Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

Age-Related Effects on MSC Immunomodulation, Macrophage Polarization, Apoptosis, and Bone Regeneration Correlate with IL-38 Expression.

Mesenchymal stem cells (MSCs) are known to promote tissue regeneration and suppress excessive inflammation caused by infection or trauma. Reported evidence indicates that various factors influence the expression of MSCs' endogenous immunomodulatory properties. However, the detailed interactions of MSCs with macrophages, which are key cells involved in tissue repair, and their regulatory mechanisms are not completely understood. We herein investigated how age-related immunomodulatory impairment of MSCs alters the interaction of MSCs with macrophages during bone healing using young (5-week old) and aged (50-week old) mice. To clarify the relationship between inflammatory macrophages (M1) and MSCs, their spatiotemporal localization at the bone healing site was investigated by immunostaining, and possible regulatory mechanisms were analyzed in vitro co-cultures. Histomorphometric analysis revealed an accumulation of M1 and a decrease in MSC number at the healing site in aged mice, which showed a delayed bone healing. In in vitro co-cultures, MSCs induced M1 apoptosis through cell-to-cell contact but suppressed the gene expression of pro-inflammatory cytokines by soluble factors secreted in the culture supernatant. Interestingly, interleukin 38 (Il-38) expression was up-regulated in M1 after co-culture with MSCs. IL-38 suppressed the gene expression of inflammatory cytokines in M1 and promoted the expression of genes associated with M1 polarization to anti-inflammatory macrophages (M2). IL-38 also had an inhibitory effect on M1 apoptosis. These results suggest that MSCs may induce M1 apoptosis, suppress inflammatory cytokine production by M1, and induce their polarization toward M2. Nevertheless, in aged conditions, the decreased number and immunomodulatory function of MSCs could be associated with a delayed M1 clearance (i.e., apoptosis and/or polarization) and consequent delayed resolution of the inflammatory phase. Furthermore, M1-derived IL-38 may be associated with immunoregulation in the tissue regeneration site.

Therapeutic effects of peptide P140 in a mouse periodontitis model.

In our search for innovative drugs that could improve periodontal treatment outcomes, autophagy and its anomalies represent a potential target for therapeutic intervention. We sought to identify autophagy defects in murine experimental periodontitis and study the effectiveness of P140, a phosphopeptide known to bind HSPA8 and inhibit its chaperone properties, and that corrects autophagy dysfunctions in several autoimmune and inflammatory diseases. Experimental periodontitis was induced by placing silk ligature around mandibular first molars. Sick mice were treated intraperitoneally with either P140 or a control, scrambled peptide. After 10 days, mandibles were harvested and bone loss was measured by micro-CT. Immune cells infiltration was studied by histological analyses. Cytokines levels and autophagy-related markers expression were evaluated by qRT-PCR and western blotting. A comparison with non-affected mice revealed significant alterations in the autophagy processes in mandibles of diseased mice, especially in the expression of sequestosome 1/p62, Maplc3b, Atg5, Ulk1, and Lamp2. In vivo, we showed that P140 normalized the dysregulated expression of several autophagy-related genes. In addition, it diminished the infiltration of activated lymphocytes and pro-inflammatory cytokines. Unexpectedly P140 decreased the extent of bone loss affecting the furcation and alveolar areas. Our results indicate that P140, which was safe in clinical trials including hundreds of autoimmune patients with systemic lupus erythematosus, not only decreases the inflammatory effects observed in mandibular tissues of ligation-induced mice but strikingly also contributes to bone preservation. Therefore, the therapeutic peptide P140 could be repositioned as a decisive breakthrough for the future therapeutic management of periodontitis.

MazF activation causes ACA sequence-independent and selective alterations in RNA levels in Escherichia coli.

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.

The ß-hairpin region of the cyanobacterial F1-ATPase γ-subunit plays a regulatory role in the enzyme activity.

