Phylogenetic Separation of Holotrichia Species (Insecta, Coleoptera, Scarabaeidae) Exhibiting Circadian Rhythm and Circa'bi'dian Rhythm.

A unique two-day rhythm, circabidian rhythm, has been reported in the black chafer, Holotrichia parallela. However, it remains unknown how widely the circabidian rhythm appears in related species. We examined the activity rhythm and phylogeny of congeneric species inhabiting Japan to investigate the appearance of circabidian rhythms in a few subgenera of the genus Holotrichia. We found that Holotrichia picea also exhibited circabidian rhythm. In addition to the regular circabidian pattern, circabidian rhythms with day-switching or with a circadian activity component were also observed. In the day-switching pattern, H. picea switched appearance from odd to even days, or vice versa. In the circadian-like activity patterns, a major night activity and a minor dusk activity appeared alternately. Holotrichia kiotonensis, Holotrichia convexopyga, and Holotrichia loochooana loochooana exhibited a circadian rhythm. Two distinct clades, A and B, were recognized in the histone H3, cytochrome c oxidase subunit 1, and 16S ribosomal RNA phylogenetic trees. This phylogenetic separation was in accordance with the subgeneric classification based on external morphology in a previous study and with behavioral rhythm in the present study: clade A included Nigrotrichia group members, H. kiotonensis, H. convexopyga, H. loochooana loochooana, and H. loochooana okinawana, while clade B included Pedinotrichia group members, H. paralella and H. picea. We suggest that after separation into Nigrotrichia and Pedinotrichia, the behavioral trait of circabidian rhythm probably appeared once in an ancestral species of the Pedinotrichia group, including H. parallela and H. picea.

Ultrasonographic observation in combination with progesterone monitoring for detection of ovulation in Labrador Retrievers.

Although it is well known that the ovulation occurs during a period of time after LH surge in dogs, there are few reports of observing the entire process of development, ovulation and luteinization of each follicle. This study aimed to detect the ovulation kinetics by ultrasonography in combination with progesterone monitoring and therefore identify the time-range of the ovulation process in a dog. Daily transabdominal ultrasonography and progesterone monitoring were conducted for 24 natural oestrus cycles of Labrador Retrievers. Ovarian follicles were observed as anechoic structure with contours before ovulation. Ovulation (follicular collapse) was defined as when follicles became cloudy and contours obscure by transabdominal ultrasonography. Ultrasound imaging was capable of identifying the day of ovulation for 94.7% (178/188) of the follicles through the appearance of collapsed follicle or corpus luteum. Ovulation was observed between LH 0 (the day of LH surge) and LH 5, with 48.0%, 33.5% and 15.0% for LH 2, LH 3 and LH 1, respectively. The total number of ovulations on LH 2 and LH 3 accounted for 81.5% (141/173) of the total ovulation in 24 cycles examined. Ovulation occurred in 12 cycles for 2 d and for 3 d in 12 cycles. Seventeen cycles (70.8%) with multiple days of ovulation showed the largest number of ovulations on LH 2. The average follicle diameter 3 d before the LH surge was less than 5 mm, then exceeded 5 mm 2 d before the LH surge. The average follicle diameter at the time of ovulation (follicular collapse) was 6.1 ± 1.0 mm (n = 118). On the day before ovulation, the average diameters of the follicles ovulated on LH 1, LH 2 and LH 3 were 5.0 ± 0.7 mm, 5.8 ± 1.2 mm and 6.2 ± 1.3 mm, respectively. There was a significant difference in the follicle diameter between LH1 and LH2 (p < .001), LH2 and LH3 (p < .05), and LH1 and LH3 (p < .001). Suggesting that it is difficult to estimate the ovulation day based on follicle size. This study showed that combination of ultrasonography with progesterone monitoring could follow follicular development, ovulation and luteinization of the ovary in Labrador Retrievers. The direct visualization of the ovulation was achieved in a non-invasive, labour-friendly way. Furthermore, the time-range of the ovulation process was clarified in a dog. These results may contribute to an accurate understanding of the optimum timing of mating and improved breeding efficiency, including artificial insemination and embryo transfer for Labrador Retrievers.

Force Plate with Simple Mechanical Springs and Separated Noncontact Sensor Elements.

This paper reports on a force plate (FP) using mechanical springs and noncontact distance sensors. The ground reaction force (GRF) is one of the factors for clarify biomechanics, and FPs are widely used to measure it. The sensor elements of conventional FPs are mainly strain gauges. Thus, the mechanical properties of FP depend on the sensor element performance. If the FP performance must change, we must redesign the FP, including changing the sensor elements. Here, we proposed an FP that uses a measuring principle based on simple springs and noncontact sensors. The shape and performance of the proposed FP are expected to change easily. As a prototype device, we designed and fabricated an FP installed with 12 springs and four sensors for human walking. A planar coil and magnet were used as the sensor elements, and the sensor output was proportional to the vertical and horizontal displacements. The FP resonance frequency was 123 Hz, which was larger than the required specification. The calibration experiments showed that vertical and horizontal forces and moments could be measured independently. The FP's resolutions were 1.9 N and 1.4 N in the anterior-posterior and vertical directions, respectively. Furthermore, the fabricated FP measured GRF similarly to the commercial FP when a human walked on the plate. These results suggest that the proposed method will be helpful for FPs with custom-made requirements.

Image Processing for the Prevention of Infectious Diseases: Determination of Mask Wearing, Measurement of Hand Washing Time, and Disinfection Support System.

The new coronavirus infection (COVID-19) has spread to numerous countries around the world since several cases of the disease were first reported in late December 2019 in China. Currently, the WHO strongly recommends infection prevention measures such as wearing masks, hand washing, and frequent disinfection of high-touch surfaces, but there were many arguments against infection prevention policy in March 2020. For example, the WHO did not recommend the use of masks for the healthy general public. In Japan, wearing a mask was required before the habit of wearing a mask was established, which gave additional works of checking whether customer was wearing a mask to the employees. To reduce the workloads of employee and ensure mask-wearing, we started providing the mask-wearing system free of charge on March 5, 2020. We also developed hand washing time estimation and disinfection support system. It is useful to accumulate data on the status of implementation of countermeasures by our application, which leads to gain useful knowledge regarding countermeasures against COVID-19 as well as other infectious diseases. In this paper, we describe the development and introduction impacts of these systems in a pandemic emergencies. In addition, because of the security and privacy issues in running these image analysis applications, we discuss the delivery methods suitable for each service.

Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

Effect of quercetin on the motility of cryopreserved canine spermatozoa.

The addition of an antioxidant to cryopreservation solutions for preventing oxidative stress to sperm from several species, including that from humans, has been studied previously. Quercetin is a flavonoid contained in subarctic trees with freeze resistance and is known to be a strong antioxidant. Therefore, the effect of quercetin on the cryopreservation of dog spermatozoa was examined in this study. The proportions of total motile spermatozoa were significantly higher at 30, 60, 90, 120, and 150 min and at 60, 120, and 150 min after thawing in groups treated with 5 µg/ml and 10 µg/ml of quercetin dissolved in 0.1% DMSO added to the second extender based on skim milk compared to that in the control group, respectively. There were no differences between the experimental groups in the proportion of total motile spermatozoa during the observation periods. The proportion of total motile spermatozoa among those treated with 5 µg/ml of quercetin in 0.1% DMSO was improved by approximately 10-20% at 30-180 min after thawing compared to that in the control group. To evaluate the fertility of cryopreserved spermatozoa treated with quercetin, 2 × 108 spermatozoa were transcervically inseminated into bitches, and a total of 18 puppies were delivered in three bitches. These results indicated that supplementation of quercetin as a cryoprotectant to the skim milk-based extender improved the motility of cryopreserved spermatozoa from dogs compared to those of the control group. And fertility of cryopreserved spermatozoa with quercetin supplementation was proven with higher efficiency.

Identification and characterization of the S-layer formed on the sheath of Thiothrix nivea.

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphological feature of proteinaceous S-layers, was newly observed on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not observed in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) solution and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was observed in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.

Plausible link between circa'bi'dian activity rhythms and circadian clock systems in the large black chafer Holotrichia parallela.

