Comparison of national strategies to reduce meticillin-resistant Staphylococcus aureus infections in Japan and England.

BACKGROUND: National responses to healthcare-associated infections vary between high-income countries, but, when analysed for contextual comparability, interventions can be assessed for transferability. AIM: To identify learning from country-level approaches to addressing meticillin-resistant Staphylococcus aureus (MRSA) in Japan and England. METHODS: A longitudinal analysis (2000-2017), comparing epidemiological trends and policy interventions. Data from 441 textual sources concerning infection prevention and control (IPC), surveillance, and antimicrobial stewardship interventions were systematically coded for: (a) type: mandatory requirements, recommendations, or national campaigns; (b) method: restrictive, persuasive, structural in nature; (c) level of implementation: macro (national), meso (organizational), micro (individual) levels. Healthcare organizational structures and role of media were also assessed. FINDINGS: In England significant reduction has been achieved in number of reported MRSA bloodstream infections. In Japan, in spite of reductions, MRSA remains a predominant infection. Both countries face new threats in the emergence of drug-resistant Escherichia coli. England has focused on national mandatory and structural interventions, supported by a combination of outcomes-based incentives and punitive mechanisms, and multi-disciplinary IPC hospital teams. Japan has focused on (non-mandatory) recommendations and primarily persuasive interventions, supported by process-based incentives, with voluntary surveillance. Areas for development in Japan include resourcing of dedicated data management support and implementation of national campaigns for healthcare professionals and the public. CONCLUSION: Policy interventions need to be relevant to local epidemiological trends, while acceptable within the health system, culture, and public expectations. Cross-national learning can help inform the right mix of interventions to create sustainable and resilient systems for future infection and economic challenges.

Defining the user role in infection control.

BACKGROUND: Health policy initiatives continue to recognize the valuable role of patients and the public in improving safety, advocating the availability of information as well as involvement at the point of care. In infection control, there is a limited understanding of how users interpret the plethora of publicly available information about hospital performance, and little evidence to support strategies that include reminding healthcare staff to adhere to hand hygiene practices. AIM: To understand how users define their own role in patient safety, specifically in infection control. METHODS: Through group interviews, self-completed questionnaires and scenario evaluation, user views of 41 participants (15 carers and 26 patients with recent experience of inpatient hospital care in London, UK) were collected and analysed. In addition, the project's patient representative performed direct observation of the research event to offer inter-rater reliability of the qualitative analysis. FINDINGS: Users considered evidence of systemic safety-related failings when presented with hospital choices, and did not discount hospitals with high ('red' flagged) rates of meticillin-resistant Staphylococcus aureus. Further, users considered staff satisfaction within the workplace over and above user satisfaction. Those most dissatisfied with the care they received were unlikely to ask staff, 'Have you washed your hands?' CONCLUSION: This in-depth qualitative analysis of views from a relatively informed user sample shows 'what matters', and provides new avenues for improvement initiatives. It is encouraging that users appear to take a holistic view of indicators. There is a need for strategies to improve dimensions of staff satisfaction, along with understanding the implications of patient satisfaction.

Identification and expression analysis of nervous wreck, which is preferentially expressed in the brain of the male silkworm moth, Bombyx mori.

Sexually dimorphic neural circuits are essential for reproductive behaviour. The molecular basis of sexual dimorphism in the silkworm moth (Bombyx mori) brain, however, is unclear. We conducted cDNA subtraction screening and identified nervous wreck (Bmnwk), a synaptic growth regulatory gene, whose expression is higher in the male brain than in the female brain of the silkworm. Bmnwk was preferentially expressed in the brain at the late pupae and adult stages. In situ hybridization revealed that Bmnwk is highly expressed in the optic lobe of the male moth brain. These findings suggest that Bmnwk has a role in the development and/or maintenance of the optic lobe in the male silkworm brain.

20-Hydroxyecdysone regulation of two isoforms of the Ets transcription factor E74 gene in programmed cell death in the silkworm anterior silk gland.

