Elucidating the structural changes of cellulose molecules and dynamics of Na ions during the crystal transition from cellulose I to II in low temperature and low concentration NaOH solution.

Low-concentration alkali treatments at low temperatures facilitate the crystal transition of cellulose I to II. However, the transition mechanism remains unclear. Hence, in this study, we traced the transition using in situ solid-state 13C CP/MAS NMR, WAXS, and 23Na NMR relaxation measurements. In situ solid-state 13C CP/MAS NMR and WAXS measurements revealed that soaking cellulose in NaOH at low temperatures disrupts the intramolecular hydrogen bonds and lowers the crystallinity of cellulose. The dynamics of Na ions (NaOH) play a crucial role in causing these phenomena. 23Na NMR relaxation measurements indicated that the Na-ion correlation time becomes longer during the crystal transition. This transition requires the penetration of Na ions (NaOH) into the cellulose crystal and a reduction in Na-ion mobility, which occurs at low temperatures or high NaOH concentrations. The interactions between cellulose and NaOH disrupt intramolecular hydrogen bonds, inducing a conformational change in the cellulose molecules into a more stable arrangement. This weakens the hydrophobic interactions of cellulose, and facilitates the penetration of NaOH and water into the crystal, leading to the formation of alkali cellulose. Our findings suggest that a strategy to control NaOH dynamics could lead to the discovery of a novel preparation method for cellulose II.

Induction and decay of seasonal acclimatization on whole body heat loss responses during exercise in a hot humid environment with different air velocities.

Whether whole body heat loss and thermoregulatory function (local sweat rate and skin blood flow) are different between summer and autumn and between autumn and winter seasons during exercise with different air flow in humid heat remain unknown. We therefore tested the hypotheses that whole body sweat rate (WBSR), evaporated sweat rate, and thermoregulatory function during cycling exercise in autumn would be higher than in winter but would be lower than in summer under hot-humid environment (32 C, 75% RH). We also tested the hypothesis that the increase of air velocity would enhance evaporated sweat rate and sweating efficiency across winter, summer, and autumn seasons. Eight males cycled for 1 h at 40% V̇o2max in winter, summer, and autumn seasons. Using an electric fan, air velocity increased from 0.2 m/s to 1.1 m/s during the final 20 min of cycling. The autumn season resulted in a lower WBSR, unevaporated sweat rate, and a higher sweating efficiency compared with summer (all P ≤ 0.05) but WBSR and unevaporated sweat rate in autumn were higher than in winter and thus sweating efficiency was lower when compared with winter only at the air velocity of 0.2 m/s (All P ≤ 0.05). Furthermore, evaporated sweat rate and core temperature (Tcore) were not different among winter, summer, and autumn seasons (All P > 0.19). In conclusion, changes in WBSR across different seasons do not alter Tcore during exercise in a hot humid environment. Furthermore, increasing air velocity enhances evaporated sweat rate and sweating efficiency across all seasons.

Effect of combination of residual glucose concentration and subsequent increment by temporal glucose feeding on oscillation of clock gene Per2 expression.

With the aim of regulating clock gene expression to control cell activities in cell processing engineering, the effect of the combination of residual glucose concentration and subsequent increment by temporal glucose feeding on the oscillation of the expression of clock gene Per2 was investigated employing rat Mesenchymal stem cell (MSC)-like cells having Per2 promoter gene with a destabilized luciferase gene (Per2-dLuc). Two experiments with several initial glucose concentrations and different times of cultures (2 and 5 days) before temporal glucose feeding (0.9 g/L) were employed to realize various concentrations of residual glucose in the medium before the feeding. In these experiments, the lower residual glucose concentrations (0.002-0.02 g/L) before temporal glucose feeding tended to induce the larger amplitude of oscillation of Per2 expression than the higher ones (0.55-0.74 g/L). When the residual glucose concentration before glucose feeding was low (0.014-0.038 g/L), the higher temporal glucose concentration (0.23-0.9 g/L) feeding tended to induce the larger amplitude of oscillation of Per2 expression than the lower ones (0.012-0.023 g/L). Taken together, we found that the amplitude of oscillation of the expression of clock gene Per2 could be controlled by the combination of residual glucose concentration and glucose concentration of subsequent temporal feeding.

