Appropriate pancreatic phase image acquisition by free-breathing dynamic contrast-enhanced pancreatic MRI using stack-of-stars radial sampling and Compressed SENSE.

PURPOSE: To evaluate the feasibility of a free-breathing sequence (4D FreeBreathing) combined with Compressed SENSE in dynamic contrast-enhanced pancreatic MRI and compare it with a breath-holding sequence (eTHRIVE). METHOD: Patients who underwent pancreatic MRI, either eTHRIVE or 4D FreeBreathing, from April 2022 to November 2023 were included in this retrospective study. Two radiologists, who were unaware of the scan sequence, independently and randomly reviewed the images at the precontrast, pancreatic, portal venous, and equilibrium phases and assigned confidence scores for motion and streaking artifacts, pancreatic sharpness, and overall image quality using a 5-point scale. Furthermore, the radiologists assessed the appropriateness of the scan timing of the pancreatic phase. Mann-Whitney U and Fisher's exact tests were conducted to compare the confidence scores and adequacy of the pancreatic phase scan timing between eTHRIVE and 4D FreeBreathing. RESULTS: Overall, 48 patients (median age, 71 years; interquartile range, 64-77 years; 24 women) were included. Among them, 20 patients (42%) were scanned using 4D FreeBreathing. The 4D FreeBreathing showed moderate streaking artifact but improved motion artifact (P <.001-.17) at all phases. Pancreatic sharpness and overall image quality were almost comparable between two sequences (P = .17-.96). All 20 examinations in 4D FreeBreathing showed appropriate pancreatic phase images, but only 16 (57%; P <.001 for reviewer 1) and 18 (64%; P = .003 for reviewer 2) examinations showed it in eTHRIVE. CONCLUSION: The use of 4D FreeBreathing combined with Compressed SENSE was feasible in pancreatic MRI and provided appropriate pancreatic phase images in all examinations.

Pancreatic cancer detection with dual-energy CT: diagnostic performance of 40 keV and 70 keV virtual monoenergetic images.

PURPOSE: To compare the diagnostic performance of 40 keV and 70 keV virtual monoenergetic images (VMIs) generated from dual-energy CT in the detection of pancreatic cancer. METHODS: This retrospective study included patients who underwent pancreatic protocol dual-energy CT from January 2019 to August 2022. Four radiologists (1-11 years of experience), who were blinded to the final diagnosis, independently and randomly interpreted 40 keV and 70 keV VMIs and graded the presence or absence of pancreatic cancer. For each image set (40 keV and 70 keV VMIs), the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated. The diagnostic performance of each image set was compared using generalized estimating equations. RESULTS: Overall, 137 patients (median age, 71 years; interquartile range, 63-78 years; 77 men) were included. Among them, 62 patients (45%) had pathologically proven pancreatic cancer. The 40 keV VMIs had higher specificity (75% vs. 67%; P < .001), PPV (76% vs. 71%; P < .001), and accuracy (85% vs. 81%; P = .001) than the 70 keV VMIs. On the contrary, 40 keV VMIs had lower sensitivity (96% vs. 98%; P = .02) and NPV (96% vs. 98%; P = .004) than 70 keV VMIs. However, the diagnostic confidence in patients with (P < .001) and without (P = .001) pancreatic cancer was improved in 40 keV VMIs than in 70 keV VMIs. CONCLUSIONS: The 40 keV VMIs showed better diagnostic performance in diagnosing pancreatic cancer than the 70 keV VMIs, along with higher reader confidence.

1.5†µm wavelength NPN-type photonic-crystal surface-emitting laser exceeding 100 mW.

A 1.5 µm laser diode has applications in eye-safe light detection and ranging (LiDAR) and optical communications via photonic integrated circuits. Photonic-crystal surface-emitting lasers (PCSELs) have lens-free applications in compact optical systems because of narrow beam divergences (<1 degree). However, the output power has still been less than 1 mW for 1.5 µm PCSELs. For higher output power, one approach is suppression of p-dopant Zn diffusion in the photonic crystal layer. Therefore, n-type doping was used for the upper crystal layer. Moreover, an NPN-type PCSEL structure was proposed to reduce intervalence band absorption in the p-InP layer. Here, we demonstrate a 1.5 µm PCSEL with 100 mW output power, which exceeds previous reported values by two orders of magnitude.

