Quantitative analysis of microscopic X‐ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining

Rapid three‐dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I₂KI and alcoholic iodine stains) combined with microscopic X‐ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I₂KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X‐ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I₂KI protocol. We present comparative microscopic X‐ray computed tomography analyses of ethanol I₂KI and I₂KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X‐ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28 days staining for I₂KI and ethanol I₂KI, respectively. Ethanol I₂KI provided superior soft tissue contrasts by reducing overstaining of fluid‐filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three‐dimensional visualization.     

Synthesis and Photoinitiated Cationic Polymerization of Substituted 2‐Cyclopropyl‐4‐methylene‐1,3‐dioxolanes

The synthesis of substituted 2‐cyclopropyl‐4‐methylene‐1,3‐dioxolanes 6 a – c is described. Photoinitiated cationic polymerization at ambient temperatures in bulk and in solution was investigated using (η⁵‐2,4‐cyclopentadiene‐1‐yl)[1,2,3,4,5,6‐η (1‐(methylethyl) benzene)iron(II) hexafluorophosphate (Irgacure 261, I‐1 ) and ditolyliodonium hexafluoro‐phosphate ( I‐2 ). Upon irradiation a single ring‐opening polymerization occured within 4 min leading to polyether ketones 6 a – c with number average molecular weights M_w between 5 000 and 33 600 g/mol. No ring‐opening polymerization of the cyclopropane ring could be detected. A volume shrinkage of about 6.4% was found.     

Solvent‐free tissue processing using supercritical carbon dioxide

Bleuel E P, Roebers T P C, Schulting E & den Dunnen W F A
(2012) Histopathology
Solvent‐free tissue processing using supercritical carbon dioxide Aims: Xylene is most often employed in tissue processing protocols for paraffin embedding, but poses a health hazard. The aim of this study was to evaluate a solvent‐free processing protocol that uses supercritical carbon dioxide (scCO₂) as an intermediate. Methods and Results: A series of tests (with bovine tissues) was run, evaluating dehydration and tissue shrinkage in our new scCO₂‐based protocol as compared with routine processing using a graded ethanol and xylene series. A series of tests was then run to evaluate the significance of processing parameters for the outcome. Finally, a validation series was performed with optimal conditions, testing various human tissues with several staining methods. The tissue water content after paraffination was the same with our new scCO₂‐based protocol and the routine xylene‐based protocol. Tissue shrinkage was similar with the two methods, at ～15%, which is also similar to values in the literature. In the validation series, the human tissues showed good morphology with strong staining, probably because of stronger antigenicity. Conclusions: This scCO₂‐based protocol has been shown to be a good solvent‐free, alternative form of tissue processing. Although not the focus of this article, the time needed for tissue processing with this new protocol is within 4 h, and there is no need to change macroscopy/sectioning protocols.     

Diffusible iodine‐based contrast‐enhanced computed tomography (diceCT): an emerging tool for rapid,high‐resolution, 3‐D imaging of metazoan soft tissues

Morphologists have historically had to rely on destructive procedures to visualize the three‐dimensional (3‐D) anatomy of animals. More recently, however, non‐destructive techniques have come to the forefront. These include X‐ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard‐tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT‐based research is the use of chemical agents to render visible, and differentiate between, soft‐tissue structures in X‐ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine‐based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine‐based, contrast‐enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting‐edge applications of diffusible iodine‐based contrast‐enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.     

Technical considerations on the validity of blood oxygenation level‐dependent‐based MR assessment of vascular deoxygenation

