Habituation of Arctic ground squirrels (Urocitellus parryii) to handling and movement during torpor to prevent artificial arousal

Hibernation is a unique physiological adaptation characterized by periods of torpor that consist of repeated, reversible, and dramatic reductions of body temperature, metabolism, and blood flow. External and internal triggers can induce arousal from torpor in the hibernator. Studies of hibernating animals often require that animals be handled or moved prior to sampling or euthanasia but this movement can induce changes in the hibernation status of the animal. In fact, it has been demonstrated that movement of animals while they are hibernating is sufficient to induce an artificial arousal, which can detrimentally alter experimental findings obtained from animals assumed to be torpid. Therefore, we assessed a method to induce habituation of torpid hibernators to handling and movement to reduce inadvertent arousals. A platform rocker was used to mimic motion experienced during transfer of an animal and changes in respiratory rate (RR) were used to assess responsiveness of torpid Arctic ground squirrels (AGS, Urocitellus parryii). We found that movement alone did not induce a change in RR, however, exposure to handling induced an increase in RR in almost all AGS. This change in RR was markedly reduced with increased exposures, and all AGS exhibited a change in RR ≤ 1 by the end of the study. AGS habituated faster mid-season compared to early in the season, which mirrors other assessments of seasonal variation of torpor depth. However, AGS regained responsiveness when they were not exposed to daily handling. While AGS continued to undergo natural arousals during the study, occurrence of a full arousal was neither necessary for becoming habituated nor detrimental to the time required for habituation. These data suggest that even when torpid, AGS are able to undergo mechanosensory habituation, one of the simplest forms of learning, and provides a reliable way to reduce the sensitivity of torpid animals to handling

Collagen overlays can inhibit leptin and adiponectin secretion but not lipid accumulation in adipocytes

Background White adipose tissue (WAT) is essential for energy storage as well as being an active endocrine organ. The secretion of adipokines by adipocytes can affect whole body metabolism, appetite, and contribute to overall health. WAT is comprised of lipid-laden mature adipocytes, as well as immune cells, endothelial cells, pre-adipocytes, and adipose-derived stem cells. In addition, the presence of extracellular matrix (ECM) proteins in WAT can actively influence adipocyte differentiation, growth, and function. Type I collagen is an abundant fibrous ECM protein in WAT that is secreted by developing adipocytes. However, the extent and overall effect of Type I collagen on adipokine secretion in mature adipocytes when added exogenously has not been established. Methods We characterized the effects of Type I collagen overlays prepared using two different buffers on adipocyte physiology and function when added at different times during differentiation. In addition, we compared the effect of collagen overlays when adipocytes were cultured on two different tissue culture plastics that have different adherent capabilities. Triglyceride accumulation was analyzed to measure adipocyte physiology, and leptin and adiponectin secretion was determined to analyze effects on adipokine secretion. Results We found that collagen overlays, particularly when added during the early differentiation stage, impaired adipokine secretion from mature adipocytes. Collagen prepared using PBS had a greater suppression of leptin than adiponectin while collagen prepared using HANKS buffer suppressed the secretion of both adipokines. The use of CellBind plates further suppressed leptin secretion. Triglyceride accumulation was not substantially impacted with any of the collagen overlays. Discussion Adipokine secretion can be selectively altered by collagen overlays. Thus, it is feasible to selectively manipulate the secretion of adipokines by adipocytes in vitro by altering the composition or timing of collagen overlays. The use of this technique could be applied to studies of adipokine function and secretion in vitro as well as having potential therapeutic implications to specifically alter adipocyte functionality in vivo

Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture

The Atlantic salmon (Salmo salar) is an economically important fish, both in aquaculture and in the wild. In vertebrates, macrophages are some of the first cell types to respond to pathogen infection and disease. While macrophage biology has been characterized in mammals, less is known in fish. Our previous work identified changes in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon adherent head kidney leukocytes (HKLs) from predominantly “monocyte-like” at Day 1 of in vitro culture to predominantly “macrophage-like” at Day 5 of culture. Therefore, to further characterize these two cell populations, we examined the mRNA transcriptome profile in Day 1 and Day 5 HKLs using a 44K oligonucleotide microarray. Large changes in the transcriptome were revealed, including changes in the expression of macrophage and immune-related transcripts (e.g. csf1r, arg1, tnfa, mx2), lipid-related transcripts (e.g. fasn, dhcr7, fabp6), and transcription factors involved in macrophage differentiation and function (e.g. klf2, klf9, irf7, irf8, stat1). The in silico target prediction analysis of differentially expressed genes (DEGs) using miRNAs known to change expression in Day 5 HKLs, followed by gene pathway enrichment analysis, supported that these miRNAs may be involved in macrophage maturation by targeting specific DEGs. Elucidating how immune cells, such as macrophages, develop and function is a key step in understanding the Atlantic salmon immune system. Overall, the results indicate that, without the addition of exogenous factors, the adherent HKL cell population differentiates in vitro to become macrophage-like

Identification of lysyl oxidase as an adipocyte-secreted mediator that promotes a partial mesenchymal-to-epithelial transition in MDA-MB-231 cells

Aim: Breast cancer (BC) is the most common cancer in women worldwide, where adiposity has been linked to BC morbidity. In general, obese premenopausal women diagnosed with triple-negative BC (TNBC) tend to have larger tumours with more metastases, particularly to the bone marrow, and worse prognosis. Previous work using a 3-dimensional (3D) co-culture system consisting of TNBC cells, adipocytes and the laminin-rich extracellular matrix (ECM) trademarked as Matrigel, demonstrated that adipocytes and adipocyte-derived conditioned media (CM) caused a partial mesenchymal-to-epithelial transition (MET). Given that MET has been associated with secondary tumour formation, this study sought to identify molecular mediators responsible for this phenotypic change. Methods: Adipocytes were cultured with and without Matrigel, where semi-quantitative proteomics was used to identify proteins whose presence in the CM was induced or enhanced by Matrigel, which were referred to as adipocyte-secreted ECM-induced proteins (AEPs). The AEPs identified were assessed for association with prognosis in published proteomic datasets and prior literature. Of these, 4 were evaluated by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), followed by a functional and MET marker analysis of 1 AEP on MDA-MB-231 cells grown on Matrigel or as monolayers. Results: The 4 AEPs showed a positive correlation between protein expression and poor prognosis. RT-qPCR analysis reported no significant change in AEPs mRNA expression. However, lysyl oxidase (LOX) was increased in CM of ECM-exposed adipocytes. Recombinant LOX (rLOX) caused the mesenchymal MDA-MB-231 TNBC cells to form less branched 3D structures and reduced the expression of vimentin. Conclusions: The data suggest that adipocyte-secreted LOX changes the mesenchymal phenotype of BC cells in a manner that could promote secondary tumour formation, particularly at sites high in adipocytes such as the bone marrow. Future efforts should focus on determining whether targeting LOX could reduce BC metastasis in obese individuals

First-in-Human Clinical Trial of Oral ONC201 in Patients with Refractory Solid Tumors

Purpose: ONC201 is a small-molecule selective antagonist of the G protein–coupled receptor DRD2 that is the founding member of the imipridone class of compounds. A first-in-human phase I study of ONC201 was conducted to determine its recommended phase II dose (RP2D). Experimental Design: This open-label study treated 10 patients during dose escalation with histologically confirmed advanced solid tumors. Patients received ONC201 orally once every 3 weeks, defined as one cycle, at doses from 125 to 625 mg using an accelerated titration design. An additional 18 patients were treated at the RP2D in an expansion phase to collect additional safety, pharmacokinetic, and pharmacodynamic information. Results: No grade \u3e 1 drug-related adverse events occurred, and the RP2D was defined as 625 mg. Pharmacokinetic analysis revealed a Cmax of 1.5 to 7.5 μg/mL (∼3.9–19.4 μmol/L), mean half-life of 11.3 hours, and mean AUC of 37.7 h·μg/L. Pharmacodynamic assays demonstrated induction of caspase-cleaved keratin 18 and prolactin as serum biomarkers of apoptosis and DRD2 antagonism, respectively. No objective responses by RECIST were achieved; however, radiographic regression of several individual metastatic lesions was observed along with prolonged stable disease (\u3e 9 cycles) in prostate and endometrial cancer patients. Conclusions: ONC201 is a selective DRD2 antagonist that is well tolerated, achieves micromolar plasma concentrations, and is biologically active in advanced cancer patients when orally administered at 625 mg every 3 weeks

