Postnatal development of mouse spermatogonial stem cells as determined by immunophenotype, regenerative capacity, and long-term culture-initiating ability: a model for practical applications.

Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs.

Highly Effective and Efficient Self-Assembled Multilayer-Based Electrode Passivation for Operationally Stable and Reproducible Electrolyte-Gated Transistor Biosensors.

To ensure the operational stability of transistor-based biosensors in aqueous electrolytes during multiple measurements, effective electrode passivation is crucially important for reliable and reproducible device performances. This paper presents a highly effective and efficient electrode passivation method using a facile solution-processed self-assembled multilayer (SAML) with excellent insulation property to achieve operational stability and reproducibility of electrolyte-gated transistor (EGT) biosensors. The SAML is created by the consecutive self-assembly of three different molecular layers of 1,10-decanedithiol, vinyl-polyhedral oligomeric silsesquioxane, and 1-octadecanethiol. This passivation enables EGT to operate stably in phosphate-buffered saline (PBS) during repeated measurements over multiple cycles without short-circuiting. The SAML-passivated EGT biosensor is fabricated with a solution-processed In2O3 thin film as an amorphous oxide semiconductor working both as a semiconducting channel in the transistor and as a functionalizable biological interface for a bioreceptor. The SAML-passivated EGT including In2O3 thin film is demonstrated for the detection of Tau protein as a biomarker of Alzheimer's disease while employing a Tau-specific DNA aptamer as a bioreceptor and a PBS solution with a low ionic strength to diminish the charge-screening (Debye length) effect. The SAML-passivated EGT biosensor functionalized with the Tau-specific DNA aptamer exhibits ultrasensitive, quantitative, and reliable detection of Tau protein from 1 × 10-15 to 1 × 10-10 M, covering a much larger range than clinical needs, via changes in different transistor parameters. Therefore, the SAML-based passivation method can be effectively and efficiently utilized for operationally stable and reproducible transistor-based biosensors. Furthermore, this presented strategy can be extensively adapted for advanced biomedical devices and bioelectronics in aqueous or physiological environments.

Dissecting the spermatogonial stem cell niche using spatial transcriptomics.

Spermatogonial stem cells (SSCs) in the testis support the lifelong production of sperm. SSCs reside within specialized microenvironments called "niches," which are essential for SSC self-renewal and differentiation. However, our understanding of the molecular and cellular interactions between SSCs and niches remains incomplete. Here, we combine spatial transcriptomics, computational analyses, and functional assays to systematically dissect the molecular, cellular, and spatial composition of SSC niches. This allows us to spatially map the ligand-receptor (LR) interaction landscape in both mouse and human testes. Our data demonstrate that pleiotrophin regulates mouse SSC functions through syndecan receptors. We also identify ephrin-A1 as a potential niche factor that influences human SSC functions. Furthermore, we show that the spatial re-distribution of inflammation-related LR interactions underlies diabetes-induced testicular injury. Together, our study demonstrates a systems approach to dissect the complex organization of the stem cell microenvironment in health and disease.

The Effects of Transforming Growth Factor-ß1 on the Differentiation of Cell Organoids Composed of Gingiva-Derived Stem Cells.

This study was aimed at evaluating the effects of transforming growth factor-ß on the differentiation and mRNA expression of organoids made out of human mesenchymal stem cells. Cell organoids composed of gingiva-derived stem cells were cultured in the presence of transforming growth factor-ß1 at concentrations ranging from 0, 1, 10, to 20 ng/ml. Evaluations of the cell morphology of the organoids were performed on days 7, 9, 11, and 14. Quantitative cellular viability was completed on day 14. Alkaline phosphatase activity assays were performed to evaluate the differentiation of stem cells on day 14. Real-time polymerase chain reactions were used to determine the expression levels of TGF-ß1, RUNX2, OCN, SOX9, and COL1A1 mRNA on day 14. The stem cells produced well-formed organoids on day 7, and the addition of transforming growth factor-ß1 did not result in relevant changes in their shape. The organoids grew in size and became more intact with longer incubation times. On day 14, the diameters were 222.2 ± 9.6, 186.1 ± 4.8, 197.2 ± 9.6, and 211.1 ± 19.2 m for transforming growth factor-ß1 at final concentrations of 0, 1, 10, and 20 ng/ml, respectively. Quantitative cell viability results from day 14 exhibited no significant difference between the groups (P > 0.05). There was significantly higher alkaline phosphatase activity with the addition of transforming growth factor-ß1 with the highest value for the 1 ng/ml group (P < 0.05). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and SOX were higher in 1 ng/ml but did not reach statistical significance. Treatment with 1 ng/ml of transforming growth factor-ß1 significantly increased COL1A1 mRNA expression at day 14. The application of transforming growth factor-ß1 increased differentiation, which was confirmed by alkaline phosphatase activity and mRNA expression while maintaining cell viability.

