Construction and Stability of All-in-One Adenovirus Vectors Simultaneously Expressing Four and Eight Multiplex Guide RNAs and Cas9 Nickase.

CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.

Acute salivary antimicrobial peptide secretion response to different exercise intensities and durations.

Antimicrobial peptides, key players of innate mucosal immunity in the oral cavity, exert antibacterial and bacteriolytic effects. This study aimed to clarify the effects of acute exercise at different intensities and durations on salivary antimicrobial peptide levels. In a randomized crossover trial, 14 young healthy untrained men performed intensity trials (cycling at 35%, 55%, and 75% of maximal oxygen uptake [VO2max] for 30 min) and duration trials (cycling at 55% VO2max for 30, 60, and 90 min). Saliva samples were collected at baseline and 0 and 60 min after exercise. In intensity trials, the change in salivary Lactoferrin levels from baseline to 0 min after 30-min exercise was greater at 75% VO2max exercise intensity compared to that at 35% VO2max. Furthermore, the change in salivary human beta defensin-2 (HBD-2) levels was greater at 75% VO2max compared to that at 35% and 55% VO2max. Salivary Lysozyme levels increased after exercise, independent of exercise intensity. However, salivary LL-37 levels did not change after exercise at any intensity. Additionally, in duration trials, the change in salivary levels of LL-37 and HBD-2 from baseline to 0 min after exercise at 55% VO2max was greater after 60 min and 90 min of exercise compared to that after 30 min of exercise. However, salivary Lactoferrin and Lysozyme levels increased after exercise, independent of exercise duration. Our findings suggest that secretory responses to acute exercise with exercise intensity and duration differ among salivary antimicrobial peptides.

From Tooth Adhesion to Bioadhesion: Development of Bioabsorbable Putty-like Artificial Bone with Adhesive to Bone Based on the New Material "Phosphorylated Pullulan".

Bioabsorbable materials have a wide range of applications, such as scaffolds for regenerative medicine and cell transplantation therapy and carriers for drug delivery systems. Therefore, although many researchers are conducting their research and development, few of them have been used in clinical practice. In addition, existing bioabsorbable materials cannot bind to the body's tissues. If bioabsorbable materials with an adhesive ability to biological tissues can be made, they can ensure the mixture remains fixed to the affected area when mixed with artificial bone or other materials. In addition, if the filling material in the bone defect is soft and uncured, resorption is rapid, which is advantageous for bone regeneration. In this paper, the development and process of a new bioabsorbable material "Phosphorylated pullulan" and its capability as a bone replacement material were demonstrated. Phosphorylated pullulan, which was developed based on the tooth adhesion theory, is the only bioabsorbable material able to adhere to bone and teeth. The phosphorylated pullulan and ß-TCP mixture is a non-hardening putty. It is useful as a new resorbable bone replacement material with an adhesive ability for bone defects around implants.

Investigation of a new implant surface modification using phosphorylated pullulan.

Various implant surface treatment methods have been developed to achieve good osseointegration in implant treatment. However, some cases remain impossible to treat with implants because osseointegration is not obtained after implantation, and the implants fail. Thus, this study focused on phosphorylated pullulan because of its adhesiveness to titanium (Ti) and bone, high biocompatibility, and early replacement with bone. In this study, the response of bone-related cells to phosphorylated pullulan was evaluated to develop a new surface treatment method. Saos-2 (human osteosarcoma-derived osteoblast-like cells), MC3T3-E1 (mouse calvaria-derived osteoblast-like cells), and RAW264.7 (mouse macrophage-like cells) were used. In evaluating cellular responses, phosphorylated pullulan was added to the culture medium, and cell proliferation and calcification induction tests were performed. The proliferation and calcification of cells on the surface of Ti disks coated with phosphorylated pullulan were also evaluated. In addition, bone morphogenetic protein-2 (BMP-2), an osteogenic factor, was used to evaluate the role of phosphorylated pullulan as a drug carrier in inducing calcification on Ti disks. Phosphorylated pullulan tended to promote the proliferation of osteoblast-like cells and the formation of calcification on Ti disks coated with phosphorylated pullulan. Ti disks coated with phosphorylated pullulan loaded with BMP-2 enhanced calcification. Phosphorylated pullulan inhibited osteoclast-like cell formation. These results are due to the properties of phosphorylated pullulan, such as adhesiveness to titanium and drug-loading function. Therefore, phosphorylated pullulan effectively promotes bone regeneration when coated on titanium implants and is useful for developing a new surface treatment method.