Sustainable pest control inspired by prey-predator ultrasound interactions.

Nocturnal moths evolved ultrasound-triggered escape maneuvers for avoiding predatory bats emitting ultrasonic echolocation calls. Using ultrasound for pest control is not a novel concept, but the technique has not been systemized because of the moths' habituation to sounds and the narrow directionality of conventional ultrasound speakers. Here, we report the use of pulsed ultrasonic white noise, which contributes to achieving ecologically concordant plant protection. An ultrasonic pulse, which is temporal mimicry of the search-phase pulse in the echolocation calls of a sympatric bat, was identified using neuroethological screening of eared moth-repelling ultrasounds; these pulses elicit flight-stopping reactions in moths but have no or little auditory adaptation. Such repellent ultrasounds broadcast from the cylindrical omni-azimuth ultrasound emitters suppressed the intrusion of gravid females of pest moths into cultivation fields. Thus, egg numbers and plant damage by hatched larvae were drastically reduced, enabling farmers to substantially skip applications of chemical insecticides for controlling moth pests.

[Effect of Soy Sauce, Fish Sauce for Nitrite Determination in Meat Products].

The diazotization method is used for nitrite determination in meat products. In this method, it is known that the presence of reducing substances such as ascorbic acid and cysteine in the sample interferes with nitrite determination. We speculated that soy sauce, fish sauce and mirin, which are used in meat products, might be the reducing substances, so investigated meat products which contain soy sauce and other ingredients. The results showed that quantitative disturbance was observed in soy sauce and fish sauce, and the recovery in spiked samples of these products decreased in proportion to the increase in soy sauce and fish sauce content. However the antioxidant of soy sauce and fish sauce depending on manufacturing methods, so quantitative disturbance dose not be observed each time, so nitrite determination in meat products, and to conduct simultaneous determination using different sample amounts to confirm whether there is any difference in the quantitative values.

Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals.

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype-phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

Heterologous expression of high-activity cytochrome P450 in mammalian cells.

The evaluation of Cytochrome P450 (CYP) enzymatic activity is essential to estimate drug pharmacokinetics. Numerous CYP allelic variants have been identified; the functional characterisation of these variants is required for their application in precision medicine. Results from heterologous expression systems using mammalian cells can be integrated in in vivo studies; however, other systems such as E. coli, bacteria, yeast, and baculoviruses are generally used owing to the difficulty in expressing high CYP levels in mammalian cells. Here, by optimising transfection and supplementing conditions, we developed a heterologous expression system using 293FT cells to evaluate the enzymatic activities of three CYP isoforms (CYP1A2, CYP2C9, and CYP3A4). Moreover, we established co-expression with cytochrome P450 oxidoreductase and cytochrome b5. This expression system would be a potential complementary or beneficial alternative approach for the pharmacokinetic evaluation of clinically used and developing drugs in vitro.

Development and application of a rapid and sensitive genotyping method for pharmacogene variants using the single-stranded tag hybridization chromatographic printed-array strip (STH-PAS).

Genetic polymorphisms contribute to inter-individual variability in the metabolism of multiple clinical drugs, including warfarin, thiopurines, primaquine, and aminoglycosides. A rapid and sensitive clinical assessment of various genome biomarkers is, therefore, required to predict the individual responsiveness of each patient to these drugs. In this study, we developed a novel genotyping method for the detection of nine pharmacogene variants that are important in the prediction of drug efficiency and toxicity. This genotyping method uses competitive allele-specific PCR and a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) that can unambiguously determine the presence or absence of the gene variant by displaying visible blue lines on the chromatographic printed-array strip. Notably, the results of our STH-PAS method were in 100% agreement with those obtained using standard Sanger sequencing and KASP assay genotyping methods for CYP4F2 gene deletion. Moreover, the results were obtained within 90 min, including the PCR amplification and signal detection processes. The sensitive and rapid nature of this novel method make it ideal for clinical genetic testing to predict drug efficacy and toxicity, and in doing so will aid in the development of individualized medicine and better patient care.

Spectral purity and tunability of terahertz quantum cascade laser sources based on intracavity difference-frequency generation.

Terahertz sources based on intracavity difference-frequency generation in mid-infrared quantum cascade lasers (THz DFG-QCLs) have recently emerged as the first monolithic electrically pumped semiconductor sources capable of operating at room temperature across the 1- to 6-THz range. Despite tremendous progress in power output, which now exceeds 1 mW in pulsed and 10 µW in continuous-wave regimes at room temperature, knowledge of the major figure of merits of these devices for high-precision spectroscopy, such as spectral purity and absolute frequency tunability, is still lacking. By exploiting a metrological grade system comprising a terahertz frequency comb synthesizer, we measure, for the first time, the free-running emission linewidth (LW), the tuning characteristics, and the absolute center frequency of individual emission lines of these sources with an uncertainty of 4 × 10-10. The unveiled emission LW (400 kHz at 1-ms integration time) indicates that DFG-QCLs are well suited to operate as local oscillators and to be used for a variety of metrological, spectroscopic, communication, and imaging applications that require narrow-LW THz sources.

Ultra-broadband room-temperature terahertz quantum cascade laser sources based on difference frequency generation.

We present ultra-broadband room temperature monolithic terahertz quantum cascade laser (QCL) sources based on intra-cavity difference frequency generation, emitting continuously more than one octave in frequency between 1.6 and 3.8 THz, with a peak output power of ~200 µW. Broadband terahertz emission is realized by nonlinear mixing between single-mode and multi-mode spectra due to distributed feedback grating and Fabry-Perot cavity, respectively, in a mid-infrared QCL with dual-upper-state active region design. Besides, at low temperature of 150 K, the device produces a peak power of ~1.0 mW with a broadband THz emission centered at 2.5 THz, ranging from 1.5 to 3.7 THz.

Broadband tuning of continuous wave quantum cascade lasers in long wavelength (> 10 µm) range.

Broadband spectral tuning in the long wavelength range (greater than 10 µm) was demonstrated with an external-cavity quantum cascade laser. The tunable wavelength of the laser ranged from 9.5 to 11.4 µm (176 cm(-1); corresponding to 18% of the center wavelength) in continuous wave (cw) operation at room temperature, without any anti-reflection coating. The gain chip based on the anti-crossed dual-upper-state (DAU) design provided a cw lasing up to 300 K, with a low threshold current density of 2.1 kA/cm2. The highly stable broadband spectral tuning and high laser performance were enabled by the spectrally homogeneous gain profile of the anti-crossed DAU active region.

Single-cell heterogeneity in suppression of PC12 differentiation by direct microinjection of a differentiation inhibitor, U0126.

Phenotypic and genomic heterogeneity among single cells in a cell population leads to inaccuracy and obscuration in research about mammalian cell differentiation. In order to address the problems regarding bulk analysis on heterogeneous cell populations, it is necessary to accurately regulate and analyze changes in differentiating cells at the single-cell level. To investigate the single-cell changes in PC12 neuronal differentiation that occur when inhibited by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), we directly injected the chemical into individual target cells and analyzed the outcomes (neurite outgrowth) at the single-cell level. As a result, we could accurately regulate the quantity of U0126 being introduced into each target cell, which was previously not possible using the common method of simply adding the inhibitor to the culture medium. It was possible to analyze the inhibitive effect of U0126 even when the injected quantity was lower than the lower limit for inhibition when added to culture medium (0.1 µM, identical to 1.2 × 10(8) molecules per cell on dish). In particular, injection of 1.5 × 10(7) molecules into each cell resulted in a 59% decrease of the mean total neurite length. Time-course analysis of neurite outgrowth at the single-cell level using fluorescence staining method showed that the changes in neurite length of differentiating PC12 cells were not homogeneous, but were largely variable across individual target cells.

Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos.

Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated α-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and α-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to α-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated α-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including α-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development.

Crystal structure of the parasporin-2 Bacillus thuringiensis toxin that recognizes cancer cells.

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

Haeme-regulated degradation of delta-aminolevulinate synthase 1 in rat liver mitochondria.

Protein turnover, which occurs at various rates, is critical for the homeostasis of cellular protein levels. However, the proteolysis systems that determine the turnover rate of mitochondrial proteins are largely unknown. Delta-aminolevulinic acid synthase (ALAS) 1, a rate-limiting enzyme in the haeme biosynthesis, is one of the mitochondrial proteins that have a very short lifetime. In this study, to reveal the regulatory mechanisms for ALAS1 degradation, we examined the turnover rates of ALAS1 in rat liver under several conditions. In primary rat hepatocytes, the degradation of ALAS1 was stimulated by haeme, and suppressed by inhibition of haeme biosynthesis. Furthermore, the haeme-stimulated degradation of ALAS1 was observed in the isolated mitochondria. These results suggested that, in mitochondria, there exists an ALAS1 degradation system that is regulated by cellular haeme level and plays a crucial role in the regulation of haeme biosynthesis.

Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer.

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.

A protein from a parasitic microorganism, Rickettsia prowazekii, can cleave the signal sequences of proteins targeting mitochondria.

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the alpha and beta subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with beta-MPP but barely contained any of the glycine-rich loop region characteristic of alpha-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast beta-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis.

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Presence of monoamine oxidase type B protein but absence of associated enzyme activity in neurons within the inferior olive nucleus of the rat.

A previous study demonstrated that monoamine oxidase type B (MAOB) mRNA is located in the inferior olive complex (IO). The purpose of the present study was to examine whether neuronal cell bodies within the IO also express MAOB protein and whether they exhibit associated MAOB enzyme activity. Using immunohistochemistry and enzyme histochemistry, we demonstrated that IO neuronal cell bodies were positive for MAOB immunohistochemistry but negative for MAOB enzyme histochemistry. These findings indicate that IO neuronal cell bodies express MAOB mRNA and produce MAOB protein but curiously do not exhibit MAOB enzyme activity, as might be expected. The mechanism responsible for the failure of MAOB protein to result in enzymatic activity in IO neuronal cell bodies is clearly of significance in terms of functionality but remains to be elucidated.

Immunohistochemical localization of monoamine oxidase type B in pancreatic islets of the rat.

Monoamine oxidase (MAO) is regarded as a mitochondrial enzyme. This enzyme localizes on the outer membrane of mitochondria. There are two kinds of MAO isozymes, MAO type A (MAOA) and type B (MAOB). Previous studies have shown that MAOB activity is found in the pancreatic islets. This activity in the islets is increased by the fasting-induced decrease of plasma glucose level. Islet B cells contain monoamines in their secretory granules. These monoamines inhibit the secretion of insulin from the B cells. MAOB is active in degrading monoamines. Therefore, MAOB may influence the insulin-secretory process by regulating the stores of monoamines in the B cells. However, it has not been determined whether MAOB is localized on B cells or other cell types of the islets. In the present study, we used both double-labeling immunofluorescence histochemical and electron microscopic immunohistochemical methods to examine the subcellular localization of MAOB in rat pancreatic islets. MAOB was found in the mitochondrial outer membranes of glucagon-secreting cells (A cells), insulin-secreting cells (B cells), and some pancreatic polypeptide (PP)-secreting cells (PP cells), but no MAOB was found in somatostatin-secreting cells (D cells), nor in certain other PP cells. There were two kinds of mitochondria in pancreatic islet B cells: one contains MAOB on their outer membranes, but a substantial proportion of them lack this enzyme. Our findings indicate that pancreatic islet B cells contain MAOB on their mitochondrial outer membranes, and this enzyme may be involved in the regulation of monoamine levels and insulin secretion in the B cells.