The γ-subunit of cyanobacterial and chloroplast ATP synthase, the rotary shaft of F1-ATPase, equips a specific insertion region that is only observed in photosynthetic organisms. This region plays a physiologically pivotal role in enzyme regulation, such as in ADP inhibition and redox response. Recently solved crystal structures of the γ-subunit of F1-ATPase from photosynthetic organisms revealed that the insertion region forms a ß-hairpin structure, which is positioned along the central stalk. The structure-function relationship of this specific region was studied by constraining the expected conformational change in this region caused by the formation of a disulfide bond between Cys residues introduced on the central stalk and this ß-hairpin structure. This fixation of the ß-hairpin region in the α3ß3γ complex affects both ADP inhibition and the binding of the ε-subunit to the complex, indicating the critical role that the ß-hairpin region plays as a regulator of the enzyme. This role must be important for the maintenance of the intracellular ATP levels in photosynthetic organisms.

Aging-Affected MSC Functions and Severity of Periodontal Tissue Destruction in a Ligature-Induced Mouse Periodontitis Model.

Mesenchymal stem cells (MSCs) are known to play important roles in the repair of lost or damaged tissues and immunotolerance. On the other hand, aging is known to impair MSC function. However, little is currently known about how aged MSCs affect the host response to the local inflammatory condition and tissue deterioration in periodontitis, which is a progressive destructive disease of the periodontal tissue potentially leading to multiple tooth loss. In this study, we examined the relationship between aging-induced impairment of MSC function and the severity of periodontal tissue destruction associated with the decrease in host immunomodulatory response using a ligature-induced periodontitis model in young and aged mice. The results of micro computerized tomography (micro-CT) and histological analysis revealed a more severe bone loss associated with increased osteoclast activity in aged (50-week-old) mice compared to young (5-week-old) mice. Immunostaining analysis revealed that, in aged mice, the accumulation of inflammatory T and B cells was higher, whereas the percentage of platelet-derived growth factor receptor α (PDGFRα)+ MSCs, which are known to modulate the apoptosis of T cells, was significantly lower than in young mice. In vitro analysis of MSC function showed that the expression of surface antigen markers for MSCs (Sca-1, CD90, CD146), colony formation, migration, and osteogenic differentiation of aged MSCs were significantly declined compared to those of young MSCs. Moreover, a significantly higher proportion of aged MSCs were positive for the senescence-associated ß galactosidase activity. Importantly, aged MSCs presented a decreased expression of FAS-L, which was associated with a lower immunomodulatory property of aged MSCs to induce T cell apoptosis in co-cultures compared with young MSCs. In summary, this is the first study showing that aging-induced impairment of MSC function, including immunomodulatory response, is potentially correlated with progressive periodontal tissue deterioration.

Acidic Pre-Conditioning Enhances the Stem Cell Phenotype of Human Bone Marrow Stem/Progenitor Cells.

A deeper understanding of the detailed mechanism of in vivo tissue healing is necessary for the development of novel regenerative therapies. Among several external factors, environmental pH is one of the crucial parameters that greatly affects enzyme activity and cellular biochemical reactions involving tissue repair and homeostasis. In this study, in order to analyze the microenvironmental conditions during bone healing, we first measured the pH in vivo at the bone healing site using a high-resolution fiber optic pH microsensor directly in femur defects and tooth extraction sockets. The pH was shown to decrease from physiological 7.4 to 6.8 during the initial two days of healing (inflammatory phase). In the same initial stages of the inflammatory phase of the bone healing process, mesenchymal stem cells (MSCs) are known to migrate to the healing site to contribute to tissue repair. Therefore, we investigated the effect of a short-term acidic (pH 6.8) pre-treatment on the stemness of bone marrow-derived MSCs (BMSCs). Interestingly, the results showed that pre-treatment of BMSCs with acidic pH enhances the expression of stem cell markers (OCT-4, NANOG, SSEA-4), as well as cell viability and proliferation. On the other hand, acidic pH decreased BMSC migration ability. These results indicate that acidic pH during the initial stages of bone healing is important to enhance the stem cell properties of BMSCs. These findings may enable the development of novel methods for optimization of stem cell function towards tissue engineering or regenerative medicine.

Different effects of an extended photoperiod treatment on growth, gonadal function, and condition of hair coats in Thoroughbred yearlings reared under different climate conditions.

