ABSTRACT Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) has emerged as an attractive target for the development of modulatory compounds, particularly in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1(PCASP-Ig3) 339–719 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained largely unexplored. We present here inaugural dynamic analyses of the apo MALT1(PCASP-Ig3) 339–719 form along with its mutated variant, E549A. This investigation harnessed an array of NMR relaxation techniques, including longitudinal and transverse 15 N auto-relaxation, heteronuclear NOE, transverse cross-correlated relaxation and NOE measurements between side-chain methyl groups. Our findings unequivocally confirm that MALT1(PCASP-Ig3) 339–719 exists solely as a monomer in solution, and demonstrate that the two domains display semi-independent movements in relation to each other. Our extensive dynamic study, covering a range of time scales, along with the assessment of diverse conformational populations for MALT1(PCASP-Ig3) 339–719 , by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, shed light at potential mechanisms underlying the allosteric regulation of this enzyme, and the specific importance of interdomain motions.

Various forms of diabetes care necessitate exogenous insulin replacement. Administered insulin must have predictable potency to avoid potentially dangerous glycaemic excursions. However, insulin is temperature sensitive, with potency reduced through rising temperatures.1Storvick WO Henry HJ Effect of storage temperature on stability of commercial insulin preparations.Diabetes. 1968; 17: 499-502Crossref PubMed Scopus (28) Google Scholar, 2Pingel M Vølund A Stability of insulin preparations.Diabetes. 1972; 21: 805-813Crossref PubMed Scopus (32) Google Scholar, 3Grajower MM Fraser CG Holcombe JH et al.How long should insulin be used once a vial is started?.Diabetes Care. 2003; 26: 2665-2666Crossref PubMed Scopus (36) Google Scholar Insulin manufacturers and regulatory agencies direct that insulin be refrigerated (at 4–6°C), never frozen, and with a maximum usage or storage period of approximately 1 month at standard room temperatures (20–25°C).3Grajower MM Fraser CG Holcombe JH et al.How long should insulin be used once a vial is started?.Diabetes Care. 2003; 26: 2665-2666Crossref PubMed Scopus (36) Google Scholar This requirement is especially challenging in hot climate settings if home refrigeration is unavailable, which is the case for some 770 million people.4Ogle GD Middlehurst AC Silink M The IDF Life for a Child Program Index of diabetes care for children and youth.Pediatr Diabetes. 2016; 17: 374-384Crossref PubMed Scopus (32) Google Scholar, 5Burki T Hot topics on insulin access: the issue of thermostability.Lancet Diabetes Endocrinol. 2022; 10: 389-390Summary Full Text Full Text PDF PubMed Scopus (2) Google Scholar Furthermore, in some situations, insulin must also be stored for a few months due to clinic visit frequency, travel costs, and intermittent pharmacy supplies.4Ogle GD Middlehurst AC Silink M The IDF Life for a Child Program Index of diabetes care for children and youth.Pediatr Diabetes. 2016; 17: 374-384Crossref PubMed Scopus (32) Google Scholar, 6Ogle GD Abdullah M Mason D Januszewski AS Besançon S Insulin storage in hot climates without refrigeration: temperature reduction efficacy of clay pots and other techniques.Diabet Med. 2016; 33: 1544-1553Crossref PubMed Scopus (30) Google Scholar In response to this, many families use evaporative cooling with clay pots to assist in reducing insulin storage temperatures, by storing insulin within an air-filled space or a sealed bag in clay pots.6Ogle GD Abdullah M Mason D Januszewski AS Besançon S Insulin storage in hot climates without refrigeration: temperature reduction efficacy of clay pots and other techniques.Diabet Med. 2016; 33: 1544-1553Crossref PubMed Scopus (30) Google Scholar Data regarding insulin stability outside the recommendations are scarce. In 1968, Storvick and Henry1Storvick WO Henry HJ Effect of storage temperature on stability of commercial insulin preparations.Diabetes. 1968; 17: 499-502Crossref PubMed Scopus (28) Google Scholar reported that animal-derived isophane insulin retained 95% or more of its potency for 12 months at 25°C. In 1972, Pingel and Volund2Pingel M Vølund A Stability of insulin preparations.Diabetes. 