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Purpose of Investigation: When carbonyl reductase 1 (CR1) is highly expressed in human ovarian cancer cells in vivo, tumor growth is reported to be inhibited. Conversely, when expression of CR1 decreases, tumor growth, invasion, and metastasis are reported to increase. Thus, the aim of the current study was to examine dynamic changes in ovarian cancer cells under different CR1 expression levels in artificial human peritoneal tissue (AHPT). Materials and Methods: Serous ovarian cancer cells with different levels of CR1 expression were produced by transfection of HRA human ovarian carcinoma cells with CR1 DNA or CR1 siRNA. The transfected cells were seeded in AHPT and observed over time until peritoneal development of carcinomatosis. Apoptotic cells in the AHPT were compared using TUNEL staining and fluorescence-based flow cytometry. Results: Cells transfected with CR1 DNA or CR1 siRNA did not differ from control cells in terms of their adherence to the mesothelium. After 24 hours, when cells had invaded the tissue below the mesothelium, proliferation of CR1-overexpressing cells was inhibited while proliferation of CR1-suppressing cells increased. At 72 hours, CR1-suppressing cells had invaded the stroma. CR1-overexpressing cells had a markedly higher rate of apoptosis than control or CR1-suppressing cells. Moreover, electron microscopy revealed apoptotic bodies in cells overexpressing CR1. Differences in tumor growth depending on the extent of CR1 expression have been noted in vivo, and similar results were obtained in the present in vitro model of AHPT. High and low levels of CR1 expression did not affect cell adherence to the mesothelium, but low levels did result in cells invading and proliferating below the mesothelium. Conclusion: The present results have also demonstrated that tumor inhibition by CR1 involves an increase in apoptosis. [ABSTRACT FROM AUTHOR]

Fibroin light chain (L-chain) mRNA (mol. wt 4.0×10 daltons) was purified from the posterior silk gland of the silkworm, Bombyx mori (J-131 strain). Double-stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development. [ABSTRACT FROM AUTHOR]

YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to investigate the impact of occupational pushing and pulling combined with improper working posture on work-related low back pain (LBP) among workers. A web-based survey was conducted in 2022 to collect data from 15,623 workers, who were categorized into proper and improper working posture groups. Multiple logistic regression analysis was used to analyze the association between pushing and pulling loads and LBP in each group. In the proper working posture group, the odds ratios (ORs) of LBP for workers who pushed and pulled were not significantly different compared with those of no-handling workers. However, in the improper working posture group, the ORs of LBP were significantly greater among workers who pushed and pulled compared with those of no-handling workers, and this association became stronger with increasing weights. Therefore, improper working posture combined with pushing and pulling were strongly associated with LBP among workers, particularly with heavier weights.

Objectives: Manual rolling of heavy objects remains in the workplace. The Health and Safety Executive (HSE) in the United Kingdom recommends load weights of <400 kg in the rolling task. However, the association of rolling weights <400 kg with work-related low back pain (LBP) has not been sufficiently investigated. This study examined the effect of rolling loads weighing <400 kg on LBP among Japanese workers., Methods: A web-based survey gathered information from 15 158 workers in 2022. Among them, 15 035 did not handle loads, whereas 123 handled rolling weights <400 kg. Load weight was categorized into 4 groups: no-handling (0 kg) and rolling weights of ≤20, 20-40, and >40 kg. Multiple logistic regression analysis examined the association between the subdivided rolling weight and LBP., Results: No significant differences in odds ratio (OR) of LBP were found for workers handling ≤40 kg rolling weights compared with that for no-handling workers. However, workers handling >40 kg rolling weights had a significantly greater OR of LBP than those not handling loads., Conclusions: Rolling weights between 40 and 400 kg could place a high stress on the lower back. Implementation in Japan of the HSE recommendations regarding rolling load should be carefully considered., (© The Author(s) [2024]. Published by Oxford University Press on behalf of Journal of Occupational Health.)