Effects of the slaughter weight of non-lean finishing pigs on their carcass characteristics and meat quality.

The present study aimed to assess the feasibility of increasing the slaughter weight (SW) of non-lean finishing pigs to improve their meat quality. A total of 36 (Landrace × Yorkshire) × Duroc gilts and barrows were slaughtered at 115 (Av), 125 (Hi), or 135 (XHi) kg, followed by physicochemical analyses and sensory evaluation on their longissimus dorsi (LD) and Semimembranosus (SM) muscles. Backfat thickness was greater (p < 0.05) for the XHi (31.2 mm) and Hi (29.3 mm) groups than for Av (25.0 mm). Dressing percentage and yield of the belly per whole carcass were also slightly greater for XHi and Hi vs. Av. The intramuscular fat (IMF) content of SM was greater for XHi (2.64%) than for Av (1.83%) and Hi (2.04%) and also was correlated with SW (r = 0.55). The pH value, lightness, redness, drip loss, shear force, and moisture and protein contents of LD and SM, as well as IMF content of LD, were unaffected by SW. Percentages of 14:0, 16:0, and total saturated fatty acids (FA) were less for Hi and XHi vs. Av in SM, those of total unsaturated FA, 18:2, 20:4, and n-6 being opposite; FA composition of LM was not influenced by SW except for a reduced 18:0 percentage for XHi vs. Av. The sensory score was less for XHi vs. Av for odor in fresh LD and SM, and less for Hi and XHi vs. Av for aroma in fresh LM; scores for color, drip loss, marbling, and acceptability were unaffected by SW. As for cooked muscles, none of the scores for color, aroma, flavor, juiciness, tenderness, and acceptability was affected by SW, except for a greater LD color score for Hi and XHi vs. Av. Collectively, the results suggested that the increased yield of the carcass and belly due to increased SW is outbalanced negatively by excessive backfat deposition in production efficiency, whereas the SW increase exerts little influence on overall sensory quality of fresh or cooked meat. Production of non-lean market pigs overweighing 115 kg therefore will be uneconomical unless consumers pay a substantial premium for the over-fattened pork.

Correction to: No prominent toxicity of polyethylene microplastics observed in neonatal mice following intratracheal instillation to dams during gestational and neonatal period.

[This corrects the article DOI: 10.1007/s43188-020-00086-7.].

Effects of noni on cellular viability and osteogenic differentiation of gingiva-derived stem cells demonstrated by RNA sequencing and quantitative PCR.

Noni fruit (Morinda citrifolia) has been widely used in traditional medicine across tropical and subtropical regions, and is now being paid more attention in Western medicine. The present study aimed to investigate the effects of noni extract on the change in the cellular morphology, maintenance of cellular viability and enhancement of osteogenic differentiation of stem cells. Stem cells obtained from gingiva were cultured where noni extracts existed at concentrations ranging from 10-200 ng/ml. Evaluations of cell morphology and cellular viability were performed. Alkaline phosphatase activity assays were performed to assess the osteogenic differentiation. Alizarin Red S staining was performed to evaluate the calcium deposits in the culture, with the addition of noni extract. Global gene expression was analyzed via next-generation mRNA sequencing. Gene ontology and pathway analyses were performed to determine the associated mechanisms. Validation procedures were performed via quantitative (q)PCR analysis. The addition of noni at concentrations ranging from 10-200 ng/ml did not produce significant morphological changes. There were significantly higher values of cellular viability, with the highest value at 100 ng/ml compared with the control (P<0.05). Furthermore, significantly higher values of alkaline phosphatase activity was noted in the 10 and 100 ng/ml groups compared with the 0 ng/ml group on day 7 (P<0.05). Alizarin Red S staining revealed calcium deposits in each group. In addition, the highest value for Alizarin Red S staining was observed at 100 ng/ml compared with the unloaded control (P<0.05). qPCR analysis demonstrated that the mRNA expression levels of RUNX2, BSP, OCN and COL1A1 increased following treatment with noni. Taken together, the results of the present study suggest that noni extract has enhancing effects on gingiva-derived mesenchymal stem cells, by enhancing cellular viability and osteogenic differentiation.

