Mechanisms of Drug Resistance in Ovarian Cancer and Associated Gene Targets.

In the United States, over 100,000 women are diagnosed with a gynecologic malignancy every year, with ovarian cancer being the most lethal. One of the hallmark characteristics of ovarian cancer is the development of resistance to chemotherapeutics. While the exact mechanisms of chemoresistance are poorly understood, it is known that changes at the cellular and molecular level make chemoresistance challenging to treat. Improved therapeutic options are needed to target these changes at the molecular level. Using a precision medicine approach, such as gene therapy, genes can be specifically exploited to resensitize tumors to therapeutics. This review highlights traditional and novel gene targets that can be used to develop new and improved targeted therapies, from drug efflux proteins to ovarian cancer stem cells. The review also addresses the clinical relevance and landscape of the discussed gene targets.

Methods to evaluate the impact of SARS-CoV-2 nucleocapsid mutations on antigen detection by rapid diagnostic tests.

Mutations in the nucleocapsid of SARS-CoV-2 may interfere with antigen detection by diagnostic tests. We used several methods to evaluate the effect of various SARS-CoV-2 nucleocapsid mutations on the performance of the Panbio™ and BinaxNOW™ lateral flow rapid antigen tests and a prototype high-throughput immunoassay that utilizes Panbio antibodies. Variant detection was also evaluated by immunoblot and BIAcore™ assay. A panel of 23 recombinant nucleocapsid antigens (rAgs) were produced that included mutations found in circulating SARS-CoV-2 variants, including variants of concern. All mutant rAgs were detected by all assays, at a sensitivity equivalent to wild-type control (Wuhan strain). Thus, using a rAg approach, we found that the SARS-CoV-2 nucleocapsid mutations examined do not directly impact antigen detection or antigen assay performance.

At the Front Lines: Effectively Training Community Stakeholders to Recognize and Report Child Abuse and Neglect.

In states with universal mandatory reporting of child abuse and neglect (CAN), it is essential that people who work with children and youth in a community be appropriately trained to recognize and report CAN. The primary goal of CAN training is early detection and intervention with a secondary purpose of impacting rates of violence, disease, drug use, and teen pregnancies in the community. The purpose of this project was to implement a standardized, community-tailored CAN training for laypersons and a train-the-trainer program in a rural Oklahoma community. The CAN training was evaluated on knowledge, confidence, training satisfaction, and willingness to participate in the train-the-trainer session. The train-the-trainer session was evaluated on confidence and training satisfaction. Participant knowledge and confidence was measured by comparing pretest scores to immediate and 4 months after the training posttest scores. Posttest scores indicated increase in knowledge at the posttest (p < .001) and posttest 2 (p < .001). There was a significant increase in confidence at the posttest (p < .001) and posttest 2 (p = .009).

Comprehensive quantitative analysis of fatty-acyl-Coenzyme A species in biological samples by ultra-high performance liquid chromatography-tandem mass spectrometry harmonizing hydrophilic interaction and reversed phase chromatography.

Fatty acyl-Coenzyme A species (acyl-CoAs) are key biomarkers in studies focusing on cellular energy metabolism. Existing analytical approaches are unable to simultaneously detect the full range of short-, medium-, and long-chain acyl-CoAs, while chromatographic limitations encountered in the analysis of limited amounts of biological samples are an often overlooked problem. We report the systematic development of a UHPLC-ESI-MS/MS method which incorporates reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) separations in series, in an automated mode. The protocol outlined encompasses quantification of acyl-CoAs of varying hydrophobicity from C2 to C20 with recoveries in the range of 90-111 % and limit of detection (LOD) 1-5 fmol, which is substantially lower than previously published methods. We demonstrate that the poor chromatographic performance and signal losses in MS detection, typically observed for phosphorylated organic molecules, can be avoided by the incorporation of a 0.1% phosphoric acid wash step between injections. The methodological approach presented here permits a highly reliable, sensitive and precise analysis of small amounts of tissues and cell samples as demonstrated in mouse liver, human hepatic (HepG2) and skeletal muscle (LHCNM2) cells. The considerable improvements discussed pave the way for acyl-CoAs to be incorporated in routine targeted lipid biomarker profile studies.

Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190.

The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/ß is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis.

Defining Akt actions in muscle differentiation.

Muscle development in childhood and muscle regeneration in adults are highly regulated processes that are necessary for reaching and maintaining optimal muscle mass and strength throughout life. Muscle repair after injury relies on stem cells, termed satellite cells, whose activity is controlled by complex signals mediated by cell-cell contact, by growth factors, and by hormones, which interact with genetic programs controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development and help coordinate muscle repair after injury, primarily by stimulating the phosphatidylinositol 3-kinase-Akt signaling pathway, and both in vitro and in vivo studies have shown that Akt kinase activity is critical for optimal muscle growth and regeneration. Here we find that of the two Akts expressed in muscle, Akt1 is essential for initiation of differentiation in culture and is required for normal myoblast motility, while Akt2 is dispensable. Although Akt2 deficiency did lead to diminished myotube maturation, as assessed by a decline in myofiber area and in fusion index, either Akt1 or Akt2 could restore these processes toward normal. Thus levels of Akt expression rather than distinct actions of individual Akt species are critical for normal myofiber development during the later stages of muscle differentiation.