Author Correction:Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts (Nature Genetics, (2021), 53, 1, (86-99), 10.1038/s41588-020-00750-6)

This paper was originally published without open access. As of the date of this correction, the paper is available online as an open-access paper under a Creative Commons Attribution 4.0 International License. *Lists of authors and their affiliations appear online

Repression of CD24 surface protein expression by oncogenic Ras is relieved by inhibition of Raf but not MEK or PI3K

CD24 is a dynamically regulated cell surface protein. High expression of CD24 leads to progression of lung, prostrate, colon, and pancreatic cancers, among others. In contrast, low expression of CD24 leads to cell proliferation and metastasis of breast cancer stem cells (BCSCs). Activating mutations in Ras are found in 30% of all human cancers. Oncogenic Ras constitutively stimulates the Raf, PI3K, and Ral GDS signaling pathways, leading to cellular transformation. Previous studies have shown that expression of oncogenic Ras in breast cancer cells generates CD24− cells from CD24+ cells. However, the molecular mechanisms involved in the generation of CD24− cells were not determined. Here, we demonstrate that oncogenic Ras (RasV12) expression suppresses CD24 mRNA, protein, and promoter levels when expressed in NIH/3T3 cells. Furthermore, activation of only the Raf pathway was sufficient to downregulate CD24 mRNA and protein expression to levels similar to those seen in with RasV12 expression. In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression. Surprisingly, inhibition of MEK with U0126 only partially restored CD24 mRNA expression but not surface protein expression. In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24. Therefore, the Raf pathway is the major repressor of CD24 mRNA and protein expression, with PI3K also able to substantially inhibit CD24 expression. Moreover, these data indicate that the levels of CD24 mRNA and surface protein are independently regulated. Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24− cells, such as BCSCs

Suppression of IFN-Induced Transcription Underlies IFN Defects Generated by Activated Ras/MEK in Human Cancer Cells

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes

戦後日本の地域的不均等発展と地域社会類型の新段階：第3章 市町村における地域基礎構造の変動と地域社会類型

Trace amines and their receptors (trace amine-associated receptors; TAARs) are an emerging pharmacological target for the treatment of human disorders. While most studies have focused on their therapeutic potential for neurologic and psychiatric disorders, TAARs are also expressed throughout the periphery, including prominent expression in human leukocytes. Furthermore, recent independent, unbiased metabolomic studies have consistently identified one or more TAAR ligands as potential etiologic factors in inflammatory bowel disease (IBD). The putative role of TAARs in diseases such as IBD that are associated with hyperactive immune responses has not, however, previously been systematically addressed. Here, we review the current state of the knowledge of the effects of TAARs on leukocyte function, in particular in the context of mucosal epithelial cells that interface with the environment; developing a model whereby TAARs may be considered as a novel therapeutic target for disorders associated with dysregulated immune responses to environmental factors. In this model, we hypothesize that altered trace amine homeostasis results in hyperactivity of the immune system. Such loss of homeostasis can occur through many different mechanisms including TAAR polymorphisms and altered trace amine load due to changes in host synthesis and/or degradative enzymes, diet, or microbial dysbiosis. The resulting alterations in TAAR functioning can then lead to a loss of homeostasis of leukocyte chemotaxis, differentiation, and activation, as well as an altered ability of members of the microbiota to adhere to and penetrate the epithelial cell layers. Such changes would generate a pro-inflammatory state at mucosal epithelial barrier layers that can manifest as clinical symptomatology such as that seen in IBD. These alterations may also have the potential to induce systemic effects, which could possibly contribute to immunomodulatory disorders in other systems, including neurological diseases