Geochemical and Dietary Drivers of Mercury Bioaccumulation in Estuarine Benthic Invertebrates.

Sediments represent the main reservoir of mercury (Hg) in aquatic environments and may act as a source of Hg to aquatic food webs. Yet, accumulation routes of Hg from the sediment to benthic organisms are poorly constrained. We studied the bioaccumulation of inorganic and methylmercury (HgII and MeHg, respectively) from different geochemical pools of Hg into four groups of benthic invertebrates (amphipods, polychaetes, chironomids, and bivalves). The study was conducted using mesocosm experiments entailing the use of multiple isotopically enriched Hg tracers and simulation of estuarine systems with brackish water and sediment. We applied different loading regimes of nutrients and terrestrial organic matter and showed that the vertical localization and the chemical speciation of HgII and MeHg in the sediment, in combination with the diet composition of the invertebrates, consistently controlled the bioaccumulation of HgII and MeHg into the benthic organisms. Our results suggest a direct link between the concentration of MeHg in the pelagic planktonic food web and the concentration of MeHg in benthic amphipods and, to some extent, in bivalves. In contrast, the quantity of MeHg in benthic chironomids and polychaetes seems to be driven by MeHg accumulation via the benthic food web. Accounting for these geochemical and dietary drivers of Hg bioaccumulation in benthic invertebrates will be important to understand and predict Hg transfer between the benthic and the pelagic food web, under current and future environmental scenarios.

A Cross-Sectional and Longitudinal Analysis of Pre-Diagnostic Blood Plasma Biomarkers for Early Detection of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death that typically presents at an advanced stage. No reliable markers for early detection presently exist. The prominent tumor stroma represents a source of circulating biomarkers for use together with cancer cell-derived biomarkers for earlier PDAC diagnosis. CA19-9 and CEA (cancer cell-derived biomarkers), together with endostatin and collagen IV (stroma-derived) were examined alone, or together, by multivariable modelling, using pre-diagnostic plasma samples (n = 259 samples) from the Northern Sweden Health and Disease Study biobank. Serial samples were available for a subgroup of future patients. Marker efficacy for future PDAC case prediction (n = 154 future cases) was examined by both cross-sectional (ROC analysis) and longitudinal analyses. CA19-9 performed well at, and within, six months to diagnosis and multivariable modelling was not superior to CA19-9 alone in cross-sectional analysis. Within six months to diagnosis, CA19-9 (AUC = 0.92) outperformed the multivariable model (AUC = 0.81) at a cross-sectional level. At diagnosis, CA19-9 (AUC = 0.995) and the model (AUC = 0.977) performed similarly. Longitudinal analysis revealed increases in CA19-9 up to two years to diagnosis which indicates a window of opportunity for early detection of PDAC.

Plasma Micro-RNA Alterations Appear Late in Pancreatic Cancer.

OBJECTIVES: The aim of this research was to study whether plasma microRNAs (miRNA) can be used for early detection of pancreatic cancer (PC) by analyzing prediagnostic plasma samples collected before a PC diagnosis. BACKGROUND: PC has a poor prognosis due to late presenting symptoms and early metastasis. Circulating miRNAs are altered in PC at diagnosis but have not been evaluated in a prediagnostic setting. METHODS: We first performed an initial screen using a panel of 372 miRNAs in a retrospective case-control cohort that included early-stage PC patients and healthy controls. Significantly altered miRNAs at diagnosis were then measured in an early detection case-control cohort wherein plasma samples in the cases are collected before a PC diagnosis. Carbohydrate antigen 19-9 (Ca 19-9) levels were measured in all samples for comparison. RESULTS: Our initial screen, including 23 stage I-II PC cases and 22 controls, revealed 15 candidate miRNAs that were differentially expressed in plasma samples at PC diagnosis. We combined all 15 miRNAs into a multivariate statistical model, which outperformed Ca 19-9 in receiver-operating characteristics analysis. However, none of the candidate miRNAs, individually or in combination, were significantly altered in prediagnostic plasma samples from 67 future PC patients compared with 132 matched controls. In comparison, Ca 19-9 levels were significantly higher in the cases at <5 years before diagnosis. CONCLUSION: Plasma miRNAs are altered in PC patients at diagnosis, but the candidate miRNAs found in this study appear late in the course of the disease and cannot be used for early detection of the disease.

Statin consumption as a risk factor for developing colorectal cancer: a retrospective case study.

BACKGROUND: Statins are the backbone of lipid-lowering therapy and are among the most commonly prescribed drugs in the elderly population in Sweden today. Colorectal cancer is the second most common cancer in men and women, after prostate and breast cancer, respectively, with a median age of 72 years at diagnosis. Statins induce mitochondrial damage leading to accumulation of reactive oxygen species in the cell. Reactive oxygen species can cause mutations in mitochondrial as well as nuclear DNA leading to the development of cancer. Our hypothesis was that statins increase the risk for colorectal cancer. METHODS: A case study was performed on consecutive cases of colorectal cancer diagnosed at Norrlands University Hospital (NUS) in Umeå between 2012 and 2015 (n = 325). Patients diagnosed with diabetes mellitus type II (DM II n = 65) were excluded in the primary endpoint analysis (occurrence of colorectal cancer). As control, three databases were used to create an age-matched population in order to calculate the proportion of inhabitants using statins in the county of Västerbotten, Sweden. A secondary endpoint was cancer-specific survival among our study group of colorectal cancer patients, including those with DM II, investigating whether there was a difference if the patient was a 'recent' statin user or not at the time of diagnosis. RESULTS: Statin use at the time of colorectal cancer diagnosis in the study group was 23.8%. The corresponding figure in an age-matched population in Västerbotten was 24.6%. Using a one-proportional one-sided z test, there was no significant difference between these (23.8%, 95% CI 18.6-29.0%, p = 0.601). When comparing groups 20-64 years of age, the difference was greater with recent statin use in 17.8% in the study population and 11.9% in Västerbotten (17.8%, 95% CI 9.0-26.6%, p = 0.059). When considering cancer-specific survival, no significant difference in survival was seen when comparing 'former/never' statin users as reference category with 'recent' users diagnosed with colorectal cancer (HR 1.39, 95% CI 0.89-2.16). CONCLUSIONS: No significant increase in risk for developing colorectal cancer among patients (type II diabetics excluded) medicated with statins was found. We found no correlation between 'recent' statin use at the time of diagnosis and cancer-specific survival.

Effects of Nutrient Loading and Mercury Chemical Speciation on the Formation and Degradation of Methylmercury in Estuarine Sediment.

Net formation of methylmercury (MeHg) in sediments is known to be affected by the availability of inorganic divalent mercury (Hg(II)) and by the activities of Hg(II) methylating and MeHg demethylating bacteria. Enhanced autochthonous organic matter deposition to the benthic zone, following increased loading of nutrients to the pelagic zone, has been suggested to increase the activity of Hg(II) methylating bacteria and thus the rate of net methylation. However, the impact of increased nutrient loading on the biogeochemistry of mercury (Hg) is challenging to predict as different geochemical pools of Hg may respond differently to enhanced bacterial activities. Here, we investigate the combined effects of nutrient (N and P) supply to the pelagic zone and the chemical speciation of Hg(II) and of MeHg on MeHg formation and degradation in a brackish sediment-water mesocosm model ecosystem. By use of Hg isotope tracers added in situ to the mesocosms or ex situ in incubation experiments, we show that the MeHg formation rate increased with nutrient loading only for Hg(II) tracers with a high availability for methylation. Tracers with low availability did not respond significantly to nutrient loading. Thus, both microbial activity (stimulated indirectly through plankton biomass production by nutrient loading) and Hg(II) chemical speciation were found to control the MeHg formation rate in marine sediments.

Type IV collagen stimulates pancreatic cancer cell proliferation, migration, and inhibits apoptosis through an autocrine loop.

BACKGROUND: Pancreatic cancer shows a highly aggressive and infiltrative growth pattern and is characterized by an abundant tumor stroma known to interact with the cancer cells, and to influence tumor growth and drug resistance. Cancer cells actively take part in the production of extracellular matrix proteins, which then become deposited into the tumor stroma. Type IV collagen, an important component of the basement membrane, is highly expressed by pancreatic cancer cells both in vivo and in vitro. In this study, the cellular effects of type IV collagen produced by the cancer cells were characterized. METHODS: The expression of type IV collagen and its integrin receptors were examined in vivo in human pancreatic cancer tissue. The cellular effects of type IV collagen were studied in pancreatic cancer cell lines by reducing type IV collagen expression through RNA interference and by functional receptor blocking of integrins and their binding-sites on the type IV collagen molecule. RESULTS: We show that type IV collagen is expressed close to the cancer cells in vivo, forming basement membrane like structures on the cancer cell surface that colocalize with the integrin receptors. Furthermore, the interaction between type IV collagen produced by the cancer cell, and integrins on the surface of the cancer cells, are important for continuous cancer cell growth, maintenance of a migratory phenotype, and for avoiding apoptosis. CONCLUSION: We show that type IV collagen provides essential cell survival signals to the pancreatic cancer cells through an autocrine loop.

Controlling and monitoring orientation of DNA nanoconstructs on lipid surfaces.

Its extraordinary self-assembly property, with potential to form nonperiodic structures with unique addressability, makes DNA ideal for fabrication of advanced nanostructures. We here demonstrate the controllable tethering of a hexagonal DNA nanostructure in two distinct orientations at the lipid bilayer of a liposome functioning as a soft-matter support. With polarized light (linear dichroism) applied to the flow-aligned liposomes, we show that the construct is preferentially in a parallel alignment with the lipid surface when two anchors are attached while with one anchor only a perpendicular orientation is observed.

Mercury methylation rates for geochemically relevant Hg(II) species in sediments.

Monomethylmercury (MeHg) in fish from freshwater, estuarine, and marine environments is a major global environmental issue. Mercury levels in biota are mainly controlled by the methylation of inorganic mercuric mercury (Hg(II)) to MeHg in water, sediments, and soils. There is, however, a knowledge gap concerning the mechanisms and rates of methylation of specific geochemical Hg(II) species. Such information is crucial for a better understanding of variations in MeHg concentrations among ecosystems and, in particular, for predicting the outcome of currently proposed measures to mitigate mercury emissions and reduce MeHg concentrations in fish. To fill this knowledge gap we propose an experimental approach using Hg(II) isotope tracers, with defined and geochemically important adsorbed and solid Hg(II) forms in sediments, to study MeHg formation. We report Hg(II) methylation rate constants, k(m), in estuarine sediments which span over 2 orders of magnitude depending on chemical form of added tracer: metacinnabar (ß-(201)HgS(s)) < cinnabar (α-(199)HgS(s)) < Hg(II) reacted with mackinawite (≡FeS-(202)Hg(II)) < Hg(II) bonded to natural organic matter (NOM-(196)Hg(II)) < a typical aqueous tracer ((198)Hg(NO(3))(2)(aq)). We conclude that a combination of thermodynamic and kinetic effects of Hg(II) solid-phase dissolution and surface desorption control the Hg(II) methylation rate in sediments and cause the large observed differences in k(m)-values. The selection of relevant solid-phase and surface-adsorbed Hg(II) tracers will therefore be crucial to achieving biogeochemically accurate estimates of ambient Hg(II) methylation rates.

Crystal structure of the TL29 protein from Arabidopsis thaliana: an APX homolog without peroxidase activity.

TL29 is a plant-specific protein found in the thylakoid lumen of chloroplasts. Despite the putative requirement in plants for a peroxidase close to the site of photosynthetic oxygen production, and the sequence homology of TL29 to ascorbate peroxidases, so far biochemical methods have not shown this enzyme to possess peroxidase activity. Here we report the three-dimensional X-ray crystal structure of recombinant TL29 from Arabidopsis thaliana at a resolution of 2.5Å. The overall structure of TL29 is mainly alpha helical with six longer and six shorter helical segments. The TL29 structure resembles that of typical ascorbate peroxidases, however, crucial differences were found in regions that would be important for heme and ascorbate binding. Such differences suggest it to be highly unlikely that TL29 functions as a peroxidase.

Circulating Tissue Polypeptide-Specific Antigen in Pre-Diagnostic Pancreatic Cancer Samples.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is challenging, and late diagnosis partly explains the low 5-year survival. Novel and sensitive biomarkers are needed to enable early PDAC detection and improve patient outcomes. Tissue polypeptide specific antigen (TPS) has been studied as a biomarker in PDAC diagnostics, and it has previously been shown to reflect clinical status better than the 'golden standard' biomarker carbohydrate antigen 19-9 (CA 19-9) that is most widely used in the clinical setting. In this cross-sectional case-control study using pre-diagnostic plasma samples, we aim to evaluate the potential of TPS as a biomarker for early PDAC detection. Furthermore, in a subset of individuals with multiple samples available at different time points before diagnosis, a longitudinal analysis was used. We assessed plasma TPS levels using enzyme-linked immunosorbent assay (ELISA) in 267 pre-diagnostic PDAC plasma samples taken up to 18.8 years before clinical PDAC diagnosis and in 320 matched healthy controls. TPS levels were also assessed in 25 samples at PDAC diagnosis. Circulating TPS levels were low both in pre-diagnostic samples of future PDAC patients and in healthy controls, whereas TPS levels at PDAC diagnosis were significantly increased (odds ratio 1.03; 95% confidence interval: 1.01-1.05) in a logistic regression model adjusted for age. In conclusion, TPS levels increase late in PDAC progression and hold no potential as a biomarker for early detection.

An Investigation of the Clinical Use of a Single-Word Procedure to Assess Intelligibility (Swedish Test of Intelligibility for Children) and an Evaluation of the Validity and Reliability of the Intelligibility in Context Scale.

Purpose Intelligibility is a core concept of speech-language pathology, central both to the assessment of speech disorders and to intervention in such disorders. One purpose of this study was to investigate the reliability and usability of a single-word assessment procedure, the Swedish Test of Intelligibility for Children (STI-CH), in a clinical setting. Another purpose was to investigate the validity and reliability of an assessment method designed to assess functional intelligibility: the Intelligibility in Context Scale (ICS). Method Thirty children aged 4;1-10;1 years;months were recruited from speech-language pathology clinics in Western Sweden. Each child's treating speech-language pathologist served as listener in the STI-CH assessment while the ICS was completed by each child's parents. External listeners (2 last-year speech-language pathology students) were used to assess the validity of the speech-language pathology. Results The mean duration of the test procedure for the STI-CH was about 19 min, and 57% of the test sessions were reported as difficult. There was a weak but statistically significant correlation between the results from the STI-CH and the ICS ( r = .40, p < .05), and both methods showed high reliability in terms of interlistener reliability (intraclass correlation exceeding .97) and internal consistency, respectively. Conclusions The STI-CH had high reliability and was time efficient but had some procedural problems. The ICS had moderate validity but high reliability. The STI-CH is promising for clinical use but needs to be developed further. The validity of the ICS can be discussed and needs to be further investigated.

Association between occurrence of urinary bladder cancer and treatment with statin medication.

OBJECTIVE: The incidence of urinary bladder cancer (UBC) has increased in Sweden despite decreased smoking, indicating that other factors might be associated. The increased use of statin medication for elevated blood lipids might be one such influencing factor. The aim of the present study was to assess whether statins are afflicted with an increased incidence of UBC. MATERIAL AND METHODS: Data from the Swedish National Register of Urinary Bladder Cancer, National Population Register, and Swedish Prescribed Drug Register were extracted. There were 22,936 patients with new diagnosed UBC between 2005 and 2014. Statin prescription was defined as any medication prescribed with the Anatomical Therapeutic Classification code C10A. For each patient, 10 control individuals were matched by age, gender, and living area, comprising 229,326 individuals. The Cochran-Mantel-Haenszel test was used to evaluate the hazards ratios. RESULTS: Statins were more frequently used in patients with UBC (33.8%) than in controls (29.8%, p<0.0001). The use of statins was afflicted with a 23% increased odds ratio (OR) for UBC (OR 1.23 (1.19-1.27), p<0.001). Subgroup analyses showed that an increased OR was found in non-muscle invasive UBC only. There was a tendency that OR was stronger for men and for younger patients. Limitations include its retrospective register-based design and potential risk of bias of confounding factors, such as smoking and body mass index. CONCLUSION: This nationwide register study suggests an association between the occurrence of UBC and patients using statins. The association was found in patients with non-muscle invasive disease only. Confounding factors, such as smoking, cannot be overruled.

Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal.

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

Terrestrial discharges mediate trophic shifts and enhance methylmercury accumulation in estuarine biota.

The input of mercury (Hg) to ecosystems is estimated to have increased two- to fivefold during the industrial era, and Hg accumulates in aquatic biota as neurotoxic methylmercury (MeHg). Escalating anthropogenic land use and climate change are expected to alter the input rates of terrestrial natural organic matter (NOM) and nutrients to aquatic ecosystems. For example, climate change has been projected to induce 10 to 50% runoff increases for large coastal regions globally. A major knowledge gap is the potential effects on MeHg exposure to biota following these ecosystem changes. We monitored the fate of five enriched Hg isotope tracers added to mesocosm scale estuarine model ecosystems subjected to varying loading rates of nutrients and terrestrial NOM. We demonstrate that increased terrestrial NOM input to the pelagic zone can enhance the MeHg bioaccumulation factor in zooplankton by a factor of 2 to 7 by inducing a shift in the pelagic food web from autotrophic to heterotrophic. The terrestrial NOM input also enhanced the retention of MeHg in the water column by up to a factor of 2, resulting in further increased MeHg exposure to pelagic biota. Using mercury mass balance calculations, we predict that MeHg concentration in zooplankton can increase by a factor of 3 to 6 in coastal areas following scenarios with 15 to 30% increased terrestrial runoff. The results demonstrate the importance of incorporating the impact of climate-induced changes in food web structure on MeHg bioaccumulation in future biogeochemical cycling models and risk assessments of Hg.

Differentiated availability of geochemical mercury pools controls methylmercury levels in estuarine sediment and biota.

Neurotoxic methylmercury (MeHg) formed from inorganic divalent mercury (Hg(II)) accumulates in aquatic biota and remains at high levels worldwide. It is poorly understood to what extent different geochemical Hg pools contribute to these levels. Here we report quantitative data on MeHg formation and bioaccumulation, in mesocosm water-sediment model ecosystems, using five Hg(II) and MeHg isotope tracers simulating recent Hg inputs to the water phase and Hg stored in sediment as bound to natural organic matter or as metacinnabar. Calculations for an estuarine ecosystem suggest that the chemical speciation of Hg(II) solid/adsorbed phases control the sediment Hg pool's contribution to MeHg, but that input of MeHg from terrestrial and atmospheric sources bioaccumulates to a substantially greater extent than MeHg formed in situ in sediment. Our findings emphasize the importance of MeHg loadings from catchment runoff to MeHg content in estuarine biota and we suggest that this contribution has been underestimated.

Can humic water discharge counteract eutrophication in coastal waters?

A common and established view is that increased inputs of nutrients to the sea, for example via river flooding, will cause eutrophication and phytoplankton blooms in coastal areas. We here show that this concept may be questioned in certain scenarios. Climate change has been predicted to cause increased inflow of freshwater to coastal areas in northern Europe. River waters in these areas are often brown from the presence of high concentrations of allochthonous dissolved organic carbon (humic carbon), in addition to nitrogen and phosphorus. In this study we investigated whether increased inputs of humic carbon can change the structure and production of the pelagic food web in the recipient seawater. In a mesocosm experiment unfiltered seawater from the northern Baltic Sea was fertilized with inorganic nutrients and humic carbon (CNP), and only with inorganic nutrients (NP). The system responded differently to the humic carbon addition. In NP treatments bacterial, phytoplankton and zooplankton production increased and the systems turned net autotrophic, whereas the CNP-treatment only bacterial and zooplankton production increased driving the system to net heterotrophy. The size-structure of the food web showed large variations in the different treatments. In the enriched NP treatments the phytoplankton community was dominated by filamentous >20 µm algae, while in the CNP treatments the phytoplankton was dominated by picocyanobacteria <5 µm. Our results suggest that climate change scenarios, resulting in increased humic-rich river inflow, may counteract eutrophication in coastal waters, leading to a promotion of the microbial food web and other heterotrophic organisms, driving the recipient coastal waters to net-heterotrophy.

Nanofabrication yields. Hybridization and click-fixation of polycyclic DNA nanoassemblies.

We demonstrate the stepwise assembly of a fully addressable polycyclic DNA hexagon nanonetwork for the preparation of a four-ring system, one of the biggest networks yet constructed from tripodal building blocks. We find that the yield exhibits a distinct upper level <100%, a fundamental problem of thermodynamic DNA assembly that appears to have been overlooked in the DNA nanotechnology literature. A simplistic model based on a single step-yield parameter y can quantitatively describe the total yield of DNA assemblies in one-pot reactions as Y = y(duplex)(n), with n the number of hybridization steps. Experimental errors introducing deviations from perfect stoichiometry and the thermodynamics of hybridization equilibria contribute to decreasing the value of y(duplex) (on average y = 0.96 for our 10 base pair hybridization). For the four-ring system (n = 31), the total yield is thus less than 30%, which is clearly unsatisfactory if bigger nanoconstructs of this class are to be designed. Therefore, we introduced site-specific click chemistry for making and purifying robust building blocks for future modular constructs of larger assemblies. Although the present yield of this robust module was only about 10%, it demonstrates a first step toward a general fabrication approach. Interestingly, we find that the click yields follow quantitatively a binomial distribution, the predictability of which indicates the usefulness of preparing pools of pure and robust building blocks in this way. The binomial behavior indicates that there is no interference between the six simultaneous click reactions but that step-yield limiting factors such as topological constraints and Cu(I) catalyst concentration are local and independent.

A new fixation strategy for addressable nano-network building blocks.

Rapid controlled self-assembly makes DNA ideal for building nanostructures. A problem using hybridized intermediates in hierarchic assembly is their thermodynamic lability. We demonstrate a click-fixation technology by which robust hexagonal DNA modules can be made. This principle is applicable to a wide variety of DNA nanoconstructs.

Stability and fibril formation properties of human and fish transthyretin, and of the Escherichia coli transthyretin-related protein.

Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease. Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amyloid fibrils. The transthyretin (TTR)-related proteins comprise a protein family of 5-hydroxyisourate hydrolases with structural similarity to TTR. In this study, we tested the amyloidogenic properties, if any, of sea bream TTR (sbTTR) and Escherichia coli transthyretin-related protein (ecTRP), which share 52% and 30% sequence identity, respectively, with hTTR. We obtained filamentous structures from all three proteins under various conditions, but, interestingly, different structures displayed different tinctorial properties. hTTR and sbTTR formed thin, curved fibrils at low pH (pH 2-3) that bound thioflavin-T (thioflavin-T-positive) but did not stain with Congo Red (CR) (CR-negative). Aggregates formed at the slightly higher pH of 4.0-5.5 had different morphology, displaying predominantly amorphous structures. CR-positive material of hTTR was found in this material, in agreement with previous results. ecTRP remained soluble at pH 2-12 at ambient temperatures. By raising of the temperature, fibril formation could be induced at neutral pH in all three proteins. Most of these temperature-induced fibrils were thicker and straighter than the in vitro fibrils seen at low pH. In other words, the temperature-induced fibrils were more similar to fibrils seen in vivo. The melting temperature of ecTRP was 66.7 degrees C. This is approximately 30 degrees C lower than the melting temperatures of sbTTR and hTTR. Information from the crystal structures was used to identify possible explanations for the reduced thermostability of ecTRP.