Placental mammals' ancestors were insectivores, suggesting that modern mammals may have inherited the ability to digest insects. Acidic chitinase (Chia) is a crucial enzyme hydrolyzing significant component of insects' exoskeleton in many species. On the other hand, herbivorous animal groups, such as cattle, have extremely low chitinase activity compared to omnivorous species, e.g., mice. The low activity of cattle Chia has been attributed to R128H mutation. The presence of either of these amino acids correlates with the feeding behavior of different bovid species with R and H determining the high and low enzymatic activity, respectively. Evolutionary analysis indicated that selective constraints were relaxed in 67 herbivorous Chia in Cetartiodactyla. Despite searching for another Chia paralog that could compensate for the reduced chitinase activity, no active paralogs were found in this order. Herbivorous animals' Chia underwent genetic alterations and evolved into a molecule with low activity due to the chitin-free diet., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Introduction: A quarter of work-related low back pain (LBP) cases result from handling heavy loads in Japan. The maximum weight male/female workers can handle is 40%/24% of their body weight but has set a constant load weight in ISO 11228-1 and NIOSH lifting equation. The preventive effect of the relative weight limit on LBP has not been clarified. This study aimed to identify the effect of relative weight limits set as body weight percentages on LBP prevalence., Methods: Data from 21924 workers were collected via a web-based survey in 2022. The workers were categorized into three groups: group A, "no handling," group B, "handling loads up to 40%/24% or less of body weight," and group C, "handling loads over 40%/24% of body weight." Moreover, they were categorized into eight groups: no handling, 1-5 kg, 5-10 kg, 10-15 kg, 15-20 kg, 20-25 kg, 25-30 kg, and ≥30 kg. Multiple logistic regression analysis was used to identify the effects of the limits set to body weight percentages and constant load weights on LBP., Results: In groups A, B, and C, 25.5%, 39.2%, and 47.3% of males or 16.9%, 26.4%, and 38.0% of females had LBP, respectively. The odds ratio (OR) of LBP was significantly greater in group B than in group A and even greater in group C. The OR of LBP among workers handling loads under 10 kg was not significantly different compared to no-handling workers., Conclusions: LBP prevalence was greater in group B than in group A but lesser than in group C. Weight limits based on body weight percentages could not eliminate the factor of handling loads. However, handling loads under 10 kg suppressed LBP. Relative weight limits set as body weight percentages were inappropriate and ineffective for preventing LBP., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Iwakiri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ym1 (chitinase-like 3, Chil3) expressed in mice is a nonenzymatic chitinase-like protein, which shows 67% identity with mouse acidic chitinase (Chia). Similar to Chia, Ym1 is overexpressed in asthma and parasitic infections in mouse lungs. Due to the lack of chitin-degrading activity, the biomedical role of Ym1 under these pathophysiological conditions remains to be determined. In this study, we investigated what region and amino acid changes in Ym1 resulted in the loss of enzymatic activity. Replacing two amino acids at the catalytic motif to obtain a Chia-like sequence (N136D and Q140E; MT-Ym1) did not activate the protein. We conducted a comparative study of Ym1 and Chia. We found that three protein segments-(i) the catalytic motif residues, (ii) exons 6 and 7, and (iii) exon 10-are responsible for chitinase activity loss in Ym1. We show that replacing each of these three segments in Chia that are also involved in substrate recognition and binding by the Ym1 sequence can fully abolish the enzymatic activity. In addition, we show that there have been extensive gene duplication events at the Ym1 locus specific to the rodent lineages. Consistent with this result, Ym1 orthologs from the rodent genome were under positive selection when analyzed through the CODEML program. These data suggest that numerous amino acid substitutions in the regions involved in the chitin recognition, binding, and degradation ability of the ancestor Ym1 molecule lead to the irreversible inactivation of the protein., (© 2023 The Protein Society.)

