Methods to improve the translation of evidence-based interventions: A primer on dissemination and implementation science for occupational safety and health researchers and practitioners.

Objective: A limited focus on dissemination and implementation (D&I) science has hindered the uptake of evidence-based interventions (EBIs) that reduce workplace morbidity and mortality. D&I science methods can be used in the occupational safety and health (OSH) field to advance the adoption, implementation, and sustainment of EBIs for complex workplaces. These approaches should be responsive to contextual factors, including the needs of partners and beneficiaries (such as employers, employees, and intermediaries). Methods: By synthesizing seminal literature and texts and leveraging our collective knowledge as D&I science and/or OSH researchers, we developed a D&I science primer for OSH. First, we provide an overview of common D&I terminology and concepts. Second, we describe several key and evolving issues in D&I science: balancing adaptation with intervention fidelity and specifying implementation outcomes and strategies. Next, we review D&I theories, models, and frameworks and offer examples for applying these to OSH research. We also discuss widely used D&I research designs, methods, and measures. Finally, we discuss future directions for D&I science application to OSH and provide resources for further exploration. Results: We compiled a D&I science primer for OSH appropriate for practitioners and evaluators, especially those newer to the field. Conclusion: This article fills a gap in the OSH research by providing an overview of D&I science to enhance understanding of key concepts, issues, models, designs, methods and measures for the translation into practice of effective OSH interventions to advance the safety, health and well-being of workers.

A gene for autosomal dominant juvenile amyotrophic lateral sclerosis (ALS4) localizes to a 500-kb interval on chromosome 9q34.

Amyotrophic lateral sclerosis (ALS) denotes a heterogeneous group of neurodegenerative disorders affecting upper and lower motor neurons. ALS4 is a juvenile-onset, autosomal dominant form of ALS that is characterized by slow progression, distal limb weakness and amyotrophy, and pyramidal signs associated with severe loss of motor neurons in the brain and spinal cord. The ALS4 locus was recently mapped by linkage analysis to a large genetic interval on chromosome 9q34. By undertaking extensive genetic linkage analysis, we have significantly refined the ALS4 locus to a critical interval of less than 3 cM, flanked by D9S149 and D9S1198. Previous physical mapping in this region has indicated that this critical interval spans approximately 500 kb. Seventeen putative transcripts have been localized within this interval including 7 characterized genes, 2 partially characterized genes, and 8 "anonymous" expressed sequence tags . These are therefore positional candidate genes for the ALS4 locus. We have also undertaken mutation analysis and genetic mapping to investigate and exclude candidate genes, including RING3L/ORFX and RALGDS, from a pathogenic role in ALS4.

Autosomal dominant juvenile amyotrophic lateral sclerosis.

Juvenile amyotrophic lateral sclerosis (ALS) is a form of chronic motor neuron disease characterized by combined upper and lower motor neuron symptoms and signs with onset prior to age 25 years. We report the clinical and electrodiagnostic findings in 49 affected family members and neuropathological findings from two autopsies of a Maryland kindred with autosomal dominant juvenile ALS linked to the chromosome 9q34 region (ALS4). Patients ranged in age from 12 to 85 years (mean 45 years) and the mean age of onset was 17 years. Distal weakness and atrophy was associated with pyramidal signs (43/49) and normal sensation (44/49). Motor conduction studies (n = 8) showed reduced evoked amplitudes and normal conduction parameters. Sensory conduction studies (n = 8), quantitative sensory testing (n = 4) and intracutaneous sensory fibres in skin biopsies (n = 6) were normal in all patients tested. Electromyography showed distal more than proximal chronic partial denervation and reinnervation (n = 8). Post-mortem spinal cord tissue demonstrated atrophic spinal cords with marked loss of anterior horn cells and degeneration of corticospinal tracts, as well as loss of neurons in the dorsal root ganglia and degeneration of the posterior columns. Axonal spheroids were present in the grey matter of the spinal cord, the dorsal root entry zones and the peripheral nerves. Motor and sensory roots, as well as peripheral nerves, showed significant axonal loss. Swellings were prominent around motor neurons, probably representing changes in presynaptic terminals. These studies define autosomal dominant juvenile ALS linked to the chromosome 9q34 region (ALS4) and extend the clinical, pathological and genetic heterogeneity of familial ALS and juvenile ALS.

Mapping of the kinesin-related gene ATSV to chromosome 2q37.

The human ATSV (axonal transporter of synaptic vesicles) gene encodes an anterograde axonal motor transport protein and demonstrates homology to the kinesin gene family in several species. The human ATSV gene was mapped to chromosome 2q37 by screening of a human/rodent somatic cell hybrid panel by the polymerase chain reaction and by fluorescent in situ hybridization analysis using genomic and cDNA clones.

Linkage of the gene for an autosomal dominant form of juvenile amyotrophic lateral sclerosis to chromosome 9q34.

We performed genetic mapping studies of an 11-generation pedigree with an autosomal dominant, juvenile-onset motor-systems disease. The disorder is characterized by slow progression, distal limb amyotrophy, and pyramidal tract signs associated with severe loss of motor neurons in the brain stem and spinal cord. The gene for this disorder, classified as a form of juvenile amyotrophic lateral sclerosis (ALS), is designated "ALS4." We performed a genomewide search and detected strong evidence for linkage of the ALS4 locus to markers from chromosome 9q34. The highest LOD score (Z) was obtained with D9S1847 (Z=18.8, recombination fraction of .00). An analysis of recombinant events identified D9S1831 and D9S164 as flanking markers, on chromosome 9q34, that define an approximately 5-cM interval that harbors the ALS4 gene. These results extend the degree of heterogeneity within familial ALS syndromes, and they implicate a gene on chromosome 9q34 as critical for motor-neuron function.

Identification and characterization of a mouse homologue of the spinal muscular atrophy-determining gene, survival motor neuron.

Spinal muscular atrophy (SMA), the second most common fatal, autosomal recessive disease of infants, manifests as generalized muscle weakness. The most severe form (Type I, Werdnig-Hoffmann disease) is associated with quadriplegia, respiratory muscle paralysis and death in infancy. Less severe forms are classified as Type II and Type III, based on age of onset and ultimate motor disability. Some spinal motor neurons show chromatolysis and the number of these cells is decreased. Recently, SMA has been mapped to chromosome 5q11.2-13.3 (Gilliam et al., 1990), a region that contains three candidate genes: Survival Motor Neuron (SMN) (Lefebvre et al., 1995); Neuronal Apoptosis Inhibitory Protein (NAIP) (Roy et al., 1995); and p44, a subunit of transcription factor II H (TFIIH) (Carter et al., 1995; Bürglen et al., 1997). Homozygous deletions or deleterious mutations in SMN are present in all SMA patients, and in some affected individuals, deletions have been identified in one or both of the other genes. These extensive deletions may be associated with a more severe phenotype. We have identified and characterized the mouse homologue of SMN, MoSMN, which is 82% identical to SMN at the amino-acid level. Unlike the duplicated human SMN, MoSMN is present in single copy. Like its human counterpart, MoSMN is ubiquitously expressed, but unlike SMN, MoSMN does not appear to be alternatively spliced. In-situ hybridization analysis of the mouse nervous system revealed that MoSMN mRNA is expressed in spinal cord and throughout the brain, with relatively higher levels of expression in the hippocampus and cerebellum.

Accumulation of the adenosine triphosphate synthase subunit C in the mnd mutant mouse. A model for neuronal ceroid lipofuscinosis.

The motor neuron degeneration (mnd) mutant mouse, initially described as an autosomal semidominant model of motor neuron disease, is characterized by progressive loss of motor activities and the accumulation of lipofuscin-like material in the cytoplasm of neurons in many regions of the nervous system. The stored material is composed of granular, multilamellar, fingerprint, and curvilinear profiles and degenerating mitochondria. These inclusions are associated with the accumulation of subunit c of mitochondrial adenosine triphosphate synthase in an age-dependent pattern. These abnormalities first appear in neurons of the thalamus, hippocampus, and cortex and eventually involve virtually all nerve cells, including those in the retina and enteric nervous system. This type of neuropathology and the presence of subunit c in neurons of mnd mutant mice are characteristic features of neuronal ceroid lipofuscinosis (NCL). The murine disease resembles Batten's disease, an autosomal recessive disorder and the most common NCL in humans. The mnd mouse should be of great value for investigations of the genetics of NCL, for studies designed to delineate the mechanism that lead to neuronal degeneration in these disorders, and for testing novel therapeutic approaches.

