The effect of nintedanib on health-related quality of life in Japanese patients with progressive fibrosing interstitial lung diseases: A subset analysis of the INBUILD trial.

BACKGROUND: In previous Japanese subgroup/subset analyses of the global INBUILD trial, nintedanib reduced the annual rate of forced vital capacity (FVC) decline and the risk of disease progression in patients with progressive fibrosing interstitial lung diseases (PF-ILDs). This exploratory subset analysis assessed the effect of nintedanib on symptoms and impacts of pulmonary fibrosis in Japanese patients with PF-ILDs, including those with usual interstitial pneumonia (UIP)-like fibrotic pattern on high-resolution computed tomography (HRCT). METHODS: This analysis included Japanese patients who received at least one dose of study treatment in the randomized, double-blind, placebo-controlled INBUILD trial. The Living with Pulmonary Fibrosis (L-PF) questionnaire was used to assess pulmonary fibrosis symptoms and impacts (higher scores indicated greater impairment) at baseline and weeks 12-52. RESULTS: In total, 108 Japanese patients (nintedanib: n = 52; placebo: n = 56) were included; 84 patients had UIP-like fibrotic pattern on HRCT. In the total Japanese subgroup and in those with UIP-like fibrotic pattern, numerically greater increases in L-PF total, symptoms total, symptoms fatigue domain, and impacts scores were observed in the placebo group than in the nintedanib group at all timepoints, starting from week 12. A numerically greater increase in the symptoms dyspnea domain score was observed with placebo versus nintedanib starting from week 36. Throughout the study, the symptoms cough domain score increased in the placebo group but decreased in the nintedanib group. CONCLUSIONS: Our findings demonstrate that nintedanib has the potential to reduce the worsening of symptoms and impacts of pulmonary fibrosis in Japanese patients with PF-ILDs.

Automated contour extraction for light-sheet microscopy images of zebrafish embryos based on object edge detection algorithm.

Embryo contour extraction is the initial step in the quantitative analysis of embryo morphology, and it is essential for understanding the developmental process. Recent developments in light-sheet microscopy have enabled the in toto time-lapse imaging of embryos, including zebrafish. However, embryo contour extraction from images generated via light-sheet microscopy is challenging owing to the large amount of data and the variable sizes, shapes, and textures of objects. In this report, we provide a workflow for extracting the contours of zebrafish blastula and gastrula without contour labeling of an embryo. This workflow is based on the edge detection method using a change point detection approach. We assessed the performance of the edge detection method and compared it with widely used edge detection and segmentation methods. The results showed that the edge detection accuracy of the proposed method was superior to those of the Sobel, Laplacian of Gaussian, adaptive threshold, Multi Otsu, and k-means clustering-based methods, and the noise robustness of the proposed method was superior to those of the Multi Otsu and k-means clustering-based methods. The proposed workflow was shown to be useful for automating small-scale contour extractions of zebrafish embryos that cannot be specifically labeled owing to constraints, such as the availability of microscopic channels. This workflow may offer an option for contour extraction when deep learning-based approaches or existing non-deep learning-based methods cannot be applied.

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion.

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides. This article has an associated interview with the co-first authors of the paper.

Near-wall rheotaxis of the ciliate Tetrahymena induced by the kinesthetic sensing of cilia.

To survive in harsh environments, single-celled microorganisms autonomously respond to external stimuli, such as light, heat, and flow. Here, we elucidate the flow response of Tetrahymena, a well-known single-celled freshwater microorganism. Tetrahymena moves upstream against an external flow via a behavior called rheotaxis. While micrometer-sized particles are swept away downstream in a viscous flow, what dynamics underlie the rheotaxis of the ciliate? Our experiments reveal that Tetrahymena slides along walls during upstream movement, which indicates that the cells receive rotational torque from shear flow to control cell orientation. To evaluate the effects of the shear torque and propelling speed, we perform a numerical simulation with a hydrodynamic model swimmer adopting cilia dynamics in a shear flow. The swimmer orientations converge to an upstream alignment, and the swimmer slides upstream along a boundary wall. The results suggest that Tetrahymena automatically responds to shear flow by performing rheotaxis using cilia-stalling mechanics.

