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We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeastSchizosaccharomyces pombe. Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II–coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoebaDictyostelium, but in this case, by detaching actin filaments from myosin II–coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.

Summary Immunogenic tumor cell death enhances anti-tumor immunity. However, the mechanisms underlying this effect are incompletely understood. We established a system to induce tumor cell death in situ and investigated its effect on dendritic cell (DC) migration and T cell responses using intravital photolabeling in mice expressing KikGR photoconvertible protein. We demonstrate that tumor cell death induces phagocytosis of tumor cells by tumor-infiltrating (Ti)-DCs, and HMGB1-TLR4 and ATP-P2X7 receptor signaling-dependent Ti-DC emigration to draining lymph nodes (dLNs). This led to an increase in anti-tumor CD8+ T cells of memory precursor effector phenotype and secondary tumor growth inhibition in a CD103+ DC-dependent manner. However, combining tumor cell death induction with lipopolysaccharide treatment stimulated Ti-DC maturation and emigration to dLNs but did not improve tumor immunity. Thus, immunogenic tumor cell death enhances tumor immunity by increasing Ti-DC migration to dLNs where they promote anti-tumor T cell responses and tumor growth inhibition.

Measuring the volatile organic compounds (VOCs) released from a human is a promising method for noninvasive disease screening and metabolism assessment. Selectively imaging multiple VOCs derived from human breath and skin gas is expected to improve current gas analysis techniques. In this study, a gas-imaging system (sniff-cam) that can be used to simultaneously image the concentration distribution of multiple VOCs, namely, ethanol (EtOH) and acetaldehyde (AcH), was developed. The sniff-cam was based on the pH-dependent redox reactions of nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH). The sniff-cam was constructed with a camera, two ADH-immobilized meshes, and a UV-LED array sheet. The ADH-immobilized mesh containing a solution of the oxidized form of NAD (NAD+) or reduced form (NADH) was used as an EtOH-imaging mesh and an AcH-imaging mesh, respectively. The distributions of the EtOH and AcH concentrations were visualized through the fluorescence of NADH (the excitation wavelength was 340 nm; the emission wavelength was 490 nm) occurring by the ADH-mediated redox reaction. First, the influence of pH on the activity of the redox reaction of ADH was measured, and then the quantitativeness and selectivity of the sniff-cam were evaluated. The ADH-mediated reactions of EtOH and AcH showed maximum activities at pH 9.0 and pH 6.5, respectively. The sniff-cam demonstrated not only a dynamic range (0.1–1000 ppm for EtOH and 0.2–10 ppm for AcH) for measuring EtOH and AcH in breath after drinking alcohol, but also displayed a high selectivity against other breath VOCs. Finally, EtOH and AcH in breath after drinking alcohol were measured simultaneously. A group with high activity of aldehyde dehydrogenase type 2 (EtOH = 143.3 ± 13.5 ppm, AcH = 1.7 ± 0.2 ppm) and a group with low activity (EtOH = 163.3 ± 28.0 ppm, AcH = 8.4 ± 0.5 ppm) displayed differences in the concentrations of EtOH and AcH contained in their breath samples, and the effectiveness of the developed method was confirmed and compared with previous results. It is suggested that the multiplexed sniff-cam in the future may be capable of selectively and simultaneously imaging various VOCs in human breath and skin gas by using multiple NADH-dependent enzymes.

Substoichiometric binding of certain actin-binding proteins induces conformational changes in a disproportionally large number of actin protomers in actin filaments. Here, we report a case in which such conformational changes in actin filaments have profound functional consequences. Rng2 is an IQGAP protein implicated in the assembly and contraction of contractile rings in Schizosaccharomyces pombe. We found that the calponin-homology actin-binding domain of Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD halved the sliding speed of actin filaments at a binding ratio of 1.3% (=1/77), and virtually stopped movement at a binding ratio of 11% (=1/9). Rng2CHD also inhibited actin movements on Dictyostelium myosin II, but in this case by inducing the detachment of actin filaments from myosin II-coated surfaces. Rng2CHD induced cooperative structural changes of actin filaments accompanied by shortening of the filament helical pitch, and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II in the presence of ADP. Intriguingly, actin-activated ATPase of S1 was hardly inhibited by Rng2CHD. We suggest that sparsely bound Rng2CHD induces global structural changes of actin filaments and interferes with the force generation by actin-myosin II.

