Fox04

Accretion rates of muscle protein are elevated in normal neonates, but this anabolic drive decreases with maturation. As this change occurs, it is not known whether development also influences muscle protein catabolism induced by sepsis. We hypothesize that protein degradation in skeletal muscle induced by endotoxemia becomes more severe as the neonate develops. Fasted 7- and 26-day-old pigs were infused for 8 h with LPS (0 and 10 μg·kg(-1)·h(-1)), while plasma amino acids (AA), 3-methylhistidine (3-MH), and α-actin concentrations and muscle protein degradation signal activation were determined (n = 5-7/group/age). Plasma full-length α-actin was greater in 7- than 26-day-old pigs, suggesting a higher baseline protein turnover in neonatal pigs. LPS increased plasma total AA, 3-MH, and full-length and cleaved α-actin in 26- than in 7-day-old pigs. In muscle of both age groups, LPS increased AMPK and NF-κB phosphorylation, the abundances of activated caspase 3 and E-3 ligases MuRF1 and atrogin1, as well as the abundance of cleaved α-actin, suggesting activation of muscle proteolysis by endotoxin in muscle. LPS decreased Forkhead box 01 (Fox01) and Fox04 phosphorylation and increased procaspase 3 abundance in muscle of 26-day-old pigs despite the lack of effect of LPS on PKB phosphorylation. The results suggest that skeletal muscle in healthy neonatal pigs maintains high baseline degradation signal activation that cannot be enhanced by endotoxin, but as maturation advances, the effect of LPS on muscle protein catabolism manifests its severity.

Background: Differentiation of Ewing sarcoma family of tumors (ESFT) and Ewing-like tumors remains problematic. Certain ESFT with morphological and immunohistochemical (IHC) profiles lack the EWSR1-ETS transcript. To improve diagnostic accuracy we investigated the presence of several specific transcripts in 200 small round cell tumors (SRCT) displaying ESFT morphology and immunophenotype in which EWSR1 FISH analysis was non-informative or negative. Design: 200 tumors (formalin-fixed, paraffin-embedded) were analyzed by RT-PCR. All tumors were tested for EWSR1-ETS, EWSR1/WT1, PAX3/7-FOX01 or SYT/SSX transcripts, and the negative tumors were subsequently analyzed for CIC/DUX4, BCOR/CCNB3 and CIC/FOX04 transcripts. Results: 133 (66.5%) ESFT displayed one of the above EWSR1-ETS translocations. Three cases (1.5%) revealed the SYT-SSX transcript for Synovial sarcoma, and one (0.5%) a EWSR1-WT1 transcript for Desmoplastic Small Round Cell tumor. The CIC-DUX4 translocation was found in six Ewing-like tumors (3%) with CD99 positivity. The BCOR-CCNB3 gene fusion was observed in 5 tumors (2.5%) displaying round or spindle cells with strong CCNB3 IHC expression in 3 tumors. Moreover, RT-PCR failed to detect any gene fusion transcripts in 19 tumors (9.5%) and were considered "undifferentiated small round cell sarcoma" (SRCS). Molecular biology results were non-informative in 33 SRCTs (16.5%) due to RNA degradation through inadequate fixation and/or decalcification. Conclusion: Our analysis of 200 SRCTs confirms the molecular heterogeneity of neoplasms with ESFT morphology and highlight that molecular studies with RT-PCR including new emerging gene fusion transcripts are mandatory for the diagnosis when EWSR1 FISH is negative or non-informative. The incidence of CIC-DUX4, BCOR-CCNB3 and CIC-FOX04 transcripts was relatively low. A small group of Ewing-like sarcomas or undifferentiated SRCS remains unclassified. Adopting appropriate tissue fixation and processing protocols is important to avoid degradation of fixed/embedded tissue when no frozen tumor is available.

Circadian rhythms regulate a wide range of cellular, physiological, metabolic and behavioral activities in mammals. The complexity of tissue- and day-time specific regulation of thousands of clock controlled genes (CCGs) suggests that many transcriptional regulators are involved. Our bioinformatic analysis is based on two published DNA-array studies from mouse liver. We search overrepresented transcription factor binding sites in promoter regions of CCGs using GC-matched controls. Analyzing a large set of CCG promoters, we find known motifs such as E-boxes, D-boxes and cAMP responsive elements. In addition, we find overrepresented GC-rich motifs (Sp1, ETF, Nrf1), AT-rich motifs (TBP, Fox04, MEF-2), Y-box motifs (NF-Y, C/EBP) and cell cycle regulators (E2F, Elk-1). In a subset of system-driven genes, we find overrepresented motifs of the serum response factor SRF and the estrogen receptor ER. The analysis of published ChIP data reveals that some of our predicted regulators (C/EBP, E2F, HNF-1, Myc, MEF-2) target relatively many clock controlled genes. Our analysis of CCG promoters contributes to an understanding of the complex transcriptional regulation of circadian rhythms in liver.