1972; 21: 805-813Crossref PubMed Scopus (32) Google Scholar found that similar isophane and soluble insulins retained 95% or more of their potency for at least 5 months at temperatures of up to 30°C. A study using human insulin showed loss of some potency during storage at 32–37°C for 28 days,7Vimalavathini R Gitanjali B Effect of temperature on the potency & pharmacological action of insulin.Indian J Med Res. 2009; 130: 166-169PubMed Google Scholar whereas a recent study showed that potency of human and analogue insulin remained unchanged at oscillating temperatures of 25–37°C for 28 days and 12 weeks.8Kaufmann B Boulle P Berthou F et al.Heat-stability study of various insulin types in tropical temperature conditions: new insights towards improving diabetes care.PLoS One. 2021; 16e0245372 Crossref Scopus (17) Google Scholar In this pilot study, we aimed to identify: (1) the potency of six 100 IU/mL insulins (vials of human soluble; Eli Lilly), human isophane (Eli Lilly), and human soluble–isophane (30:70; Novo Nordisk); and 3 mL cartridges of insulin aspart (Novo Nordisk), and two preparations of insulin glargine (Sanofi and Eli Lilly) stored unopened for 1–4 months in non-refrigerated conditions in a real-world setting during the summer in India, all compared with control samples of each insulin, which remained refrigerated; and (2) whether there was any difference in storage temperatures and changes in potency between samples stored within and outside clay pots. These insulins were chosen to include commonly used insulins. Six families with a person younger than 25 years with insulin-requiring diabetes attending the Diabetes Research Education and Management (DREAM) Trust in Nagpur, India, between March and June, 2021, participated in this study. Each received two different types of insulin. Insulin vials were stored in watertight bags. Bags were placed in either an open plastic container stored on a high shelf or in a cupboard, or in clay pots with a separate water compartment. All storage containers were placed in shaded areas (appendix). Control samples of all insulins were also refrigerated at the DREAM Trust, and these samples were used to measure relative potency of unrefrigerated samples. The temperature was measured every 15 min by electronic data loggers. The methodology of this study is described in detail in the appendix (pp 1–5). The monthly mean open box temperature across the six families ranged from 29·4°C to 32·0°C, with mean maximum temperatures of 30·4–34·9°C and minimum temperatures of 28·3–29·8°C (appendix pp 6–7). Compared with the open boxes, the clay pots significantly reduced temperatures by mean 2·6°C (SD 1·8, range 0·4–5·7, p<0·0001) in ten of the 12 clay pots (appendix p 8). Insulin analyses were done at the University of Florida, FL, USA, with high-performance liquid chromatography, and at the University of Gothenburg, Gothenburg, Sweden, with nuclear magnetic resonance spectroscopy (appendix pp 8–21). The potency was measured with US Pharmacopeia monographs for each insulin as described in the appendix (pp 8–11). Potency is defined in terms of insulin units per millilitre (IU/mL) and be around 100 IU/mL (±5%).9Garrett TJ Atkinson P Quinlivan EP et al.Commercially available insulin products demonstrate stability throughout the cold supply chain across the US.Diabetes Care. 2020; 43: 1360-1362Crossref PubMed Scopus (4) Google Scholar In the analysis at the University of Florida, all human insulin samples maintained 95% or more of the refrigerated potency except for one vial each of human soluble, human soluble–isophane (30:70), and human isophane (range 92·4–94·1%), all at 4 months (figure). Human soluble and human soluble–isophane (70:30) relative potencies drifted downward over the 4-month period. The analogue insulins (Aspart [Kalundborg, Denmark], Glargine [Basaglar, Indianapolis, IN, USA], and Glargine [Lantus, Frankfurt, Germany]), tested all assayed 95% or more relative potency at all timepoints. All relative and raw sample measurements are included in the appendix (pp 8–11). Clay pot storage resulted in less decline in relative potency compared with refrigerated samples at 4 months than open box storage (0·5% vs 3·6%, p=0·001; appendix p 20). In the analysis done at the University of Gothenburg, there was an apparent subtle line width increase and slight peak shifts in nuclear magnetic resonance spectra, which correlated with storage time for non-refrigerated samples (appendix pp 16-17). This is consistent with subtle, well known changes due to altered conformation (eg, relaxed to tense transition of the insulin hexamer) or multimerisation (eg, formation of dimers of insulin hexamer) in a minor fraction of the insulin molecules. A decrease in total concentration in any of the insulin types was not observed as being larger than the precision of measurements (<1%), regardless of storage time or type of storage (appendix pp 12–14). This study of insulin thermostability outside refrigeration, during the summer in India, showed that acceptable insulin concentrations were maintained up to 2 months for all samples of all insulin preparations. At 4 months, all samples from three analogue insulin preparations and three of four samples for each of the human insulins also maintained a relative concentration of 95% or more. The US Pharmacopeia stipulates that insulin potency should be 100 IU/mL (±5%) to be safe to use.9Garrett TJ Atkinson P Quinlivan EP et al.Commercially available insulin products demonstrate stability throughout the cold supply chain across the US.Diabetes Care. 2020; 43: 1360-1362Crossref PubMed Scopus (4) Google Scholar No loss of relative concentration was found using nuclear magnetic resonance. These results are consistent with the findings of Kaufman and colleagues,8Kaufmann B Boulle P Berthou F et al.Heat-stability study of various insulin types in tropical temperature conditions: new insights towards improving diabetes care.PLoS One. 2021; 16e0245372 Crossref Scopus (17) Google Scholar which noted preservation of insulin concentration of human and analogue insulins at 4 weeks and then 12 weeks, at cycled laboratory temperatures similar to the current study. Study limitations include the small number of samples, and that the samples were re-refrigerated after the period in the open box or clay pot. Furthermore, insulin potency and concentration were only tested by analytical methods and not by changes in glucose concentration in vivo. The employed methods do not establish the exact nature of the observed insulin following this method of storage, so additional studies will be required to address this. The vials remained unused during the study, whereas in a real-world situation insulin would be witdrawn frequently. The sterility of vials stored in clay pots was not tested. If these results are confirmed in real-world and laboratory studies with larger sample sizes, and the further in vivo and biochemical tests show reassuring findings, then regulatory agencies could be prompted to review the requirement to dispose of unrefrigerated insulin after 1 month or a similar period at room temperature (20–25°C).6Ogle GD Abdullah M Mason D Januszewski AS Besançon S Insulin storage in hot climates without refrigeration: temperature reduction efficacy of clay pots and other techniques.Diabet Med. 2016; 33: 1544-1553Crossref PubMed Scopus (30) Google Scholar Potentially, usage could be extended to 2–4 months in situations in which daily temperatures cycle from 25°C to 35°C when refrigeration is not available. This would reduce cost, waste, and family anxiety about whether the insulin is still effective and safe to use. In addition, it could help to provide health professionals with guidance and reassurance; and likely improve insulin access in under-resourced settings. Pande and Thakur10Pande AR Thakur AK Perceptions of alternative methods of insulin storage: an ethical dilemma.Lancet Diabetes Endocrinol. 2022; 10: 558Summary Full Text Full Text PDF PubMed Scopus (2) Google Scholar described the ethical dilemma commonly faced in these situations about what advice the doctor should provide about storing insulin according to the recommendations, in a situation where replacement insulin is unavailable or unaffordable. Clay pots and other traditional evaporative cooling techniques are used by families and recommended by many health professionals to keep insulin cool in several countries, and such devices have been shown to reduce storage temperatures by up to 8°C. Clay pots are especially effective in lower-humidity situations.6Ogle GD Abdullah M Mason D Januszewski AS Besançon S Insulin storage in hot climates without refrigeration: temperature reduction efficacy of clay pots and other techniques.Diabet Med. 2016; 33: 1544-1553Crossref PubMed Scopus (30) Google Scholar Our results strengthen evidence for the use of these basic devices by showing that clay pot storage can reduce the decline in insulin potency. Finally, it would be benefical if, concurrently, insulin manufacturers would release any other relevant data on potency and safety of insulin stored for periods and in temperatures beyond product guidelines, so this could be considered in guidelines for less-resourced situations. Notably, the European Medicines Agency has released a recommendation that permits storage of two human insulin preparations for 30 days at temperatures less than 30°C before use. In conclusion, the results of this study suggest that unrefrigerated insulins might be sufficiently thermostable for 2 months, and possibly even up to 4 months. In the absence of refrigeration, clay pot storage appears to be effective in reducing storage temperatures and declines in insulin potency. JM and GDO work for the Life for a Child programme at Diabetes NSW & ACT, which does receive non-salary support from Eli Lilly. MAA reports cooperation (consultative, educational, and research) with companies and entities (CodeBio, Diamyd Medical, Endsulin, IM therapeutics, and Repitoire) interested in type 1 diabetes prevention and reversal (not directly insulin thermostability), as well as receiving an independent award from the Novo Nordisk Foundation. MAA is also President of Insulin for Life USA. All other authors declare no competing interests. Download .pdf (1.71 MB) Help with pdf files Supplementary appendix

