A necrotic stimulus is required to maximize matrix-mediated myogenesis in mice

Biomaterials that are similar to skeletal muscle extracellular matrix have been shown to augment regeneration in ischemic muscle. In this study, treatment with a collagen-based matrix stimulated molecular myogenesis in an mdx murine model of necrosis. Matrix-treated animals ran ≥ 40% further, demonstrating functional regeneration, and expressed increased levels of myogenic transcripts. By contrast, matrix treatment was unable to induce transcriptional or functional changes in an MLC/SOD1G93A atrophic mouse model. In vitro, satellite cells were cultured under standard conditions, on matrix, in the presence of myocyte debris (to simulate a necrotic-like environment) or with both matrix and necrotic stimuli. Exposure to both matrix and necrotic stimuli induced the greatest increases in mef2c, myf5, myoD and myogenin transcripts. Furthermore, conditioned medium collected from satellite cells cultured with both stimuli contained elevated levels of factors that modulate satellite cell activation and proliferation, such as FGF-2, HGF and SDF-1. Application of the conditioned medium to C2C12 myoblasts accelerated maturation, as demonstrated by increased mef2c, myf5 and myogenin transcripts and fusion indexes. In summary, the collagen matrix required a necrotic stimulus to enhance the maturation of satellite cells and their secretion of a myogenic cocktail. Considering that matrix treatment supports myogenesis only in in vivo models that exhibit necrosis, this study demonstrates that a necrotic environment is required to maximize matrix-mediated myogenesis

Metabolites, Metabolic Hormones and Hematological Profiles in Mountain Goats Before the Breeding Season and During the First Trimester of Pregnancy

Objectives were to evaluate the relationships among energy-related metabolites, hormones, and hematological variables in mountain goats (Oreamnos Americans) before the breeding season and during the 1st trimester of pregnancy. Does were from herds in the Palisades (PAL) and NE Yellowstone (NEY) areas. Samples were collected from July to Aug. (before breeding season) and mid-Jan. (1st trimester of pregnancy). Sera was assayed for insulin (I), thyroxine (T4), triiodothyronine (T3), b-OH-butyrate (bOHB), blood urea nitrogen (BUN), and total protein (TP). Concentrations of TP did not differ (P > 0.05) between pregnant (P) and non-breeding season (NB) does. bOHB, I, BUN, and T3 concentrations, and the T3:T4 ratios were greater (P < 0.05) in NB does than in P does. Whereas, T4 concentrations were greater (P < 0.05) in P does than in NB does. Obviously, NB does have a different profiles of metabolites, metabolic hormones and select hematological variables compared to P does.  In conclusion, these differences may be related to P does utilizing and partitioning nutrients to support placental and fetal growth and development. These differences may also be related to the effect of season, since there were no non-pregnant does were sampled in Jan. Another factor that may be important for interpretation of these differences is location. All NB does were sampled in the PAL, while all P does were sampled in the NEY

Characteristics of temporal patterns of cortisol and luteinizing hormone in primiparous, postpartum, anovular, suckled, beef cows exposed acutely to bulls