A Study on the Relationship between Mental Well-Being and Cultural Tourism Guides Based on the Interview Methodology.

The purpose of this study was to find a way for modern tourists to enjoy increased well-being while being provided with high-quality information about cultural assets. In order for tourists to enjoy well-being, cultural tourism guides must provide quality services while using various storytelling techniques. As the number of tourists who are interested in cultural assets and use their leisure time for this purpose increases, quality cultural tourism commentary can be directly connected to the well-being of tourists. Modern tourists can experience richness of life and emotional stability while being provided with cultural tourism commentary services through various storytelling techniques rather than professional knowledge. In order for tourists to effectively experience well-being through cultural tourism commentary, cultural tourism guides need to implement the following effective commentary. First, culture tourism guides should try to have sense of unity with visitors. Second, they should encourage humanistic imagination through related information. Third, they should provide customized explanations for tourists' understanding because tourists consist of various classes and ages. Cultural tourism guides who attract tourists' interest, have appropriate wit, skillful responses to cope with unexpected situations, cheerful laughter, and a loud voice gained satisfaction from many tourists. In modern society, cultural tourism commentators are not limited to simply explaining tourist destinations, but play an important role in satisfying the well-being of modern tourists who seek leisure and emotional stability. The external environment that refers to outdoor atmospherics is also crucial as it influences visitor experiences. In museums, external physical environment factors such as architectural style, positioning of entrances, and exterior décor and signage can be a crucial facet of external ambiance affecting visitor experiences. The external environment (e.g., spacious design, pretty landscape design, outdoor natural surroundings) is a constituent of the tangibility aspect of museum performances. These external environment factors and internal factors together create perceived physical environments that visitors and staff cognitively/emotionally/physiologically respond to. The significance of this study is that various cultural storytelling activities performed by tour guides are examining the possibility of experiencing increased psychological well-being while making tourists as well as themselves aware of the happiness and joy of life.

Vitamin D Enhanced the Osteogenic Differentiation of Cell Spheroids Composed of Bone Marrow Stem Cells.

Background and Objectives: Vitamin D is a bone modulator widely used in regenerative medicine. This study aimed to analyze the effects of vitamin D on the osteogenic differentiation and mineralization of human mesenchymal stem cells. Materials and Methods: Spheroids were fabricated using human bone marrow-derived stem cells, and were cultured in the presence of vitamin D at concentrations of 0, 0.1, 1, 10, and 100 nM. Stem cell spheroids were fabricated and the morphological evaluation was conducted on days 1, 3, 7 and 14. Determination of qualitative cellular viability was performed with Live/Dead Kit assay on days 1 and 7. Quantitative cellular viability was evaluated with Cell Counting Kit-8 on days 1, 3, 7, and 14. To analyze the osteogenic differentiation of cell spheroids, alkaline phosphatase activity assays were performed with commercially available kit on days 7 and 14. Real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, and COL1A1 on days 7 and 14. Results: The stem cells produced well-formed spheroids, and addition of vitamin D did not result in any noticeable changes in the shape. The addition of vitamin D did not significantly change the diameter of the spheroids at 0, 0.1, 1, 10, or 100 nM concentrations. Quantitative cell viability results from days 1, 3, 7 and 14 showed no significant difference between groups (p > 0.05). There was significantly higher alkaline phosphatase activity in the 0.1 nM group when compared with the control group on day 14 (p < 0.05). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and COL1A1 were significantly increased when vitamin D was added to the culture. Conclusions: Based on these findings, we concluded that vitamin D could be applied to the increased osteogenicity of stem cell spheroids.