Cationic dioctadecyldimethylammonium bromide and chloride (DODAB and DODAC) lipids assemble in water as unilamellar vesicles or more complex bilayer structures depending on the lipid concentration, counter-ion, solvent characteristics, etc. By using differential scanning calorimetry (DSC) and cryogenic transmission electron microscopy (cryo-TEM) techniques we investigated the structural and thermal properties of these two widely investigated molecules at 1-20 mM in water. Changes in the DSC thermogram profile indicate that DODAB bilayers undergo more structural changes than DODAC, while Cryo-TEM micrographs show that, in this range of concentration, DODAB assembles as more complex bilayer structures than DODAC, which assemble predominantly as unilamellar vesicles. These findings thus indicate that DODAC is preferable to DODAB at 1-20 mM for the preparation of unilamellar vesicles.

Known and consistent bioactivity between samples of insulin is essential to correctly estimate the dose. Insulin concentration is not the same thing as bioactivity, however, and methods to correctly determine both are required. Here we show that one dimensional nuclear magnetic resonance (1D NMR), in contrast to, for example, reverse phase high pressure liquid chromatography, can be used to determine both insulin concentration as well as confirm the structural integrity required for activity. In response to the report by Carter and Heinemann, we decided to investigate insulin intended for public use. Insulin from several manufacturers was investigated. Correct insulin concentrations were found in all tested samples although the general sample variability, which possibly could influence the bioactivity, varied depending on insulin type and manufacturer.

Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.

Metabolomics represents a powerful tool for exploring modulation of the human metabolome in response to food intake. However, the choice of multivariate statistical approach is not always evident, especially for complex experimental designs with repeated measurements per individual. Here we have investigated the serum metabolic responses to two breakfast meals: an egg and ham based breakfast and a cereal based breakfast using three different multivariate approaches based on the Projections to Latent Structures framework. In a cross over design, 24 healthy volunteers ate the egg and ham breakfast and cereal breakfast on four occasions each. Postprandial serum samples were subjected to metabolite profiling using 1H nuclear magnetic resonance spectroscopy and metabolites were identified using 2D nuclear magnetic resonance spectroscopy. Metabolic profiles were analyzed using Orthogonal Projections to Latent Structures with Discriminant Analysis and Effect Projections and ANOVA-decomposed Projections to Latent Structures. The Orthogonal Projections to Latent Structures with Discriminant Analysis model correctly classified 92 and 90% of the samples from the cereal breakfast and egg and ham breakfast, respectively, but confounded dietary effects with inter-personal variability. Orthogonal Projections to Latent Structures with Effect Projections removed inter-personal variability and performed perfect classification between breakfasts, however at the expense of comparing means of respective breakfasts instead of all samples. ANOVA-decomposed Projections to Latent Structures managed to remove inter-personal variability and predicted 99% of all individual samples correctly. Proline, tyrosine, and N-acetylated amino acids were found in higher concentration after consumption of the cereal breakfast while creatine, methanol, and isoleucine were found in higher concentration after the egg and ham breakfast. Our results demonstrate that the choice of statistical method will influence the results and adequate methods need to be employed to manage sample dependency and repeated measurements in cross-over studies. In addition, 1H nuclear magnetic resonance serum metabolomics could reproducibly characterize postprandial metabolic profiles and identify discriminatory metabolites largely reflecting dietary composition. Registered with ClinicalTrials.gov, identifier: NCT02039596 . Date of registration: January 17, 2014.