One- to two-year-old Thoroughbred colts and fillies being reared in Miyazaki (warm climate) and Hidaka (cold climate), Japan, were administered extended photoperiod (EP) treatment between December 20 and the following April 10, and its effect on growth, endocrine changes, gonadal activation, and hair coat condition was investigated. In colts reared in Miyazaki, no effect of EP treatment was noted on the growth indices, including body weight (BW), height at withers (HW), girth, and cannon circumference (CC), whereas the BWs and CCs of fillies were significantly higher in the EP treatment group than the control. In Hidaka, the BWs and HWs of colts and HWs of fillies were significantly higher in the EP treatment group. Gonadal activation characterized by an increase in circulating hormone concentrations was earlier in the EP treatment group for fillies reared in Miyazaki [luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), and estradiol-17ß (E2)] and in colts (LH, testosterone, and E2) and fillies (LH, FSH, P4, and E2) reared in Hidaka. Regardless of sex and climate, prolactin was significantly higher in the EP treatment group, whereas insulin-like growth factor (IGF-I) was not. Initial ovulation occurred before April in more of the EP treatment group than the control regardless of the climate. Molting of the hair coat, examined in March, was advanced in the EP treatment group regardless of sex and climate. These results suggest that EP treatment may promote growth and gonadal activation in fillies reared in Miyazaki and in colts and fillies reared in Hidaka and that the effect may be mediated by prolactin.

Comparison of growth and endocrine changes in Thoroughbred colts and fillies reared under different climate conditions.

Development and endocrine changes in Thoroughbreds colts and fillies were compared between those reared at two facilities of the Japan Racing Association, the Hidaka Training and Research Center (Hidaka) and Miyazaki Yearling Training Farm (Miyazaki). Thoroughbred colts and fillies born in Japan between 2003 and 2010 were used. Each colt group and filly group was divided into 2 groups, respectively, and raised in Hidaka or Miyazaki for 7 months from September at 1 year old to April at 2 years old. For the growth parameters, the body weight, height at withers, and girth and cannon circumferences were measured once a month. For parameters of endocrine function, circulating prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin-like growth factor-1 (IGF-1), testosterone, progesterone, and estradiol-17ß levels were measured. Regarding growth, the rate of increase over the 7-month period was significantly higher in both colts and fillies raised in Miyazaki than in Hidaka in all 4 parameters: body weight, height at withers, and girth and cannon circumferences. The endocrine changes of the colts and fillies born in 2007 were as follows. In colts, although circulating prolactin tended to be higher in colts reared in Hidaka from October to April, circulating LH, FSH, testosterone, estradiol-17ß and IGF-1 tended to be higher in colts reared in Miyazaki than in Hidaka, suggesting that the gonadotropin-releasing hormone-LH/FSH system and the growth hormone-IGF-1 system were more active in colts reared in Miyazaki as compared with those reared in Hidaka. In fillies, circulating prolactin tended to be higher in fillies reared in Hidaka in February and March, but no significant difference was noted in the serum LH, FSH, IGF-1, or progesterone level between the 2 groups. Circulating estradiol-17ß tended to be higher in fillies reared in Miyazaki than in Hidaka in October and November. Regarding ovarian function, the initial ovulation occurred by the end of March in 2 (16.7%) of 12 fillies reared in Hidaka and 7 (38.9%) of 18 fillies reared in Miyazaki, suggesting that the ovarian function was more active in fillies reared in Miyazaki as compared with those reared in Hidaka. Based on these findings, it was clarified that development of the body and gonads was faster in Miyazaki compared with Hidaka in both colts and fillies.

IFN-γ and TNF-α synergistically induce mesenchymal stem cell impairment and tumorigenesis via NFκB signaling.

An inflammatory microenvironment may cause organ degenerative diseases and malignant tumors. However, the precise mechanisms of inflammation-induced diseases are not fully understood. Here, we show that the proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) synergistically impair self-renewal and differentiation of mesenchymal stem cells (MSCs) via nuclear factor κB (NFκB)-mediated activation of mothers against decapentaplegic homolog 7 (SMAD7) in ovariectomized (OVX) mice. More interestingly, a long-term elevated levels of IFN-γ and TNF-α result in significantly increased susceptibility to malignant transformation in MSCs through NFκB-mediated upregulation of the oncogenes c-Fos and c-Myc. Depletion of either IFN-γ or TNF-α in OVX mice abolishes MSC impairment and the tendency toward malignant transformation with no NFκB-mediated oncogene activation. Systemic administration of aspirin, which significantly reduces the levels of IFN-γ and TNF-α, results in blockage of MSC deficiency and tumorigenesis by inhibition of NFκB/SMAD7 and NFκB/c-FOS and c-MYC pathways in OVX mice. In summary, this study reveals that inflammation factors, such as IFN-γ and TNF-α, synergistically induce MSC deficiency via NFκB/SMAD7 signaling and tumorigenesis via NFκB-mediated oncogene activation.