Dabigatran or Aspirin in East Asian Patients With Embolic Stroke of Undetermined Source: RE-SPECT ESUS Subgroup Analysis.

BACKGROUND AND PURPOSE: We assessed the outcomes of dabigatran versus aspirin in a prespecified subgroup analysis of East Asian patients with embolic stroke of undetermined source in the RE-SPECT ESUS trial (Randomized, Double-Blind, Evaluation in Secondary Stroke Prevention Comparing the Efficacy and Safety of the Oral Thrombin Inhibitor Dabigatran Etexilate Versus Acetylsalicylic Acid in Patients With Embolic Stroke of Undetermined Source). METHODS: Patients with a recent embolic stroke of undetermined source were randomized to dabigatran (150 or 110 mg BID) or aspirin (100 mg QD). The primary efficacy outcome was recurrent stroke; the primary safety outcome was major bleeding. The East Asia cohort was compared with patients from all other countries (non-East Asia cohort). RESULTS: Overall, 988 of 5390 patients (18%) were randomized in East Asia. During a median follow-up of 18.8 months, there was no statistically significant difference in recurrent stroke (hazard ratio, 0.65 [95% CI, 0.41-1.03]) or major bleeding (hazard ratio, 1.04 [95% CI, 0.57-1.91]) in East Asian patients receiving dabigatran versus aspirin. Death from any cause occurred more often in the dabigatran versus the aspirin group (hazard ratio, 3.98 [95% CI, 1.32-12.01]). CONCLUSIONS: The treatment effect of dabigatran versus aspirin was consistent between cohorts, with no apparent superiority for dabigatran over aspirin in preventing recurrent stroke in patients with embolic stroke of undetermined source. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02239120.

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development.

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.

Dabigatran vs. Aspirin for Secondary Prevention After Embolic Stroke of Undetermined Source　- Japanese Subanalysis of the RE-SPECT ESUS Randomized Controlled Trial.

BACKGROUND: The international Randomized, Double-Blind, Evaluation in Secondary Stroke Prevention Comparing the EfficaCy and Safety of the Oral Thrombin Inhibitor Dabigatran Etexilate versus Acetylsalicylic Acid in Patients with Embolic Stroke of Undetermined Source (RE-SPECT ESUS) trial did not demonstrate superiority of dabigatran over aspirin for reduction of recurrent strokes in patients with embolic strokes of undetermined source (ESUS). Based on pre-defined subanalyses, the safety and efficacy of dabigatran vs. aspirin in Japanese patients was assessed.Methods and Results:ESUS patients were randomized to receive either dabigatran (150 or 110 mg twice daily) or aspirin (100 mg once daily). Of 5,390 patients randomized, 594 were Japanese. Most Japanese patients (99.8%) underwent brain magnetic resonance imaging for trial screening, compared to 76.8% of non-Japanese (P<0.0001). In the Japanese cohort, over a 19.4-month median follow-up period, recurrent stroke as the primary outcome occurred in 20/294 patients (4.3%/year) in the dabigatran group and 38/300 (8.3%/year) in the aspirin group (hazard ratio [HR], 0.55; 95% confidence interval [CI], 0.32-0.94). Major bleeding occurred in 12 patients (2.5%/year) and 17 patients (3.5%/year), respectively (HR, 0.72; 95% CI, 0.34-1.52). In contrast, in the non-Japanese cohort, recurrent stroke occurred in 4.1%/year and 4.3%/year, respectively, showing no apparent difference in recurrent stroke for dabigatran vs. aspirin (HR, 0.91; 95% CI, 0.74-1.14). The P-interaction for treatment and region did not reach statistical significance (P=0.09). CONCLUSIONS: Dabigatran was putatively associated with a lower relative risk of recurrent stroke compared with aspirin in Japanese ESUS patients.