No prominent toxicity of polyethylene microplastics observed in neonatal mice following intratracheal instillation to dams during gestational and neonatal period.

Microplastics (MPs) have been recently recognized as a global environmental threat and its exposure as a risk factor to human health. Health effects through MPs exposure have been recently reported, especially through oral route of exposure. Since MPs could be exposed to humans through routes other than oral, this study was designed to evaluate whether MPs exposed through the inhalation route could be delivered to fetal mice and exhibit systemic toxicity. Polyethylene (PE) with 10-45 µm diameter were administered at 0 (distilled water, vehicle control), 6 (low administration), and 60 (high administration) µg/mouse/day to 3 pregnant dams per group from gestational day 9 to postnatal day (PND) 7 through intratracheal instillation. Dams and neonates were sacrificed at PND 7 and blood was collected. Various neonatal organs including brain, lung, heart, stomach, intestine, kidneys, and ovaries were collected for histopathological observation and weight measurement. No influence of PE-MPs administration was observed on the number of offsprings born, but the body and organs' weight were heavier overall in the high administration group of dams and neonates than the other groups with statistical significance achieved in the heart and spleen weight. Level of serum acetylcholinesterase and glutathione peroxidase activity was decreased in the high administration group of dams and neonates compared with the other groups. Lung was the organ with highest number of PE-MPs present in the both administration groups of dams, and PE-MPs were also detected in liver and intestine of the high administration dams. Whereas, PND7 neonates showed accountable numbers of PE-MPs only in kidneys of the high administration group. Overall, the present study indicates that PE-MPs instilled intratracheally could be delivered to neonates from dams. Even though adverse effects from PE-MPs exposure during pregnant and lactational period are less prominent on both dam and neonate, potential of second-generation toxicity could be considered for further investigation.

NELL-1 Increased the Osteogenic Differentiation and mRNA Expression of Spheroids Composed of Stem Cells.

Background and objectives: NELL-1 is a competent growth factor and it reported to target cells committed to the osteochondral lineage. The secreted, osteoinductive glycoproteins are reported to rheostatically control skeletal ossification. This study was performed to determine the effects of NELL-1 on spheroid morphology and cell viability and the promotion of osteogenic differentiation of stem cell spheroids. Materials and Methods: Cultures of stem cell spheroids of gingiva-derived stem cells were grown in the presence of NELL-1 at concentrations of 1, 10, 100, and 500 ng/mL. Evaluations of cell morphology were performed using a microscope, and cell viability was assessed using a two-color assay and Cell Counting Kit-8. Evaluation of the activity of alkaline phosphatase and calcium deposition assays involved anthraquinone dye assay to determine the level of osteogenic differentiation of cell spheroids treated with NELL-1. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expressions of RUNX2, BSP, OCN, COL1A1, and ß-actin mRNAs. Results: The applied stem cells produced well-formed spheroids, and the addition of NELL-1 at tested concentrations did not show any apparent changes in spheroid shape. There were no significant changes in diameter with addition of NELL-1 at 0, 1, 10, 100, and 500 ng/mL concentrations. The quantitative cell viability results derived on Days 1, 3, and 7 did not show significant disparities among groups (p > 0.05). There was statistically higher alkaline phosphatase activity in the 10 ng/mL group compared with the unloaded control on Day 7 (p < 0.05). A significant increase in anthraquinone dye staining was observed with the addition of NELL-1, and the highest value was noted at 10 ng/mL (p < 0.05). qPCR results demonstrated that the mRNA expression levels of RUNX2 and BSP were significantly increased when NELL-1 was added to the culture. Conclusions: Based on these findings, we conclude that NELL-1 can be applied for increased osteogenic differentiation of stem cell spheroids.

Evaluation of the Effects of Cuminum cyminum on Cellular Viability, Osteogenic Differentiation and Mineralization of Human Bone Marrow-Derived Stem Cells.

