TGFß carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Contributions of TGFß to cancer progression are well documented. However, plasma TGFß levels often do not correlate with clinicopathological data. We examine the role of TGFß carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFß expression levels during oral carcinogenesis. In human HNSCC, TGFß and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFß levels were evaluated by ELISA and TGFß bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFß content was quantified using bioassays and bioprinted microarrays. RESULTS: During 4-NQO carcinogenesis, TGFß levels in tumour tissues and in serum increased as the tumour progressed. The TGFß content of circulating exosomes also increased. In HNSCC patients, TGFß, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFß levels. Neither TGFß expression in tumours nor levels of soluble TGFß correlated with clinicopathological data or survival. Only exosome-associated TGFß reflected tumour progression and correlated with tumour size. CONCLUSIONS: Circulating TGFß+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

Medical student judgments of adolescents with alcohol use disorders (AUD).

UNLABELLED: The clinical encounter presents opportunities for detection and intervention of adolescent alcohol use disorders (AUDs). AIMS: Investigate (a) identification rate of AUDs, (b) whether AUD identification predicts clinical judgment, and (c) patient characteristics influences on clinical judgment. Medical students (n=123) read a case study and completed questions on diagnosis and clinical judgment. Twenty-five percent of participants identified AUD adolescents, who were more negatively rated than non-AUD adolescents. Prior clinical experience and addiction training predicted AUD identification. Patient race and gender influenced clinical judgment ratings. Addictions training is needed to improve identification rates. Study limitations are noted.

Regulation of skeletal muscle fiber type and slow myosin heavy chain 2 gene expression by inositol trisphosphate receptor 1.

Innervation-dependent signaling cascades that control activation of downstream transcription factors regulate expression of skeletal muscle fiber type-specific genes. Many of the innervation-regulated signaling cascades in skeletal muscle are dependent on intracellular calcium and the mechanisms by which calcium is released from the sarcoplasmic reticulum (SR). We report that the inositol trisphosphate receptor 1 (IP3R1), responsible for calcium release from the SR as a slow wave, was more abundant in fast contracting compared to slow contracting avian muscle fibers. Furthermore, inhibition of IP3R1 activity by 2-aminoethoxydiphenylborate (2-APB) and xestospongin D induced a fiber type transition and expression of the slow myosin heavy chain 2 (slow MyHC2) gene in innervated fast muscle fibers. Activation of the slow MyHC2 promoter by IP3R1 inhibition was accompanied by a reduction in protein kinase C activity. In addition, inhibition of IP3R1 activity resulted in a reduction of nuclear factor of activated T cells (NFAT)-dependent transcription and nuclear localization, indicating that IP3R1 activity regulated NFAT transcription factor activity in skeletal muscle fibers. Myocyte enhancer factor 2 (MEF2)-dependent transcriptional activity was increased by innervation, but unaffected by IP3R1 activity. The results indicate that IP3R1 activity regulates muscle fiber type-specific gene expression in innervated muscle fibers.

Inhibition of ryanodine receptor 1 in fast skeletal muscle fibers induces a fast-to-slow muscle fiber type transition.

Skeletal muscle fiber type is regulated by innervation-induced cell signaling including calcium release mechanisms that lead to transcriptional activation of fiber type-specific genes. Avian fast pectoralis major (PM) and slow medial adductor (MA) muscles differentially control expression of the slow myosin heavy chain 2 (slow MyHC2) gene. We report here that slow MyHC2 gene expression in fast PM muscle fibers is repressed by endogenous activity of the ryanodine receptor 1 (RyR1). Inhibition of RyR1 with ryanodine led to expression of the slow MyHC2 gene in innervated PM muscle fibers in vitro. Administration of ryanodine to innervated PM muscle fibers also decreased protein kinase C (PKC) activity, the reduction of which is necessary for slow MyHC2 gene expression in both PM and MA muscle fibers. Furthermore, RyR1 inhibition increased slow MyHC2 promoter activity in innervated PM muscle fibers and enhanced transcriptional activities of nuclear factor of activated T cells (NFAT) and myocyte enhancer factor 2 (MEF2), as well as their interactions with their respective binding sites of the slow MyHC2 promoter. These results indicate that RyR1 activity in innervated fast PM muscle fibers contributes to the cell type-specific repression of slow muscle specific genes.

Innervation-dependent and fiber type-specific transcriptional regulation of the slow myosin heavy chain 2 promoter in avian skeletal muscle fibers.

Skeletal muscle fiber type is regulated, in part, by innervation leading to transcriptional regulation of fiber type-specific genes. Here, we report the initial characterization of the transcriptional regulation of the slow myosin heavy chain 2 (MyHC2) promoter in innervated and noninnervated slow medial adductor (MA) and fast pectoralis major (PM) muscle fibers in cell culture. The proximal 1358 bp of slow MyHC2 upstream DNA contains a functional E-box and binding sites for myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT). Mutagenesis studies indicated that both MEF2 and NFAT binding sites are required for innervation-induced slow MyHC2 promoter activity in MA muscle fibers. However, MEF2 transcription factor activity was unaffected by innervation and did not demonstrate fiber type-specific interactions with the slow MyHC2 MEF2 binding site. NFAT transcription factor activity did increase in innervated MA muscle fibers and not in PM muscle fibers, indicating innervation and muscle fiber type-specific regulation. However, transfection of constitutively active NFAT indicated that NFAT is insufficient to induce slow MyHC2 gene expression in either fast PM or slow MA muscle fibers without innervation. These results indicate the requirement for MEF2 and NFAT in innervation-induced slow MyHC2 gene expression and suggest that additional innervation-dependent and fiber type-specific control of slow MyHC2 gene expression resides in MA and PM muscle fibers, respectively.

Repression of slow myosin heavy chain 2 gene expression in fast skeletal muscle fibers by muscarinic acetylcholine receptor and G(alpha)q signaling.

Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and G(alpha)q. Both mAchR and G(alpha)q are abundant in medial adductor (MA) and PM fibers, and mAchR and G(alpha)q interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 expression, inhibition of mAchR activity with atropine in innervated PM fibers induced slow MyHC2 expression. Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers. Reduced mAchR activity decreased PKC activity in PM fibers, and increased G(alpha)q activity increased PKC activity in PM and MA fibers. Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression. These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.

A lack of dimorphism of sex or sexual orientation in the human anterior commissure.

Four studies have examined the cross-sectional area of the anterior commissure (AC) for variation with sex, with conflicting results. One also reported the AC to be larger in homosexual as opposed to heterosexual men. We examined the cross-sectional area of the AC in postmortem material from 120 individuals, and found no variation in the size of the AC with age, HIV status, sex, or sexual orientation.