and TOP2B will be useful for detection of mutations in clinical samples from patients with drug-resistant malignant disease.

Interdisciplinary rural health training for health professional students: strategies for curriculum design.

Interdisciplinary rural health program offer a promising solution to the challenge of preparing graduates for rural practice, with the ultimate goal of promoting better health care for rural populations. This article focuses on the three-year experience of a model interdisciplinary rural health curriculum implemented in eastern North Carolina. Ten strategies are presented as a framework for the design and implementation of an effective practice-based curriculum for interdisciplinary rural health training. Allied health educators should examine existing curriculum models to build upon their strengths and explore new models to meet evolving delivery system and consumer needs.

Bipartite nuclear localization signals in the C terminus of human topoisomerase II alpha.

DNA topoisomerase II alpha is the intracellular target for several important chemotherapeutic agents, and drug-resistant human tumor cell lines have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal (NLS) sequences in this region using a modified definition of the motif, and in the present study, we have expressed five of these as fusion proteins with beta-galactosidase. Only one sequence (spanning amino acids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bipartite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basic domain with glutamine resulted in a fusion protein that localized inefficiently to the nucleus, indicating that all three basic residues in this domain are necessary. Our results confirm that a broader definition is required to detect all potential bipartite NLS motifs in a polypeptide sequence, although functional tests are still essential for identification of those sequences actually capable of directing nuclear localization.

Long-term results of laser treatment in the ocular histoplasmosis syndrome.

OBJECTIVE: To determine the long-term visual outcome, rate of persistent choroidal neovascularization, and rate of recurrent choroidal neovascularization in eyes undergoing laser photocoagulation for choroidal neovascularization secondary to ocular histoplasmosis syndrome. DESIGN AND PATIENTS: One hundred one eyes with 5 to 16 years of follow-up that presented with choroidal neovascularization secondary to ocular histoplasmosis were retrospectively evaluated. Patients were grouped according to location of choroidal neovascularization and assignment to observation or laser photocoagulation. MAIN OUTCOME MEASURES: Visual acuity outcome and loss for all groups were compared. The rates of persistent and recurrent choroidal neovascularization for the treated eyes were also evaluated. RESULTS: Visual acuity of 20/40 or better was obtained in 71% of eyes with treated extrafoveal choroidal neovascularization and 68% with treated juxtafoveal choroidal neovascularization. Recurrent choroidal neovascularization was observed in 23% of treated eyes during a mean follow-up of 9.6 years. CONCLUSION: Results support the long-term benefit of photocoagulation and need for careful follow-up.

Characterization and expression of a gene encoding serine tRNA5 from Escherichia coli.

The genes for translational components frequently are located together on the Escherichia coli genome. We have reported previously that the gene for a serine tRNA lies directly downstream from infA, the gene encoding initiation factor IF1. Here we characterize this tRNA gene, named serW. The serW gene expresses a minor form of serine tRNA(GGA) which recognizes the most frequently used serine codons, UCC and UCU. Two promoters were identified by S1 nuclease mapping: P1, which lies about 72 bp upstream from the structural gene; and P2, which lies about 35 bp upstream. Expression from P1 and P2 is comparable under conditions of rapid growth. The P2 promoter is followed by a GC-rich element characteristic of promoters regulated by ppGpp. A putative hairpin structure followed by a stretch of U residues about 25 nucleotides following the mature tRNA sequence resembles a rho-independent termination signal. The upstream gene, infA, is followed by a transcriptional terminator, but S1 mapping shows considerable readthrough. This serW expression appears to rely both on its own promoters and on promoters further upstream. The downstream gene, encoding an unidentified protein of about 100 kDa, is expressed in the opposite orientation and also is followed by a termination signal. Therefore serW is expressed both as a monocistronic gene and in combination with infA.

Translation initiation factor IF1 is essential for cell viability in Escherichia coli.