Efficient bone formation in a swine socket lift model using Escherichia coli-derived recombinant human bone morphogenetic protein-2 adsorbed in ß-tricalcium phosphate.

Several preclinical studies have shown that Escherichia coli-derived bone morphogenetic protein-2 (E-BMP-2) is as effective as mammalian cell-derived bone morphogenetic protein-2 (C-BMP-2) in the treatment of bone defects. However, further investigation of the effectiveness and determination of the optimal dosage of E-BMP-2 in large animals are still necessary before its full application in humans. This study investigated the efficiency of different concentrations of E-BMP-2 adsorbed in ß-TCP for bone augmentation and osseointegration of immediate dental implants in a swine socket lift model. Following exposure of the maxillary sinus lateral wall, a 3.4-mm (diameter) cavity was drilled and filled with 0.1 g of ß-TCP containing different doses of E-BMP-2 (0, 10, 30, or 100 µg/site) to lift the Schneiderian membrane. A dental implant was then immediately inserted. Bone-to-implant contact (BIC) and bone density (BD) examined via histological analysis were used as parameters to assess E-BMP-2 efficiency in bone formation. The implant stability quotient (ISQ) was measured using Osstell to determine the effect of E-BMP-2/ß-TCP on implant stability. After 8 weeks, the groups that received 30 and 100 µg of E-BMP-2 showed substantial new bone formation in the elevated space, while no bone formation was observed with ß-TCP alone. Accordingly, BIC and BD presented a dose-dependent response to increasing doses of E-BMP-2. However, there was no increase in implant stability with E-BMP-2 treatment. In conclusion, the E-BMP-2/ß-TCP combination was efficient in bone formation and osseointegration of dental implants in a socket lift model in mini-pigs.

Comparison of the Occurrence of Transportation-associated Fever in 2 Years Old Thoroughbreds before and after Introduction of Prophylactic Marbofloxacin Administration.

In order to reveal the preventive effect of marbofloxacin (MRFX) administration just before transportation, we compared the occurrence of transportation-associated fever before and after introduction of MRFX administration. After the introduction of prophylactic MRFX administration, the rectal temperatures of horses after transportation were significantly lower than before the introduction of MRFX administration (P<0.01) and the number of febrile horses was significantly lower than before the introduction of MRFX administration (P<0.01). In conclusion, these results show that prophylactic MRFX administration just before transportation is clinically effective at preventing transportation-associated fever.

Individual identification method using samples associated with doping tests: A comparison of mitochondrial and nuclear genetic data.

Doping offenses involve the use or attempted use of any prohibited method or substance as well as substituting samples. Consequently, it has been recommended that short tandem repeat (STR) analysis be used to determine if the doping control samples are from the same athlete. However, it has been recognized that it may be difficult to obtain full STR analysis using negligible amounts of DNA samples. Mitochondrial DNA (mtDNA) is characterized by its stability and high cellular copy number. Therefore, mtDNA testing in urine is expected to be used to analyze samples that cannot be analyzed using STR analysis. The objective of this study was to compare mtDNA testing with STR analysis by conducting sensitivity, concordance (whole blood, dried blood spot, and urine), and case-type studies. In sensitivity studies, mtDNA testing exhibited greater sensitivity compared with STR analysis. Concordance studies indicated that all samples were consistent with the mtDNA sequences and STR profiles. Allelic dropout occurred in some urine samples that were examined for STR analysis. Case-type sample studies demonstrated that mtDNA testing could be used to obtain DNA profiles of all the samples tested, including blood, dried blood spots, urine, blood residues on needles, and blood stains. In conclusion, mtDNA testing is valuable for analyzing highly degraded DNA samples, such as urine samples, compared with STR analysis. Urine testing should be performed for the initial testing procedure, because mtDNA is inherited maternally. In situations where the DNA match is detrimental to the athlete, additional blood STR analysis may be required.