A blood oxygenation level‐dependent (BOLD)‐based apparent relative oxygen extraction fraction (rOEF) as a semi‐quantitative marker of vascular deoxygenation has recently been introduced in clinical studies of patients with glioma and stroke, yielding promising results. These rOEF measurements are based on independent quantification of the transverse relaxation times T₂ and T₂* and relative cerebral blood volume (rCBV). Simulations demonstrate that small errors in any of the underlying measures may result in a large deviation of the calculated rOEF. Therefore, we investigated the validity of such measurements. For this, we evaluated the quantitative measurements of T₂ and T₂* at 3 T in a gel phantom, in healthy subjects and in healthy tissue of patients with brain tumors. We calculated rOEF maps covering large portions of the brain from T₂, T₂* and rCBV [routinely measured in patients using dynamic susceptibility contrast (DSC)], and obtained rOEF values of 0.63 ± 0.16 and 0.90 ± 0.21 in healthy‐appearing gray matter (GM) and white matter (WM), respectively; values of about 0.4 are usually reported. Quantitative T₂ mapping using the fast, clinically feasible, multi‐echo gradient spin echo (GRASE) approach yields significantly higher values than much slower multiple single spin echo (SE) experiments. Although T₂* mapping is reliable in magnetically homogeneous tissues, uncorrectable macroscopic background gradients and other effects (e.g. iron deposition) shorten T₂*. Cerebral blood volume (CBV) measurement using DSC and normalization to WM yields robust estimates of rCBV in healthy‐appearing brain tissue; absolute quantification of the venous fraction of CBV, however, is difficult to achieve. Our study demonstrates that quantitative measurements of rOEF are currently biased by inherent difficulties in T₂ and CBV quantification, but also by inadequacies of the underlying model. We argue, however, that standardized, reproducible measurements of apparent T₂, T₂* and rCBV may still allow the estimation of a meaningful apparent rOEF, which requires further validation in clinical studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Acrylate‐Terminated Macromonomers by Michael Addition

A series of diacrylate macromonomers bearing alkoxysilyl units was prepared by convenient Michael addition of aminopropyl methyl diethoxysilane to 1,2‐ethylene glycol diacrylate (EGDA), p‐phenylene diacrylate (PDA) and 1,4‐cyclohexanediol diacrylate (CHDA). The resulting macromonomers have been characterized in detail by NMR spectroscopy, vapor pressure osmometry (VPO) measurements and fast‐atom bombardment mass spectroscopy (FAB‐MS). Average molecular weights M̄_n ranged between 530 and 1 300 g·mol^–1 (VPO). FAB‐MS and size exclusion chromatography (SEC) showed the formation of a homologous macromonomer series. Viscosities of the liquid monomers are relatively low, ranging from 0.082 to 8.30 Pa·s. This renders these compounds interesting as reactive diluents in dental composite formulations. Upon polymerization of the macromonomers, low volumetric shrinkage occurred, which was in the range of ΔV = 2.4 and 3.9 vol.‐% at high conversion. Crosslinking was monitored by photo‐differential scanning calorimetry (photo‐DSC). Furthermore, composites were prepared by mixing 2,2‐bis‐[p‐(2‐hydroxy‐3‐methacryloxypropoxy)‐phenyl]propane (Bis‐GMA) with the new macromonomers, initiator and glass filler. The composites showed compressive strengths up to 244 MPa, flexural strengths from 22 to 42 MPa and Young's moduli between 870 and 3 070 MPa. The composite materials exhibited low volume shrinkage of about 2 vol.‐% in comparison to over 3 vol.‐% shrinkage of commercially available composites.     

Design and synthesis of a two compartment micellar system based on the self‐association behavior of poly(N‐acylethyleneimine) end‐capped with a fluorocarbon and a hydrocarbon chain

The synthesis, characterization, and association behavior of a new class of amphiphilic polymer surfactants are described. Living cationic polymerization of 2‐methyl‐2‐oxazoline was utilized to create a series of water‐soluble polymer surfactants FP_xL_n, where F denotes a well defined flourinated end group C₈F₁₇CH₂CH₂, P_x denotes the degree of polymerization and L_n a hydrocarbon end group of variable length C_nH_2n+1, n = 6, 8, ..., 18. The molecular weights and polydispersities of the polymers were determined by gel permeation chromatography (GPC) in chloroform. ¹H NMR spectroscopy was used to calculate the number average degree of polymerization and the degree of end group modification with the hydrocarbon chain containing termination agent. The molecular weights and degree of polymerization corresponded closely to the values expected from the monomer/initiator ratio. The polydispersities were low, ranging from 1.15 to 1.20. The extent of end group modification was calculated to be a minimum of 85% for all polymers. Fluorescence experiments employing pyrene as a probe showed that these polymers form aggregates in water through self‐association in the concentration range from 0.018 to 0.061 wt.‐%. In addition, the micropolarity experienced by the peak ratio I₁/I₃ in the vibronic fluorescence spectrum of pyrene above the cmc up to 1.0 wt.‐% changed from hydrocarbon‐like for FPL₁₂, FPL₁₄, FPL₁₆ and FPL₁₈ to fluorocarbon‐like for FPL₆ and FPL₈, and intermediate behavior for FPL₁₀. Dynamic light scattering of FP₂₅L₁₆ showed single micelles at a concentration of 0.50 wt.‐% and 2.0 wt.‐% with a hydrodynamic radius of R_h = 5.5 and 6.7 nm, respectively. ¹⁹F NMR experiments were applied for FP₂₅L₁₆ to study the aggregation behavior of the fluorocarbon end group. T₂ relaxation experiments indicated that the degree of aggregation increased linearly with polymer concentration. Moreover, the results of the T₂ relaxation experiments suggested the presence of pure fluorocarbon phases consistent with those found by fluorescence spectroscopy for FP₂₅L₁₆.     