This study examined the effects of short sleep duration (SSD) on hemodynamic and psychological responses under long working hours (LWH) in a laboratory experiment. Sixteen subjects participated in a crossover design experiment consisting of two conditions: normal (7-hours) sleep and short (5-hours) sleep. In each condition, participants engaged in simulated LWH (13 hours a day), comprising 12 task sessions. Hemodynamic and psychological responses were measured in each session. Results showed that there were significant main effects of condition and session but no interaction for hemodynamic and psychological responses. Systolic blood pressure and fatigue were higher in the later sessions than the first one. Stroke volume, sleepiness, fatigue, and stress were higher in the 5-hour than the 7-hour sleep condition (all p<0.05). These results suggest that although the combined effect of LWH and SSD was not significant, both LWH and SSD caused a hemodynamic and psychological burden.

This study focused on everyday furniture and computers used in work from home and aimed to investigate how improper postures increase the risk of musculoskeletal disorders using different combinations of tables, chairs, and computers. Twenty-one healthy participants were asked to perform a visual display terminal task for 30 minutes in a laboratory modeled on the work from home concept. Seven experimental conditions were set up according to the different combinations of desks, chairs, and computers. Three-dimensional body posture was measured using a magnetic tracking device. The results showed that when using a low table, floor chair, and laptop computer, the body posture above the hip was similar to that when using a dining table, chair, and desktop computer. When using a sofa, and tablet computers, or laptop computer, severe neck flexion, which is stressful to the neck, was observed. Moreover, excessive low back flexion was observed when using a floor cushion and laptop computer. We suggest that computer work while sitting on a sofa or floor cushion without a backrest is harmful to the neck and low back.

In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at K D 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N -acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and random-type chitosan appeared to be the best sources of GlcNAc dimers and broad-scale chitooligosaccharides, respectively. In addition, the pattern of the products from the block-type chitosan was different between pH conditions (pH 2.0 and pH 5.0). Thus, monkey CHIA can degrade chitin and chitosan efficiently without inactivation under high-temperature or low pH conditions. Our results show that certain chitooligosaccharides are enriched by using different substrates under different conditions. Therefore, the reaction conditions can be adjusted to obtain desired oligomers. Crab-eating monkey CHIA can potentially become an efficient tool in producing chitooligosaccharide sets for agricultural and biomedical purposes.

Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides' formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N -acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was from random-type chitosan than from the block-type form of the molecule. The data presented here indicate that Chia digests chitosan acquired by homogenous deacetylation of chitin in vitro and in vivo. The degradation products may then influence different physiological or pathological processes. Our results also suggest that bioactive chitooligosaccharides can be obtained conveniently using homogenously deacetylated chitosan and Chia for various biomedical applications.

Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0-7.0) and temperature (30-70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications., (© 2021. The Author(s).)

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been attracting research interest due to their involvement in various pathological conditions such as Gaucher's disease and asthma, respectively. Both enzymes are highly expressed in mice, while the level of AMCase mRNA was low in human tissues. In addition, the chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. Here, we revealed a substantially higher chitinolytic and transglycosylation activity of human Chit1 against artificial and natural chitin substrates as compared to the mouse enzyme. We found that the substitution of leucine (L) by tryptophan (W) at position 218 markedly reduced both activities in human Chit1. Conversely, the L218W substitution in mouse Chit1 increased the activity of the enzyme. These results suggest that Chit1 may compensate for the low of AMCase activity in humans, while in mice, highly active AMCase may supplements low Chit1 activity., Competing Interests: Declaration of competing interest All authors have no competing interests to declare., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Di- N -acetylchitobiase (Ctbs) degrades β-1,4 glycoside bonds of the chitobiose core of free asparagine-linked glycan. This study examined whether Ctbs degrades chitin-oligosaccharides to GlcNAc in mammals. We analyzed Ctbs mRNA and protein expression in mouse tissues and characterized enzymatic activity using recombinant mouse Ctbs expressed in Escherichia coli . Ctbs mRNA and protein were expressed in various tissues of mouse, including the stomach. Optimal conditions for recombinant Ctbs were pH 3.0 and 45°C, and the recombinant enzyme was retained more than 94% activity after incubation at pH 3.0-7.0 and below 37°C. The recombinant Ctbs hydrolyzed (GlcNAc) 3 and (GlcNAc) 6 at pH 3.0 and produced GlcNAc. The K m of Ctbs was lowest with (GlcNAc) 3 as a substrate. k cat / K m was fourfold as high with (GlcNAc) 3 and (GlcNAc) 4 as substrates than with (GlcNAc) 2 . These results suggest that Ctbs digests chitin-oligosaccharides or (GlcNAc) 2 of reducing-end residues of oligosaccharides and produces GlcNAc in mouse tissues.

