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Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)—a master transcriptional regulator of lysosomal biogenesis and autophagy—is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.Nakamura et al. find that the master transcriptional regulator of lysosomal biogenesis and autophagy TFEB is activated following LC3 lipidation during lysosomal damage and show the importance of this mechanism during kidney injury.

Assessment of liver fibrosis stage is important in the management of patients with metabolic dysfunction-associated steatotic liver disease (MASLD). However, non-invasive methods to determine the changes in liver fibrosis stage are unknown. We investigated whether Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA+-M2BP), a serum fibrosis marker, can evaluate the changes in liver fibrosis stage. We observed the course of liver fibrosis stage and five serum fibrosis markers of 196 MASLD patients who had a paired biopsy performed. The changes in fibrosis markers, including WFA+-M2BP, were compared according to the changes in fibrosis stage. Factors associated with improvement of fibrosis stage were examined in a multivariate analysis. The changes in WFA+-M2BP (ΔWFA+-M2BP) were significantly correlated with changes in fibrosis stage (p < 0.01). The median of ΔWFA+-M2BP was −0.21, −0.08, −0.04, and 0.19 in 2 fibrosis stage regression group, 1 fibrosis stage regression group, fibrosis stage unchanged group, and fibrosis stage progression group, respectively (p < 0.01). However, other non-invasive markers did not reflect changes in fibrosis. ΔWFA+-M2BP was a significant factor for the regression of liver fibrosis stage in multivariate analysis (odds ratio: 3.54, 95% confidence interval: 1.55–8.12, p < 0.01). Time-course changes in WFA+-M2BP levels indicate the changes in liver fibrosis.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

Current smartphones equipped with high-sensitivity and high-resolution sensors in the camera can respond to the needs of low-light imaging, streaming acquisition, targets of various scales, etc. Therefore, a smartphone has great potential as an imaging device even in the scientific field and has already been introduced into biomolecular imaging using fluorescence tags. However, owing to the necessity of an excitation light source, fluorescence methods impair its mobility. Bioluminescence does not require illumination; therefore, imaging with a smartphone camera is compact and requires minimal devices, thus making it suitable for personal and portable imaging devices. Here, we report smartphone-based methods to observe biological targets in various scales using bioluminescence. In particular, we demonstrate, for the first time, that bioluminescence can be observed in an organelle in a single living cell using a smartphone camera by attaching a detachable objective lens. Through capturing color changes with the camera, changes in the amount of target molecules was detected using bioluminescent indicators. The combination of bioluminescence and a mobile phone makes possible a compact imaging system without an external light source and expands the potential of portable devices.

Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca 2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

Background and aimDouble-balloon enteroscopy (DBE) performed to investigate overt small bowel bleeding can miss the source of bleeding. We investigated the clinical outcomes of patients with negative DBE results for suspected overt small bowel bleeding, which is defined in the current guidelines as obscure gastrointestinal bleeding.MethodsWe reviewed the prospectively collected medical records of patients who underwent DBE at our hospital between May 1, 2004 and April 30, 2016. During this period, 297 patients underwent DBE for suspected overt small bowel bleeding. The first DBE yielded negative results for 83 patients (27.9%). Written interviews, telephone interviews, and medical records of these patients were reviewed in April 2017. Follow-up data were collected for 63 patients (75.9%).ResultsDuring a mean follow-up period of 83.5 months, re-bleeding occurred in 21 of 63 patients (33.3%) after a mean of 23.0 months after the first DBE yielded negative results. The bleeding source was identified in 19 of 21 patients (90.5%). In 15 of these 19 patients (78.9%), the source was the small intestine. Among these 15 patients, 14 (93.3%) had bleeding sites within reach of the first DBE and 3 (20%) experienced their first incidence of re-bleeding more than 3 years after the first DBE. The need for transfusion for the first bleeding episode was a predictor of re-bleeding (odds ratio 7.5; 95% confidence interval 1.7–33.0).ConclusionsFalse-negative DBE results for overt small bowel bleeding are not rare, and the first re-bleeding episode can occur 3 years later. Repeat DBE when re-bleeding occurs should be considered, even if the first DBE results were negative.

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca 2+ , cyclic adenosine monophosphate and adenosine 5′-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants. Luminescent proteins are important tools for biomedical imaging but tend to emit fairly little light. Saito et al. . describe a brighter version of a bioluminescent protein that can visualize intracellular dynamics of various signalling molecules with high spatial and temporal resolution.

BackgroundDouble-balloon enteroscopy (DBE) is a safe and useful procedure for managing small bowel bleeding. However, there are limited studies regarding the preferable timing of DBE and its impact on long-term outcomes.AimWe aimed to evaluate the association between the timing of DBE and the long-term outcomes of patients suspected of having overt small bowel bleeding who underwent DBE.MethodsWe retrospectively reviewed a prospectively collected database of patients who underwent DBE procedures between May 2004 and April 2016. The electronic medical records were reviewed, and interviews were conducted via mail and telephone.ResultsOne-hundred sixty-five patients could be followed up. The bleeding source was detected during the initial DBE (DBE-positive group) for 102 patients. Sixty-three patients had no definite lesion during the initial DBE (DBE-negative group). Urgent DBE (DBE within 24 h after the last bleeding episode) was performed more often for the DBE-positive group (50/102; 49.0%) than for the DBE-negative group (10/63; 16.1%) (p < 0.0001). Nine patients in the DBE-positive group underwent curative surgery after diagnosis. Among the remaining DBE-positive patients, 38 of 93 (40.9%) had recurrent bleeding during 2675 days of follow-up. Twenty-one of 63 patients (33.3%) in the DBE-negative group had recurrent bleeding during 2490 days of follow-up. There was no significant difference between the two groups in terms of intervals without rebleeding (p = 0.17).ConclusionUrgent DBE at the initial bleeding episode was useful for detecting lesions. However, the rebleeding rate was not dependent on the initial DBE results.

Background Photosensitizing fluorescent proteins, which generate reactive oxygen species (ROS) upon light irradiation, are useful for spatiotemporal protein inactivation and cell ablation. They give us clues about protein function, intracellular signaling pathways and intercellular interactions. Since ROS generation of a photosensitizer is specifically controlled by certain excitation wavelengths, utilizing colour variants of photosensitizing protein would allow multi-spatiotemporal control of inactivation. To expand the colour palette of photosensitizing protein, here we developed SuperNova Green from its red predecessor, SuperNova. Results SuperNova Green is able to produce ROS spatiotemporally upon blue light irradiation. Based on protein characterization, SuperNova Green produces insignificant amounts of singlet oxygen and predominantly produces superoxide and its derivatives. We utilized SuperNova Green to specifically inactivate the pleckstrin homology domain of phospholipase C-δ1 and to ablate cancer cells in vitro. As a proof of concept for multi-spatiotemporal control of inactivation, we demonstrate that SuperNova Green can be used with its red variant, SuperNova, to perform independent protein inactivation or cell ablation studies in a spatiotemporal manner by selective light irradiation. Conclusion Development of SuperNova Green has expanded the photosensitizing protein toolbox to optogenetically control protein inactivation and cell ablation.

Pressureless sinter joining of bare Cu substrates using submicron Cu particles was successfully achieved at 250–300 °C in N 2 –3%H 2 forming gas atmosphere via the in-situ generation of Cu nanoparticles reduced from a thin oxide layer present on the surface of Cu particles. The joining strength of the Cu joints at 300 °C reached 26.2 MPa in the forming gas atmosphere, while it was 7.4 MPa under N 2 gas atmosphere. Thermogravimetry–differential thermal analysis and X-ray diffraction showed that the natural oxide layer (Cu 2 O) started to reduce to Cu at approximately 220 °C only under the N 2 –H 2 gas atmosphere. Cu sintering progressed rapidly above the reduction temperature, and the joining strength improved with increasing temperature. Transmission electron microscopy demonstrated the generation of Cu nanoparticles around the submicron Cu particles and the subsequent promotion of sintering behavior. These results suggest that reduction of the initially present oxide layer on submicron Cu particles is crucial for pressureless sinter joining. Based on the reduction and sintering behavior of submicron Cu particles, pressureless sinter joining was realized at 250 °C by increasing the holding time, and the joining strength was > 18 MPa.