Effects of an extended photoperiod on body composition of young Thoroughbreds in training.

The effects of an extended photoperiod (EP) on body composition of Thoroughbreds colts and fillies from December at one year old to April at two years old were investigated. Seventy-three Thoroughbreds reared and trained in Hidaka Training and Research Center, Japan Racing Association, Hokkaido were used. Forty-one horses were under the EP conditions from December 20 to April 15, and the 32 horses were under natural light alone as the control group. Body weight (BW), rump fat thickness (RFT), fat free mass (FFM) and percentage of fat (%F) were used as parameters of body composition. The present study revealed that BW and FFM increased with age in the EP group in colts. In fillies, BW increased with age in both the EP and the control group, however FFM increased with age only in the EP group. From December to April, only colts had a higher rate of increase in both BW and FFM in the EP group than in the control group. However, according to the mean rates of increase in FFM from January to March, the EP group was significantly higher than the control group in both sexes. Furthermore, monthly increase rate of FFM in March was significantly higher in the EP group than in the control group in both sexes. These results suggests that EP treatment to young Thoroughbreds in training at Hokkaido, which is shorter daylength in winter, accelerate the increase of FFM, representing muscle mass.

Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors.

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

Technetium-99 conjugated with methylene diphosphonate ameliorates ovariectomy-induced osteoporotic phenotype without causing osteonecrosis in the jaw.

Technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP) is a novel bisphosphonate derivative without radioactivity and has been successfully used to treat arthritis in China for years. Since bisphosphonate therapy has the potential to induce bisphosphonate-related osteonecrosis of the jaw (BRONJ), we examined whether (99)Tc-MDP represents a new class of bisphosphonate for antiresorptive therapy to ameliorate estrogen deficiency-induced bone resorption with less risk of causing BRONJ. We showed that (99)Tc-MDP-treated, ovariectomized (OVX) mice had significantly improved bone mineral density and trabecular bone volume in comparison to the untreated OVX group by inhibiting osteoclasts and enhancing osteogenic differentiation of bone marrow mesenchymal stem cells. To determine the potential of inducing BRONJ, (99)Tc-MDP/dexamethasone (Dex) or zoledronate/Dex was administered into C57BL/6J mice via the tail vein, followed by extraction of maxillary first molars. Interestingly, (99)Tc-MDP treatment showed less risk to induce osteonecrosis in the maxillary bones compared to zoledronate treatment group, partially because (99)Tc-MDP neither suppressed adaptive regulatory T cells nor activated the inflammatory T-helper-producing interleukin-17 cells. Taken together, our findings demonstrate that (99)Tc-MDP therapy may be a promising approach in the treatment of osteoporosis with less risk of causing BRONJ.

Double allogenic mesenchymal stem cells transplantations could not enhance therapeutic effect compared with single transplantation in systemic lupus erythematosus.

The clinical trial of allogenic mesenchymal stem cells (MSCs) transplantation for refractory SLE patients has shown significant safety and efficacy profiles. However, the optimum frequency of the MSCs transplantation (MSCT) is unknown. This study was undertaken to observe whether double transplantations of MSCs is superior to single transplantation. Fifty-eight refractory SLE patients were enrolled in this study, in which 30 were randomly given single MSCT, and the other 28 were given double MSCT. Patients were followed up for rates of survival, disease remission, and relapse, as well as transplantation-related adverse events. SLE disease activity index (SLEDAI) and serologic features were evaluated. Our results showed that no remarkable differences between single and double allogenic MSCT were found in terms of disease remission and relapse, amelioration of disease activity, and serum indexes in an SLE clinical trial with more than one year followup. This study demonstrated that single MSCs transplantation at the dose of one million MSCs per kilogram of body weight was sufficient to induce disease remission for refractory SLE patients.

Alginate hydrogel as a promising scaffold for dental-derived stem cells: an in vitro study.