Background: Postoperative ileus is a major complication of abdominal surgical procedures. The purpose of this study was to investigate preventive effect of daikenchuto (DKT) on onset of ileus in patients who received gynecological surgery for malignant tumors., Methods: A total of 904 patients who received gynecological surgery for malignant tumors by opening retroperitoneum along with retroperitoneal lymph node dissection during a period between 2004 and 2018 were included in this retrospective study. The retroperitoneum was not sutured in all patients. Comparisons were made for proportion of patients developing ileus (frequency of postoperative ileus onset), timing of ileus onset, and treatment types for ileus among following three groups: a group treated with enema or laxatives to release gas if they did not pass the intestinal gas for 3 days postoperatively (Group A, n = 152); a group treated with adhesion-inhibitory absorptive barrier at the opening to the retroperitoneum (Group B, n = 188); and a group treated with adhesion-inhibitory absorptive barrier and oral intake of DKT 7.5 g per day (Group C, n = 564)., Results: The frequency of ileus onset significantly decreased in both Groups B (4.8%) and C (3.5%) compared to Group A (16.4%). Furthermore, the frequency of ileus onset was significantly less in Group C compared to Group B. For the treatment types, frequency of ileus, which was successfully treated only with conservative therapy, was the same for Groups B and C. However, incidence of serious ileus that required surgery decreased by 45% in Group C (2/564) compared to Groups A (2/152) and B (3/188)., Conclusions: Results suggest that DKT prevents development of serious ileus after gynecological surgery for malignant tumors and therefore contributes to improvement in patients' QOL., (© 2020 John Wiley & Sons Australia, Ltd.)

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequently observed treatment-related adverse effect, particularly associated with taxane-based chemotherapy, which affects the quality of life of the patients. To date, CIPN has been subjectively evaluated by patients or physicians. Intraepidermal electrical stimulation (IES) may be applied to evaluate the function of small fibers by measuring pain threshold, and assess the degree of diabetic peripheral neuropathy. The aim of the present study was to evaluate CIPN objectively by using IES. The pain threshold measured by IES in patients with gynecological cancer who underwent taxane-based chemotherapy was compared with the clinical grading scale of peripheral neuropathy (National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0). A total of 57 patients were evaluated (151 measurements). The median age of the patients was 63 years. The number of measurements with clinical grades of 0, 1 and ≥2 was 49, 57 and 45, respectively. The mean pain threshold was 0.1, 0.14 and 0.18 mA for grades 0, 1 and ≥2, respectively. Therefore, the mean pain threshold significantly increased with the progression of the clinical grade. The measurement of pain threshold by using IES may be a reliable indicator for quantitative evaluation of CIPN., (Copyright © 2020, Spandidos Publications.)

Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this "FACS-array" with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Miyazaki et al.)

Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of degradation products from artificial and natural chitin substrates such as 4-NP-(GlcNAc) 2 , (GlcNAc) 4 and colloidal chitin. The FACE method has been improved by our group for analysis of chitooligosaccharides in the presence of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities of enzymes at pH 2.0-8.0. FACE is a very sensitive technique detecting picomolar amounts of molecules. We optimized the detection conditions as follows: exposure type, precision; sensitivity, high resolution; exposure time, 5 s. We evaluated the (GlcNAc) 2 levels using a standard curve that allows chitooligosaccharides quantification at up to 10 nmol amounts. Using the method presented here, the chitinolytic properties of different chitinases can be compared directly. Serratia chitinase A (ChiA) and chitinase B (ChiB), two well-studied bacterial chitinases, have been shown by HPLC to have a synergistic effect on the chitin degradation rate. Using the FACE method, we determined the combinatory effects of mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) in natural chitin substrates processing.•FACE is a simple and quantitative method.•Our improved procedure enables the quantification of chitooligosaccharides produced by chitinases at pH 2.0-8.0.•FACE is able to quantify chitooligosaccharides at up to 10 nmol amounts., (© 2020 The Author(s). Published by Elsevier B.V.)

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a K D of 16.4 nm We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans , without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris ) in culture supernatant in vitro We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections., (© 2020 Yamanaka et al.)

Commercially available porcine pepsin preparations have been used for the production of chitooligosaccharides with various biomedical activities. However, the origin of this activity is not well understood. Here we show that the chitosan-degrading activity is conferred by residues with chitinolytic activity of truncated forms of acidic chitinase (Chia) persisting in the pepsin preparation. Chia is an acid-stable and pepsin-resistant enzyme that degrades chitin to produce N-acetyl-D-glucosamine dimer. We found that Chia can be truncated by pepsin under stomach-like conditions while maintaining its enzymatic activity. Similarly to the full-length protein, truncated Chia as well as the pepsin preparations digested chitosan with different degrees of deacetylation (DD: 69-84%) with comparable degradation products. The efficiency was DD-dependent with a marked decrease with higher DD, indicating that the chitosan-degrading activity in the pepsin preparation is due to the chitinolytic activity rather than chitosanolytic activity. We suggest that natural or recombinant porcine Chia are suitable for producing chitooligosaccharides for biomedical purposes.

Objectives: This study aimed to clarify haemodynamic responses of different age groups to simulated long working hours., Methods: Men of three age groups participated in this study (16 in their 30s (mean 33.9±2.7 years old), 15 in their 40s (45.5±2.9) and 16 in their 50s (54.1±2.7)). All participants conducted 12 45-min personal computer-based tasks from 09:00 to 22:00. Nine 10-min to 15-min breaks between task periods, a 1-hour break at noon, and a 50-min break in the evening were provided. Haemodynamic responses were measured during task periods. All participants had normal resting systolic blood pressure (SBP <140 mm Hg) and diastolic blood pressure (DBP<90 mm Hg), which were measured before tasks started in the morning. Two-way repeated-measures analysis of variances and multiple comparisons (Bonferroni) were conducted., Results: No haemodynamic indices were significantly different among groups at baseline. Compared with baseline, SBP was almost unchanged for the 30s group but increased for the 40s and 50s groups during task periods. The 50s group showed higher SBP compared with the 30s group especially in the latter half of the working hours (p<0.05). In addition, the 50s group also showed higher total peripheral resistance (TPR) than the 30s group (p<0.1)., Conclusion: The 50s group showed higher SBP and TPR responses than the 30s group, especially in the latter half of working hours. These results suggest that older workers might suffer more cardiovascular damage related to long working hours., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food processing and various pathophysiological conditions such as chronic inflammatory diseases. By combination of the colorimetric analysis and fluorophore-assisted carbohydrate electrophoresis (FACE) method, we directly compared the chitinolytic properties of mouse Chit1 and AMCase and determined their combinatory effects in artificial and natural chitin substrates processing. Chit1 and AMCase display different dynamics of chitinolytic properties through acidic to neutral conditions. At pH2.0, the activity of AMCase was higher than that of Chit1 and stronger or comparable with that of Serratia marcescens chitinase B, a well-characterized bacterium chitinase. Changes of degradation products using different substrates indicate that AMCase and Chit1 have diverse properties under various pH conditions. Exposure of the chitin substrates to both Chit1 and AMCase did not indicate any mutual interference of these enzymes and showed no synergistic effect, in contrast to observations regarding some bacterial chitinases. Our results suggest that Chit1 and AMCase have no synergistic effect under physiological conditions., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase from Listeria innocua has a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.

