The bacterial peptidoglycan layer plays an important role in protecting the bacteria from turgor pressure, viruses, and predators. However, it also acts as a barrier in transmitting forces generated on the cell membrane to adhesion proteins on the surface during gliding locomotion. In this study, peptidoglycan layers were isolated from two species of gliding diderm, i.e., gram-negative bacteria, and their structures were visualized by quick-freeze deep-etch replica electron microscopy. The horizontal bonding of peptidoglycan did not differ obviously among the three species. However, the diameter of pores in the peptidoglycan layer of M. xanthus and the area of surface pores were 51 nm and 14.6%, respectively, which were significantly larger than those of E. coli (32 nm and 5.8%) and F. johnsoniae (29 nm and 7.0%). Based on this, we discussed the mechanism by which diderm bacteria transmit forces across the PG layer to the bacterial surface.

Chrimson is cation-conducting channelrhodopsin (CCR) with the most red-shifted absorption spectrum, rendering itself as one of the most promising optogenetic tools. However, the molecular mechanisms underlying its red-shifted absorption have not been completely clarified yet. Here, we found a CCR gene showing high sequence similarity to Chrimson from Lake Hula through freshwater metatranscriptome sampling. Interestingly, despite its high similarity to Chrimson, this CCR—named HulaChrimson—showed significantly blue-shifted action and absorption spectra compared to those of Chrimson. Mutations of amino acid residues, which are prominently different from those in Chrimson, in HulaChrimson did not reproduce the red-shifted absorption of Chrimson, suggesting the color-tuning between these proteins achieved by organizing the entire protein architecture, particularly in the broad hydrogen bonding network around the retinal Schiff base counterion, rather than by the difference in several specific residues. The optical characteristics of HulaChrimson distinct from those of Chrimson provide a basis for understanding the color-tuning mechanisms of channelrhodopsins.

A time-resolved small-angle X-ray scattering (SAXS) system for protein solution samples using an X-ray free-electron laser (XFEL) was established by developing a SAXS diffractometer by integrating a helium path into the DAPHNIS system initially designed for Serial Femtosecond Crystallography (SFX) experiments at BL2 of SACLA. This modification enabled us to successfully capture the SAXS profiles of ovalbumin under conditions without any reaction trigger, using both the newly developed system and the sample solution flow device that was originally designed for SFX experiments. Furthermore, we conducted acid denaturation experiments on cytochrome c, using a T-junction-type solution mixing flow system, and observed the denaturation-induced changes in the SAXS profiles.

Single epidermal keratocytes, which are responsible for wound repair in fish, migrate while maintaining their characteristic shape: a frontal crescent-shaped lamellipodium and a posterior rugby-ball-shaped cell body. These cells are widely used in cell migration studies, especially to examine the role of actin polymerization at the leading edge of the cell. In the posterior part of the cell, stress fibers, which are bundles of actomyosin, are aligned along the seam of the ‘rugby ball.’ The ball rotates with the stress fibers during migration. The linear contraction of stress fibers appears to drive the rotation of the cell body. This review describes a conversion mechanism from linear motion to rotation driven by stress fiber contraction and soft cell body deformation, which is not found in man-made machines. We also describe a unique research method that is able to demonstrate this machinery by creating robot models. Due to their high migration rate and ease of culturing, fish keratocytes appear to be one of the best materials for studying both single cell and collective cell migration. In this review, we will also give some recent research examples of collective migration using keratocytes.

The malignancies of prostate tumour cells are assessed by pathologists as grade groups (GGs) from 1 (least aggressive) to 5 (most aggressive) on histopathology images. GGs are associated with the degree of tumour cell differentiation and may have different self-similarities depending on GG and tumour-related cell types, which are neoplastic epithelial, inflammatory, connective tissue, necrotic, and non-neoplastic epithelial cells. We investigated the associations between GGs and fractal dimensions (FDs) for five types of prostate tumour-related cells using a multiple-threshold box counting algorithm (MTBC). We showed the association of FDs of 9 channel images (eosin, hematoxylin, normalised images for red, green, and blue colour channels) with multiple threshold values on histopathology images (patch images) and the feasibility of FD-threshold images in an artificial intelligence model to classify patients into low (GG≤3) and high (GG≥4) GGs. We constructed FD-threshold images based on MTBC algorithm for characterizing prostate tumour cells. A shallow-convolutional neural network (sCNN) model to classify patients into low and high GGs was trained with input data of the FD-threshold images for all 9 channels and evaluated using the area under receiver operating characteristic curve (AUC). There were statistically significant correlations between the FD of non-neoplastic epithelial cells and GG [Pearson correlation coefficient=-0.849, p=0.001]. Significant correlations also existed for connective tissue and the original images. The AUC for the sCNN classification model into high and low GGs was 0.811. FD can characterise physical properties of prostate tumour-related cells for low and high GGs.

Quality assessment and characterization of liquid milk by means of electronic sensor remains an intensive area of research. In this work, cylinder-in-cylinder type sample holder is fabricated to measure the bioimpedance of milk as a function of frequency. Experiments on bioimpedance spectroscopy were conducted during adulteration (at control temperature, 23°C) of branded (pasteurized) milk as well as raw milk. Cole equivalent circuit is considered as a characterizing model for milk. Cole parameters were extracted from the experimental data. From Cole parameters, relaxation-time was estimated and state of the milk sample has been expressed in terms of relaxation-time. Analysis of variance was performed on relaxation-time of the samples to gain statistical significance. Our method is capable to discriminate liquid milk from different commercial brands. It was found that the relaxation-time decreases monotonically with the progression of adulteration time for all kinds of milk considered in this study. From the changes in relaxation-time, the adulteration was found to be significant in the first three hours. Hence it is not advisable to consume milk after two hours of adulteration if kept at 23°C. Dominant biochemical pathways responsible for adulteration of milk are also presented.

Enzyme function is often regulated by weak metal-ion binding, which results from conformational changes while maintaining conformational fluctuations. We analyzed the structure and function of cutinase-like enzyme, Cut190, using biophysical methods such as X-ray crystallography and molecular dynamics (MD) simulations, showing that its structure and function are finely regulated by weak Ca2+ binding and release. We succeeded to stabilize the enzyme by introducing a disulfide-bond which can degrade polyethylene terephthalate (PET) to PET monomers at the glass transition temperature of PET, ≈70°C. In this study, using the stabilized Cut190 mutants, Cut190**SS and Cut190**SS_F77L, we evaluated the requirement of Ca2+ for catalytic activity at 70°C, showing that the enzyme expressed the activity even in the absence of Ca2+, in contrast to that at 37°C. These results were supported by multicanonical MD analysis, which showed that the respective forms of the enzyme, such as closed, open, and engaged forms, were exchangeable, possibly because the potential energy barriers between the respective forms were lowered. Taken together, the conformational equilibrium to express the catalytic activity was regulated by weak Ca2+ binding at 37°C, and was also regulated by increasing temperature. The respective conformational states of Cut190**SS and Cut190**SS_F77L correlated well with their different catalytic activities for PET.