**2.3 Iodine analyses**

36 Aquaculture

foodstuffs are commonly used to avoid iodine-deficiency pathologies such as hyperthyroidism and goiter. Nevertheless, environmental conditions such as poor water quality or malnutrition can lead to marked proliferation of thyroid tissue in some fishes (Hoover, 1984) indicating that care must be taken to maintain a minimum iodine level in artificial environments including zebrafish aquaculture facilities. Dietary supplements have been shown to prevent thyroid hyperplasia in the saltwater cyprinodont *Fundulus* (Pickford, 1954) and goiters in the Chinook salmon (Woodall & La Roche, 1964), but no study to date has effectively assessed the minimum iodine level for maintaining normal development and

While experimenting with varied diets in our facility, we observed that conditions of reduced iodine result in less robust somatic growth and an elevated incidence of jaw dysmorphogenesis. This prompted us to evaluate the developmental consequences of rearing zebrafish on a single live food or a mixed food diet with varying levels of iodine. Herein we demonstrate that a strict single-food *Artemia* diet, although high in the protein necessary for robust egg production, may also result in a morphological alteration of the zebrafish jaw architecture formed in response to swelling and hypervascularization of

Adult WIK wild-type zebrafish were raised, maintained, and spawned in the Zebrafish Model Organism Laboratory, located at the University of Cincinnati (UC) Genome Research Institute according to previously published protocols (Craig et al., 2006; Kimmel et al., 1995; Westerfield, 2000). Zebrafish fry were reared in a multi-rack system from Aquatic Habitats (Apopka, FL) with automated control of water chemistry (YSI Model 5200 control system, Yellow Springs, OH) utilizing a 14-h light/10-h dark cycle. Water chemistry was maintained at the following conditions to ensure optimal fish health: temperature: 28.5 ± 1 oC; pH: 7.5 ± 0.2; ammonia: 0 ± 0.25 mg/l; nitrite: 0 ± 0.20 mg/l; nitrate: 5 ± 5 mg/l; total hardness: 102 ±17 mg/l; alkalinity 50 ± 17 mg/l. Conductivity was maintained at 750 ± 50 μS. Adult breeding stock fish were fed a standard diet of 3 day old HUFA-enriched (Super SELCO, Aquatic Ecosystems) *Artemia* in the morning plus a balanced flake food (Aquatox, Aquatic

Fry were fed a mixed diet of *Paramecium*, rotifers, and Zeigler's larval diet (Zeigler Bros, Inc.) until they were capable of eating *Artemia* (approximately 1 month of age) and to ensure proper early development. Beginning at 1 month of age, fish were placed on either a live study diet of three *Artemia* feedings or a mixed control diet of two Aquatox flake and one

Dietary *Artemia* intake was measured by feeding *Artemia* to overnight fasted fish (17.2 mg per tank, 15 minutes feeding time to approach satiety). Fish were euthanized in concentrated 250 mg/l MS-222, eviscerated and the alimentary tract was removed. The gut was everted, and the total number of ingested shrimp counted under a stereomicroscope. Aquatox flake

thyroid health in zebrafish.

**2. Experimental methods**

Ecosystems) in the afternoon.

**2.2 Dietary paradigm**

*Artemia* feedings per day.

**2.1 Animal housing and maintenance**

adjacent tissues.

Quantification of iodine in the foodstuffs was performed using ICP-MS as described previously for other heavy metals (Figueroa et al., 2008). Briefly, *Artemia* samples were cultured for 72 h, harvested and freeze-dried overnight in a lyophilizer (FreeZone 4.5 Liter Benchtop System, Labconco Corporation, Kansas City, MI). Three replicates of dried *Artemia*  and system water samples (~30 mg) were placed in septum sealed glass tubes, treated with HNO3 and microwave digested: power, 130 W; ramp, 1:00 min; hold, 1:00 min; temp, 75 °C. Subsequently, 0.1 mL of 30 % H2O2 was added and the samples were processed through the second digestion stage: power, 130 W; ramp, 1:00 min; hold, 1:00 min; temp, 110 °C. Samples were diluted with DDW to 10 mL and analyzed for total iodine content in continuous flow mode using an Agilent 7500ce ICP-MS (Agilent Technologies, Tokyo, Japan) equipped with a quadrupole mass analyzer.


**6.1 Thioflavin T binding assay**

The use of Thioflavin T (ThT) and its derivatives is perhaps the simplest and most widely used method for monitoring the aggregation of amyloidogenic proteins. ThT is a low molecular weight dye exhibiting fluorescence emission when fibrillar protein aggregates bind to the β-layer groove structure. Traditionally, ThT has been used to detect amyloid fibrils because of the characteristic sigmoidal increase in fluorescence that occurs between the monomeric state and the ends of the fibrils [69, 70] beside ThT also binds to fibrillar aggregates containing β-sheet groove binding sites (fibrillar oligomers and fibrils). Prefibrillar aggregates of β-sheet structure contain few binding sites than fibril thereby showing low frequency of fluorescence [71]. Thus, ThT could indicate the presence of toxic circular fibrin and fibrin oligomers, but not the presence of pre-fibrin oligomers that do not have a clear β-sheet structure [72].


**3. Discussion**

Histopathological features of the esophagus in SSc and MCTD patients are similar, but muscular change in SSc is more progressive than in MCTD patients in our study. It has been suggested that there is no association between manometric abnormality and cutaneous symptoms in MCTD patients, and the characteristics of SSc are not always linked to esophageal dysfunction [5]. The pathological mechanism of esophageal dysfunction in MCTD may be similar but not always identical to that in SSc.

In patients with CTDs, autoimmune inflammation occurs in systemic organs such as kidney, lung, skin and blood vessels, and so on. The gastrointestinal tract is also involved though the histological features and grades are different from disease to disease even in the same CTD.

In CTDs, many kinds of autoantibodies may play an important role in causing the various symptoms and diseases, whether they are fatal or not. These differ from disease to disease and from tissue to tissue. We showed that IgG from MCTD patients reacts to various tissues such as kidney and lung (unpublished data) (Figure 7). It is well known that pulmonary hyperten‐ sion is the fatal cause of MCTD. Anti-endothelial cell antibody (AECA) was identified in the serum of MCTD patients, and was especially high in patients with pulmonary hypertension [21]. We now examine the antigen of AECA in endothelial cells of small pulmonary vascular vessels [22]. As for the autoantibody of MCTD against esophagus, our study revealed that IgG extracted from MCTD patients showed a positive immunohistochemical reaction not only for the smooth muscle cells of esophagus, but also for the ganglion cells in Auerbach's plexus, the vascular walls in esophageal muscular tissues, and squamous epithelium of the esophagus. Dysphagia in MCTD and SSc patients may be one of the symptoms often occurring as an autoimmune reaction.

The reason why the inner layer of the lower portion incurs more severe damage than other portions has not been clarified. Esophageal manometry shows that this portion sustains more intense mechanical stress in peristalsis than the outer layer or upper portions. Thus autoanti‐ bodies, mechanical stress and regurgitation may induce the severe dysphagia in MCTD and other CTDs.

Motility dysfunction is not a direct cause of death, but a strong association between esophageal dysmotility and interstitial lung disease in patients with MCTD is indicated [23]. Therefore, care must be taken with diagnosis.

**Figure 7.** Reaction of IgG from MCTD patients with various tissues. (A) kidney, (B) lung


**4. Results**

### *4.1. Consistent Interpretation of Relationship between Hazard and Top Event*

Regarding the inconsistency of usage of the hazard and top event, we propose the following categorisation.

Format A: In cases where the scope of analysis is limited to one situation, the correspondence between hazard and the top event is not problematic. If the Bowtie diagram is too large, it may be represented as multiple smaller diagrams, providing the hazard and top event are used in the same way. The case study presented in this paper follows this approach.

Format B: The top event may have multiple hazard dimensions. In which case the top event should be consistent across all the diagrams, while the hazard changes. The hazard thus corresponds to a di fferent contextualisation for the same top event; see Figure 3.

**Figure 3.** Consistent top event with multiple hazard dimensions.

Format C: A hazard may have di fferent top event dimensions. Each dimension may represent a di fferent step in a process. Figure 4 provides an example for human factors in di fferent areas of the maintenance process.

**Figure 4.** Hazard investigation with di fferent top events.

We propose that the Bowtie analyst should decide beforehand how the problem is to be structured, and select one of the above formats, and then use it consistently throughout the analysis. This has the potential to avoid some of the inconsistencies seen in practice.

### *4.2. Proposal to Use 6M Structure*

The challenge with selecting the most suitable categorisation was that Bowtie contains di fferent risk elements including hazards, root causes (threats), consequences, barriers and controls. For each of these elements, di fferent approaches and categorisations exist. Some are suitable to categorise risks (causes and e ffects), but less suitable for categorising barriers. Hence, we decided to choose a risk categorisation that works for both risks (threats and consequences) and means of risk prevention and mitigation (barriers).

The 6M approach was seen as most suitable for the planned research and case study for the following reasons. First of all, the 6M approach was chosen due to the familiarity of the Bowtie and the Ishikawa diagram, with both being a cause and e ffect diagram. The 6M has already been successfully applied to structure such a diagram [23]. Secondly, it has been successfully applied outside the manufacturing environment such as health care [69,70], management [71], and education [72]. Moreover, the 6M categorisation was well known at our industry partner, as the cause and e ffect diagram from Ishikawa is part of the measure phase of the DMAIC, a continuous improvement process often used in Six Sigma and know by industry practitioners [73]. The 6M structure can be used for both vertical categorisation of the threats and consequences, as well as horizontal categorisation of barriers.

The 6M aligns with other categorisations presented in the broader literature in Section 2.3 including the five accident categories used by IATA [53], and the four main risk categories by Lester [44]. Both are quite similar and a terminology adjustment, following the 'category starts with the letter M' concept, would make these categories match the selected 6M approach.

### *4.3. Integration of 6M with Bowtie (Contextualisation)*

The MRO environment di ffers to the manufacturing environment, and hence the original 6M structure by Ishikawa required modification. The contextualisation was carried out in the light of organisational quality factors that influence the maintenance and inspection result. In a production environment, the measurement category commonly includes the inspection and measurement of the parts to check if they meet the quality requirements. However, in an inspection environment the 'measurement' category can overlap with 'machine' and 'method', since the inspection tool and followed processes would fit into all three categories. The measurement category was therefore less useful to apply in an inspection environment. Instead of the 'measurement' category, we included 'management', which is one of the additional categories from the 8M approach by Burch et al. [58] and aligns to the work by Gwiazda [74] and Vaanila [73]. This was carried out as it matches the area under investigation being the highly regulated aviation and maintenance industry (see below) [75,76]. Management refers to organisational and regulatory factors. The other category from the 8M we could have chosen was maintenance, but since we wanted to investigate risks in a maintenance

environment, maintenance as a threat and a hazard at the same time would have caused problems with the consistency of the Bowtie structure.

In Table 1 below, we demonstrate how the categories and their interpretation may change according to the industry. This is demonstrated based on three examples, namely manufacturing, maintenance and health care. The latter was chosen to demonstrate the integration to a quite different industry.

### *4.4. Threats and Consequence Structure Using 6M*

### 4.4.1. Threat Structure in MRO

We started with integration of the 6M structure for the threats. A description of each category together with an example is given in the Table 2 below.

**Table 2.** The 6M categories for threats with description and example.