Programmed cell death of larval-specific tissues in insects is under the control of 20-hydroxyecdysone (20E). The ecdysteroid-regulated early genes are conserved in the programmed cell death of anterior silk glands (ASGs) in Bombyx mori and salivary glands in Drosophila melanogaster. We identified and characterized two isoforms of the Ets transcription factor E74 gene in B. mori (BmE74). In ASGs of B. mori last instar larvae, the Bm74A mRNA level increased concomitantly with an increase in haemolymph ecdysteroid titre after gut purge. The optimal 20E concentration for stimulation of Bm74A in ASGs was 4 microM, a similar value to the peak haemolymph ecdysteroid concentration after gut purge. In contrast, BmE74B expression peaked on day 5 of the feeding period, after which it did not increase again. These findings suggest that the BmE74 isoforms play different roles in the regulation of programmed cell death in B. mori ASGs.

Coordinate responses of transcription factors to ecdysone during programmed cell death in the anterior silk gland of the silkworm, Bombyx mori.

Programmed cell death (PCD) in Bombyx mori anterior silk glands (ASGs) is triggered by 20-hydroxyecdysone (20E). We examined the expression profiles and effects of 20E on 11 transcription factor genes in the fifth instar to determine whether they demonstrate the hierarchical control seen in Drosophila PCD. Results indicate that EcR-A and usp-2, but not EcR-B1 or usp-1, may be components of the ecdysone receptor complex. Up-regulation of E75A, BHR3, and three BR-C isoforms, but not E75B, appeared to be associated with the induction of PCD. betaFTZ-F1 was not expressed during PCD execution. Thus, gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis.

Observation of fluorapatite formation under hydrolysis of tetracalcium phosphate in the presence of KF by means of soft X-ray emission and absorption spectroscopy.

The effect of fluoride on the hydrolysis of tetracalcium phosphate (TTCP; Ca4(PO4)2O) in 0.1 mol/l KH2PO4 containing 62-83 mmol/l KF was studied with the help of X-ray fluorescence measurements. Fluorine X-ray emission and absorption spectra of the final product of hydrolysis and reference samples (CaF2 and Ca5(PO4)3F) were measured at Beamline BL-2C of Photon Factory (PF, Tsukuba). Based on these measurements we concluded that hydrolysis of TTCP in the presence of KF converts it into fluorapatite. Formation of CaF2, which is often found in the hydrolysis of hydroxyapatite at high fluoride concentration, was not observed.

Pupal commitment and its hormonal control in wing imaginal discs.

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.

Expression of arginine vasopressin and vasopressin V1a receptor mRNA in diabetic (db/db) mice.

To assess the involvement of arginine vasopressin (AVP) in genetical diabetic (db/db) mice, we examined the mRNA expression levels of AVP and vasopressin V(1a) receptors (V(1a)R) in brain and liver of db/db mice. In 10 week-old db/db mice, a significant elevation in blood sugar levels and plasma osmolality were observed, showing obvious diabetic symptoms. There was a significant increase in brain AVP mRNA levels in db/db mice. The expression level of liver V(1a)R mRNA in db/db mice was significantly down-regulated, presumably as a consequence of ligand-receptor interaction. This is in contrast to results that show no significant reduction in brain V(1a)R mRNA levels when comparing db/db and control mice. Thus, it is possible that in the progress of genetic diabetes mellitus, AVP acts in liver than in brain through V(1a)R.

Ecdysteroid-inducible genes in the programmed cell death during insect metamorphosis.

The anterior silk gland of the silkworm, Bombyx mori, undergoes programmed cell death (PCD) during pupal metamorphosis and PCD is triggered by 20-hydroxyecdysone (20E) in vitro. In order to identify the genes responsible for the PCD, we subtracted cDNAs prepared from the anterior silk glands incubated in the presence or absence of 20E in vitro. After a series of screenings by dot blot hybridization, DNA sequencing and reverse transcription polymerase chain reaction (RT-PCR), we obtained seven novel genes that were activated by 20E in vitro. Nucleotide sequence analysis indicated that two cDNAs (EN78 and EC08) did not have any obvious region to encode proteins, while five genes, designated EC74, EN86, EN03, EN10 and EN16, encoded proteins that are similar to inorganic phosphate cotransporter, TIA-1-like protein, chitinase-related protein, translation-initiation-factor subunit and annexin, respectively. Expression profiles of the genes after 20E stimulation indicated that four genes could be classified as early genes, while two are delayed early genes. The genes identified may provide insight into the PCD induced by a steroid hormone.