Background and Objectives: Cuminum cyminum L. has long been used in the treatment of various diseases in multiple geographical regions. This study was performed to determine the effects of C. cyminum methanolic extract (CCT) on the cellular viability, alkaline phosphatase activity and mineralization of human mesenchymal stem cells. Materials and Methods: Bone marrow-derived stem cells were cultured in the presence of CCT at concentrations of 0, 0.001, 0.01, 0.1 and 1 µg/mL. Evaluations of cell morphology were performed on days 1, 3, 7 and 14. Cellular viability was evaluated on days 1, 3, 5 and 7. On the 7th and 14th day, alkaline phosphatase activity measurements and Alizarin red S staining were conducted to assess the osteogenic differentiation of stem cells. A real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, COL2A1 and ß-catenin mRNAs. Results: Stem cells in the control group showed fibroblast-like morphology and the addition of CCT at 0.001, 0.01, 0.1 and 1 µg/mL did not generate noticeable changes in morphology compared with the untreated control group. The application of CCT did not produce significant changes in cellular viability or alkaline phosphatase activity compared with controls. Alizarin Red S staining was significantly increased with the application of CCT. Treatment with CCT increased the expressions of RUNX2, BSP and OCN. Conclusions: These results indicate that CCT enhanced the osteogenic differentiation of stem cells derived from bone marrow by regulating the expressions of RUNX2, BSP and OCN. Thus, the use of CCT may be applied to achieve beneficial effects on the mineralization of stem cells.

Saccadic adaptation in the presence of artificial central scotomas.

Saccadic adaptation can occur over a short period of time through a constant adjustment of the saccade target during the saccade, resulting in saccadic re-referencing, which directs the saccade to a location different from the target that elicited the saccade. Saccade re-referencing could be used to help patients with age-related macular degeneration to optimally use their residual visual function. However, it remains unknown whether saccade adaptation can take place in the presence of central scotomas (i.e., without central vision). We tested participants in two experiments in a conventional double-step paradigm with a central gaze-contingent artificial scotoma. Experiment 1 (N = 12) comprised a backward adaptation paradigm with no scotoma control, visible, and invisible 3° diameter scotoma conditions. Experiment 2 (N = 13) comprised a forward adaptation paradigm with no scotoma control, invisible 2°, and 4° diameter scotoma conditions. In Experiment 1, we observed significant adaptation in both the visible and invisible scotoma conditions comparable to the control condition with no scotoma. This was the case even when the saccade landed such that the target was occluded by the scotoma. We observed that adaptation occurred based on peripheral viewing of the stepped target during the deceleration period. In Experiment 2, we found that both scotoma conditions showed adaptation again comparable to the control condition with no scotoma. We conclude that saccadic adaptation can occur with central scotomas, showing that it does not require central vision and can be driven primarily by peripheral retinal error.

Morphological stability, cellular viability and stem cell marker expression of three-dimensional cultures of stem cells from bone marrow and periodontium.

The aim of the present study was to evaluate the morphology, cellular viability and stem cell marker expression of three-dimensional cultures of bone marrow and gingiva-derived stem cells in different ratios. Stem cell spheroids were made with bone marrow and gingiva-derived stem cells using ratios of 6:0 (Group 1), 4:2 (Group 2), 3:3 (Group 3), 2:4 (Group 4) and 0:6 (Group 5), respectively. The viability of cell spheroids was analyzed using a Live/Dead kit assay and a Cell Counting Kit-8 assay. Total RNA extraction and reverse transcription-quantitative PCR were performed to detect the mRNA expression levels of Nanog and ß-actin in each group. Stem cell spheroids were well formed in silicone elastomer-based concave microwells with different ratios of bone marrow and gingiva-derived stem cells. The shape of the spheroids and their viability were maintained throughout the entirety of the experimental procedure. Statistically significant increases in spheroid diameters were noted in Groups 4 and 5 on day 1 when compared with Group 1 on day 1. There was a significant increase in the cell viability values seen in Group 3 on day 1 when compared with Group 1 on day 1. Highest levels of Nanog expression was seen in Group 3 on day 10, but the increase was not significant when compared with Group 1 on day 1. Co-culturing with higher ratios of gingiva-derived stem cells produced stem cell spheroids with larger diameters and increased cellular viability. This co-culture technique may be used in stem cell therapy with allogenic stem cell transplantation.

Bone morphogenetic protein-7 upregulates genes associated with osteoblast differentiation, including collagen I, Sp7 and IBSP in gingiva-derived stem cells.