Effects of different exercise intensities or durations on salivary IgA secretion.

PURPOSE: This study aimed to examine changes in salivary immunoglobulin A (s-IgA) secretion at different intensities or durations of acute exercise. METHODS: Twelve healthy untrained young males were included in randomized crossover trials in Experiment 1 (cycling exercise for 30 min at a work rate equivalent to 35%, 55%, and 75% maximal oxygen uptake [ V ˙ O2max]) and Experiment 2 (cycling exercise at 55% V ˙ O2max intensity for 30, 60, and 90 min). Saliva samples were collected at baseline, immediately after, and 60 min after each exercise. RESULTS: Experiment 1: The percentage change in the s-IgA secretion rate in the 75% V ˙ O2max trial was significantly lower than that in the 55% V ˙ O2max trial immediately after exercise (- 45.7%). The percentage change in the salivary concentration of cortisol, an s-IgA regulating factor, immediately after exercise significantly increased compared to that at baseline in the 75% V ˙ O2max trial (+ 107.6%). A significant negative correlation was observed between the percentage changes in saliva flow rate and salivary cortisol concentration (r = - 0.52, P < 0.01). Experiment 2: The percentage change in the s-IgA secretion rate in the 90-min trial was significantly lower than that in the 30-min trial immediately after exercise (-37.0%). However, the percentage change in salivary cortisol concentration remained the same. CONCLUSION: Our findings suggest that a reduction in s-IgA secretion is induced by exercise intensity of greater than or equal to 75% V ˙ O2max for 30 min or exercise duration of greater than or equal to 90 min at 55% V ˙ O2max in healthy untrained young men.

Changes in roller skiing economy among Nordic combined athletes leading up to the competition season.

The purpose of this study was to compare roller skiing economy during different training phases in Nordic combined (NC) athletes and determine the aerobic and anaerobic factors responsible for changes in skiing economy. Seven elite NC athletes underwent incremental load tests on a large buried treadmill in both spring and autumn using roller skis. Measurements included oxygen uptake, respiratory exchange ratio, and blood lactate concentration. Roller skiing economy was calculated from aerobic and anaerobic energy system contributions, and overall roller skiing economy was determined by combining the two. Comparisons were made between the skiing economies obtained in the two measurement sessions. Physical characteristics and incremental test performance remained consistent between the two measurement sessions. The overall skiing economy at each speed significantly improved toward the competition season (p < 0.05). Similarly, the contribution of anaerobic energy system at each speed showed significant improvement (p < 0.05). In contrast, the contribution of aerobic energy system did not change between the two measurement sessions. This study reveals that NC athletes enhance their skiing economy at the same speed during submaximal efforts in preparation for the competition season. This improvement is predominantly associated with an improvement in the contribution of anaerobic energy system.

Duloxetine improves chronic orofacial pain and comorbid depressive symptoms in association with reduction of serotonin transporter protein through upregulation of ubiquitinated serotonin transporter protein.

ABSTRACT: Chronic orofacial pain (COP) is relieved by duloxetine (DLX) and frequently causes depressive symptoms. The aim of this study was to confirm effects of DLX on pain and depressive symptoms, and to associate with their effectiveness in platelet serotonin transporter (SERT) expression, which is a target molecule of DLX and plasma serotonin concentration in COP patients with depressive symptoms. We assessed for the severity of pain and depressive symptoms using the Visual Analog Scale (VAS) and 17-item Hamilton Depression Rating Scale (HDRS), respectively. Chronic orofacial pain patients were classified into 2 groups based on their HDRS before DLX-treatment: COP patients with (COP-D) and without (COP-ND) depressive symptoms. We found that the VAS and HDRS scores of both groups were significantly decreased after DLX treatment compared with those before DLX treatment. Upregulation of total SERT and downregulation of ubiquitinated SERT were observed before DLX treatment in both groups compared with healthy controls. After DLX treatment, there were no differences in total SERT of both groups and in ubiquitinated SERT of COP-D patients compared with healthy controls; whereas, ubiquitinated SERT of COP-ND patients remained downregulated. There were positive correlations between changes of serotonin concentrations and of VAS or HDRS scores in only COP-D patients. Our findings indicate that DLX improves not only pain but also comorbid depressive symptoms of COP-D patients. Duloxetine also reduces platelet SERT through upregulation of ubiquitinated SERT. As the result, decrease of plasma serotonin concentrations may be related to the efficacy of DLX in relieving pain and depression in COP patients.