We previously reported that the moderate knockdown of chitin synthase 1 gene of the model beetle Tribolium castaneum impairs the host defense against entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, which infect host insects via the direct penetration of cuticular integuments (Hayakawa et al., 2017). In this study, we focused on the antifungal roles of laccase2 (Lac2) as well as yellow-e (Y-e) genes, both of which are shown to be important to the establishment of stable cuticular structures in this beetle species. The expression profiles of the two genes somewhat resembled each other, peaking in late prepupae and mid to late pupae, while the transcript levels of Lac2 were higher than Y-e throughout. The knockdown of Lac2 gene at the prepupal and pupal peaks with relatively small amounts of dsRNA resulted in pupae with a lighter color and adults with a lighter color and dimpled/wrinkled elytra, respectively. Meanwhile, similar gene knockdown of Y-e but with 10 times more dsRNA compared to Lac2 resulted in pupae having a normal appearance and adults with a darker color. We conducted fungal infection assays with B. bassiana and M. anisopliae using these knockdown animals. The knockdown of Y-e gene had no or limited effects in both pupae and adults in terms of the antifungal host defense. Similarly, the knockdown of Lac2 gene did not change significantly the defense phenotypes of the resulting pupae. By sharp contrast, the host defense of the adult beetles against the two fungal species was almost totally destroyed by the moderate knockdown of Lac2 gene, suggesting its indispensable role in antifungal host defense presumably through the construction of sound cuticles of the adults. Finally, we investigated the maturation of host defense against fungal infection in the Lac2 knockdown adults and found that while the day 10 adults were still susceptible to M. anisopliae infection with some delay of death in comparison with day 1 adults, they exhibited complete refractoriness to B. bassiana., ファイル公開：2019-01-01

The importance of the insect cuticle as a primary protective barrier against entomopathogens has long been noted. In the present study, we addressed this issue by utilizing an experimental infection system composed of the model beetle T. castaneum and two entomopathogenic fungal species, Beauveria bassiana and Metarhizium anisopliae. The pupae were relatively susceptible to these fungi by the natural route of infection, with some refractoriness developed with age, while the adults exhibited much higher refractoriness. Whereas M. anisopliae exhibited seemingly higher infectivity to the pupae compared to B. bassiana when the natural conidium infection was employed, direct inoculation of cultured hyphal body cells into the hemocoel was found highly and equally virulent in the pupae for the both fungal species. These results collectively suggest an important role of the cuticular integument in antifungal host defense, and we subsequently conducted the knockdown of chitin synthase 1 gene (CHS1). We targeted the prepupal and mid-pupal peaks of its expression respectively by using injection of the dsRNA at very low dosages to avoid lethality. The resulting pupae looked normal, but the adults showed a mild phenotype with dimpled/wrinkled elytra. The CHS1 gene knockdown compromised significantly host defense against the fungal infection via the natural route, except the configuration of knockdown pupae and M. anisopliae, suggesting an indispensable role of CHS1.