Chitin is a polymer of N-acetyl-D-glucosamine (GlcNAc) and a main constituent of insects' exoskeleton. Insects are rich in protein with high energy conversion efficiency. Recently, we have reported that acidic chitinases (Chia) act as digestive enzymes in mouse, pig and chicken (omnivorous) but not in dog (carnivorous) and bovine (herbivorous), indicating that feeding behavior affects Chia expression levels, and determines chitin digestibility in the particular animals. Common marmoset (Callithrix jacchus) belongs to New World monkey family and provides a potential bridge between mouse models and human diseases. Common marmoset is an insectivorous nonhuman primate with unknown expression levels and enzymatic functions of the Chia homologue, CHIA. Here, we report that common marmoset highly expresses pepsin-, trypsin- and chymotrypsin-resistant CHIA in the stomach. We show that CHIA is most active at pH 2.0 and degrades chitin and mealworm shells into GlcNAc dimers under gastrointestinal conditions. Although common marmoset and crab-eating monkey (Old World monkey) have two CHIA genes in their genomes, they primarily express one gene in the stomach. Thus, this study is the first to investigate expression levels and enzymatic functions of CHIA in a New World primate, contributing to the understanding of dietary adaptation and digestion in this taxon.

Objectives The present study examined hemodynamic responses of normotensive and untreated hypertensive participants under simulated long working hours (LWH) - 13 hours - in an experimental laboratory study. Methods Thirty-five men participated in this study. Twenty-two of these participants were categorized into the normotensive group (systolic blood pressure (SBP) ≤140 mmHg and diastolic blood pressure (DBP) ≤90 mmHg); one participant was excluded due to missing data, leaving twenty-one participants with a mean age of 49.2 years. Another thirteen participants were categorized into the high blood pressure group (SBP=140-160 mmHg or DBP=90-100 mmHg) with a mean age of 51.9 years. The hemodynamic responses at the resting state from 09:00-09:10 hours (baseline) and during LWH from 09:10-22:00 hours (12 sessions) were measured. In each session, participants performed mental tasks. Changes in the hemodynamic response (Δ) were calculated by subtracting the individual values at each session from the baseline values. Results The values for the ΔSBP, ΔDBP, and Δmean arterial pressure increased with work time. Additionally, we found a significant interaction between the group and sessions for the ΔSBP (P<0.05, partial η 2 =0.086). Although ΔSBP values did not differ between the groups at first (09:10-14:30 hours), the values in later sessions (14:40-21:50 hours) were significantly higher in the high blood pressure compared to the normotensive group. Conclusions Our study found that LWH increases BP, with a larger increase in SBP in the later working hours among individuals with untreated hypertension, whereas other hemodynamic responses did not differ between groups as a function of the LWH.

This study aimed to examine hemodynamic responses and the necessity of breaks under long working hours. Thirty-eight healthy males conducted PC-based work from 9:10 to 22:00. Nine 10-minute short breaks and two long breaks (a 1-hour break and a 50-minute break) were provided, and hemodynamic responses were measured regularly during this period. The results showed that systolic blood pressure increased during the working hours and cardiovascular burden increased under long working hours. Cardiac responses decreased, but vascular responses increased continually during work periods without long breaks. The long breaks, however, benefitted workers by preventing excessive decreases in cardiac responses and increases in vascular responses, but this effect may decrease with the extension of working hours. In conclusion, long working hours increase cardiovascular burden, and taking long breaks is important for reducing these burdens when long working hours cannot be avoided.