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2?inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239?274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239?256 and aa 257?274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242?268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.

Metabolomics provide an unbiased tool for exploring the modulation of the human metabolome in response to food intake. This study applied metabolomics to capture the postprandial metabolic response to breakfast meals corresponding to vegan (VE), lacto ovo-vegetarian (LOV), and omnivore (OM) diets. In a cross over design 32 healthy volunteers (16 men and 16 females) consumed breakfast meals in a randomized order during three consecutive days. Fasting and 3 h postprandial serum samples were collected and then subjected to metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy. Changes in concentration of identified and discriminating metabolites, between fasting and postprandial state, were compared across meals. Betaine, choline, and creatine displayed higher concentration in the OM breakfast, while 3-hydroxyisobutyrate, carnitine, proline, and tyrosine showed an increase for the LOV and unidentified free fatty acids displayed a higher concentration after the VE breakfast. Using 1H NMR metabolomics it was possible to detect and distinguish the metabolic response of three different breakfast meals corresponding to vegan, lacto-ovo vegetarian, and omnivore diets in serum.

A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the μM to nM range for which no small molecule ligands are known.

The ability of ectotherms to respond to changes in their thermal environment through plastic mechanisms is central to their adaptive capability. However, we still lack knowledge on the physiological and functional responses by which ectotherms acclimate to temperatures during development, and in particular, how physiological stress at extreme temperatures may counteract beneficial acclimation responses at benign temperatures. We exposed Drosophila melanogaster to 10 developmental temperatures covering their entire permissible temperature range. We obtained metabolic profiles and reaction norms for several functional traits: egg-to-adult viability, developmental time, and heat and cold tolerance. Females were more heat tolerant than males, whereas no sexual dimorphism was found in cold tolerance. A group of metabolites, mainly free amino acids, had linear reaction norms. Several energy-carrying molecules, as well as some sugars, showed distinct inverted U-shaped norms of reaction across the thermal range, resulting in a positive correlation between metabolite intensities and egg-to-adult viability. At extreme temperatures, low levels of these metabolites were interpreted as a response characteristic of costs of homeostatic perturbations. Our results provide novel insights into a range of metabolites reported to be central for the acclimation response and suggest several new candidate metabolites. Low and high temperatures result in different adaptive physiological responses, but they also have commonalities likely to be a result of the failure to compensate for the physiological stress. We suggest that the regulation of metabolites that are tightly connected to the performance curve is important for the ability of ectotherms to cope with variation in temperature.

It is challenging to measure dietary exposure with techniques that are both accurate and applicable to free-living individuals. We performed a cross-over intervention, with 24 healthy individuals, to capture the acute metabolic response of a cereal breakfast (CB) and an egg and ham breakfast (EHB). Fasting and postprandial urine samples were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Metabolic profiles were distinguished in relation to ingestion of either CB or EHB. Phosphocreatine/creatine and citrate were identified at higher concentrations after consumption of EHB. Beverage consumption (i.e., tea or coffee) could clearly be seen in the data. 2-furoylglycine and 5-hydroxymethyl-2-furoic acid - potential biomarkers for coffee consumption were identified at higher concentrations in coffee drinkers. Thus 1H NMR urine metabolomics is applicable in the characterization of acute metabolic fingerprints from meal consumption and in the identification of metabolites that may serve as potential biomarkers.

By clinical experience, rhinitis has been suggested as caused by some endocrine disorders, but the evidence for this is vague, and the few descriptions almost anecdotal. Rhinitis of the menstrual cycle has been more described, although a solid picture is still lacking. Pregnancy rhinitis is therefore so far the only clearly defined "hormonal rhinitis." However, the cause of pregnancy rhinitis is not simply estrogen or progesterone, but seems multifactorial, and may possibly be associated with the PGH. Treatment consists mainly of information, physiological measures, and nasal saline washings.