The objectives of this study were to: (1) develop an injectable and biodegradable scaffold based on oxidized alginate microbeads encapsulating periodontal ligament (PDLSCs) and gingival mesenchymal stem cells (GMSCs); and (2) investigate the stem cell viability, and osteogenic differentiation of the stem cells in vitro. Stem cells were encapsulated using alginate hydrogel. The stem cell viability, proliferation and differentiation to adipogenic and osteogenic tissues were studied. To investigate the expression of both adipogenesis and ontogenesis related genes, the RNA was extracted and RT-PCR was performed. The degradation behavior of hydrogel based on oxidized sodium alginate with different degrees of oxidation was studied in PBS at 37 °C as a function of time by monitoring the changes in weight loss. The swelling kinetics of alginate hydrogel was also investigated. The results showed that alginate is a promising candidate as a non-toxic scaffold for PDLSCs and GMSCs. It also has the ability to direct the differentiation of these stem cells to osteogenic and adipogenic tissues as compared to the control group in vitro. The encapsulated stem cells remained viable in vitro and both osteo-differentiated and adipo-differentiated after 4 weeks of culturing in the induction media. It was found that the degradation profile and swelling kinetics of alginate hydrogel strongly depends on the degree of oxidation showing its tunable chemistry and degradation rate. These findings demonstrate for the first time that immobilization of PDLSCs and GMSCs in the alginate microspheres provides a promising strategy for bone tissue engineering.

Effects of different winter climates in Japan on body composition of young Thoroughbreds in training.

Changes in the body composition of 50 Thoroughbreds colts and fillies, born between 2004 and 2010, were compared between those reared at the Hidaka Training and Research Center (Hidaka), Hokkaido, which is extremely cold in winter, and those reared at the Miyazaki Yearling Training Farm (Miyazaki), Kyushu, which is mildly cold in winter. The horses were divided into two sex groups and reared and trained in Hidaka or Miyazaki for 7 months from October of one year of age to April of two years of age. Body weight (BW), rump fat thickness (RFT), fat-free mass (FFM), and percentage of fat (%F) were used as parameters of body composition. This study revealed that BW and FFM were higher, and %F was lower in colts than in fillies at both training sites. Among colts, Miyazaki colts tended to have higher FFM values than Hidaka colts, and %F was significantly lower in Miyazaki colts than in Hidaka colts. Furthermore, from October to April, Miyazaki horses had a higher rate of increase in BW than Hidaka horses in both sexes and a higher rate of increase in FFM in colts. The higher rate of increase in FFM in Miyazaki colts suggests that training young Thoroughbreds in winter under mildly cold climate is more effective, than severely cold climate, particularly in colts.

Doping control analyses during the Tokyo 2020 Olympic and Paralympic Games.

The doping control analyses at the XXXII Olympic Games (July 23 to August 8, 2021) and the XVI Paralympic Games (August 24 to September 5, 2021) held in Tokyo, Japan, after a year of delay due to the COVID-19 pandemic are summarized in this paper. A new satellite facility at the existing World Anti-Doping Agency (WADA)-accredited Tokyo laboratory was established and fully operated by 278 staff, including 33 Tokyo laboratory staff, 49 international experts, and 196 Japanese temporary staff. The numbers of urine samples were 5081 (Olympics) and 1519 (Paralympics), and the numbers of blood samples were 1103 (Olympics) and 500 (Paralympics). The laboratory could prepare for analysis in advance using a paperless chain-of-custody system, allowing for faster turnaround time reporting. For the first time, a new polymerase chain reaction method for detecting erythropoietin (EPO) gene doping was used. The laboratory also analyzed blood samples for detecting steroid esters following the spotting of collected venous EDTA blood onto dried blood spot cards. Moreover, full-scan data acquisition using high-resolution mass spectrometers was performed for all urine samples, allowing for detecting traces of doping substances, which are not currently analyzed in the subsequent data processing. The presence of some prohibited substances was confirmed, resulting in 8 atypical findings (ATFs) and 11 adverse analytical findings (AAFs), including homologous blood transfusion (2 cases) and recombinant EPO in the blood (1 case), at the Olympics, whereas 2 ATFs and 10 AAFs were reported at the Paralympics.