A gas-imaging system (sniff-cam) that allows fluorometric visualization of a two-dimensional (2-D) distribution of gaseous acetaldehyde (AcH) was developed. It employed a reverse reaction of a nicotinamide adenine dinucleotide (NADH) dependent enzyme that led to consumption of NADH in that reaction. The system was constructed with a highly sensitive camera, an ultraviolet light emitting diode array sheet, two band pass filters and an alcohol dehydrogenase (ADH)-immobilized mesh that was used for AcH detection. The reverse reaction of the ADH catalyzed the reduction of AcH to ethanol and the oxidation of NADH to NAD+, which occurred when gaseous AcH was applied to the ADH immobilized mesh that was wetted with a slightly acidic NADH solution. As NADH has an autofluorescence property [emission (λem) at 490 nm; excitation (λex) at 340 nm], the presence of gaseous AcH was visualized by a decrease of fluorescence of the NADH at the ADH immobilized mesh. After constructing the gaseous AcH imaging system, optimizations of pH, and concentration of the NADH solution were performed. As a result of the optimizations (500 μM of NADH in 0.1 M of Tris hydrochloride (Tris-HCl) buffer at pH 6.5), the AcH sniff-cam showed a wide dynamic range (0.1-10 ppm) for gaseous AcH with a high correlation coefficient (R = 0.999). Furthermore, a fluorescence gradient with a rounded shape centered in a gas outlet was observed. These results demonstrated that the AcH sniff-cam utilizing the fluorescence decrease of NADH could be used to quantitatively evaluate the 2-D distribution of gaseous AcH.

Understanding concentration distributions, release sites, and release dynamics of volatile organic compounds (VOCs) from the human is expected to lead to methods for noninvasive disease screening and assessment of metabolisms. In this study, we developed a visualization system (sniff-cam) that enabled one to identify a spatiotemporal change of gaseous acetaldehyde (AcH) in real-time. AcH sniff-cam was composed of a camera, a UV-LED array sheet, and an alcohol dehydrogenase (ADH)-immobilized mesh. A reverse reaction of ADH was employed for detection of gaseous AcH where a relationship between fluorescence intensity from nicotinamide adenine dinucleotide and the concentration of AcH was inversely proportional; thus, the concentration distribution of AcH was measured by detecting the fluorescence decrease. Moreover, the image differentiation method that calculated a fluorescence change rate was employed to visualize a real-time change in the concentration distribution of AcH. The dynamic range of the sniff-cam was 0.1-10 ppm which encompassed breath AcH concentrations after drinking. Finally, the sniff-cam achieved the visualization of the concentration distribution of AcH in breath and skin gas. A clear difference of breath AcH concentration was observed between aldehyde dehydrogenase type 2 active and inactive subjects, which was attributed to metabolic capacities of AcH. AcH in skin gas showed a similar time course of AcH concentration to the breath and a variety of release concentration distribution. Using different NADH-dependent dehydrogenases in the sniff-cam could lead to a versatile method for noninvasive disease screening by acquiring spatiotemporal information on various VOCs in breath or skin gas.

Migration of software between systems requires the migrated software to be compatible with the new platform. The original machine code in the source machine cannot always be directly ported. The program's source code may not be available for compilation to the new platform or the machine code may not be compatible to the new platform due to architectural differences. An alternative solution to porting software can be achieved through assembly language translation. This study presents the essential considerations in assembly language translation such as handling of the registers, data segment, and code segment. Possible approaches towards are also discussed.

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum.

Purpose: Our purpose was to investigate the changes in the dynamic property of vergence eye movements caused by changes in the co-existing stationary background in the central visual field. Methods: Disparity-driven target movement was presented virtually by a head-mounted liquid-crystal display. Two targets were used: a bar-shaped target that moved between 2 and 0.5 m along the mid-sagittal line at a speed of 50 cm/s (vergence target) and a background image of a cross-shaped target that stayed at a distance of 2 m (background target). Eight normal subjects participated in the experiments. The subject was asked to follow the vergence target while the configuration of the background target was randomly changed among four conditions in each experiment: the length (experiment 1) or the width (experiment 2) of the horizontal and vertical lines composing the cross of the background target was each randomly changed among four conditions. A limbus tracker was used to measure eye movements. Results: In experiment 1, there was a negative correlation between the amplitude of the vergence eye movements and the lengths of the lines of the cross in each of five subjects (mean r = 0.018, n = 48 in each subject). Similarly, in experiment 2, there was a negative correlation between the amplitude of the vergence eye movements and the width of the lines of the cross in each of 8 subjects (mean r = –0.12, n = 48 in each subject). Conclusion: The vergence response to a target object significantly differs depending on the texture of background objects on the visual axis.