Two-day rhythms, referred to as circa'bi'dian rhythms, have been reported in humans and mosquitos. However, these rhythms only appear under constant conditions, and the functional mechanisms of 2-day rhythms were unknown. Here, we report clear circabidian rhythms of large black chafers (Holotrichia parallela, Coleoptera: Scarabaeidae) in both the laboratory and field. Under 12 h:12 h light:dark (L:D) conditions at 25°C, H. parallela appeared on the ground at the beginning of the dark phase every 2 days. Under constant darkness, H. parallela exhibited free-running with a period of 47.9±0.3 h, suggesting the existence of a clear circabidian rhythm entrained to two 12 h:12 h L:D cycles. Phase responses of the circabidian rhythm to light pulses occurred under constant darkness in a phase-dependent manner. Phase responses suggest that there are two circadian cycles, each consisting of a less-responsive and more-responsive period, in a circabidian oscillation, and the circabidian rhythm is driven by the circadian clock. A mark-recapture study showed that beetles repeatedly appeared on the same tree approximately every 2 days. However, the periodicity was not as rigid as that observed under laboratory conditions in that individuals often switched appearance days. For instance, a large precipitation made the 2-day rhythm shift phase by half a cycle of the rhythm at a time. We propose a novel function of the circadian clock characterized by the release of an output signal every two cycles to produce the 2-day rhythm.

[Development of Magnetic Stimulator].

Transcranial magnetic stimulation (TMS) has been used in research into brain function. Recently, TMS has also become an option in treating brain dysfunction. The equipment consists of a coil and a driving circuit. The most widely used coil is a figure-8 coil because of its good resolution, and the most widely used circuit generates a biphasic pulse for repetitive stimulation. To assess the induced electric field distribution in the brain, we conducted simulations using a coil and constructed a brain model from a MR image. Finally, we introduce the coils we have developed for treatment in the home. These include an eccentric figure-8 coil and a dome-shaped coil. Reducing the size of the equipment is one of the chief technical challenges of using rTMS therapeutically in the home. An eccentric figure-8 coil can be driven with a lower current than a conventional figure-8 coil because of its good efficiency, which results in a smaller driving circuit. Conventional figure-8 coils stimulate a highly localized area, therefore making it difficult to ensure that a particular area receives stimulation without the assistance of an infrared camera support system, which is large and expensive. A dome-shaped coil is capable of stimulating a larger area and is robust against errors of positioning caused by the simple navigation system used in the home.

Elongation pattern and fine structure of the sheaths formed by Thiothrix nivea and Thiothrix fructosivorans.

Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans, are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [→4)-ß-d-GlcN-(1→4)-ß-d-Glc-(1→]n modified with unusual deoxy sugars. In an attempt to elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were determined in this study. Prior to analysis, both sheaths were confirmed to be highly de-N-acetylated. Sheaths in viable filaments were N-biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addition, the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chemically fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N-acetylation.

Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans.

Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1→4)-Glc-(1→]n with side chains of Rha4N-(1→3)-Fuc(1→ at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N-substitution. The sheath was hydrolyzed with concentrated HCl at 0°C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N-lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l-isomer by chiral HPLC analysis. To estimate the N-lactylation degree, the sheath was N-acetylated. N-Acetyl-Rha4N-Fuc-ABEE and N-lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40°C, GlcNAc-ABEE was obtained. For estimation of the N-acetylation degree, the sheath was N-acetylated with deuterated acetic anhydride and then N-acetyl-GlcN-ABEE was prepared. The content of deuterated N-acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D-(1)H NMR spectrum. It was concluded that Rha4N is mostly N-l-lactylated and GlcN is substoichiometrically N-acetylated.

Small geographic variation in photoperiodic entrainment of the circannual rhythm in the varied carpet beetle, Anthrenus verbasci.