Translation initiation factor IF1 is a highly conserved element of the prokaryotic translational apparatus. It has been demonstrated earlier that the factor stimulates in vitro the initiation phase of protein synthesis. However, no mutation in its gene, infA, has been identified, and a role for IF1 in translation has not been demonstrated in vivo. To elucidate the function of IF1 and determine if the protein is essential for cell growth, the chromosomal copy of infA was disrupted. Cell viability is maintained only when infA is expressed in trans from a plasmid, thereby demonstrating that IF1 is essential for cell growth in Escherichia coli. Cells depleted of IF1 exhibit few polysomes, suggesting that IF1 functions in the initiation phase of protein synthesis.

The nurse extern practicum: a new partnership between education and service.

An externship was developed for academic credit but also offered financial remuneration for students. It is believed that a partnership between an educational institution and a health-care institution might offer students the opportunity not only for reality-based clinical practice, but also to enhance their education. Benefits to the agency include recruitment of students for future employment; benefits to the university include additional credit generation without additional revenues, because instructional costs are borne by the contracting health-care agency. Evaluations of this joint endeavor have been enthusiastic, with positive outcomes for all.

Merkel cell carcinoma of the eyelid: a report of two new cases and a review of the literature.

Merkel cell carcinoma is a cutaneous neoplasm that rarely occurs in the eyelid. The tumor has an aggressive nature with high local recurrence and metastases rates. Early diagnosis and prompt complete excision with frozen section monitoring of margins are recommended.

Structure and expression of the infA operon encoding translational initiation factor IF1. Transcriptional control by growth rate.

The cellular levels of the three translational initiation factors, IF1, IF2, and IF3, increase as a function of growth rate in parallel with those of ribosomes. Therefore both ribosomal and initiation factor gene expression is under metabolic control. To address how expression of the Escherichia coli gene for IF1, infA, is regulated, a 3-kilobase region of the genome surrounding infA was sequenced. The 5' and 3' termini of in vivo infA transcripts were defined by S1 nuclease mapping, and mRNA size was measured by Northern blot hybridization. The infA gene is transcribed by two promoters, P1 and P2, which generate transcripts of 525 and 330 nucleotides, apparently ending at the same rho-independent terminator. Analyses of operon and protein fusions to lacZ demonstrate that neither infA transcription nor translation is affected by high cellular levels of IF1. However, P2, but not P1, increases in activity as a function of the growth rate of the cell and is the dominant promoter in rich medium. Therefore, metabolic control of infA expression occurs exclusively at the level of transcription by the P2 promoter.

Effects of topical flurbiprofen on the intraocular pressure lowering effects of apraclonidine and timolol.

We performed a prospective study that evaluated whether pretreatment with topical flurbiprofen alters the intraocular pressure (IOP) lowering effects of either topical 1% apraclonidine hydrochloride or 0.5% timolol maleate. Eighteen normal volunteers participated in this six-armed, randomized, double-masked, crossover study. All subjects received the first study medication, either bilateral 0.3% flurbiprofen or placebo (its vehicle), every 30 minutes for four applications. They next received the second study medication: either 0.5% timolol maleate (Timoptic), 1% apraclonidine hydrochloride, or placebo in both eyes. We measured IOP before the instillation of the first study medication and the second study medication (baseline), and then at 1, 3, and 6 hours later. All subjects underwent all six treatment arms. Flurbiprofen alone had no effect on IOP. Maximum IOP lowering occurred between 3 and 6 hours after timolol and apraclonidine administration. There was no difference in IOP lowering between timolol- and apraclonidine-treated eyes. Pretreatment with flurbiprofen did not affect the IOP lowering that was obtained with timolol or apraclonidine administration.

The existence of two genes between infB and rpsO in the Escherichia coli genome: DNA sequencing and S1 nuclease mapping.