Purpose: The functional organization of the human cerebellum involved in saccadic eye movements was investigated using functional magnetic resonance imaging (fMRI). Methods: The subjects were 7 normal volunteers aged 18–34 years. Visual stimuli were back-projected onto a screen placed at the subjects’ feet. The stimulation period of 30 s consisted of a saccade target jumping back and forth horizontally by 20° once per second. The control period of 30 s consisted of a fixed target. The stimulation and control periods were alternated 10 times during the presentation. Functional images were collected with a 1.5-tesla clinical MRI scanner. The significance of activation was determined by Statistical Parametric Mapping (SPM 99) at a threshold of p < 0.001 (uncorrected), and significantly activated areas were superimposed on the T1-weighted images. Results: Significantly activated areas related to visually guided saccades were observed in the cerebellar vermis (declive and folium), in the bilateral cerebellar hemispheres (mainly the superior semilunar lobule) of the cerebellum, in the frontal eye field, in the supplementary eye field and in parts of the parietal lobule of the cerebrum. Conclusion: Our results suggest that the cerebellar posterior vermis and bilateral hemispheres are related to saccades in humans. These results are consistent with neurophysiological data obtained in primates.

Superior oblique myokymia is a microtremor of the eye that causes monocular torsional oscillopsia. A modified Harada-Ito procedure was used to treat a case of the disease in a 20-year-old woman. The authors used video-image analysis pre- and postoperatively to evaluate the effect of the surgery on abnormal torsional eye movements. This analysis revealed that before surgery, the abnormal torsional movement had a very regular cycle (duration of attack, 8.0 +/- 0.5 s; time interval between attacks, 18.7 +/- 3.2 s; n = 9). After the surgery, amplitude of the abnormal torsional eye movement was reduced, and the oscillopsia had subjectively improved, although the movement cycle remained unchanged. The authors' video-image analysis, which used iris striation, proved to be a useful method for clinical measurement of torsional eye movements.

Purpose To report two patients with glaucoma who exhibited severe damage to the corneal endothelium after a trabeculectomy with mitomycin C (MMC). Methods This study includes clinical histories and specular microscopic pictures of the cases. Results Both patients were middle-aged women, underwent trabeculectomy with MMC, had moderate to severe cornea guttata preoperatively, and developed a shallow to flat anterior chamber, classified as grade 2 according to Spaeth early in the postoperative period. Stromal opacity caused by corneal edema associated with severe Descemet's membrane folds appeared within 2 to 5 days in both cases. The density of the corneal endothelium was decreased on specular microscopic examination. The severe corneal endothelial damage seen after the trabeculectomy with MMC was likely owing to a combination of the preexisting cornea guttata, the flat anterior chamber, and possibly the administration of MMC. Conclusion Severe endothelial damage after trabeculectomy with MMC may occur in patients with glaucoma and associated cornea guttata. The use of tight sutures on the scleral flap or a modified operative method, nonpenetrating trabeculectomy, may be effective in preventing a shallow to flat anterior chamber postoperatively.

Purpose: We attempted to apply a newly developed image-analysis system for measurement and analysis of nystagmus. Method: Eye movements were recorded by digital video through a head-mounted charge coupled device (CCD) camera. The recorded movie was converted into black and white in order to detect the area of the pupil. Horizontal and vertical eye positions were determined by calculating the centroid of the pupil. Torsional angle was calculated using the iris striate pattern around the pupillary margin. Results: The parameters (amplitude, cycle, etc.) of nystagmus were calculated easily by the new image-analysis system from the recorded images. As examples, the foveation period was measured accurately in a case of jerky-type congenital nystagmus. Very regular cycles of intorsional attack period were revealed in a case of superior oblique myokymia. A case of cork-screw-like nystagmus showed a characteristic combination of large and small cycles unassociated with torsion. Conclusion: This image-analysis system was useful for quantitative analysis of nystagmus, and especially for measurement of torsion. Detailed waveforms and specific rhythms of nystagmus, which could not be recognized by observation, were demonstrated by this system.