Gut microbiota alternation with training periodization and physical fitness in Japanese elite athletes.

Introduction: The gut microbiome plays a fundamental role in host homeostasis through regulating immune functions, enzyme activity, and hormone secretion. Exercise is associated with changes in gut microbiome composition and function. However, few studies have investigated the gut microbiome during training periodization. The present study aimed to investigate the relationship between training periodization and the gut microbiome in elite athletes. Methods: In total, 84 elite athletes participated in the cross-sectional study; and gut microbiome was determined during their transition or preparation season period. Further, 10 short-track speed skate athletes participated in the longitudinal study, which assessed the gut microbiome and physical fitness such as aerobic capacity and anaerobic power in the general and specific preparation phase of training periodization. The gut microbiome was analyzed using 16S rRNA sequencing. Results: The cross-sectional study revealed significant differences in Prevotella, Bifidobacterium, Parabacteroides, and Alistipes genera and in enterotype distribution between transition and preparation season phase periodization. In the longitudinal study, training phase periodization altered the level of Bacteroides, Blautia, and Bifidobacterium in the microbiome. Such changes in the microbiome were significantly correlated with alternations in aerobic capacity and tended to correlate with the anaerobic power. Discussion: These findings suggest that periodization alters the gut microbiome abundance related to energy metabolism and trainability of physical fitness. Athlete's condition may thus be mediated to some extent by the microbiota in the intestinal environment.

Inhalation adherence for asthma and COPD improved during the COVID-19 pandemic: a questionnaire survey at a university hospital in Japan.

Background: Good adherence to an inhaled medication protocol is necessary for the management of asthma and chronic obstructive pulmonary disease (COPD), and several interventions to improve adherence have been reported. However, the impact of patient life changes and psychological aspects on treatment motivation is obscure. Here, we investigated changes in inhaler adherence during the COVID-19 pandemic and how lifestyle and psychological changes affected it.Methods: Seven-hundred sixteen adult patients with asthma and COPD who had visited Nagoya University Hospital between 2015 and 2020 were selected. Among them, 311 patients had received instruction at a pharmacist-managed clinic (PMC). We distributed one-time cross-sectional questionnaires from January 12 to March 31, 2021. The questionnaire covered the status of hospital visits, inhalation adherence before and during the COVID-19 pandemic, lifestyles, medical conditions, and psychological stress. The Adherence Starts with Knowledge-12 (ASK-12) was used to assess adherence barriers.Results: Four-hundred thirty-three patients answered the questionnaire. Inhalation adherence was significantly improved in both diseases during the COVID-19 pandemic. The most common reason for improved adherence was fear of infection. Patients with improved adherence were more likely to believe that controller inhalers could prevent COVID-19 from becoming more severe. Improved adherence was more common in patients with asthma, those not receiving counseling at PMC, and those with poor baseline adherence.Conclusions: Inhalation adherence for asthma and COPD improved in the COVID-19 pandemic. The patients seemed to realize the necessity and benefits of the medication more strongly than before the pandemic, which motivated them to improve adherence.

[Promoting Appropriate Use of Medications by "Lectures and Practices Regarding Medicine" for the Regional Community Residents].

We held lectures and practices for the regional community residents with the aim of promoting appropriate use of medications. To evaluate the usefulness of our activity, we conducted medicine-related questionnaire surveys (pre- and post-questionnaires) before and after the lectures and practices, and satisfaction with the activity questionnaire surveys (post-questionnaires) after them. We also evaluated the effect of age difference on the results by dividing the participants into elderly (age ≥65) and non-elderly (age <65), before the analysis. In the both (elderly and non-elderly) groups, the ratio of positive answers in several items of post-questionnaire regarding appropriate use of medications was higher than that of pre-questionnaire. These results suggest that the basic medicine-related knowledge improved by attending the lectures and practices, and indicate that the knowledge of the regional community residents is higher than we imagined. Furthermore, there was no difference in the ratio of positive answers between the both groups, suggesting that our activity is applicable to a wide range of age groups. Our activity benefitted the participants, and not only provided education to the regional community residents regarding appropriate drug use, but also lead to the development of regional community activity, by the coming together of the regional community residents of various age.