Heterotopia in microcephaly induced by cytosine arabinoside: hippocampus in the neocortex.

Pregnant mice were injected intraperitoneally with cytosine arabinoside (Ara-C) on days 13.5 and 14.5 of pregnancy. The brains of their offspring were studied histologically and histochemically. In addition to dysgenic microcephaly, nodular structures consisting of cells with a relatively homogeneous morphology were observed in the depths of the cerebral cortex. The cell clusters were first seen around postnatal day 4, and had a cellular continuity with the disarrayed pyramidal cell layer in the CA 1 region of the hippocampus. Golgi-Cox staining showed a number of pyramidal-shaped cells in the clusters. Morphologically, they resembled the pyramidal neurons of the hippocampus. Immunohistochemical examination, using anti-serotonin or anti-tyrosine hydroxylase antibodies, also indicated similarities between the cell clusters and the pyramidal cell layer. It is, therefore, proposed that the cell clusters consisted of heterotopic pyramidal cells of the hippocampus. A few synaptic structures could already be detected in the heterotopic cell clusters on postnatal day 3 by electron microscopy. This early establishment of synaptic contact with related neurons may have caused the heterotopic localization of the pyramidal cells.

Development of hyperfimbriated strains of Vibrio cholerae O1.

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.

Programmed cell death triggered by insect steroid hormone, 20-hydroxyecdysone, in the anterior silk gland of the silkworm, Bombyx mori.

Silk gland is a larval specific tissue of lepidopteran insects and begins to degenerate shortly before pupation. Programmed cell death (PCD) of the anterior silk gland of Bombyx mori last instar larvae was studied in vivo and in vitro, focusing on the effects of 20- hydroxyecdysone (20E). The glands began to exhibit signs of PCD in vivo 2 days after gut purge and completed PCD by 48 h. In vitro, 20E prematurely induced PCD, and its completion took 144 h (6 days). An oligo-nucleosomal ladder pattern was observed in DNA extracted at the end of PCD. Caspase 3 inhibitor inhibited attainment of full PCD, but it did not block chromatin condensation as revealed by acridine orange staining. alpha-Amanitin inhibited the PCD induced by 20E in vitro if added to the culture in the first 8 h. Similarly, cycloheximide and emetine completely blocked PCD when applied in the first 18 h of culture with 20E. These results indicate that 20E-stimulated transcription and protein synthesis for PCD are completed in 8 h and 18 h, respectively. Nevertheless, withdrawal of 20E from the medium at different times showed that 20E must be present in vitro for 42 h to elicit full PCD. Current results indicate that the effects of 20E on the progression of PCD are mediated by two distinct processes - one through nuclear hormone receptors, and the other independent from de novo gene expression.

Involvement of adipokinetic hormone in the homeostatic control of hemolymph trehalose concentration in the larvae of Bombyx mori.

Prior to wandering, 5th instar larvae of the silkworm, Bombyx mori, maintain constant hemolymph titers of trehalose. Head ligation of day 3, 5th instar larvae significantly decreased the hemolymph trehalose concentrations, but the concentrations did not decrease in starved larvae. After being diluted by replacement of larval hemolymph with insect Ringer's solution, the trehalose concentrations recovered the initial levels in 90 min in the non-ligated larvae, while they were not restored in 90 min in the neck-ligated larvae. These results suggest that a head factor(s) with hypertrehalosemic activity is involved in the homeostatic control of hemolymph trehalose concentration. When adipokinetic hormone (AKH) was injected into neck-ligated larvae, the trehalose concentrations increased in 2 h and decreased thereafter. Repeated injections of AKH every 4 h maintained the concentrations for 12 h. These findings suggest that AKH induces a hypertrehalosemic response and is involved in the homeostasis of hemolymph trehalose concentration in the larval feeding period.

Bombyxin: An Insect Brain Peptide that Belongs to the Insulin Family.