The circannual pupation rhythm of Anthrenus verbasci is entrained to an environmental cycle by changes in photoperiod. Exposure of larvae reared under short-day conditions to long days induced a clear phase delay of the circannual rhythm. There was no notable difference in the initial phase or period of the circannual rhythm among four geographically distinct populations of A. verbasci in Japan: Takanabe (32.1°N), Osaka (34.7°N), Sendai (38.3°N), and Sapporo (43.1°N) populations. The range of photoperiodic changes effective for phase delay in the circannual pupation rhythm was compared among the four populations. Although larvae did not show a typical threshold response, but responded quantitatively to the photophase duration in intermediate conditions, the critical daylengths were calculated as those under which the pupation was delayed by 50%: 12.8 h in the Takanabe population, 13.2 h in the Osaka population, and 13.6 h in the Sendai and Sapporo populations. Thus, the critical daylength for entrainment of the circannual rhythm in A. verbasci was correlated to the habitat latitude, but the differences among the populations were much smaller than those reported in photoperiodism for induction of diapause in various insects. Consequently, the difference in the pupation time among the four geographic populations was very small under the natural photoperiod in Osaka at 20°C, and absent under the natural photoperiod and temperature in Osaka. These results suggest that A. verbasci survives and successfully produces the next generation in different geographic regions without changing the parameters of the circannual rhythm.

A spatial relationship between sheath elongation and cell proliferation in Sphaerotilus natans.

Sphaerotilus natans is a filamentous sheath-forming bacterium. A method of selective fluorescent-labeling of its sheath using conventional reagents was developed. Terminal expansion of the sheath was confirmed by this method. In addition, ubiquitous cell growth was revealed by sequential phase-contrast microscopy of a filament. Based on this and earlier reports, a model of the sheath formation is proposed.

Patterns of sheath elongation, cell proliferation, and manganese(II) oxidation in Leptothrix cholodnii.

Leptothrix cholodnii is a Mn(II)-oxidizing and sheath-forming member of the class ß-Proteobacteria. Its sheath is a microtube-like filament that contains a chain of cells. From a chemical perspective, the sheath can be described as a supermolecule composed of a cysteine-rich polymeric glycoconjugate, called thiopeptidoglycan. However, the mechanism that controls the increase in sheath length is unknown. In this study, we attempted to detect sheath elongation through microscopic examination by using conventional reagents. Selective fluorescent labeling of preexisting or newly formed regions of the sheath was accomplished using combinations of biotin-conjugated maleimide, propionate-conjugated maleimide, and a fluorescent antibiotin antibody. Epifluorescence microscopy indicated that the sheath elongates at the terminal regions. On the bases of this observation, we assumed that the newly secreted thiopeptidoglycan molecules are integrated into the preexisting sheath at its terminal ends. Successive phase-contrast microscopy revealed that all cells proliferate at nearly the same rate regardless of their positions within the sheath. Mn(II) oxidation in microcultures was also examined with respect to cultivation time. Results suggested that the deposition of Mn oxides is notable in the aged regions. The combined data reveal the spatiotemporal relationships among sheath elongation, cell proliferation, and Mn oxide deposition in L. cholodnii.

Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase.

A typical filamentous bacterium, Sphaerotilus natans, secretes a thiolic glycoconjugate which is assembled into a microtube, so called sheath. The glycoconjugate is known to consist of a pentasaccharide-dipeptide repeating unit, but its chemical structure has not been completely elucidated. In order to determine its chemical structure, the sheath was broken down by performic acid oxidation. The released sulfonated derivative was water soluble which was suitable for detailed NMR analysis. The data exhibited the presence of two stoichiometric and one substoichiometric (relative abundance was about 0.5) acetylations, suggesting that the glycoconjugate is composed of two equimolar pentasaccharide-dipeptide repeating units each having either two or three acetyl groups. However, the position of substoichiometric acetylation could not be defined. To determine the position, the sheath was derivatized with a thiol selective fluorescent reagent followed by digestion with a specific polysaccharide lyase prepared from a sheath-degrading bacterium, Paenibacillus koleovorans. As expected, two fluorescent digests were recovered by reverse-phase HPLC and were subjected to NMR analysis. The data revealed that both digests are pentasaccharide-dipeptides which have unsaturated glucuronic acid and galactosamine residues at their reducing and non-reducing ends, respectively. It was also confirmed that one digest has 3-O-acetylated glucose residue while the other has non-derivatized glucose residue. The substoichiometric acetylation was thus identified with the 3-O-acetylation, and structural determination of the thiolic glycoconjugate was completed. By virtue of the clarification of the two digests' structures, the cleavage site was specified as (1→4)-α-galactosaminic bond to glucuronic acid. Based on the present and earlier findings, we propose a novel glycoconjugate category named thiopeptidoglycan and a novel polysaccharide lyase named thiopeptidoglycan lyase.