Flow independent black blood imaging with a large FOV from the neck to the aortic arch: A feasibility study at 3 tesla.

PURPOSE: To investigate the feasibility of obtaining black-blood imaging with a large FOV from the neck to the aortic arch at 3 T using a newly modified Relaxation-Enhanced Angiography without Contrast and Triggering for Black-Blood Imaging (REACT-BB). MATERIALS AND METHODS: REACT-BB provides black-blood images by adjusting the inversion time (TI) in REACT to the null point of blood. The optimal TI for REACT-BB was investigated in 10 healthy volunteers with TI varied from 200 ms to 1400 ms. Contrast ratios were calculated between muscle and three branch arteries of the aortic arch. Additionally, a comparison between REACT-BB and MPRAGE involved evaluating the depiction of high-intensity plaques in 222 patients with stroke or transient ischemic attack. Measurements included plaque-to-muscle signal intensity ratios (PMR), plaque volumes, and carotid artery stenosis rates in 60 patients with high-intensity plaques in carotid arteries. RESULTS: REACT-BB with TI = 850 ms produced the black-blood image with the best contrast between blood and background tissues. REACT-BB outperformed MPRAGE in depicting high-intensity plaques in the aortic arch (55.4% vs 45.5%) and exhibited superior overall image quality in visual assessment (3.31 ± 0.70 vs 2.89 ± 0.73; p < 0.05). Although the PMR of REACT-BB was significantly lower than MPRAGE (2.227 ± 0.601 vs 2.285 ± 0.662; P < 0.05), a strong positive correlation existed between REACT-BB and MPRAGE (ρ = 0.935; P < 0.05), and all high-intensity plaques that MPRAGE detected were clearly detected by REACT-BB. CONCLUSION: REACT-BB provides black-blood images with uniformly suppressed fat and blood signals over a large FOV from the neck to the aortic arch with comparable or better high-signal plaque depiction than MPRAGE.

Diffusion weighted imaging combining respiratory triggering and navigator echo tracking in the upper abdomen.

OBJECTIVES: To evaluate a new motion correction method, named RT + NV Track, for upper abdominal DWI that combines the respiratory triggering (RT) method using a respiration sensor and the Navigator Track (NV Track) method using navigator echoes. MATERIALS AND METHODS: To evaluate image quality acquired upper abdominal DWI and ADC images with RT, NV, and RT + NV Track in 10 healthy volunteers and 35 patients, signal-to-noise efficiency (SNRefficiency) and the coefficient of variation (CV) of ADC values were measured. Five radiologists independently performed qualitative image-analysis assessments. RESULTS: RT + NV Track showed significantly higher SNRefficiency than RT and NV (14.01 ± 4.86 vs 12.05 ± 4.65, 10.05 ± 3.18; p < 0.001, p < 0.001). RT + NV Track was superior to RT and equal or better quality than NV in CV and visual evaluation of ADC values (0.033 ± 0.018 vs 0.080 ± 0.042, 0.057 ± 0.034; p < 0.001, p < 0.001). RT + NV Track tends to acquire only expiratory data rather than NV, even in patients with relatively rapid breathing, and can correct for respiratory depth variations, a weakness of RT, thus minimizing image quality degradation. CONCLUSION: The RT + NV Track method is an efficient imaging method that combines the advantages of both RT and NV methods in upper abdominal DWI, providing stably good images in a short scan time.

Circadian Clock Genes Regulate Temperature-Dependent Diapause Induction in Silkworm Bombyx mori.

The bivoltine strain of the domestic silkworm, Bombyx mori, exhibits a facultative diapause phenotype that is determined by maternal environmental conditions during embryonic and larval development. Although a recent study implicated a circadian clock gene period (per) in circadian rhythms and photoperiod-induced diapause, the roles of other core feedback loop genes, including timeless (tim), Clock (Clk), cycle (cyc), and cryptochrome2 (cry2), have to be clarified yet. Therefore, the aim of this study was to elucidate the roles of circadian clock genes in temperature-dependent diapause induction. To achieve this, per, tim, Clk, cyc, and cry2 knockout (KO) mutants were generated, and the percentages of diapause and non-diapause eggs were determined. The results show that per, tim, Clk, cyc, and cry2 regulated temperature-induced diapause by acting upstream of cerebral γ-aminobutyric acid (GABA)ergic and diapause hormone signaling pathways. Moreover, the temporal expression of the clock genes in wild-type (wt) silkworms was significantly different from that of thermosensitive transient receptor potential ankyrin 1 (TRPA1) KO mutants during embryonic development. Overall, the findings of this study provide target genes for regulating temperature-dependent diapause induction in silkworms.

Three-dimensional multicontrast blood imaging with a single acquisition: Simultaneous non-contrast-enhanced MRA and vessel wall imaging in the thoracic aorta.

PURPOSE: To evaluate MRA and vessel wall imaging (VWI) image quality in the thoracic aorta using a novel method named BRIDGE (bright and dark blood images with multishot gradient-echo EPI). METHODS: The BRIDGE method consists of 3D multishot gradient-echo EPI acquisition using pulse gating, navigator gating, and magnetization preparation with a T2 -preparation pulse and a nonselective inversion-recovery pulse. The BRIDGE and conventional methods (noncontrast MRA based on 3D turbo-field-echo [TFE] and VWI based on 3D turbo spin echo with variable refocusing flip angle [VRFA-TSE]) were performed in 10 healthy volunteers and 10 patients. The SNR, contrast-to-noise ratio (CNR), and sharpness in the thoracic aorta were compared for MRA evaluation. The values of SNRlumen , SNRwall , CNRwall-lumen , contrast ratio (CR)lumen-muscle , coefficient of variation, sharpness, lumen area, and wall area in the thoracic aorta were compared for VWI evaluation. Two radiologists independently performed qualitative image-analysis assessments. RESULTS: When MRA and VWI were acquired, the acquisition time was 26.6% to 27.8% shorter with BRIDGE than the conventional method. In the MRA evaluation, BRIDGE and TFE methods were comparable. In the VWI evaluation, BRIDGE was superior to the VRFA-TSE method in blood suppression and evaluation of the ascending aorta. Because the blood signal suppression of BRIDGE is based on the T1 value of blood, the blood signal can be suppressed more uniformly than with the VRFA-TSE method, regardless of age, blood flow velocity, or vascular anatomy. CONCLUSION: The BRIDGE method can provide both MRA, to assess vascular anatomy and luminal changes, and VWI, to assess the vessel wall and detect vulnerable plaques, in a single scan.

Involvement of the Clock Gene Period in the Photoperiodism of the Silkmoth Bombyx mori.

We established a knockout strain of a clock gene, period (per), by using TALEN in a bivoltine strain (Kosetsu) of Bombyx mori (Insecta, Lepidoptera), and examined the effect of per knockout on the circadian rhythm and photoperiodism. The generated per knockout allele was considered to be null, because a new stop codon was present in the insertion allele. The wild type (Kosetsu) showed clear circadian rhythms in eclosion and hatching, whereas the per knockout strain showed arrhythmic eclosion and hatching under constant darkness. In this strain, moreover, temporal expression changes of clock genes per and timeless were disrupted. The wild type showed a clear long-day response for induction of embryonic diapause: when larvae were reared under long-day and short-day conditions at 25°C, adults produced nondiapause and diapause eggs, respectively. However, the per knockout strain lost the sensitivity to photoperiod and laid nondiapause eggs under both conditions. We conclude that per plays an important role both in circadian rhythms and in photoperiodism of B. mori, indicating the involvement of the circadian clock consisting of per in the photoperiodism.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Involvement of the Clock Gene period in the Circadian Rhythm of the Silkmoth Bombyx mori.

In Lepidoptera, the roles of period ( per) and the negative feedback involving this gene in circadian rhythm are controversial. In the present study, we established a per knockout strain using TALEN in Bombyx mori, and compared eclosion and hatching rhythms between the per-knockout and wild-type strains to examine whether per is actually involved in these rhythms. The generated per knockout allele was considered null, because it encoded an extensively truncated form of PERIOD (198 aa due to a 64-bp deletion in exon 7, in contrast to 1113 aa in the wild-type protein). In this per knockout strain, circadian rhythms in eclosion and hatching were disrupted. Under LD cycles, however, a steep peak existed at 1 h after lights-on in both eclosion and hatching, and was considered to be produced by a masking effect-a direct response to light. In the per-knockout strain, temporal expression changes of per and timeless ( tim) were also lost. The expression levels of tim were continuously high, probably due to the loss of negative feedback by per and tim. In contrast, the expression levels of per were much lower in the per knockout strain than in the wild type at every time point. From these results, we concluded that per is indispensable for circadian rhythms, and we suggest that the negative feedback loop of the circadian rhythm involving per functions for the production of behavioral rhythms in B. mori.

High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment.

Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy.

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.