TGF-ß inhibits muscle differentiation by blocking autocrine signaling pathways initiated by IGF-II.

Skeletal muscle differentiation and regeneration are regulated by interactions between exogenous hormone- and growth factor-activated signaling cascades and endogenous muscle-specific transcriptional programs. IGF-I and IGF-II can promote muscle differentiation in vitro and can enhance muscle maintenance and repair in vivo. In contrast, members of the TGF-ß superfamily prominently inhibit muscle differentiation and regeneration. In this study, we have evaluated functional interactions between IGF- and TGF-ß-regulated signaling pathways during skeletal muscle differentiation. In the mouse C2 muscle cell line and in human myoblasts in primary culture, addition of TGF-ß1 blocked differentiation in a dose-dependent way, inhibited expression of muscle-specific mRNAs and proteins, and impaired myotube formation. TGF-ß1 also diminished stimulation of IGF-II gene expression in myoblasts, decreased IGF-II secretion, and reduced IGF-I receptor activation. To test the hypothesis that TGF-ß1 prevents muscle differentiation primarily by blocking IGF-II production, we examined effects of IGF analogues on TGF-ß actions in myoblasts. Although both IGF-I and IGF-II restored muscle gene and protein expression, and stimulated myotube formation in the presence of TGF-ß1, they did not reduce TGF-ß1-stimulated signaling, as measured by no decline in phosphorylation of SMA and mothers against decapentaplegic homolog (Smad)3, or in induction of TGF-ß-activated target genes, including a Smad-dependent promoter-reporter plasmid. Our results demonstrate that TGF-ß disrupts an IGF-II-stimulated autocrine amplification cascade that is necessary for muscle differentiation in vitro. Because this inhibitory pathway can be overcome by exogenous IGFs, our observations point toward potential strategies to counteract disorders that reduce muscle mass and strength.

Targeting mediators of Wnt signalling pathways by GnRH in gonadotropes.

The binding of GnRH to its receptor on pituitary gonadotropes leads to the targeting of a diverse array of signalling mediators. These mediators drive multiple signal transduction pathways, which in turn regulate a variety of cellular processes, including the biosynthesis and secretion of the gonadotropins LH and FSH. Advances in our understanding of the mechanisms and signalling pathways that are recruited to regulate gonadotrope function are continually being made. This review will focus on the recent demonstration that key mediators of the canonical Wnt signalling pathway are targeted by GnRH in gonadotropes, and that these may play essential roles in regulating the expression of many of the key players in gonadotrope biology, including the GnRH receptor and the gonadotropins.

Emerging targets of the GnRH receptor: novel interactions with Wnt signalling mediators.

The diversity of signalling pathways that gonadotropin-releasing hormone (GnRH) receptors can target has been extended markedly in recent years, ranging from classical heterotrimeric G-protein-coupled second-messenger mobilisation to MAPK cascade activation and epidermal growth factor receptor transactivation. More recently, the targeting of non-classical signalling pathways has been demonstrated, including the activation of monomeric G-proteins and integrin/focal adhesion complexes to mediate cytoskeletal remodelling, androgen receptor nuclear translocation, and luteinising hormone beta-subunit gene transcription. These have been further extended to include c-Src-mediated activation of diacylglycerol kinase, and a novel mechanism of cross-talk to prostaglandin receptor signalling. Here, we review these recent advances and highlight several emerging dimensions in GnRH receptor-mediated signal transduction. These include the targeting of mediators of classical insulin signalling, and Wnt signalling pathways. Collectively, this diverse nature of GnRH receptor signalling is suggestive of an important role for GnRH in a variety of normal physiological and pathophysiological processes in the reproductive system.

Nuclear stabilization of beta-catenin and inactivation of glycogen synthase kinase-3beta by gonadotropin-releasing hormone: targeting Wnt signaling in the pituitary gonadotrope.

The GnRH receptor is a G protein-coupled receptor (GPCR), and its ligand GnRH is the central regulator of the reproductive system. GnRH receptors are known to target a wide variety of signal transduction pathways. Several recent studies have shown that activation of GPCRs can impact on beta-catenin signaling. beta-Catenin is the main effecter of the Wnt signaling pathway where it acts with the transcription factors T cell factor/lymphoid enhancer factor to mediate the transcription of Wnt target genes. We show that GnRH treatment promotes the nuclear accumulation of beta-catenin, activation of T cell factor-dependent transcription, and up-regulation of Wnt target genes, c-Jun, Fra-1, and c-Myc. These results are observed in human embryonic kidney 293/GnRH receptor-expressing cells and have been recapitulated in LbetaT2 and alphaT3-1 mouse gonadotrope cells. In addition to these findings, we show that GnRH treatment mediates the inactivation of glycogen synthase kinase-3, a protein serine/threonine kinase that regulates beta-catenin degradation within the Wnt signaling pathway. Our findings extend the number of GPCRs that can target beta-catenin signaling through diverse pathways. Furthermore, this is the first demonstration of the targeting of Wnt/beta-catenin signaling by a peptide hormone GPCR.