Safety and tolerability of empagliflozin and linagliptin combination therapy in patients with type 2 diabetes mellitus: a pooled analysis of data from five randomized, controlled clinical trials.

OBJECTIVES: The fixed-dose combination of empagliflozin and linagliptin, two glucose-lowering drugs prescribed for type 2 diabetes mellitus, has demonstrated good tolerability in phase III clinical trials. To further evaluate the safety profile of this combination, the data from these trials were pooled and analyzed. METHODS: This was a post-hoc pooled analysis of five randomized, double-blind, clinical trials of the empagliflozin/linagliptin fixed-dose combination. Data for adverse events and laboratory parameters were evaluated. RESULTS: The analysis included 2895 patients: 1410, 1015, and 470 receiving the empagliflozin/linagliptin combination, empagliflozin monotherapy, and linagliptin monotherapy, respectively. Overall, the incidence of adverse events with the empagliflozin/linagliptin combination was similar to that with empagliflozin or linagliptin alone. Fewer than 2% of patients experienced hypoglycemia, and its incidence was similar across treatment groups. Genital infections occurred in more patients receiving empagliflozin/linagliptin (3.0%) or empagliflozin monotherapy (5.1%) than in those receiving linagliptin monotherapy (1.9%). No cases of Fournier's gangrene, diabetic ketoacidosis, or pemphigoid occurred, and no clinically relevant mean changes in laboratory parameters were noted. CONCLUSION: The safety profile of the fixed-dose combination of empagliflozin and linagliptin was similar to the individual monotherapies. No new safety signals were identified.

Dabigatran for Japanese Patients with Atrial Fibrillation and Prior Stroke: A Subgroup Analysis of the J-Dabigatran Surveillance Program.

BACKGROUND: The study objective was to evaluate long-term safety and effectiveness of dabigatran 110 mg and 150 mg twice daily (bid) in patients with nonvalvular atrial fibrillation (NVAF) with a focus on secondary stroke prevention. METHODS: In J-Dabigatran Surveillance, 6772 patients newly initiated on dabigatran to prevent ischemic stroke and systemic embolism were enrolled in Japan (1042 sites, December 2011 to November 2013). This subgroup analysis included patients with (1302) and without (5071) previous stroke/transient ischemic attack (TIA). Case report forms were reviewed to determine incidence of outcome events. RESULTS: In patients with previous stroke/TIA, the incidence rate for recurrent stroke/TIA was 2.48/100 patient-years (ischemic stroke 2.22, hemorrhagic stroke 0.18, TIA 0.12) and for major bleeding was 1.79/100 patient-years, including intracranial bleeding (0.55). Event rates for recurrent stroke/TIA or major bleeding were 1.2% (for both) for patients who started dabigatran less than 30 days after stroke onset and 0.3% (for both) for patients who started dabigatran more than or equal to 30 days after stroke onset, and were independent of dabigatran dose. For patients with previous stroke/TIA, 17% who received 110 mg bid did not meet dose reduction recommendations (DRRs) and 28% who received 150 mg bid met at least 1 DRR, but the dabigatran dose was not reduced. Use of DRRs did not have a major impact on the incidence rates of recurrent stroke/TIA and major bleeding. CONCLUSION: Findings from this subgroup analysis support the real-world safety and effectiveness of long-term dabigatran in Japan, particularly for patients with NVAF in secondary prevention settings.

Skeleton construction upon local regression of the sponge body.