The effect of seasonal acclimatization on whole body heat loss response during exercise in a hot humid environment with different air velocity.

Seasonal acclimatization from winter to summer is known to enhance thermoeffector responses in hot-dry environments during exercise whereas its impact on sweat evaporation and core temperature (Tcore) responses in hot-humid environments remains unknown. We, therefore, sought to determine whether seasonal acclimatization is able to modulate whole body sweat rate (WBSR), evaporated sweat rate, sweating efficiency, and thermoregulatory function during cycling exercise in a hot-humid environment (32°C, 75% RH). We also determined whether the increase in air velocity could enhance evaporated sweat rate and sweating efficiency before and after seasonal acclimatization. Twelve males cycled for 1 h at 40% V̇o2max in winter (preacclimatization) and repeated the trial again in summer (after acclimatization). For the last 20 min of cycling at a steady-state of Tcore, air velocity increased from 0.2 (0.04) m/s to 1.1 (0.02) m/s by using an electric fan located in front of the participant. Seasonal acclimatization enhanced WBSR, unevaporated sweat rate, local sweat rate and mean skin temperature compared with preacclimatization state (all P < 0.05) whereas sweating efficiency was lower (P < 0.01) until 55 min of exercise. Tcore and evaporated sweat rate were unaltered by acclimatization status (all P > 0.70). In conclusion, seasonal acclimatization enhances thermoeffector responses but does not attenuate Tcore during exercise in a hot-humid environment. Furthermore, increasing air velocity enhances evaporated sweat rate and sweating efficiency irrespective of acclimated state. NEW & NOTEWORTHY Seasonal acclimatization to humid heat enhances eccrine sweat gland function and thus results in a higher local and whole body sweat rate but does not attenuate Tcore during exercise in a hot-humid environment. Sweating efficiency is lower after seasonal acclimatization to humid heat compared with preacclimatization with and without the increase of air velocity. However, having a lower sweating efficiency does not mitigate the Tcore response during exercise in a hot-humid environment.

Polymer fraction including exosomes derived from Chinese hamster ovary cells promoted their growth during serum-free repeated batch culture.

While continuous (perfusion) culture of mammalian cells might reduce the reactor size owing to the high cell density, there is the problem of higher medium cost; however, this problem is expected to be solved by the reuse of growth-promoting components in the culture supernatant. The polymer fraction (PF, 10 kDa-220 nm) collected from the supernatant of serum-free repeated-batch culture of Chinese hamster ovary (CHO) cells in not only adhesion but also suspension promoted the cell growth in respective serum-free cultures. PF contained CD81-positive exosomes and proteins, both of which were necessary for its growth-promoting activity. Consequently, the medium cost for the continuous (perfusion) serum-free suspension culture of CHO cells may be decreased by the repeated collection and addition of PF that contains exosomes and growth factor proteins.

Comparison of percentage of CD90-positive cells and osteogenic differentiation potential between mesenchymal stem cells grown on dish and nonwoven fabric.

Although nonwoven fabric (NWF) has been reported to be a candidate scaffold for the large-scale expansion of mesenchymal stem cells (MSCs), the quality of cells grown in NWF has not been well clarified. In this report, MSCs grown in an NWF disc for 3 weeks showed higher osteogenic differentiation potential and percentage of CD90 (+) cells than MSCs grown on the bottom surface of dish. The amount of the extracellular matrix (ECM) per unit surface area of fibers was larger than that on the bottom surface of the dish in the first 2 weeks of culture. The osteogenic differentiation potential of MSCs inoculated onto cell-free ECM increased with increasing amount of ECM. The higher percentage of CD90 (+) cells and osteogenic differentiation potential of cells grown in an NWF disc than of cells grown on a dish might, at least in part, be due to the higher amount of ECM.