The present study was performed to evaluate the effects of short-term application of bone morphogenetic protein-7 (BMP-7) on human gingiva-derived mesenchymal stem cells with next-generation sequencing. Human gingiva-derived stem cells were treated with a final concentration of 100 ng/ml BMP-7 and the same concentration of a vehicle control. mRNA sequencing and data analysis were performed along using gene ontology and pathway analysis. RT-qPCR of mRNA of collagen I, Sp7, IBSP and western blot analysis of collagen I, osterix and bone sialoprotein was also performed. A total of 25,737 mRNAs were identified to be differentially expressed. Regarding osteoblast differentiation, 14 mRNAs were upregulated and 10 were downregulated when the results of the BMP-7 at 3 h were compared with the control at 3 h. The expression of collagen I was increased following the application of BMP-7 at 3 h, and this increase was also observed following western blot analysis. The effects of BMP-7 on stem cells were evaluated with mRNA sequencing, and the expression was validated with RT-qPCR and western blot analysis. The short-term application of BMP-7 produced an increased expression of collagen I, which was associated with target genes selected for osteoblast differentiation. This study may provide novel insights into the role of BMP-7 using mRNA sequencing.

Effects of the plane of nutrition for grower pigs on their grow-finish performance and meat quality in winter.

Little is known about the effects of the plane of nutrition on growth performance and meat quality of grow-finish pigs under commercial production conditions. The present study was thus addressed to this virtually unanswered question. One hundred and two barrows and 102 gilts weighing approximately 24 kg were fed phase I and II grower diets with a high, medium, or low plane of nutrition (HP, MP, or LP) to approximately 43 and 70 kg, respectively, in 6 replicates (pens). Subsequently, the HP and MP groups were fed the HP and MP1 finisher diets, respectively, the LP group being fed a second MP (MP2) finisher diet (LP1 group). Moreover, 68 LP-grower-fed barrows and gilts were added to the feeding trial and fed the MP1 and LP finisher diets to approximately 95 kg and thereafter, respectively (LP2 group). All MP diets had the lysine:calorie ratios comparable to the RNC recommendations, with < 18% differences between those of the HP and LP diets. The finisher pigs were reared in 16 pens and slaughtered at approximately 115 kg. The gain:feed ratio, but not average daily gain (ADG), was greater for the HP group than for the MP and LP during the grower phase I whereas during the grower phase II, ADG was greater (p < 0.05) for the HP and LP groups vs. MP. During the finisher phase I, ADG was less for the LP (LP1 + LP2) group vs. HP and MP, with no difference between the HP and MP groups; the gain:feed ratio was less for the LP vs. MP group. Backfat thickness was greater for the LP vs. HP group. The water holding capacity of fresh longissimus dorsi muscle (LM) and the sensory juiciness score for cooked LM were greatest for the LP group, the sensory flavor and tenderness scores being greater for the LP group vs. MP. In conclusion, results suggest that compensatory growth occurred for the LP and MP groups during the grower phase II and finisher phase I, respectively, with fat deposition increased for the LP group and that meat quality could be improved by the use of LP.

Effects of the plane of nutrition during the latter grower and entire finisher phases on grow-finish pig performance in summer.

The present study was undertaken to investigate the effects of the plane of nutrition (PN) for growing-finishing pigs on growth performance and meat quality in summer. One hundred and two barrows and 102 gilts weighing approximately 44 kg were placed on a high-, medium-, or low-plane grower diet (HPG, MPG, or LPG) with ME and lysine concentrations ranging from 3.33 to 3.40 Mcal/kg and 0.93% to 1.15%, respectively, for 29 days in 6 replicates (pens) in total. Pigs from each grower pen were divided into two finisher pens and provided with a high-plane finisher diet (HPF) containing 3.40 Mcal ME and 9.5 g lysine/kg and a low-plane finisher diet (LPF; 3.25 Mcal ME and 8 g lysine/kg), respectively, up to approximately 110 kg, and slaughtered. Growth performance of the pigs, including average daily gain (ADG), average daily feed intake (ADFI), and gain:feed ratio, was not influenced by the grower-phase PN during any of the grower phase, a 31-d finisher phase I, and ensuing phase II. However, both the ADG and gain:feed ratio were greater (p < 0.05) for the HPF group than for the LPF group during the finisher phase I (748 vs. 653 g with SEM = 13 g and 0.333 vs. 0.299 with SEM = 0.008, respectively). The ADG, but not gain:feed ratio, was greater for the HPF group vs. LPF during the finisher phase II (673 vs. 623 g with SEM = 15 g for ADG and 0.322 vs. 0.323 with SEM = 0.005 for the gain:feed ratio). The carcass backfat thickness (BFT) was greater for the LPF group vs. HPF within the pigs which had been placed on LPG during the grower phase, but not within the pigs from the HPG or MPG group. Physicochemical characteristics of the longissimus dorsi muscle (LM) and sensory quality attributes of fresh and cooked LM were not influenced by either the grower-phase or finisher-phase PN. In conclusion, high PN is necessary for finishing pigs during the hot season to minimize the reduced rate of weight gain and also to prevent the increase of BFT that could result from low PN.