Bombyxin is a 5 kDa secretory brain peptide that belongs to the insulin family. Bombyxin of the silkmoth Bombyx mori can induce adult development when injected into brain-removed dormant pupae of the saturniid moth Samia cynthia ricini by activating the prothoracic glands to synthesize and release ecdysone. Bombyx bombyxin has been shown to lower the concentration of the major haemolymph sugar, trehalose, and to elevate the trehalase activity in the midgut and muscles in Bombyx, but the doses required to be effective are higher than the amounts in the feeding larvae. The exact physiological function of bombyxin in Bombyx itself is therefore still obscure, but its insulin-like structure suggests it has important roles. Bombyxin comprises a mixture of highly heterogeneous molecular forms whose amino acid sequences have 40% identity with human insulin. The Bombyx bombyxin gene encodes a precursor consisting of the signal peptide, B chain, C peptide, and A chain, in that order from the N terminus. So far, 32 bombyxin genes have been identified in Bombyx, and they are classified into 7 families, A to G, according to their sequence similarity. The bombyxin genes have no introns and cluster in unique distribution patterns. The gene arrangement in the cluster has been classified into three categories: gene pairs, gene triplets, and single genes. Nucleotide sequence analysis indicates that equal and unequal crossings-over and duplications may have generated these unique distribution patterns. The Bombyx bombyxin genes are expressed predominantly in the brain and at low levels in a number of other tissues. Genes of all 7 families are expressed in four pairs of the medial neurosecretory cells of the brain. Detailed examination indicated that only a limited number of genes in the A, B and C family members are expressed and that their expression shows a gene-arrangement-dependent pattern.

Nonpeptide mimic of bradykinin with long-acting properties.

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, have been implicated in a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells and activation of sensory neurons. However, investigation of the physiological actions of kinins have been greatly hampered because its effects are curtailed by rapid proteolytic degradation. We examined the pharmacological characteristics of the first nonpeptide bradykinin receptor agonist 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl+ ++]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinolin e (FR190997). FR190997, whose structure is quite different from the natural peptide ligand, but is similar to the nonpeptide antagonists FR165649, FR167344 and FR173657, potently and selectively interacts with the human B2 receptor and markedly stimulates inositol phosphate formation in transfected Chinese hamster ovary (CHO) cells. FR190997 induces concentration-dependent contraction of isolated guinea pig ileum. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration, presumably as a consequence of its resistance to proteolytic degradation. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity at the bradykinin B2 receptor. This compound represents a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.

Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.

Gene transfer into insect brain and cell-specific expression of bombyxin gene.

A transgene reporter consisting of the bombyxin gene promoter and the green fluorescent protein coding region was introduced into intact brains of the silkworm Bombyx mori by in vitro electroporation. After in vitro culture of the brains, the fluorescence derived from the introduced reporter gene was observed in all cases in eight neurosecretory cells that had previously been identified as bombyxin-producing cells (BPCs). Although the fluorescence was not always observed in all cells, it was specific to BPCs, indicating that the reporter was under the control of the bombyxin gene promoter in a BPC-specific manner. Electroporatical introduction of a reporter gene was therefore found to be a suitable method for analyzing cell-specific expression in intact tissues and to be substitute for germ-line transmission of reporters in the transgenic system. Application of this technique enables us to analyze the cell-specific expression of transgene reporters within a few days and treat more than several dozens of the reporters within 1 month, which is difficult to do with the transgenic system.

3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme.

The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.

A novel member of the bombyxin gene family: structure and expression of bombyxin G1 gene, an insulin-related peptide gene of the silkmoth Bombyx mori.

Bombyxin G1 gene, a novel insulin-related peptide gene of the silkmoth Bombyx mori, has been identified. The G1 gene encodes a precursor peptide which shows 41-56% and 28% sequence identities with preprobombyxins previously characterized and human preproinsulin, respectively. The G1 gene forms a pair with bombyxin C2 gene with opposite transcriptional orientation in a bombyxin gene cluster. The bombyxin G1 mRNA in Bombyx brain was shown to locate in four pairs of medial neurosecretory cells.