Direct Detection of miR-122 in Hepatotoxicity Using Dynamic Chemical Labeling Overcomes Stability and isomiR Challenges.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions.

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2(0), whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.

Ultrasound of the normal canine supraspinatus tendon: comparison with gross anatomy and histology.

BACKGROUND: This study aimed to compare the ultrasonographic and histological features of the supraspinatus tendon (ST) and its peculiar appearance in contrast with the biceps brachii tendon. METHODS: For this purpose, 19 non-lame dog cadavers were subjected to an ultrasonographic and histologic evaluation of both shoulders after postmortem examination. RESULTS: Close to their insertion on the greater tubercle, all STs displayed a widened portion with a deep central hypoechoic area lacking a fibrillar pattern, when compared with its more proximal aspect and adjacent biceps brachii. Histologically this deep portion corresponded to poorly organised collagen bundles interspersed within a myxoid substance mainly composed of mucopolysaccharides. This central myxoid area with collagen disarray was responsible for the reduced echogenicity on ultrasound. CONCLUSION: The focal widening of the ST insertion and its central mucopolysaccharidic composition could be an anatomical adaptation to marked forces specifically applied to this tendon. However, the ultrasound and histological appearances are very similar to those described in tendinosis, which represents a confounding factor in diagnosing tendonitis at the insertion of the supraspinatus. In the absence of other ultrasonographic criteria of tendinopathy, a hypoechoic central area in the ST near its insertion should be considered normal.

Coccygeal chordoma in a degu: case report and review of the literature.

An 8-y-old, intact female degu ( Octodon degus) was presented with a slow-growing mass on the tail tip. The mass was completely removed by partial caudectomy. Histologically, the last coccygeal vertebra was replaced by a lobulated neoplasm composed of large clear polygonal cells embedded in a myxoid alcian blue-positive matrix with highly vacuolated cytoplasm (physaliferous cells) and intracytoplasmic periodic acid-Schiff-positive granules. The neoplasm exhibited the morphologic features of a "classic" chordoma of humans, which is 1 of 3 distinct chordoma subtypes. Immunohistochemistry revealed dual expression of cytokeratin AE1/AE3 and vimentin, consistent with a diagnosis of chordoma. Chordomas are uncommon slow-growing neoplasms in humans and animals, arising from notochordal remnants. Depending on their subtype and location, they can have a high local recurrence rate and metastatic risk. Chordoma should be included in the differential diagnosis of a soft tissue mass on the tail of a degu, similar to the clinical situation in ferrets.