A number of genes encoding proteins involved in transcription and translation are clustered between 68 and 69 minutes on the Escherichia coli genome map and are transcribed clockwise as two operons: the metY operon, containing metY, P15A, nusA, infB; and about a kilobase further downstream, the rpsO and pnp operon. The DNA sequence between infB and rpsO was determined and two open reading frames were detected which code for proteins of 15,200 (P15B) and 35,091 (P35) daltons. Maxicell analysis showed a relatively strong expression of P15B whereas P35 was synthesized more weakly. An overlap of the termination codon of P15B and the initiator codon for P35 suggests that translation of P15B and P35 may be coupled. S1 nuclease mapping of in vivo transcripts between infB and rpsO provided no evidence for major promoters but detected a moderately efficient rho-independent terminator between infB and P15B. The results indicate that P15B and P35 are expressed as part of the metY operon, but that some transcriptional read through into the rpsO operon also occurs, thereby, functionally linking the expression of these two complex systems.

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.

A monoclonal antibody to the epsilon-aminocaproic acid binding site on the kringle 4 region of human plasminogen that accelerates the activation of Glu1-plasminogen by urokinase.

A monoclonal antibody, 10-F-1, previously shown [V. A. Ploplis, H. S. Cummings, and F. J. Castellino (1982) Biochemistry 21, 5891-5897] to interact with a particular epsilon-aminocaproic acid (EACA)3 binding site on the kringle 4 (K4) region of human Glu1-plasminogen (Glu1-Pg), has been employed to assess the contribution of this particular EACA site toward the enhancement, by EACA and its analogs, of the urokinase (UK)-catalyzed activation of Glu1-Pg. As is the case with EACA-like compounds, the presence of antibody 10-F-1 accelerates the activation of Glu1-Pg by UK, but does not enhance the similar activation of Lys77-plasminogen. In the presence of concentrations of antibody 10-F-1 which saturate its binding site on Glu1-Pg, the Km of Glu1-Pg activation by UK is raised from 1.4 +/- 0.2 microM, a value obtained in the absence of antibody, to 17.0 +/- 2.0 microM. On the other hand, the kcat for this activation, 0.038 +/- 0.005 s-1, is elevated to 2.45 +/- 0.2 s-1 at saturating concentrations of antibody 10-F-1. The kcat/Km for activation under these conditions is 0.027 s-1 microM-1 in the absence of antibody, and 0.144 s-1 microM-1 in the presence of saturating levels of antibody 10-F-1. This demonstrates that the interaction of this antibody with its epitope results in a fivefold stimulation of the activation rate of Glu1-Pg by UK. The availability of antibody 10-F-1 allows for a specific means of probing the function of one of the four to five thermodynamically equivalent weak EACA sites on human plasminogen. From this particular study, it is concluded that the weak binding site for EACA on the K4 domain of Glu1-Pg is either in-part or in-whole responsible for the enhancing effect of EACA on human Glu1-Pg activation by UK.

Interspecies cross-reactivity of monoclonal antibodies to various epitopes of human plasminogen.

The immunological cross-reactivities of three conformationally specific monoclonal antibodies to distinct epitopes on human plasminogen toward plasminogens purified from 14 additional species have been examined. Antibody 10-F-1, which is produced against an epitope on the kringle 4 region of human plasminogen, shows a high degree (greater than 80%) of cross-reactivity against baboon, goat, monkey, ovine, and rabbit plasminogens; more limited (20-50%) cross-reactivity against bovine, equine, goose, guinea pig, mouse, rat, and porcine plasminogens; and little comparable cross-reactivity against canine and chicken plasminogens. Antibody 10-H-2, generated to an epitope of the kringles 1-3 region of human plasminogen, shows extensive cross-reactivity (72%) only toward monkey plasminogen, more limited (22-35%) cross-reactivity toward equine and rabbit plasminogens, and much less cross-reactivity toward any other of the above plasminogens. Antibody 10-V-1, also produced against an epitope on the kringle 1-3 region of human plasminogen, which is distinct from the 10-H-2 epitope, shows extensive cross-reactivity (72-100%) with baboon, monkey, and rabbit plasminogens; more limited cross-reactivity with equine (48%) and mouse (28%) plasminogens; and a low level of such reactivity with the remaining plasminogens. These studies show that the extent of interspecies cross-reactivity of various plasminogens greatly depends upon the epitope in question. The K4 region of these molecules appears more extensively conserved than the K1-3 region, at least in regard to the particular epitopes examined in this study.