Characterization of the binding sites for the interactions between FKBP12 and intracellular calcium release channels.

FKBP12, an FK506 binding protein, interacts with type 1 ryanodine receptor (RyR1) and modulates its calcium channel activity. However, there are many opposing reports of FKBP12's interaction with other related calcium channels, such as type 1 IP(3) receptor and type 3 ryanodine receptor (IP(3)R1 and RyR3). In addition, the involvement of the prolyl-dipeptide motif in the calcium channels and the corresponding binding residues in FKBP12 remain controversial. Through pulldown assays with recombinant proteins, we provide biochemical evidence of the interaction between FKBP12 and RyR1, RyR3 and IP(3)R1. Using NMR chemical shift mapping, we show that the important binding residues in FKBP12 are located in its hydrophobic FK506 binding region. Consistently, we demonstrate that FK506 can competitively inhibit the interaction between FKBP12 and the dipeptide motifs of the calcium channels. We believe our results shed lights on the binding mechanism of calcium channel-FKBP12 interaction.

Global dynamic conformational changes in the suppressor domain of IP3 receptor by stepwise binding of the two lobes of calmodulin.

The roles of calmodulin (CaM) have been key points of controversy in the regulation of inositol-1,4,5-trisphosphate receptor (IP(3)R). To address the issue, we studied the interaction between CaM and the suppressor domain of IP(3)R, a key allosteric regulatory domain. First, by means of a pulldown and a fluorescence titration experiment, we confirmed the interaction. Through subsequent NMR binding experiments, we observed dramatic peak disappearances of the suppressor domain on interaction with apo-CaM. The data indicated that apo-CaM induces large-scale dynamic conformational changes in the suppressor domain, involving partial unfolding and subdomain rearrangement. Analysis of the NMR data of CaM surprisingly revealed that its C lobe alone can cause such changes. Further binding experiments showed that calcium allows the free N lobe to bind to the suppressor domain, which induces extra conformational changes in both of the proteins. These results were also confirmed with CaM deletion mutants with either the N or C lobe. On the basis of this novel binding mechanism, we propose a model in which the partial unfolding of the suppressor domain by apo-CaM and the stepwise binding of the N lobe of CaM to the suppressor domain are important elements of calcium/CaM inhibition of IP(3)R. We believe that our working model encompasses previous regulation mechanisms of IP(3)R by calcium/CaM and provides new insights into the CaM-target interaction.

Differentiation of antlers from deer on different feeds using an NMR-based metabolomics approach.

Deer antler has been widely used as a dietary supplement for hundreds of years in Asian countries. The chemical composition of deer antlers strongly depends on the growth conditions of the deer, especially the feeds, but the effects of different feeds on deer antlers have not been studied. To expand our knowledge of the chemical constituents of deer antler and establish an efficient way of differentiating antlers obtained with different feeds, we applied an NMR-based metabolomics approach and OPLS-DA multivariate analysis. We show that the antlers from one species on two different feeds, made from grass or mulberry trees, can be reliably differentiated by our metabolomics approach. We identified chemical constituents of the deer antlers and the marker compounds that contribute to the difference between the feed groups. We also rigorously validated our differentiation approach by showing that it can correctly classify blind samples into their respective feed groups. Our approach is expected to help design feeds to produce antlers with more defined constituents, especially those with higher bioactivities.