We previously revealed that the mechanism of demosponge skeleton construction is self-organization by multiple rounds of sequential mechanical reactions of player cells. In these reactions, "transport cells" dynamically carry fine skeletal elements (spicules) on epithelia surrounding the inner body space of sponges (basal epithelium (basopinacoderm) and the endodermal epithelium (ENCM)). Once spicules pierce ENCM and apical pinacoderm, subsequently they are cemented to the substratum under the sponge body, or connected to other skeleton-constructing spicules. Thus, the "pierce" step is the key to holding up spicules in the temporary periphery of growing sponges' bodies. Since sponges can regress as well as grow, here we asked how skeleton construction occurs during local regression of the body. We found that prior to local basopinacoderm retraction (and thus body regression), the body became thinner. Some spicules that were originally carried outward stagnated for a while, and were then carried inwards either on ENCM or basopinacoderm. Spicules that were carried inwards on ENCM pierced epithelia after a short transport, and thus became held up at relatively inward positions compared to spicules carried on outwardly extending basopinacoderm. The switch of epithelia on which transport cells migrate efficiently occurred in thinner body spaces where basopinacoderm and ENCM became close to each other. Thus, the mechanisms underlying this phenomenon are rather mechanical: the combination of sequential reactions of skeleton construction and the narrowed body space upon local retraction of basopinacoderm cause spicules to be held up at more-inward positions, which might strengthen the basopinacoderm's attachment to substratum.

System level analysis of motor-related neural activities in larval Drosophila.

The way in which the central nervous system (CNS) governs animal movement is complex and difficult to solve solely by the analyses of muscle movement patterns. We tackle this problem by observing the activity of a large population of neurons in the CNS of larval Drosophila. We focused on two major behaviors of the larvae - forward and backward locomotion - and analyzed the neuronal activity related to these behaviors during the fictive locomotion that occurs spontaneously in the isolated CNS. We expressed a genetically-encoded calcium indicator, GCaMP and a nuclear marker in all neurons and then used digitally scanned light-sheet microscopy to record (at a fast frame rate) neural activities in the entire ventral nerve cord (VNC). We developed image processing tools that automatically detected the cell position based on the nuclear staining and allocate the activity signals to each detected cell. We also applied a machine learning-based method that we recently developed to assign motor status in each time frame. Our experimental procedures and computational pipeline enabled systematic identification of neurons that showed characteristic motor activities in larval Drosophila. We found cells whose activity was biased toward forward locomotion and others biased toward backward locomotion. In particular, we identified neurons near the boundary of the subesophageal zone (SEZ) and thoracic neuromeres, which were strongly active during an early phase of backward but not forward fictive locomotion.

WITHDRAWN: Effectiveness and safety of long-term dabigatran among patients with non-valvular atrial fibrillation in clinical practice: J-Dabigatran Surveillance.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Journal of Cardiology, 73/6 (2018) 507­514, https://doi.org/10.1016/j.jjcc.2018.12.013. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal

Characteristics of Japanese Patients with Nonvalvular Atrial Fibrillation on Anticoagulant Treatment: A Descriptive Analysis of J-dabigatran Surveillance and JAPAF Study.

INTRODUCTION: Following approval of dabigatran and other antithrombotics in Japan, few studies have specifically evaluated the clinical characteristics of patients prescribed these antithrombotics for nonvalvular atrial fibrillation (NVAF) in real-world practice. METHODS: We conducted a descriptive analysis of data from two real-world studies [J-dabigatran surveillance and Japanese study on current Anticoagulation therapies for Patients with nonvalvular Atrial Fibrillation (JAPAF); conducted at sites common to both studies] to determine the characteristics of patients with NVAF initiated on dabigatran etexilate [110 mg twice daily (BID; DE110) or 150 mg BID (DE150)], warfarin, rivaroxaban, or antiplatelets as their first antithrombotic treatment. Inferential statistical analyses were not performed, and no statistical hypothesis was tested. RESULTS: Data for 1270 and 3011 eligible patients from the J-dabigatran surveillance (DE110, 976; DE150, 273) and JAPAF study (warfarin, 82.5%; rivaroxaban, 10.3%; antiplatelets, 21%), respectively, were extracted. In the J-dabigatran surveillance, 31.8% (full cohort, 28.1%) of patients had been switched from warfarin to dabigatran. Among patients prescribed DE110/DE150, 41.4%/57.5% and 41.5%/18.7% of patients had low-to-intermediate risk for ischemic stroke (CHADS2 score of 0 or 1) and high risk for bleeding (HAS-BLED score ≥ 3), respectively. Similarly, 33.7%/41.3%/40.2% and 48.7%/42.6%/75.7% of patients taking warfarin/rivaroxaban/antiplatelets had a CHADS2 score of 0 or 1 and HAS-BLED score ≥ 3, respectively. Dabigatran was favored in patients with creatinine clearance > 50 ml/min. CONCLUSIONS: In Japan, physicians who attempt stroke prevention in patients with atrial fibrillation choose appropriate anticoagulant treatment, taking into consideration the individual patient backgrounds as well as the features of each antithrombotic agent. TRIAL REGISTRATION: ClinicalTrials.gov Identifier, NCT01491178 and University Hospital Medical Information Network (UMIN) Clinical Trial Registry Identifier, UMIN000009644. FUNDING: Nippon Boehringer Ingelheim Co., Ltd. Plain language summary available for this article.

Effectiveness and safety of long-term dabigatran among patients with non-valvular atrial fibrillation in clinical practice: J-dabigatran surveillance.

BACKGROUND: The effectiveness and safety of dabigatran etexilate (DE) have not been elucidated thoroughly in clinical practice for Japanese patients with non-valvular atrial fibrillation (NVAF). In particular, those of DE at a reduced dose due to dose reduction recommendations (DRR) remain unknown. METHODS: NVAF patients who had newly initiated DE treatment for prevention of thromboembolic events between December 2011 and November 2013 were enrolled. They were followed until August 2016. Outcome events included thromboembolism, major bleeding, and all-cause death. RESULTS: The study group consisted of 6443 patients (mean age, 70.9 years; male, 66.9%; and mean CHADS2 score, 1.8). During a follow-up period of 610 days (median), stroke, transient ischemic attack (TIA), and systemic embolism (SE) occurred at 1.4%/year for DE 110mg twice daily (BID) (DE220, n=4759) and 0.8% for dabigatran 150mg BID (DE300, n=1571, unadjusted p=0.0317). Major bleeding occurred at 1.3 and 0.6%/year for DE220 and DE300, respectively (unadjusted p=0.0097). All-cause death occurred at 1.5 and 0.5%/year for DE220 and DE300, respectively (unadjusted p=0.0005). When patients were divided into four groups based on DRR and DE doses (DE300 groups meeting and not meeting DRR, and DE220 groups meeting and not meeting DRR), incidence rates of stroke/TIA/SE and major bleeding differed among the four groups (unadjusted p=0.0026 and 0.0194 for trend, respectively); DE220 group meeting DRR had the highest rates (1.7% and 1.4%/year, respectively). However, in multivariate analysis, no differences between doses were observed regarding any outcomes. CONCLUSIONS: The present results are indicative of the favorable benefit-risk profile of dabigatran in Japanese clinical practice. Dabigatran dose was not independently associated with thromboembolic and bleeding events in Japanese NVAF patients.

Preferential prescribing of linagliptin in type 2 diabetes patients in an expanded post-marketing surveillance study in Japan.

AIMS/INTRODUCTION: To evaluate linagliptin prescribing in type 2 diabetes mellitus patients with different comorbidities, an expanded Japanese post-marketing surveillance also collected baseline data for patients initiating other glucose-lowering drugs. MATERIALS AND METHODS: Patients initiating linagliptin monotherapy were enrolled, then the next patient starting monotherapy with another glucose-lowering drug was enrolled (2012-2014). Baseline data were collected and analyzed by the Medical Dictionary for Regulatory Activities system organ class. Analyses were descriptive, and meaningful differences defined as absolute standardized difference >10%. RESULTS: Over 4,200 type 2 diabetes mellitus patients were enrolled. Most system-organ class comorbidities were more common in patients initiating linagliptin versus other glucose-lowering drugs, with meaningful differences observed for metabolism/nutritional (50.5 vs 45.5%, respectively), cardiac (12.2 vs 8.6%, respectively), vascular (56.4 vs 51.3%, respectively) and renal/urinary disorders (9.9 vs 5.7%, respectively). CONCLUSIONS: Expanding the linagliptin Japanese post-marketing surveillance revealed linagliptin prescribing to a type 2 diabetes mellitus population with more comorbidities versus other glucose-lowering drugs. Although such preferential prescribing might be expected, as linagliptin requires no dose adjustment or monitoring in renally or hepatically impaired patients, this innovative post-marketing surveillance approach generated important evidence that could only be shown in such a non-randomized comparative study. These data generated insights important for the design and interpretation of observational studies and spontaneous reports, which are key for public health.

Influence of cellular shape on sliding behavior of ciliates.

Some types of ciliates accumulate on solid/fluid interfaces. This behavior is advantageous to survival in nature due to the presence of sufficient nutrition and stable environments. Recently, the accumulating mechanisms of Tetrahymena pyriformis at the interface were investigated. The synergy of the ellipsoidal shape of the cell body and the mechanosensing feature of the cilia allow for cells to slide on interfaces, and the sliding behavior leads to cell accumulation on the interfaces. Here, to examine the generality of the sliding behavior of ciliates, we characterized the behavior of Paramecium caudatum, which is a commonly studied ciliate. Our experimental and numerical results confirmed that P. caudatum also slid on the solid/fluid interface by using the same mechanism as T. pyriformis. In addition, we evaluated the effects of cellular ellipticity on their behaviors near the wall with a phase diagram produced via numerical simulation.

Rotation of stress fibers as a single wheel in migrating fish keratocytes.

Crawling migration plays an essential role in a variety of biological phenomena, including development, wound healing, and immune system function. Keratocytes are wound-healing cells in fish skin. Expansion of the leading edge of keratocytes and retraction of the rear are respectively induced by actin polymerization and contraction of stress fibers in the same way as for other cell types. Interestingly, stress fibers in keratocytes align almost perpendicular to the migration-direction. It seems that in order to efficiently retract the rear, it is better that the stress fibers align parallel to it. From the unique alignment of stress fibers in keratocytes, we speculated that the stress fibers may play a role for migration other than the retraction. Here, we reveal that the stress fibers are stereoscopically arranged so as to surround the cytoplasm in the cell body; we directly show, in sequential three-dimensional recordings, their rolling motion during migration. Removal of the stress fibers decreased migration velocity and induced the collapse of the left-right balance of crawling migration. The rotation of these stress fibers plays the role of a "wheel" in crawling migration of keratocytes.

Simple mechanosense and response of cilia motion reveal the intrinsic habits of ciliates.

An important habit of ciliates, namely, their behavioral preference for walls, is revealed through experiments and hydrodynamic simulations. A simple mechanical response of individual ciliary beating (i.e., the beating is stalled by the cilium contacting a wall) can solely determine the sliding motion of the ciliate along the wall and result in a wall-preferring behavior. Considering ciliate ethology, this mechanosensing system is likely an advantage in the single cell's ability to locate nutrition. In other words, ciliates can skillfully use both the sliding motion to feed on a surface and the traveling motion in bulk water to locate new surfaces according to the single "swimming" mission.

Axially-confined in vivo single-cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes.

Green-to-red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave-length illumination with blue and red/near-infrared light. By modifying a confocal laser-scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide-spread use of this method. Here, we investigated whether spatially-restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half-width half-maximum for the axial dimension was below 5 µm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single-cell resolution.