Relationships between muscle morphology and insulin sensitivity are improved after adjustment for intra‐individual variability in 70‐year‐old men

The purpose of this investigation was to examine to what extent variability in the muscle morphology and insulin sensitivity influence the correlation between them. Reproducibility of muscle characteristics was estimated in duplicate biopsies from the same thigh of 23 subjects from a cohort of 70‐year‐old men. The coefficient of variation (CV) for different characteristics of muscle morphology was between 11 and 42% in duplicate biopsies. Coefficient of variation for markers of insulin sensitivity ranged between 12 and 39%. The variability reflected by intra‐class correlation ranged from 0.23 to 0.60 for muscle morphology and from 0.68 to 0.96 for estimates of insulin sensitivity. The correlation analysis between muscle morphology and insulin resistance was performed in a sample of 515 men from the cohort, correlation coefficients were calculated with (r_true) and without (r) adjustment for intra‐individual variation. Insulin sensitivity showed a positive relationship with percentage of type I fibres (r_true=0.33, r=0.21; P < 0.0001) and capillary density (r_true=0.43, r=0.21; P < 0.0001) and negative correlations with percentage of type IIB fibres (r_true=–0.35, r=–0.24; P < 0.0001). Capillary density was inversely correlated to insulin. Thus, an obvious improvement of the correlation was seen after correcting intra‐individual variation. In conclusion, owing to the low degree of reproducibility of muscle morphology variables and insulin sensitivity, implying a noticeable underestimation of correlations, the r‐values should be adjusted for within‐subject variation in order to demonstrate a more accurate estimate of the strength of the relationships studied.     

Near‐infrared spectroscopy for monitoring muscle oxygenation

Near‐infrared spectroscopy (NIRS) is a non‐invasive method for monitoring oxygen availability and utilization by the tissues. In intact skeletal muscle, NIRS allows semi‐quantitative measurements of haemoglobin plus myoglobin oxygenation (tissue O₂ stores) and the haemoglobin volume. Specialized algorithms allow assessment of the oxidation–reduction (redox) state of the copper moiety (CuA) of mitochondrial cytochrome c oxidase and, with the use of specific tracers, accurate assessment of regional blood flow. NIRS has demonstrated utility for monitoring changes in muscle oxygenation and blood flow during submaximal and maximal exercise and under pathophysiological conditions including cardiovascular disease and sepsis. During work, the extent to which skeletal muscles deoxygenate varies according to the type of muscle, type of exercise and blood flow response. In some instances, a strong concordance is demonstrated between the fall in O₂ stores with incremental work and a decrease in CuA oxidation state. Under some pathological conditions, however, the changes in O₂ stores and redox state may diverge substantially.     

Effect of Surfactant and Various Salts on Aqueous Solution Properties of Naphthalene‐Labeled Poly(hydrochloride‐quaternized 2‐norbornene‐5‐methylamine) made by Ring‐Opening Metathesis Polymerization (ROMP)

Naphthalene‐labeled cationic poly(hydrochloride‐quaternized 2‐norbornene‐5‐methyleneamine), poly(HCQNBMA)/NA, has been prepared by ring‐opening metathesis polymerization (ROMP), using {RuCl₂(CHC₆H₅)[P(C₆H₁₁)₃]₂} as a catalyst in methylene chloride, followed by hydrogenation, hydrolysis, and quaternization. The effect of salt addition on the aqueous solution properties of poly(HCQNBMA)/NA was examined in terms of reduced viscosity, surface tension and fluorescence studies. The reduced viscosity for poly(HCQNBMA)/NA is dependent on the type and concentration of salts added. The viscosity behavior of cationic poly(HCQNBMA)/NA is in contrast with polybetaines. An increase in KCl concentration causes the reduced viscosity of poly(HCQNBMA)/NA to decrease at constant poly(HCQNBMA)/NA concentration. The general shape of the surface tension versus logarithm of concentration curve for poly(HCQNBMA)/NA shows only a slow decrease with increasing polymer concentration upon addition of various salts. The surface tension increases in the series KF > KCl > KBr > KI in a given polymer concentration. When the naphthalene label was introduced into the poly(HCQNBMA), the behavior of the solution properties of the poly(HCQNBMA)/NA could be clearly defined in terms of fluorescence analysis. The quenching efficiency of NaI or CH₃NO₂ was reduced with increasing KCl concentration arising from compacted conformation of polymer chains. A model of the interaction between poly(HCQNBMA)/NA and surfactant or salt in aqueous solution is proposed.

     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.     

Heme oxygenase-2 products activate I<Subscript>KCa</Subscript>: role of CO and iron in guinea pig

Hemin (10\mu M) and carbon monoxide (CO) increased iberiotoxin-blockable I_KCa in portal vein smooth muscle cells. CO-induced I_KCa activation was abolished by 10 μM ODQ, 10 μM cyclopiazonic acid and 1 μM KT5823. The hemin-induced effect on I_KCa was abolished by pretreatment with Sn-protoporphyrin IX, a heme oxygenase inhibitor and Fe²⁺ chelator but was insensitive to inhibitors of soluble guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG). There was no effect of hemin on I_KCa in the presence of 3 μM dithiotreitol into the bath or 3 mM glutathione into the pipette solution. Superoxide dismutase (1000 U/ml) or catalase (3000 U/ml) added into the pipette solution also abolished the effect of hemin on I_KCa in this tissue. Additionally, 10 μM hemin could not influence I_KCa in Ca₂₊-free external solution or in the presence of 30 μM SKF 95356. It was concluded that CO increases I_KCa via its “conventional” signaling pathway, which involves soluble GC and PKG activation and subsequent stimulation of sarcoplasmic reticulum Ca²⁺ pump activity resulting in Ca²⁺-dependent activation of I_KCa due to the accumulation of Ca²⁺ into the space near the plasma membrane. On the other hand, internally produced CO could not yield the same I_KCa increase, while Fe²⁺ derived from heme oxygenase 2-dependent degradation of hemin in portal vein smooth muscle cells gives rise to reactive oxygen species namely hydroxyl and superoxide radicals. Both radicals are responsible for the SKF 95356-sensitive non-selective cation channel activation, the Ca²⁺ influx and the subsequent increase of Ca²⁺ concentration near the plasma membrane that augments the K_Ca channel activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Serosal application of Ba2+ induces oscillatory chloride secretion via activation of submucosal cholinergic neurones in guinea‐pig distal colon

The enteric nervous system regulates ion and fluid secretion in the mammalian intestine at both resting and stimulated conditions. To determine the type and activation mechanism of neurones involved, mucosa–submucosa sheets isolated from guinea‐pig distal colon were studied in vitro in Ussing chambers. Serosal addition of 0.5–1 mM barium (Ba²⁺), a potassium (K⁺) channel inhibitor, caused oscillatory increases in short‐circuit current (I_sc). Mean values of the size and frequency of I_sc were 369.1 μA cm^–2 and 2.3 min^–1. The oscillatory I_sc induced by the low concentrations of Ba²⁺ was blocked by either higher concentrations of Ba²⁺ (2–5 mM ) or other K⁺ channel inhibitors, such as tetraethylammonium (TEA) (1 mM ) and quinine (20 mM ). The Ba²⁺‐induced oscillatory I_sc was also inhibited by tetrodotoxin (TTX) and atropine. In a nominally Ca²⁺ free solution plus serosal addition of 0.1 mM ethylene glycol‐bis (β‐aminoethyl ether) N,N,N′,N′‐tetraacetic acid (EGTA), a Ca²⁺ chelator, the oscillatory I_sc slowed and diminished. Further, the Ba²⁺‐induced oscillatory I_sc was partially inhibited by apical addition of 100 μM 5′‐nitro‐2‐(3‐phenylpropylamino)benzoic‐acid (NPPB), a Cl^– channel inhibitor, and completely disappeared in a low Cl^– solution (11 mM ) on both sides. On the other hand, application of either cimetidine, a histamine H₂ receptor antagonist, or hexamethonium, a nicotinic antagonist, to the serosal side did not affect the Ba²⁺‐induced oscillatory I_sc. In conclusion, the Ba²⁺‐induced oscillatory I_sc is the transepithelial Cl^– current which is stimulated by activation of cholinergic neurones in submucosal plexus of guinea‐pig distal colon.     

Age‐dependent changes in metabolite profile and lipid saturation in dystrophic mice

Duchenne Muscular Dystrophy (DMD) is a fatal X‐linked genetic disorder. In DMD, the absence of the dystrophin protein causes decreased sarcolemmal integrity resulting in progressive replacement of muscle with fibrofatty tissue. The effects of lacking dystrophin on muscle and systemic metabolism are still unclear. Therefore, to determine the impact of the absence of dystrophin on metabolism, we investigated the metabolic and lipid profile at two different, well‐defined stages of muscle damage and stabilization in mdx mice. We measured NMR‐detectable metabolite and lipid profiles in the serum and muscles of mdx mice at 6 and 24 weeks of age. Metabolites were determined in muscle in vivo using ¹H MRI/MRS, in isolated muscles using ¹H‐HR‐MAS NMR, and in serum using high resolution ¹H/¹³C NMR. Dystrophic mice were found to have a unique lipid saturation profile compared with control mice, revealing an age‐related metabolic change. In the 6‐week‐old mdx mice, serum lipids were increased and the degree of lipid saturation changed between 6 and 24 weeks. The serum taurine‐creatine ratio increased over the life span of mdx, but not in control mice. Furthermore, the saturation index of lipids increased in the serum but decreased in the tissue over time. Finally, we demonstrated associations between MRI‐T₂, a strong indicator of inflammation/edema, with tissue and serum lipid profiles. These results indicate the complex temporal changes of metabolites in the tissue and serum during repetitive bouts of muscle damage and regeneration that occur in dystrophic muscle.     

Repeatability of a dual gradient‐recalled echo MRI method for monitoring post‐isometric contraction blood volume and oxygenation changes

The purpose of this study was to assess the repeatability of a dual gradient‐recalled echo (GRE) muscle functional MRI technique. On 2 days, subjects (n = 8) performed 10 s isometric dorsiflexion contractions under conditions of: (1) maximal voluntary contraction (MVC), (2) 50% MVC (50% MVC), or (3) 50% MVC with concurrent proximal arterial cuff occlusion (50% MVC_cuff). Functional MRI data were acquired using single‐slice dual GRE (TR/TE = 1000/6, 46 ms)‐echo planar imaging for 20 s before, during, and for 180 s after each contraction. The mean signal intensity (SI) time courses at each TE (SI₆ and SI₄₆, reflecting variations in blood volume and %HbO₂, respectively) from the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were characterized with the post‐contraction change in SI and the time‐to‐peak SI (ΔSI and TTP, respectively). ΔSI₆ following an MVC was 36% higher than that obtained after a 50% MVC (p = 0.048). For ΔSI₆, the highest intraclass correlation coefficients (ICCs) were observed for the TA muscle in the 50% MVC and MVC conditions, with values of 0.83 (p = 0.01) and 0.88 (p = 0.005), respectively. Bland–Altman plots revealed repeatability coefficients (RCs) for the 50% MVC and MVC conditions in the TA muscle of 1.9 and 1.4, respectively. The most repeatable measures for ΔSI₄₆ were obtained for the 50% MVC and MVC conditions in the EDL muscle (p = 0.01 and p = 0.04, respectively). Bland–Altman plots revealed RC's for 50% MVC and MVC conditions in the EDL muscle of 3.9 and 5.7, respectively. ΔSI₆ and ΔSI₄₆ increased as a function of the contraction intensity. The repeatability of the method depends on the muscle and contraction condition being evaluated, and in general, is higher following an MVC. Copyright © 2009 John Wiley & Sons, Ltd.     

The feasibility of quantitative MRI of extra‐ocular muscles in myasthenia gravis and Graves' orbitopathy

Although quantitative MRI can be instrumental in the diagnosis and assessment of disease progression in orbital diseases involving the extra‐ocular muscles (EOM), acquisition can be challenging as EOM are small and prone to eye‐motion artefacts. We explored the feasibility of assessing fat fractions (FF), muscle volumes and water T2 (T2_water) of EOM in healthy controls (HC), myasthenia gravis (MG) and Graves' orbitopathy (GO) patients. FF, EOM volumes and T2_water values were determined in 12 HC (aged 22‐65 years), 11 MG (aged 28‐71 years) and six GO (aged 28‐64 years) patients at 7 T using Dixon and multi‐echo spin‐echo sequences. The EOM were semi‐automatically 3D‐segmented by two independent observers. MANOVA and t‐tests were used to assess differences in FF, T2_water and volume of EOM between groups (P < .05). Bland–Altman limits of agreement (LoA) were used to assess the reproducibility of segmentations and Dixon scans. The scans were well tolerated by all subjects. The bias in FF between the repeated Dixon scans was ?0.7% (LoA: ±2.1%) for the different observers; the bias in FF was ?0.3% (LoA: ±2.8%) and 0.03 cm³ (LoA: ± 0.36 cm³) for volume. Mean FF of EOM in MG (14.1% ± 1.6%) was higher than in HC (10.4% ± 2.5%). Mean muscle volume was higher in both GO (1.2 ± 0.4 cm³) and MG (0.8 ± 0.2 cm³) compared with HC (0.6 ± 0.2 cm³). The average T2_water for all EOM was 24.6 ± 4.0 ms for HC, 24.0 ± 4.7 ms for MG patients and 27.4 ± 4.2 ms for the GO patient. Quantitative MRI at 7 T is feasible for measuring FF and muscle volumes of EOM in HC, MG and GO patients. The measured T2_water was on average comparable with skeletal muscle, although with higher variation between subjects. The increased FF in the EOM in MG patients suggests that EOM involvement in MG is accompanied by fat replacement. The unexpected EOM volume increase in MG may provide novel insights into underlying pathophysiological processes.     

Polymers of carbonic acid, 28. SnOct2‐initiated polymerizations of trimethylene carbonate (TMC, 1,3‐dioxanone‐2)

SnOct₂ (Sn(II) 2‐ethylhexanoate) initiated polymerizations of TMC were studied either in conc. chlorobenzene solution at 80°C or in bulk at temperatures ≥ 120°C. Benzyl alcohol added as coinitiator accelerated the polymerization process and allowed a control of the number‐average molecular weight (M_n) via the monomer/coinitiator ratio (M/C). The formation of benzyl carbonate endgroups allowed in turn the determination of (M_n) via ¹H NMR endgroup analyses. Model reactions suggest that at 80°C intermediately formed Sn‐alkoxide groups play the role of the true initiator. At 140, 160 or 180°C neat SnOct₂ polymerized TMC in such a way that polyTMC having octanoate endgroups was formed. Variation of the monomer/initiator (M/I) ratio at 160°C enabled again a proper control of M_n via the M/I‐ratio and high molecular weight polyTMC was obtained. The influence of back‐biting degradation on the molecular weight was also studied at 160°C to find the optimum time‐temperature window.     

Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

Aim

Junctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca²⁺ entry and intracellular Ca²⁺ release. The small‐conductance Ca²⁺‐activated K⁺ channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes.

Methods

Association of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real‐time PCR. The expression of proteins was analysed by Western blotting. Ca²⁺‐activated K⁺ current (I_K,Ca) in infected adult mouse cardiac myocytes was recorded using whole‐cell voltage‐clamp technique. The intracellular Ca²⁺ transient was measured using an IonOptix photometry system.

Results

We showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N‐terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I_K,Ca and reduced the amplitude of the Ca²⁺ transient in infected cardiomyocytes.

Conclusion

The present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.     

Early‐life antibiotic exposure increases the risk of developing allergic symptoms later in life: A meta‐analysis

This study systematically reviewed and quantified the relationship between exposure to antibiotics during the first 2 years of life and the risk of allergies/atopies including hay fever, eczema, food allergy, positive skin prick testing (SPT), or elevated allergen‐specific serum/plasma immunoglobulin (Ig) E levels later in life. PubMed and Web of Science databases were searched for observational studies published from January 1966 through November 11, 2015. Overall pooled estimates of the odds ratios (ORs) were obtained using fixed or random‐effects models. Early‐life exposure to antibiotics appears to be related to an increased risk of allergic symptoms of hay fever, eczema, and food allergy later in life. The summary OR for the risk of hay fever (22 studies) was 1.23, 95% confidence interval (CI):1.13‐1.34; I²: 77.0%. The summary OR for the risk of eczema (22 studies) was 1.26, 95% CI: 1.15‐1.37; I²: 74.2%, and the summary OR for food allergy (3 studies) was 1.42, 95% CI: 1.08‐1.87; I²: 80.8%. However, no association was found for antibiotics exposure early in life and objective atopy measurements including positive SPT or elevated allergen‐specific serum/plasma IgE levels.