DNA ligase from Drosophila melanogaster embryos. Substrate specificity and mechanism of action.

DNA ligase has been purified to homogeneity from 6-12 h Drosophila melanogaster embryos (Rabin, B. A., Hawley, R. S., and Chase, J. W. (1986) J. Biol. Chem. 261, 10637-10645). This enzyme had an apparent Km for ATP of 1.6 microM. Of a variety of nucleotides tested, only adenosine 5'-O-(3-thio)triphosphate could substitute for ATP in the joining reaction. The enzyme was competitively inhibited by dATP, with an apparent Ki of 2.3 microM. The apparent Km for DNA using p(dT)20 annealed with poly(dA) as substrate was 1.0 microM. Studies utilizing synthetic homopolymers showed that in addition to joining DNA to DNA, this enzyme could join the 5'-phosphoryl termini of RNA to the 3'-hydroxyl termini of DNA or RNA, when they were annealed with DNA. In addition, p(dT)7U could be joined when annealed with poly(dA). No joining was detected when RNA served as the template. Drosophila DNA ligase also catalyzed the joining of oligonucleotides containing a single mismatched nucleotide at their 3'-hydroxyl termini, as well as DNA containing short, complementary 5'-protruding ends, and in the presence of polyethylene glycol 6000, blunt-ended duplex DNA. The overall reaction mechanism was shown to be identical to that of the homologous prokaryotic DNA ligases. The joining reactions catalyzed by the Drosophila and T4 DNA ligases were shown to be reversible. Incubation of superhelical closed circular DNA molecules with the purified enzymes and AMP resulted in the production of a population of DNA molecules which had lost most, if not all, of their superhelical density.

The sevenless+ protein is expressed apically in cell membranes of developing Drosophila retina; it is not restricted to cell R7.

In the sevenless (sev) mutants of Drosophila, a single cell type, photoreceptor R7, does not develop. We made monoclonal antibody against a sev+-beta-galactosidase fusion protein, and used it to determine the ultrastructural localization of the sev+ protein in the larval eye disc. The protein is expressed on the apical surface of the developing retina. It is not restricted to cell R7; it is expressed in all the presumptive photoreceptor cells, cone cells, and possibly others. The protein localizes to the cell membranes of the apical tips and their microvilli, away from the bulk of the cell-cell contacts. Possible mechanisms for generating the specificity of the sev phenotype are discussed in light of these results.

Escherichia coli exonuclease VII. Cloning and sequencing of the gene encoding the large subunit (xseA).

We have determined the sequence of the gene encoding the large subunit of Escherichia coli exonuclease VII (xseA) and the amino acid sequence of the protein it encodes. The coding region of the xseA gene is 1368 base pairs. The protein encoded by the gene contains 456 amino acids and has a calculated molecular weight of 51,823. The promoter for xseA is close to that for guaB, and these two genes are transcribed in opposite directions: xseA clockwise and guaB counterclockwise on the standard E. coli genetic map. The cloned xseA gene can complement an xseA deletion mutant strain. In an xseA+ genetic background production of large quantities of the xseA gene product appeared to decrease the amount of exonuclease VII activity in cell extracts. In fact, no exonuclease VII activity at all could be detected following induction of strains in which the xseA gene was under lambda pL regulation. These observations suggest that the proper ratio of the large and small exonuclease VII subunits must be maintained in order to produce active enzyme.

DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization.

A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.

Isolation and preliminary characterization of Escherichia coli mutants deficient in exonuclease VII.

Strains of Escherichia coli containing reduced levels of exonuclease VII activity due to mutations in the xseB gene have been isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Seven mutants of independent origin deficient in exonuclease VII activity were obtained. Four of these contained defects in xseA, a locus which has been previously identified, and three others contained mutations in a gene distinct from xseA, which we have designated xseB. Genetic mapping studies place the xseB locus between proC and dnaZ. Exonuclease VII purified from KLC835 (xseA+ xseB3) is more heat labile than enzyme purified from the parent strain PA610 (xse+), showing that xseB is a structural gene for exonuclease VII. The isolation of lambda transducing phage carrying xseA is also described.

Subunit structure of Escherichia coli exonuclease VII.

Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure. The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons. This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits. The structure of the native enzyme consists of one large subunit and four small subunits. We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci. Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes.