Talbot-defocus multiscan tomography using the synchrotron X-ray microscope to study the lacuno-canalicular network in mouse bone

The three-dimensional network of lacunae and canaliculi that regulates metabolism in bone contains osteocytes and their dendritic processes. We constructed a synchrotron radiation X-ray microscope for sequential tomography of mouse tibia first by using a Talbot interferometer to detect the degree of bone mineralization and then by using absorption contrast under a slightly defocused setting to enhance outline contrast thereby visualizing structures of the osteocyte lacuno-canalicular network. The resultant pair of tomograms was precisely aligned with each other, allowing evaluation of mineral density in the vicinity of each osteocyte lacuna and canaliculus over the entire thickness of the cortical bone. Thus, multiscan microscopic X-ray tomography is a powerful tool for analyzing bone mineralization in relation to the lacuno-canalicular network at the submicron resolution level.OCIS codes: (070.6760) Talbot and self-imaging effects, (110.6960) Tomography, (180.7460) X-ray microscopy, (340.6720) Synchrotron radiation     

RGD-based active targeting of novel polycation liposomes bearing siRNA for cancer treatment

For the purpose of systemic delivery of siRNA, we previously developed polycation liposomes (PCLs) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as an effective siRNA carrier. In the present study, to endow these PCLs (TEPA-PCL) actively target cancer cells and angiogenic vessels, we decorated the PCLs with cyclic RGD, by using cyclic RGD-grafted distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and investigated the usefulness of this type of carrier (RGD-PEG-PCL) for active targeting. Firstly, the gene-silencing efficacy of siRNA for luciferase (siLuc2) formulated in RGD-PEG-PCL (RGD-PEG-PCL/siLuc2) was examined in vitro by using B16F10-luc2 murine melanoma cells stably expressing the luciferase 2 gene, where the siRNA was grafted with cholesterol at the 3'-end of the sense strand (siRNA-C) for the stable association of the siRNA with the PCL. RGD-PEG-PCL/siLuc2 showed high knockdown efficiency compared with siLuc2 formulated in PEGylated TEPA-PCL without cyclic RGD (PEG-PCL). Next, the gene-silencing efficacy of RGD-PEG-PCL/siLuc2 was examined in vivo by use of B16F10-luc2 lung metastatic model mice. The intravenous injection of RGD-PEG-PCL/siLuc2 showed high knockdown efficiency against metastatic B16F10-luc2 tumors in the lungs of the mice, as assessed with an in vivo imaging system. These data strongly suggest that systemic and active targeting siRNA delivery using RGD-PEG-PCL is useful for cancer RNAi therapy.     

Neointimal hyperplasia after stent placement across size-discrepant vessels in an animal study

Purpose

To examine differences in neointimal hyperplasia (NIH) after placing a self-expanding (SE) stent across size-discrepant vessels.

Materials and methods

The subjects were 6 beagles, and the target vessels were the abdominal aorta and the external iliac artery (EIA). A nitinol SE stent was placed which was normal-sized in the aorta and oversized in the EIA. Angiography and intravascular ultrasound (IVUS) were performed immediately and after 1 and 3 months; histopathologic examinations were then performed. Furthermore, the chronic outward force (COF) on the same type of stent was investigated in vitro.

Results

On IVUS, thickened intima was seen on the EIA at 1 month (5.1 ± 4.2 mm²) and at 3 months (7.8 ± 2.5 mm²). For the aorta, thickening of the intima was negligible at any time. Histopathologically, the percentage of the vessel obliterated by NIH was significantly greater on the iliac side than on the aortic side (33.2 ± 10.4 vs. 13.4 ± 4.4 %). The COF exerted when stent diameter reached that of the EIA and the aorta was 0.73 and 0.17 N/mm², respectively.

Conclusions

When a non-tapered stent is placed in vessels with a large discrepancy in diameter, attention must be paid to increased NIH in the oversized side.     

An approach to normalizing micro-CT depth profiles of mineral density for monitoring enamel remineralization progress

To indicate the possibility of a new approach to creating mineral density profiles, and to examine longitudinal changes in 'the rate of remineralization (RA)' and 'the mineral density (DAs) at 4 different depths' (surface zone: SZ, lesion body: LB, middle zone: MZ, deep zone near to sound area: DZ) in enamel subsurface lesions, eight demineralized bovine enamel-dentin blocks were remineralized for 1 to 4 week and investigated using Micro-focus X-ray CT (micro-CT). After CT scanning, mineral density profiles were created.Mineral densities at each depth after demineralization were SZ?LBMZ>DZ. This study indicated a new approach to create a mineral density profile and suggested the greater the value of the mineral density before the remineralization, the smaller the mineral density increments.     

An animal model for type II endoleaks with use of a tsuzumi drum-shaped stent-graft

PURPOSE: To create an animal model of type II endoleaks after endoluminal deployment of a specially designed stent-graft (SG). MATERIALS AND METHODS: Five swine were used. A tsuzumi drum-shaped SG consisting of a covered Z stent with its diameter narrowed at the center was deployed in the midthoracic aorta. In this way, a residual space (RS) was created between the aortic wall and the graft to simulate an aneurysm sac. A 5-F catheter was placed into the RS, and then aortography, RS angiography, and pressure measurements were performed. Follow-up was performed at 3 and 10 days after the procedure. Mean pressure indexes (MPIs) were calculated as the ratio of the mean RS pressure to the aortic pressure. Histologic examination was also performed. RESULTS: RSs with two or three pairs of intercostal arteries were successfully created in all cases. Aortography showed two type II endoleaks in five swine just after SG deployment and four type II endoleaks at 10 days. RS angiography showed circulation between the RS and the intercostal arteries in all cases. The mean MPI was 69.4% +/- 10.4% just after SG deployment and increased to 87.8% +/- 5.2% at 10 days. By gross examination, RS patency was retained. CONCLUSIONS: A swine model of type II endoleaks was successfully created endoluminally. This model does not require direct surgery to the aorta and its side branches and promises to be useful to study the mechanism of and therapy for type II endoleaks.     

Effects of axial ligands on the electron transfer catalyzed by manganese tatrakis(pentafluorophenyl)porphyrin in poly(γ-methyl L-glutamate) membrane

Poly(γ-methyl L -glutamate) (PMLG) membrane containing manganese tetrakis(pentafluorophenyl)porphyrin

1 Systematic name: Tetrakis(pentafluorophenyl)porphyrinatomanganese.

(MnPFPP) has been studied for the transmembrane redox reaction between dithionite and ferricyanide in separated solutions. The effects of the ligand species, namely, OH^?, Cl^?, imidazole (Im), pyrazine (Pyz), triethylamine (TEA), and pyridine (Py), on the electron transfer rate in the membrane were examined. UV/VIS spectroscopy and sorption measurements confirm that these ligands are sorbed by the membrane from the external buffer solution and coordinate to MnPFPP. With the membrane containing 160 μmol · g^?1 of MnPFPP, the apparent rate factor, estimated as the pseudo-first-order rate constant of the bleach of ferricyanide times membrane thickness, follows the order Im ? Cl^? > Pyz > TEA, OH^?, Py > 0, suggesting that Im, Cl^? and Pyz are effective ligands for the electron transfer. Such a ligand-specific transfer stems from the electron-transferability of the ligands between MnPFPP's.     

Systemic artery to pulmonary artery fistula associated with mitral regurgitation: successful treatment with endovascular embolization

We present the case of a 60-year-old woman with symptomatic mitral regurgitation caused by a left-to-right shunt via anastomoses consisting of microfistulae, most likely of inflammatory origin, between the right subclavian artery and the right pulmonary artery. The three arteries responsible for fistulous formation, including the internal mammary, thyrocervical, and lateral thoracic arteries, were successfully occluded by transcatheter embolization using superabsorbent polymer microsphere (SAP-MS) particles combined with metallic coils. No complications have been identified following treatment with SAP-MS particles. This approach significantly reduced the patient's mitral regurgitation and she has remained asymptomatic for more than 4 years.     

Detection of portal perfusion abnormalities: comparison of 3 ferucarbotran-enhanced magnetic resonance imaging sequences

OBJECTIVE: To estimate the accuracy, sensitivity, and specificity of 3 ferucarbotran-enhanced magnetic resonance (MR) imaging sequences prospectively for the detection of nontumoral portal perfusion abnormalities. METHODS: Thirty-nine noncirrhotic patients with liver metastases underwent computed tomography during arterial portography (CTAP) and MR imaging comprising T1-weighted gradient recalled echo (GRE), T2-weighted fast spin echo (FSE), and T2*-weighted GRE sequences with and without ferucarbotran. Magnetic resonance images were reviewed by 4 blinded observers for rating based on the confidence scale. The accuracy, sensitivity, and specificity for each sequence were measured by receiver operating characteristic analysis. Contrast-to-noise ratio (CNR) and relative signal-to-noise ratio changes were statistically compared. RESULTS: Thirty-nine nontumoral perfusion defects were observed in 22 patients by CTAP. Receiver operating characteristic analysis showed the accuracy was higher for T2*-weighted GRE (0.884) than for T1-weighted GRE (0.572) and T2-weighted FSE (0.597). T2*-weighted imaging achieved the highest sensitivity (81.4%) and the lowest specificity (86.6%). Postenhanced T2*-weighted imaging achieved the highest CNR (19.3 +/- 9.2). CONCLUSIONS: T2*-weighted imaging was the most accurate and sensitive method for detecting portal perfusion abnormalities compared with T1- or T2-weighted imaging, whereas T1- or T2-weighted imaging is superior in specificity to T2*-weighted imaging during ferucarbotran-enhanced MR imaging.     

Experimental venous thrombi: MRI characteristics with histopathological correlation

Objectives: The purpose of this study was to evaluate the MRI characteristics of venous thrombus over set time thresholds with histopathological correlation in a porcine model. Methods: Inferior vena cava thrombi were induced in 12 pigs. MRI was performed in three pigs 2 h, 1 day, 3 days and 2 weeks after thrombus induction. Results: The MRI characteristics were analysed in correlation with histopathological findings. The thrombi after 2 hours, which consisted of red blood cells (RBCs), showed isointensity on T(1 )weighted images (T(1)WIs) and hyperintensity on both T(2 )weighted images (T(2)WIs) and diffusion-weighted images (DWIs). The mean apparent diffusion coefficient (ADC) value was 1.93 × 10(-3) mm(2) s(-1). The thrombi after Day 1, which consisted of RBCs and migrating neutrophils at the periphery, showed isointensity on T(1)WIs, slight hyperintensity on T(2)WIs and hypointensity on DWIs. The mean ADC value was 1.62 × 10(-3) mm(2) s(-1) [corrected]. The thrombi after Day 3, which consisted of RBCs and peripheral inflammatory cells including macrophages, showed isointensity with peripheral hyperintense regions on T(1)WIs and hypointensity on both T(2)WIs and DWIs. The mean ADC value was 1.67 × 10(-3) mm(2) s(-1). After 2 weeks, the thrombi, which revealed RBC lysis surrounded by granulation tissues, showed isointensity on T(1)WIs and hyperintensity on T(2)WIs and DWIs. The mean ADC value was 2.48 × 10(-3) mm(2) s(-1). Conclusion: The temporal MRI characteristics seemed to be related to chemical and physical changes in RBC and organisation of granulation tissues. Free radicals generated by macrophages might also be related to some extent.     

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.Since the invention of the Haber–Bosch process, the amount of fixed nitrogen in soils and waters has been increasing, and this trend has significant impact on the global environment (1, 2). Fixed nitrogen is oxidized to nitrite (NO₂⁻) or nitrate (NO₃⁻) by nitrification and then converted to gaseous dinitrogen (N₂) by microbial denitrification, which closes the nitrogen cycle. Microorganisms involved in denitrification couple their respiratory systems to stepwise reduction of nitrogen oxides to N₂ (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (3, 4). The reduction of NO₂⁻ to toxic nitric oxide (NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O) is referred to as the key step in denitrification and catalyzed by either cd₁-heme nitrite reductase (cd₁NiR) or copper nitrite reductase (CuNiR) (3, 4). Although the catalytic mechanism of cd₁NiR is well understood (5, 6), that of CuNiR is controversial (7). CuNiR is a homotrimeric protein containing two distinct Cu sites per monomer (SI Appendix, Fig. S1). Type 1 Cu (T1Cu) with a Cys–Met–His₂ ligand set is an electron acceptor incorporated near the molecular surface, whereas type 2 Cu (T2Cu) with a His₃ ligand set is a catalytic center, which is ∼12 Å distant from the molecular surface and located between two adjacent monomers (7, 8). Spaced ∼12.5 Å apart, the two Cu sites are linked by a Cys–His bridge and a sensor loop. Whereas the Cys–His bridge is an electron pathway, the sensor loop is thought to control electron distribution between T1Cu and T2Cu (9).Two conserved residues, Asp98 and His255 (Alcaligenes faecalis numbering), are located above the T2Cu site and bridged by a water molecule called bridging water (SI Appendix, Fig. S1). They are essential to the CuNiR activity because they assist proton transfer (PT) to the substrate (10–12). Although intramolecular electron transfer (ET) from T1Cu to T2Cu can occur in the resting state (RS) (13, 14), the differences in the redox potentials of T2Cu minus T1Cu are small and sometimes negative in the absence of NO₂⁻, meaning that intramolecular ET before NO₂⁻ binding is not energetically favorable (15, 16). By contrast, intramolecular ET is dramatically accelerated in the presence of NO₂⁻ (15, 17). An explanation for this gating-like phenomenon is that substrate binding raises the redox potential of T2Cu and shifts the equilibrium of the ET reaction (16). However, pH dependence of intramolecular ET in the presence of NO₂⁻ cannot be explained by such a change of redox potentials (15). Instead, Kobayashi et al. (15) proposed that reduction-induced structural change of His255 is responsible for the gating-like mechanism. Because it has been recently proven that intramolecular ET in CuNiR is accompanied by PT and hence proton-coupled ET (PCET) (17, 18), one can readily speculate that intramolecular ET contributes PT to NO₂⁻ and that the structural change of His255 is involved in PCET. Crystal structures of CuNiR from Rhodobacter sphaeroides (RhsNiR) implies this possibility because His287 in RhsNiR, which corresponds to His255, seems to show pH- and redox-dependent conformational changes (19, 20). However, presumably because of X-ray radiation damages implied by rerefinement of RhsNiR structures (21), electron density around His287 was so unusual that interpretation of it is difficult (SI Appendix, Fig. S2).Crystal structures determined by synchrotron radiation crystallography (SRX) have provided insights into the enzymatic mechanism of CuNiR (22–25), and these studies are summarized elsewhere (7). High-resolution nitrite complex (NC) structures revealed an O-coordination of NO₂⁻ showing a near face-on binding mode (22, 23), whereas Cu(II)-NO₂⁻ model complexes show a vertical binding mode (7, 26–29). The near face-on coordination manner is thought to facilitate its conversion to side-on NO, which was observed in the crystal structures of CuNiR exposed to NO (22, 23, 25). Skeptical eyes have, however, been cast on these CuNiR structures because SRX data might be affected by some problems connected to the high radiation dose delivered on the crystals. First, strong synchrotron X-rays cause not only radiation damages to amino acid residues but also photoreduction of metalloproteins (30, 31). Although a comparison between oxidized and reduced states is necessary to closely investigate redox reactions, completely oxidized structures are almost impossible to determine by SRX. Indeed, the Cu centers in CuNiR are rapidly reduced by exposure to synchrotron X-rays (21, 32). Second, following the photoreduction of T2Cu, NO₂⁻ is easily reduced and produces NO and water in SRX (21). Consequently, electron density at the catalytic site of an NC structure is derived from the mixture of both substrate and product, making interpretation of data complicated and unreliable. Third, cryogenic manipulations for reducing radiation damages in SRX have also been focused as a factor that changes the population of amino acid residues (33, 34) and enzyme–substrate complexes (35). Crystallographic (36), computational (37), and spectroscopic (38–40) studies actually show that binding modes of NO₂⁻ and NO in CuNiR crystal structures can differ from those in physiological environments.We here ventured to use photoreduction in SRX to initiate a chemical reaction and to trap an enzymatically produced intermediary state (30, 31). Furthermore, to visualize intact CuNiR structures in the resting and NC states, we applied serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) (41), which enables damage-free structural determination of metalloproteins (42, 43) and evaluation of the native conformational population at room temperature (RT) (44). By comparing SRX and SFX data, we discuss PCET and nitrite reduction in CuNiR.