High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection.

Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

Endothelin-1 induces a strong pressor effect in ball pythons (Python regius).

Endothelin-1 (ET-1) is a very potent vasoactive peptide released from endothelial cells, and ET-1 plays an important role in the maintenance and regulation of blood pressure in mammals. ET-1 signaling is mediated by two receptors: ETA and ETB. In mammals, ETA receptors are located on vascular smooth muscle where they mediate vasoconstriction. ETB receptors located on the endothelium mediate vasodilatation through the release of nitric oxide, whereas stimulation of ETB receptors placed on vascular smooth muscle leads to vasoconstriction. Less is known about ET-1 signaling in reptiles. In anaesthetized alligators, ET-1 elicits a biphasic blood pressure with a long-lasting initial decrease followed by a smaller increase in systemic blood pressure. In anaesthetized freshwater turtles, ET-1 causes a dose-dependent systemic vasodilatation mediated through ETB receptors. In the present study, we investigated the cardiovascular effects of ET-1 on the systemic and pulmonary vasculature of pythons. The presence of ETA and ETB receptors in the vasculature of pythons was verified by means of immunoblotting. Myography on isolated vessels revealed a dose-dependent vasoconstrictory response to ET-1 in both mesenteric and pulmonary arteries. Pressure measurements in recovered specimens revealed an ET-1-induced rise in systemic blood pressure supporting our in vitro findings. In conclusion, our study shows that ET-1 induces a strong pressor effect in the systemic circulation.

Survival and revision causes of hip resurfacing arthroplasty and the Mitch proximal epiphyseal replacement: results from the Danish Hip Arthroplasty Register.

Background and purpose - The Mitch proximal epiphyseal replacement (PER) was developed to preserve proximal femoral bone and minimize femoral neck fracture associated with hip resurfacing arthroplasty (HRA). We studied the survival and risk of revision of HRA compared with cementless metal-on-polyethylene (MoP) total hip arthroplasty (THA) and the survival and risk of revision of the Mitch PER compared with MoP THA.Patients and methods - Using propensity score, we matched 1,057 HRA to 1,057 MoP THA and 202 Mitch PER to 1,010 MoP THA from the Danish Hip Arthroplasty Register. To estimate the relative risk (RR) of revision, we used regression with the pseudo-value approach and treated death as a competing risk.Results - The cumulative incidence for any revision of HRA at 10 years' follow-up was 11% (95% confidence interval [CI] 9.1-13) and 6.4% (CI 5.8-7.0) for MoP THA. The RR of any revision was 1.5 (CI 1.1-2.1) for HRA at 10 years' follow-up. By excluding the ASR components, the RR of revision at 10 years was 1.2 (CI 0.8-1.7). The cumulative incidence of revision was 9.6% (CI 4.2-18) for Mitch PER and 5.4% (CI 5.1-5.7) for MoP THA at 8 years. The RR of revision was 2.0 (CI 0.9-4.3) for Mitch PER at 8 years' follow-up.Interpretation - The HRA had increased risk of revision compared with the MoP THA. When excluding ASR, the HRA group had similar risk of revision compared with MoP THA. The Mitch PER did not have a statistically significant increased risk of revision compared with MoP THA.

The structure of the second CysD domain of MUC2 and role in mucin organization by transglutaminase-based cross-linking.

The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.

Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27.

This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4+ T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.

Genetic analysis identifies the SLC4A3 anion exchanger as a major gene for short QT syndrome.

BACKGROUND: A variant in the SLC4A3 anion exchanger has been identified as a novel cause of short QT syndrome (SQTS), but the clinical importance of SLC4A3 as a cause of SQTS or sudden cardiac death remains unknown. OBJECTIVE: The purpose of this study was to investigate the prevalence of potential disease-causing variants in SQTS patients using gene panels including SLC4A3. METHODS: In this multicenter study, genetic testing was performed in 34 index patients with SQTS. The pathogenicity of novel SLC4A3variants was validated in a zebrafish embryo heart model. RESULTS: Potentially disease-causing variants were identified in 9 (26%) patients and were mainly (15%) located in SLC4A3: 4 patients heterozygous for novel nonsynonymous SLC4A3 variants-p.Arg600Cys, p.Arg621Trp, p.Glu852Asp, and p.Arg952His-and 1 patient with the known p.Arg370His variant. In other SQTS genes, potentially disease-causing variants were less frequent (2× in KCNQ1, 1× in KCNJ2, and CACNA1C each). SLC4A3 variant carriers (n = 5) had a similar heart rate but shorter QT and J point to T wave peak intervals than did noncarriers (n = 29). Knockdown of slc4a3 in zebrafish resulted in shortened heart rate-corrected QT intervals (calculated using the Bazett formula) that could be rescued by overexpression of the native human SLC4A3-encoded protein (AE3), but neither by the mutated AE3 variants p.Arg600Cys, p.Arg621Trp, p.Glu852Asp nor by p.Arg952His, suggesting pathogenicity of these variants. Dysfunction in slc4a3/AE3 was associated with alkaline cytosol and shortened action potential of cardiomyocytes. CONCLUSION: In about a quarter of patients with SQTS, a potentially disease-causing variant can be identified. Nonsynonymous variants in SLC4A3 represent the most common cause of SQTS, underscoring the importance of including SLC4A3 in the genetic screening of patients with SQTS or sudden cardiac death.

Health biomarkers in a rat model after intake of organically grown carrots.

BACKGROUND: Organic food is perceived as being of better quality and healthier than conventional foods although the scientific research on organic foodstuffs is highly contradictory. The aim of the present study was to investigate if intake of carrots from four different cultivation systems grown in two consecutive years would influence various biomarkers of health in a rat model. All rats were fed a diet with 40% carrot content. The carrots were grown under conventional (C), 'minimalistic' organic (O1), organic (O2), or 'very' organic cultivation systems (O3). A control group (CO) being fed standard rat chow was included. RESULTS: The plasma α-tocopherol concentration was higher in the O2 carrot-based diet group than in the C carrot based-diet group in one year, while all other health biomarkers or nutrient content differences were observed between the CO diet and the carrot-based diets. CONCLUSION: This well-controlled field study demonstrated no clear influence of cultivation methods or harvest year on the nutritional quality of carrots or effect of cultivation methods on health-related biomarkers in a sensitive rat model. However, the experimental set-up and selected biomarkers could be used as a framework for further studies of health in relation to organic foodstuff.

Cohesin-Mediated Chromatin Interactions and Autoimmunity.

Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.

Histamine exerts both direct H2-mediated and indirect catecholaminergic effects on heart rate in pythons.

The vertebrate heart is regulated by excitatory adrenergic and inhibitory cholinergic innervations, as well as non-adrenergic non-cholinergic (NANC) factors that may be circulating in the blood or released from the autonomic nerves. As an example of NANC signaling, an increased histaminergic tone, acting through stimulation of H2 receptors, contributes markedly to the rise in heart rate during digestion in pythons. In addition to the direct effects of histamine, it is also known that histamine can reinforce the cholinergic and adrenergic signaling. Thus, to further our understanding of the histaminergic regulation of the cardiovascular response in pythons, we designed a series of in vivo experiments complemented by in vitro experiments on sinoatrial and vascular ring preparations. We demonstrate the tachycardic mechanism of histamine works partly through a direct binding of cardiac H2 receptors and in part through a myocardial histamine-induced catecholamine release, which strengthens the sympathetic adrenergic signaling pathway.

Effect of selective IK,ACh inhibition by XAF-1407 in an equine model of tachypacing-induced persistent atrial fibrillation.

BACKGROUND AND PURPOSE: Inhibition of the G-protein gated ACh-activated inward rectifier potassium current, IK,ACh may be an effective atrial selective treatment strategy for atrial fibrillation (AF). Therefore, the anti-arrhythmic and electrophysiological properties of a novel putatively potent and highly specific IK,ACh inhibitor, XAF-1407 (3-methyl-1-[5-phenyl-4-[4-(2-pyrrolidin-1-ylethoxymethyl)-1-piperidyl]thieno[2,3-d]pyrimidin-6-yl]azetidin-3-ol), were characterised for the first time in vitro and investigated in horses with persistent AF. EXPERIMENTAL APPROACH: The pharmacological ion channel profile of XAF-1407 was investigated using cell lines expressing relevant ion channels. In addition, eleven horses were implanted with implantable cardioverter defibrillators enabling atrial tachypacing into self-sustained AF. The electrophysiological effects of XAF-1407 were investigated after serial cardioversions over a period of 1 month. Cardioversion success, drug-induced changes of atrial tissue refractoriness, and ventricular electrophysiology were assessed at baseline (day 0) and days 3, 5, 11, 17, and 29 after AF induction. KEY RESULTS: XAF-1407 potently and selectively inhibited Kir 3.1/3.4 and Kir 3.4/3.4, underlying the IK,ACh current. XAF-1407 treatment in horses prolonged atrial effective refractory period as well as decreased atrial fibrillatory rate significantly (~20%) and successfully cardioverted AF, although with a decreasing efficacy over time. XAF-1407 shortened atrioventricular-nodal refractoriness, without effect on QRS duration. QTc prolongation (4%) within 15 min of drug infusion was observed, however, without any evidence of ventricular arrhythmia. CONCLUSION AND IMPLICATIONS: XAF-1407 efficiently cardioverted sustained tachypacing-induced AF of short duration in horses without notable side effects. This supports IK,ACh inhibition as a potentially safe treatment of paroxysmal AF in horses, suggesting potential clinical value for other species including humans.

Molecular engineering of safe and efficacious oral basal insulin.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

Activation of the insulin receptor (IR) by insulin and a synthetic peptide has different effects on gene expression in IR-transfected L6 myoblasts.

Single-chain peptides have been recently produced that display either mimetic or antagonistic properties against the insulin and IGF-1 (insulin-like growth factor 1) receptors. We have shown previously that the insulin mimetic peptide S597 leads to significant differences in receptor activation and initiation of downstream signalling cascades despite similar binding affinity and in vivo hypoglycaemic potency. It is still unclear how two ligands can initiate different signalling responses through the IR (insulin receptor). To investigate further how the activation of the IR by insulin and S597 differentially activates post-receptor signalling, we studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore, we found that half of the differentially regulated genes interact with the genes involved with the MAPK (mitogen-activated protein kinase) pathway. These findings support our signalling results obtained previously and confirm that the main difference between S597 and insulin stimulation resides in the activation of the MAPK pathway. In conclusion, we show that insulin and S597 acting via the same receptor differentially affect gene expression in cells, resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications.

A practical guide to developing virtual and augmented reality exercises for teaching structural biology.

Although virtual and augmented reality (VR and AR) techniques have been used extensively in specialized laboratories, only recently did they become affordable, reaching wider consumer markets. With increased availability, it is timely to examine the roles that VR and AR may play in teaching structural biology and in experiencing complex data sets such as macromolecular structures. This guide is suitable for those teachers of structural biology who do not have a deep knowledge of information technologies. This study focuses on three questions: 1) How can teachers of structural biology produce and disseminate VR/AR-ready educational material with established and user-friendly software tools?; 2) What are the positive and negative experiences reported by test participants when performing identical learning tasks in the VR and AR environments?; and 3) How do the test participants perceive prerecorded narration during VR/AR exploration? © 2018 International Union of Biochemistry and Molecular Biology, 47(1):16-24, 2018.

3D reconstruction of carotid atherosclerotic plaque: comparison between spatial compound ultrasound models and anatomical models.

This study deals with the creation of 3D models that can work as a tool for discriminating between tissue and background in the development of tissue classification methods. Ten formalin-fixed atherosclerotic carotid plaques removed by endarterectomy were scanned with 3D multi-angle spatial compound ultrasound (US) and subsequently sliced and photographed to produce a 3D anatomical data set. Outlines in the ultrasound data were found by means of active contours and combined into 10 3D ultrasound models. The plaque regions of the anatomical photographs were outlined manually and then combined into 10 3D anatomical models. The volumes of the anatomical models correlated with the volume found by a water displacement method (r = 0.95), except for an offset. The models were compared in three ways. Visual inspection showed quite good agreement between the models. The volumes of the ultrasound models correlated with the volumes of the anatomical models (r = 0.93), again with an offset. Finally, the overlap between the anatomical models and the ultrasound models showed, on average, that the intersection comprised 90%(vol) of the anatomical models and 73%(vol) of the ultrasound models.

Assessment of automated screening for treatment-requiring diabetic retinopathy.

PURPOSE: To evaluate fundus photographic image analysis combining automated detection of red lesions, bright lesions, and image quality as a means of identifying treatment-requiring diabetic retinopathy in a screening population of diabetic patients. METHODS: This was a retrospective cross-sectional study of 106 patients from a diabetic retinopathy screening clinic referred for photocoagulation treatment in the period from January 1996 to May 2002 on the basis of mydriatic 60-degree 35-mm color transparency fundus photography. One fovea-centered fundus photograph and one centered nasal of the optic disk from each of a subject's two eyes was selected for digitization and analyzed using a previously tested computerized red-lesion detection algorithm in combination with a new algorithm for detection of bright lesions and image quality. The algorithm was calibrated on an independent set of fundus photographs. RESULTS: Automated red-lesion detection identified 104 of 106 patients requiring photocoagulation treatment, whereas bright-lesion detection identified only 91 of the 106 patients. Two patients who were not identified by either lesion detection algorithm were automatically detected as having poor image quality in one or both eyes. In the study sample, the risk of missing treatment-requiring retinopathy patients from being detected was 0.0% (estimated CI(95) 0.0-3.4%). CONCLUSIONS: The combination of automated detection of red lesions and poor image quality identified all treatment-requiring diabetic retinopathy patients in the study sample. No additional information was contributed by the automated bright-lesion detection.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin.

A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase.

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.

Effect of maternal intake of organically or conventionally produced feed on oral tolerance development in offspring rats.

The aim of this study was to investigate the effect of maternal consumption of organically or conventionally produced feed on immunological biomarkers and their offsprings' response to a novel dietary antigen. First-generation rats were fed plant-based diets from two different cultivation systems (organic or conventional) or a chow. Second-generation rats were exposed to ovalbumin (OVA) via their mother's milk and subsequently challenged with OVA after weaning onto the chow diet. In the chow diet group feeding the dams OVA resulted in suppression of the pups' anti-OVA antibody response to the OVA challenge (total OVA-specific IgG was 197 for the OVA-treated chow diet group and 823 for the control chow diet group (arbitrary ELISA units)). In contrast, OVA exposure of the dams from the plant-based dietary groups did not result in a similar suppression. Cultivation system had no effect on the immunological biomarkers, except for a higher spleen prostaglandin E2 (PGE2) concentration in pups originating from dams fed the conventional plant-based diet (223 ng/L) than from those fed the organic plant-based diet (189 ng/L).

IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor.

Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor.