Fluorescence Imaging Using Enzyme-Activatable Probes for Detecting Diabetic Kidney Disease and Glomerular Diseases

A clear identification of the etiology of glomerular disease is essential in patients with diabetes. Renal biopsy is the gold standard for assessing the underlying nephrotic pathology; however, it has the risk for potential complications. Here, we aimed to investigate the feasibility of urinary fluorescence imaging using an enzyme-activatable probe for differentiating diabetic kidney disease and the other glomerular diseases. Hydroxymethyl rhodamine green (HMRG)-based fluorescent probes targeting gamma-glutamyl transpeptidase (GGT) and dipeptidyl-peptidase (DPP) were used. Urinary fluorescence was compared between groups which were classified by their histopathological diagnoses (diabetic kidney disease, glomerulonephritis, and nephrosclerosis) as obtained by ultrasound-guided renal biopsy. Urinary fluorescence was significantly stronger in patients with diabetic kidney disease compared to those with glomerulonephritis/nephrosclerosis after DPP-HMRG, whereas it was stronger in patients with nephrosclerosis than in patients with glomerulonephritis after GGT-HMRG. Subgroup analyses of the fluorescence performed for patients with diabetes showed consistent results. Urinary fluorescence imaging using enzyme-activatable fluorescence probes thus represents a potential noninvasive assessment technique for kidney diseases in patients with diabetes.     

Numerical experiment of measurement-integrated simulation to reproduce turbulent flows with feedback loop to dynamically compensate the solution using real flow information

Reproduction of the exact structure of real turbulent flows is crucial in many applications. Four Dimensional variation (4D-VAR) is widely used in numerical weather forecasting, but it requires huge computational power to repeatedly solve flow dynamics and its adjoint, and, therefore, is not suitable to apply to problems of real-time flow reproduction such as feedback flow control. Kalman filter and observer, in which numerical solution converges to the real state asymptotically by means of the feedback signal proportional to the difference between the calculated state and the real state, requiring much less computational load than the variational method, are potential candidates to solve the problem. By comparing Kalman filter and observer, the latter has simpler structure retaining essential part of the state estimation. This study deals with a special type of observer, or measurement-integrated simulation (MI simulation), in which a SIMPLER-based flow solver is used as the mathematical model of the system in place of approximate small dimensional linear differential equations usually used in observers. Reproduction of the exact structure of a turbulent flow was investigated by a MI simulation. A numerical experiment was performed for a fully developed turbulent flow in a pipe with a square cross section. The MI simulation was performed with the feedback from the standard solution in the flow domain for the cases using: (1) all velocity components at all grid points, (2) partial velocity components at all grid points, or (3) all velocity components at partial grid points. Convergence of the MI simulation to the standard solution was investigated using the steady error norm for the convergent state and the time constant for the transient state. The result of the MI simulation using all the velocity information exponentially converges to the standard solution with a steady state error reduced from that of the ordinary simulation in a range of the feedback gain. Decreasing the feedback gain reduces the effect of feedback, and a feedback gain which is too large destabilizes the closed loop system, resulting in large error. The time constant decreases almost inversely proportional to the feedback gain as long as the feedback system is stable. For the MI simulation with the feedback using limited information, feedback using two velocity components by omitting one transverse velocity component showed a good result, although the other results were not satisfactory. For the MI simulation with the feedback using limited grid points, the result of the MI simulation applying the feedback at the grid points on every 20th plane in the x₁ direction was almost the same as that using all grid points at some feedback gain, while the result with the feedback on the planes skipped in the x₂ direction requires 10 times more planes to achieve the same reduction rate.     

Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.     

KOMPEITO,an Atypical Arabidopsis Rhomboid-Related Gene,Is Required for Callose Accumulation and Pollen Wall Development

Fertilization is a key event for sexually reproducing plants. Pollen–stigma adhesion, which is the first step in male–female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a β-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen–stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.     

Strain Anisotropy Driven Spontaneous Formation of Nanoscrolls from 2D Janus Layers

2D Janus transition metal dichalcogenides (TMDs) have attracted attention due to their emergent properties arising from broken mirror symmetry and self-driven polarization fields. While it has been proposed that their vdW superlattices hold the key to achieving superior properties in piezoelectricity and photovoltaic, available synthesis has ultimately limited their realization. Here, the first packed vdW nanoscrolls made from Janus TMDs through a simple one-drop solution technique are reported. The results, including ab initio simulations, show that the Bohr radius difference between the top sulfur and the bottom selenium atoms within Janus ${\mathrm{M}}_{\mathrm{Se}}^{\mathrm{S}}$ (M = Mo, W) results in a permanent compressive surface strain that acts as a nanoscroll formation catalyst after small liquid interaction. Unlike classical 2D layers, the surface strain in Janus TMDs can be engineered from compressive to tensile by placing larger Bohr radius atoms on top (${\mathrm{M}}_{\mathrm{S}}^{\mathrm{Se}})$to yield inverted C scrolls. Detailed microscopy studies offer the first insights into their morphology and readily formed Moiré lattices. In contrast, spectroscopy and FETs studies establish their excitonic and device properties and highlight significant differences compared to 2D flat Janus TMDs. These results introduce the first polar Janus TMD nanoscrolls and introduce inherent strain-driven scrolling dynamics as a catalyst to create superlattices.     

Evaluation of the contents and ratios of five fractionated proteins to elucidate the protein composition in soybean seeds

Selecting suitable soybean cultivars is important for food processing and exploring their endowment with desirable physiological functions. We applied the fractionation method previously established to compare soy protein composition among cultivars to promote such a selection. More than 95% of the proteins in soybean cotyledons were extracted from 13 soybean cultivars using a high-concentration salt solution. The extracted proteins were fractionated into five fractions, namely oil body-associated protein (OBAP), polar lipid-associated protein (PLAP), globulins (11S and 7S), and whey by centrifugation after tuning the solubility behavior of the proteins with various solutions. Protein species in each fraction were analyzed by SDS-PAGE. Protein content in the total extract and five fractions was quantified to characterize the protein composition of soybean cultivars. The correlation between the protein content of each fraction and the total protein in cotyledon was investigated. A strong positive correlation was found only for the 11S fraction (r = 0.82), followed by a positive correlation in the 7S fraction (r = 0.65). Thus, we surmised that the increased protein content in soybean was due to increased globulin content. Furthermore, the calculation of the average ratio of protein content in each fraction indicated the globulin fraction (7S and 11S) to be 52%, the lipophilic protein fraction (OBAP and PLAP) to be 33%, and the whey fraction to be 13%. The preparation method employed in this study is a promising tool for efficiently comparing the protein composition of soybean cultivars to evaluate the potential use of cultivars for food production.     

Elucidation of mosaic patterns in gravel riverbeds using classifying flow velocity regimes obtained from a planar two-dimensional analysis

Gravel riverbeds in the middle reaches of Japanese rivers are essential habitats for various plants and animals. Disturbance from flooding is necessary for the formation of gravel riverbeds, but human control of rivers, such as dams and channelization, has altered flow and sediment regimes, thereby reducing disturbance. The flooding generates a mosaic pattern characterized by varying frequencies and intensities of disturbance in gravel riverbeds. Understanding the disturbance regimes that form mosaic patterns is important for the conservation of biodiversity in rivers. In this study, we proposed a method to extract mosaic patterns from flow velocity regimes obtained by planar two-dimensional analysis by classifying them with time-series clustering. Based on the distribution of Anaphalis margaritacea var. yedoensis on gravel riverbanks, we compared several past flooding events to identify mosaic patterns that are important for A. margaritacea var. yedoensis. The study site is the Echi River, which flows through Shiga Prefecture in Japan and into Lake Biwa. Using a unmanned aerial vehicle (UAV), orthomosaic images with an average ground resolution of 3.3 mm/pixel were created, and colony polygons of A. margaritacea var. yedoensis were created using image detection and visual correction. Hydraulic analysis was performed using iRIC ver2.3 (Nays2DH ver1.0). Time-series clustering was used to classify the flow velocity regimes for each computed mesh into 30 clusters. The relationship between the clusters of each flooding event and the distribution of A. margaritacea var. yedoensis was evaluated. Mosaic patterns were created by classifying the flow velocity regimes of each computational mesh calculated by planar 2D analysis into clusters using time-series clustering. After analyzing the relationship between each cluster and the area of distribution of A. margaritacea var. yedoensis, the first flooding event was determined to be the mosaic pattern that best explained the distribution of A. margaritacea var. yedoensis. Cluster 1, the “low peak, short duration type,” was considered the growth center of A. margaritacea var. yedoensis. The method used in this study is an innovative approach for obtaining mosaic patterns that quantifies these five elements of the disturbance regime.