Effects of Interventions to Prevent Work-Related Asthma, Allergy, and Other Hypersensitivity Reactions in Norwegian Salmon Industry Workers (SHInE): Protocol for a Pragmatic Allocated Intervention Trial and Related Substudies

Background: Workers in the salmon processing industry have an increased risk of developing respiratory diseases and other hypersensitivity responses due to occupational exposure to bioaerosols containing fish proteins and microorganisms, and related allergens. Little is known about effective measures to reduce bioaerosol exposure and about the extent of skin complaints among workers. In addition, while identification of risk factors is a core activity in disease prevention strategies, there is increasing interest in health-promoting factors, which is an understudied area in the salmon processing industry. Objective: The overall aim of this ongoing study is to generate knowledge that can be used in tailored prevention of development or chronification of respiratory diseases, skin reactions, protein contact dermatitis, and allergy among salmon processing workers. The main objective is to identify effective methods to reduce bioaerosol exposure. Further objectives are to identify and characterize clinically relevant exposure agents, identify determinants of exposure, measure prevalence of work-related symptoms and disease, and identify health-promoting factors of the psychosocial work environment. Methods: Data are collected during field studies in 9 salmon processing plants along the Norwegian coastline. Data collection comprises exposure measurements, health examinations, and questionnaires. A wide range of laboratory analyses will be used for further analysis and characterization of exposure agents. Suitable statistical analysis will be applied to the various outcomes of this comprehensive study. Results: Data collection started in September 2021 and was anticipated to be completed by March 2023, but was delayed due to the COVID-19 pandemic. Baseline data from all 9 plants included 673 participants for the health examinations and a total of 869 personal exposure measurements. A total of 740 workers answered the study’s main questionnaire on demographics, job characteristics, lifestyle, health, and health-promoting factors. Follow-up data collection is not completed yet. Conclusions: This study will contribute to filling knowledge gaps concerning salmon workers’ work environment. This includes effective workplace measures for bioaerosol exposure reduction, increased knowledge on hypersensitivity, allergy, respiratory and dermal health, as well as health-promoting workplace factors. Together this will give a basis for improving the work environment, preventing occupational health-related diseases, and developing occupational exposure limits, which in turn will benefit employees, employers, occupational health services, researchers, clinicians, decision makers, and other stakeholders.publishedVersio

Clinical highlights from the 2016 European Respiratory Society International Congress

This article contains highlights and a selection of the scientific advances from the European Respiratory Society (ERS) Clinical Assembly (Assembly 1) and its six respective groups (Groups 1.1–1.6) that were presented at the 2016 ERS International Congress in London, UK. The most relevant topics for clinicians will be discussed, covering a wide range of areas including clinical problems, rehabilitation and chronic care, thoracic imaging, interventional pulmonology, diffuse and parenchymal lung diseases, and general practice and primary care. In this comprehensive review, the newest research and actual data will be discussed and put into perspective

Insights into bacterial protection and survival. A study of three enzymes from cold-adapted bacteria

Bacteria are the most abundant organisms and can be found in different habitats, from polar regions, deserts and volcanoes, deep ocean trenches to the upper atmosphere. In all these environments, they are exposed to various chemical hazards, such as antibacterial chemicals (antibiotics) produced by other organisms that are found in the same habitat. In addition, they are exposed to threats from infection by bacteriophages. In order to protect themselves, bacteria have evolved several mechanisms towards avoiding antibiotics and phage infection. In this work, enzymes involved in these protection mechanisms have been attempted characterized both functionally and structurally. Specifically two DNA modifying enzymes have been studied. They are involved in bacterial protection from phages, namely the type II restriction enzyme DpnI and the C5-DNA-MTase ParI, both originating from the psychrophilic bacterium Psychrobacter arcticus. The third enzyme that has been investigated is a metallo-β-lactamase (ALI-1) from the cold adapted marine bacterium Aliivibrio salmonicida. Since all these enzymes originate from organisms adapted to a cold environment, the presence of cold adapted enzyme features like heat lability and high activity at low temperature, were investigated. In Manuscript I and in Manuscript III we partially characterized the type II restriction enzyme DpnI and the C5-DNA-MTase ParI. In addition to being important for the bacterial cell, these types of enzymes are valuable in several molecular biology techniques and the development of more efficient enzymes is necessary in terms of reducing cost and increasing efficiency. Due to their characteristics such as higher activity at lower temperatures and heat lability cold adapted enzymes became very attractive targets. Both DpnI and ParI were recombinantly expressed, but with some difficulties, that were overcome by usage of specialized E. coli strains. Both enzymes were partially characterize with regards to function, DpnI showed activity in vitro, while MTase was shown to have methylating activity in vivo. One of the mechanisms in antibiotic resistance is the acquirement and development of β-lactamases, enzymes that can cleave the amide bond in the β-lactam ring, a common structure in all β-lactam antibiotics, and thus inactivate it. This presents a huge problem in usage of β-lactam antibiotics, which are widely used against bacterial infections in both humans and animals. In Paper II, the metallo-β-lactamase ALI-1, from the cold adapted marine bacterium Aliivibrio salmonicida, was characterised. When compared to a mesophilic counterpart (VIM-1 from Pseudomonas aeruginosa) it was shown to be adapted to its natural habitat, in terms of being more active at lower temperatures and in higher salt concentrations. In addition, in this work it was found that the gene encoding ALI-1 is present in environmental bacterial samples. This could point towards a broader role for ALI-1, such as in regulating quorum sensing signalling in bacteria

Properties and distribution of a metallo-β-lactamase (ALI-1) from the fish pathogen Aliivibrio salmonicida LFI1238

Objectives. To characterize the chromosome-encoded metallo-β-lactamase (MBL) from the psychrophilic, marine fish-pathogenic bacterium Aliivibrio salmonicida LFI1238 and check for the presence of the gene in other Aliivibrio isolates both connected to the fish-farming industry and from the environment. Methods. The MBL gene was cloned and intracellularly expressed in Escherichia coli. Kinetic parameters, NaCl dependence, pH optimum and temperature optimum were determined using purified enzyme. The VIM-2 enzyme from a Pseudomonas aeruginosa hospital isolate was used as a counterpart in comparative analysis. PCRs with degenerate MBL primers were used to screen different A. salmonicida isolates for the presence of the gene. Results. A. salmonicida MBL (ALI-1) is an Ambler class B β-lactamase sharing 39% and 29% amino acid identity with IMP-1 and VIM-2, respectively. ALI-1 hydrolysed all β-lactam antibiotics tested, except for the monobactam aztreonam and the penicillin piperacillin. A profound increase in activity was observed when adding NaCl to the assay mixture (60% active without addition of NaCl, increasing to 100% at 0.5 M NaCl). The increase was less noticeable for VIM-2 (100% active at 0.2 M NaCl). ALI-1 appears to be ubiquitous in nature as it is found in Aliivibrio isolates not affected by human activity. Conclusions. This work provides more data for the ever-expanding MBL group of enzymes. These periplasmic enzymes are activated by addition of NaCl, and the marine enzyme is highly salt tolerant and cold active. The observed enzyme properties very likely reflect the conditions that the enzymes face in situ

DNA binding with a minimal scaffold: structure-function analysis of Lig E DNA ligases

DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5′P and 3′OH ends in an adenylation-dependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker

Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes

Background: Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting eforts to fnd new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identifcation of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efcient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. Results: We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNAsequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difcult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that coldadapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identifed a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of coldactive enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. Conclusions: Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273–4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.</p