The class IId bacteriocin lactococcin A and the pediocin-like bacteriocins induce membrane leakage and cell death by specifically binding the mannose phophotransferase system (man-PTS) on their target cells. The bacteriocins' cognate immunity proteins that protect the producer cell from its own bacteriocin recognize and bind to the bacteriocin-man-PTS complex and thereby block membrane leakage. In this study, we have determined the three-dimensional structure of the lactococcin A immunity protein (LciA) by the use of nuclear magnetic resonance spectroscopy. LciA forms a four-helix bundle structure with a flexible C-terminal tail. Despite the low degree of sequence similarity between LciA and the pediocin-like immunity proteins, they share the same fold. However, there are certain differences between the structures. The C-terminal helix in LciA is considerably shorter than that observed in the pediocin-like immunity proteins, and the surface potentials of the immunity proteins differ. Truncated variants of LciA in which 6 or 10 of the C-terminal residues were removed yielded a reduced degree of protection, indicating that the unstructured C-terminal tail is important for the functionality of the immunity proteins.

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

Hereditary factors are thought to play a role in at least one third of patients with colorectal cancer (CRC) but only a limited proportion of these have mutations in known high-penetrant genes. In a relatively large part of patients with a few or multiple colorectal polyps the underlying genetic cause of the disease is still unknown. Using exome sequencing in combination with linkage analyses together with detection of copy-number variations (CNV), we have identified a duplication in the regulatory region of the GREM1 gene in a family with an attenuated/atypical polyposis syndrome. In addition, 107 patients with colorectal cancer and/or polyposis were analyzed for mutations in the candidate genes identified. We also performed screening of the exonuclease domain of the POLE gene in a subset of these patients. The duplication of 16 kb in the regulatory region of GREM1 was found to be disease-causing in the family. Functional analyses revealed a higher expression of the GREM1 gene in colorectal tissue in duplication carriers. Screening of the exonuclease domain of POLE in additional CRC patients identified a probable causative novel variant c.1274A>G, p.Lys425Arg. In conclusion a high penetrant duplication in the regulatory region of GREM1, predisposing to CRC, was identified in a family with attenuated/atypical polyposis. A POLE variant was identified in a patient with early onset CRC and a microsatellite stable (MSS) tumor. Mutations leading to increased expression of genes can constitute disease-causing mutations in hereditary CRC syndromes.

Laboratory work in science education is essential for students' conceptual understanding of natural phenomena. Computer-simulated laboratory experiments have been proposed to facilitate traditional laboratory work. A virtual laboratory was designed to enable students to collaboratively discover the concept of gas solubility in water at different physiological conditions. The virtual laboratory was developed through a design experiment involving three successive versions with different guiding structures. Analysis of 12 dyads' reasoning about gas solubility in water revealed that the problem was not primarily for the students to realise how the volume of gas changed, but rather to understand the concept of solubility of gases. It was also observed how the guiding structures within the three different versions influenced the students' reasoning about the concept. The analysis indicates that the affordances of virtual laboratories might, to a certain extent, enhance joint discovery of a scientific concept.

Mitochondria play a crucial role in the intrinsic apoptotic pathway. The Bcl-2 family proteins, which interact with the mitochondrial outer membrane to modulate membrane permeability, are key regulators of this pathway. Intracellular oxidative stress is one major factor leading to apoptosis accompanied by the permeabilization of the mitochondrial outer membrane; a process causing release of apoptotic factors such as cytochrome c. Upon onset of intracellular stress, phospholipids can become oxidized in their unsaturated fatty acid region. These oxidized phospholipids (OxPls) can severely alter the properties of these mitochondrial membranes, and can therefore have i) a direct effect on the membrane properties and its perforation and ii) can have an indirect effect by altering the function of membrane-associated Bcl-2 proteins (such as the anti-apoptotic Bcl-2 and the apoptotic Bax). We therefore devised a model system that mimics oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into mitochondria-mimicking vesicles, and studied the OxPls’ impact on Bax-membrane interactions. Conformational changes in the protein upon contact with the lipid membranes were monitored using far-UV circular dichroism (CD) spectroscopy. Differential scanning calorimetry (DSC) and solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy was used to study membrane organization. In a biophysical study combining CD with surface plasmon resonance techniques we also investigated the putative interaction of solubilized full-length human Bcl-2 with Bax. There, we found a direct Bcl-2 interaction with Bax in the presence of sub-CMC concentration of detergent and could observe a high affinity between both partners (KD 35.8 nM).