BACKGROUND: The physiological mechanism by which bulls stimulate resumption of ovarian cycling activity in postpartum, anovular, suckled cows after calving may involve the concurrent activation of the hypothalamic-hypophyseal-ovarian (HPO) axis and hypothalamic-hypophyseal-adrenal (HPA) axis. Thus, the objectives of this experiment were to determine if characteristics of temporal patterns of cortisol and luteinizing hormone (LH) in postpartum, anovular, beef cows are influenced by acute exposure to bulls. The null hypotheses were that daily, temporal characteristics of cortisol and LH concentration patterns do not differ between cows exposed acutely to bulls or steers. METHODS: Sixteen cows were assigned randomly 67 +/- 4 (+/- SE) after calving to be exposed to bulls (EB, n = 8) or steers (ES, n = 8) 5 h daily for 9 d (D 0 to 8). Blood samples were collected daily from each cow via jugular catheters at 15-min intervals for 6 h from 1000 to 1600 h each day. The 5-h exposure period began 1 h after the start of the intensive bleeding period. Characteristics of cortisol and LH concentration patterns (mean, baseline, pulse frequency, pulse amplitude, and pulse duration) were identified by PULSAR analyses. RESULTS: Mean cortisol concentrations decreased (P < 0.05) in cows in both treatments from D 0 to D 2. Thereafter, mean cortisol concentrations stabilized and did not differ (P > 0.10) between EB and ES cows. The decrease in mean cortisol concentrations in EB and ES cows from D 0 to D 2 was attributed to cows acclimatizing to intensive blood sampling and handling procedures. Consequently, analyses for characteristics of cortisol and LH concentration patterns included D 2 through 8 only. Cortisol mean and baseline concentrations, and pulse amplitude did not differ (P > 0.10) between EB and ES cows. However, cortisol pulse duration tended to be longer (P = 0.09) and pulse frequency was lower (P = 0.05) in EB than ES cows. LH pulse frequency was greater (P = 0.06) in EB than ES cows. All other characteristics of LH concentration patterns did not differ (P > 0.10) between EB and ES cows. Characteristics of cortisol concentration patterns were not related to characteristics of LH concentration patterns for ES cows (P > 0.10). However, as cortisol pulse amplitude increased, LH pulse amplitude decreased (b1 = -0.04; P < 0.05) for EB cows. CONCLUSIONS: In conclusion, exposing primiparous, postpartum, anovular, suckled cows to bulls for 5-h daily over a 9-d period did not alter mean concentrations of cortisol or LH compared to mean concentrations of cortisol and LH in cows exposed to steers. However, exposing cows to bull in this manner altered characteristics of temporal patterns of both LH and cortisol by increasing LH pulse frequency and decreasing cortisol pulse frequency. Interestingly, in cows exposed to bulls, as amplitude and frequency of cortisol pulses decreased, amplitudes of LH pulses increased and frequency of LH pulses tended to increase. Thus, the physiological mechanism of the biostimulatory effect of bulls may initially involve modification of the HPA axis and these changes may facilitate activation of the HPO axis and resumption of ovulatory cycles in postpartum, anovular, suckled cows

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond; 35597280

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by ß3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER

Relationship between Fusobacterium nucleatum, inflammatory mediators and microRNAs in colorectal carcinogenesis

AIM To examine the effect of Fusobacterium nucleatum (F. nucleatum) on the microenvironment of colonic neoplasms and the expression of inflammatory mediators and microRNAs (miRNAs). METHODS Levels of F. nucleatum DNA, cytokine gene mRNA (TLR2, TLR4, NFKB1, TNF, IL1B, IL6 and IL8), and potentially interacting miRNAs (miR-21-3p, miR-22-3p, miR-28-5p, miR-34a-5p, miR-135b-5p) were measured by quantitative polymerase chain reaction (qPCR) TaqMan® assays in DNA and/or RNA extracted from the disease and adjacent normal fresh tissues of 27 colorectal adenoma (CRA) and 43 colorectal cancer (CRC) patients. KRAS mutations were detected by direct sequencing and microsatellite instability (MSI) status by multiplex PCR. Cytoscape v3.1.1 was used to construct the postulated miRNA:mRNA interaction network. RESULTS Overabundance of F. nucleatum in neoplastic tissue compared to matched normal tissue was detected in CRA (51.8%) and more markedly in CRC (72.1%). We observed significantly greater expression of TLR4, IL1B, IL8, and miR-135b in CRA lesions and TLR2, IL1B, IL6, IL8, miR-34a and miR-135b in CRC tumours compared to their respective normal tissues. Only two transcripts for miR-22 and miR-28 were exclusively downregulated in CRC tumour samples. The mRNA expression of IL1B, IL6, IL8 and miR-22 was positively correlated with F. nucleatum quantification in CRC tumours. The mRNA expression of miR-135b and TNF was inversely correlated. The miRNA:mRNA interaction network suggested that the upregulation of miR-34a in CRC proceeds via a TLR2/TLR4-dependent response to F. nucleatum. Finally, KRAS mutations were more frequently observed in CRC samples infected with F. nucleatum and were associated with greater expression of miR-21 in CRA, while IL8 was upregulated in MSI-high CRC. CONCLUSION Our findings indicate that F. nucleatum is a risk factor for CRC by increasing the expression of inflammatory mediators through a possible miRNA-mediated activation of TLR2/TLR4We thank Lucas Trevizani Rasmussen for kindly donating some miRNA probes. We are grateful to the São Paulo Research Foundation (FAPESP, NO. 2015/21464-0) for the support for English revision, the Coordination for the Improvement of Higher Education Personnel (CAPES) for the doctoral scholarship, and the National Council for Scientific and Technological Development (CNPq, NO. 310120/2015-2) for the productivity research scholarship.info:eu-repo/semantics/publishedVersio