Autonomic and perceptual thermoregulatory responses to voluntarily engaging in a common thermoregulatory behaviour.

We examined whether partial clothing removal is an effective thermoregulatory behaviour to attenuate both thermoregulatory and perceptual strain in a moderate environment (23 °C, 65% RH) during and after exercise. Ten healthy males (age: 21.9 (0.9) years; height: 173.9 (6.2) cm; mass: 62.3 (8.2) kg; body surface area: 1.8 (0.1) m2; VO2max: 51.8 (13.3) mL.kg-1.min-1) wore a long sleeve polyester shirt and performed two randomized cycling trials for 40 min at 40% VO2max followed by 20 min recovery. In one trial, they were permitted to roll up their sleeves at any time they wanted (Roll) whereas in the other trial, they were instructed to remain with long sleeves (No Roll) until the end of the recovery. Thermoregulatory variables were measured continuously whilst thermal perceptions (forearm wettedness perception (WPForearm), forearm and whole-body thermal discomfort (TDForearm, TDWhole), local and whole-body thermal sensation (TSForearm, TSWhole) and whole-body wettedness perception (WPwhole)) were measured every 10 min. All subjects behaved by rolling up their sleeves at 21.6 (4.7) minutes. Tskin (32.3 (0.2) °C, vs 32.0 (0.1) °C, p = 0.03), local sweat rate on the forearm (0.24 (0.08) mg.cm-2.min-1 vs 0.2 (0.04) mg.cm-2.min-1, p = 0.05), WPForearm, TDForearm, TSForearm and WPWhole were all lower in Roll than No Roll (all p < 0.05) whilst Tcore and cutaneous vascular conductance (CVC) on the forearm were not different (all p > 0.7) throughout the entire trial. We conclude that this behavioural response is an effective thermoregulatory behaviour to modulate local sudomotor function and thermal perceptions, WPWhole during exercise but only Tsk, TDForearm WPForearm and WPWhole persisted throughout the recovery in a moderate environment.

Protocol of mesenchymal stem cell inoculation to nonwoven fabric scaffold.

To obtain a large number of human mesenchymal stem cell (hMSCs) for allograft, nonwoven fabrics (NWF) were used as a cell culture scaffold. NWF are three-dimensional fiber aggregates formed by heat bonding and have a high surface area for cell adhesion and elongation. Inoculation hMSC was done to a center of NWF disc (diameter, 15.1 mm; depth, 0.1 mm). A cell suspension inoculum had a volume of 10 µL, which was close to the void volume of the disc, and resulted in a high initial (24 h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10 µL of medium at 0-2 h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10 µL of the medium after 1 and 2 h of hMSC inoculation (0.15 × 103 cells/cm2 NWF-fiber) markedly increased the final cell density (21 days) from 2.48 to 7.45 × 103 cells/cm2 NWF-fiber and fold increase in cell density by 16-48-fold. In conclusion, the addition of an additional medium after inoculation made the initial cells distribution in NWF more uniform, which might result in higher final cell density.

Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells.

An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3 weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-ß3 (TGF-ß3), and fibroblast growth factor-2, and the transfection of TGF-ß3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs.

Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells.

High-expression plasmid vectors for human mesenchymal stem cells (MSCs) were constructed by combination of cytomegalovirus immediate-early enhancer with cellular promoters. MSCs transfected with the vector showed higher transgene production of a cytokine, which increased the differentiation level to chondrocytes.

Fetal calf serum-free suspension culture of Chinese hamster ovary cells employing fish serum.

The effects of heat treatment and concentration of fish serum (FS) on cell growth in a suspension culture of recombinant Chinese hamster ovary (CHO) 1-15(500) (ATCC CRL-9606) cells were investigated. An increase in FS concentration from 1% to 4% markedly increased cell density. On the other hand, heat treatment of FS showed nearly no effect on cell density.

Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells.

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 degrees C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.

Correlation between cell morphology and aggrecan gene expression level during differentiation from mesenchymal stem cells to chondrocytes.

A morphological parameter of polygonal index was defined as the ratio of cell adhesion area versus the square of the major cell axis, and cells that had an adhesion area larger than 4000 mum(2) and a polygonal index larger than 0.3 were considered large polygonal cells. Cell morphology tended to change from fibroblast-like to polygonal and the percentage of the large polygonal cells increased almost in proportion to aggrecan mRNA expression level during the differentiation culture of mesenchymal stem cells (MSCs) to chondrocytes. Approximately 80% of the large polygonal cells were negative for MSC marker (CD90, CD166) expression and the aggrecan mRNA expression level of the large polygonal cells was markedly higher than that of cells with other morphologies.

Noninvasive measurement of three-dimensional morphology of adhered animal cells employing phase-shifting laser microscope.

Noninvasive measurement of 3-D morphology of adhered animal cells employing a phase-shifting laser microscope (PLM) is investigated, in which the phase shift for each pixel in the view field caused by cell height and the difference in refractive indices between the cells and the medium is determined. By employing saline with different refractive indices instead of a culture medium, the refractive index of the cells, which is necessary for the determination of cell height, is determined under PLM. The observed height of Chinese hamster ovary (CHO) cells cultivated under higher osmolarity is lower than that of the cells cultivated under physiological osmolarity, which is in agreement with previous data observed under an atomic force microscope (AFM). Maximum heights of human bone marrow mesenchymal stem cells and human umbilical cord vein endothelial cells measured under PLM and AFM agree well with each other. The maximum height of nonadherent spherical CHO cells observed under PLM is comparable to the cell diameter measured under a phase contrast inverted microscope. Laser irradiation, which is necessary for the observation under PLM, did not affect 3-D cell morphology. In conclusion, 3-D morphology of adhered animal cells can be noninvasively measured under PLM.

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells.

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.

Effect of static pressure on intracellular pH of adhesive Chinese hamster ovary cells.

The effect of static pressure on the intracellular pH of the Chinese hamster ovary (CHO) cell line DR1000L4N was investigated. In cultivation of CHO cells at 0.9 MPa, two distinct populations were observed in the histogram of a flow cytometer, while single population was observed in cultivation at 0.1 MPa. The intracellular pH of the major population at 0.9 MPa was markedly lower than that of the single population at 0.1 MPa.

CP/MAS (13)C NMR study of cellulose and cellulose derivatives. 1. Complete assignment of the CP/MAS (13)C NMR spectrum of the native cellulose.

The precise assignments of cross polarization/magic angle spinning (CP/MAS) (13)C NMR spectra of cellulose I(alpha) and I(beta) were performed by using (13)C labeled cellulose biosynthesized by Acetobacter xylinum (A. xylinum) ATCC10245 strain from culture medium containing D-[1,3-(13)C]glycerol or D-[2-(13)C]glucose as a carbon source. On the CP/MAS (13)C NMR spectrum of cellulose from D-[1,3-(13)C]glycerol, the introduced (13)C labeling were observed at C1, C3, C4, and C6 of the biosynthesized cellulose. In the case of cellulose biosynthesized from D-[2-(13)C]glucose, the transitions of (13)C labeling to C1, C3, and C5 from C2 were observed. With the quantitative analysis of the (13)C transition ratio and comparing the CP/MAS (13)C NMR spectrum of the Cladophora cellulose with those of the (13)C labeled celluloses, the assignments of the cluster of resonances which belong to C2, C3, and C5 of cellulose, which have not been assigned before, were performed. As a result, all carbons of cellulose I(alpha) and I(beta) except for C1 and C6 of cellulose I(alpha) and C2 of cellulose I(beta) were shown in equal intensity of doublet in the CP/MAS spectrum of the native cellulose, which suggests that two inequivalent glucopyranose residues were contained in the unit cells of both cellulose I(alpha) and I(beta) allomorphs.