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Kathryn L Garner

All living cells are sensors of their environment: they sense signals, hormones, cytokines, and growth factors, among others. Binding of these signals to cell surface receptors initiates the transmission of messages along intracellular signalling pathways through protein-protein interactions, enzymatic modifications and conformational changes. Typically, the activation of signalling pathways are monitored in whole populations of cells, giving population average measures, often using experimental methods that destroy and homogenise the cell population. High content imaging is an automated, high-throughput fluorescence microscopy method that enables measurements of signal transduction pathways to be taken from live cells. It can be used to measure signalling dynamics, how the abundance of particular proteins of interest change over time, or to record how particular proteins move and change their localisation in response to a signal from their environment. Using this, and other single cell methods, it is becoming increasingly clear that cells are in fact very variable in their response to a given stimulus and in the quantities of cellular components they express, even in clonal (isogenic) cell lines. This review will discuss how high content imaging has contributed to our growing understanding of cellular heterogeneity. It will discuss how data generated has been combined with information theoretic approaches to quantify the amount of information transferred through noisy signalling pathways. Lastly, the relevance of heterogeneity to our understanding and treatment of disease will be considered, highlighting the importance of single cell measurements.

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Frank Simmen, Iad Alhallak, and Rosalia C.m. Simmen

Malic enzyme 1 (ME1) is a cytosolic protein that catalyzes the conversion of malate to pyruvate while concomitantly generating NADPH from NADP. Early studies identified ME1 as a mediator of intermediary metabolism primarily through its participatory roles in lipid and cholesterol biosynthesis. ME1 was one of the first identified insulin-regulated genes in liver and adipose and is a transcriptional target of thyroxine. Multiple studies have since documented that ME1 is pro-oncogenic in numerous epithelial cancers. In tumor cells, the reduction of ME1 gene expression or the inhibition of its activity resulted in decreases in proliferation, epithelial-to-mesenchymal transition and in vitro migration, and conversely, in promotion of oxidative stress, apoptosis and/or cellular senescence. Here, we integrate recent findings to highlight ME1’s role in oncogenesis, provide a rationale for its nexus with metabolic syndrome and diabetes, and raise the prospects of targeting the cytosolic NADPH network to improve therapeutic approaches against multiple cancers.

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Shuai Shao, Hui Wang, Wei Shao, and Na Liu

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and one of the most common causes of infertility in women. PCOS patients have been found with dysregulated microRNAs (miRNAs or miRs), which is indicative of their roles as noninvasive biomarkers and novel therapeutic targets in PCOS. Herein, this study sets out to explore the mechanism of action of miR-199a-5p in PCOS in relation to the janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway via Wilms' tumor 1 (WT1) regulation in a rat model of PCOS. The expression of miR-199a-5p was highly expressed in ovarian cortical tissues and serum of PCOS patients as examined by RT-qPCR. Ovarian granulosa cells (GCs) were harvested from PCOS rat model, followed by subsequent purification. Gain-and loss-of-function experiments of miR-199a-5p were performed to determine its functions in PCOS. Cell viability, cell apoptosis and serum hormone levels were assessed, the results of which showed that down-regulation of miR-199a-5p contributed to the promotion of GC viability and inhibition of apoptosis, while simultaneously inducing the elevation of serum E2 level and reduction of serum AMH, PG, LH and FSH levels in the PCOS rat model. WT1 was identified as a target gene of miR-199a-5p by dual-luciferase reporter gene assay, and inhibition of miR-199a-5p resulted in the activation of WT1-mediated JAK/STAT3 pathway. The activated JAK/STAT3 pathway suppressed the development of PCOS by miR-199a-5p, indicating a mechanism by which miR-199a-5p could potentially prevent PCOS through the WT1-mediated JAK/STAT3 pathway.

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Rachel Njeim, William S Azar, Angie H Fares, Sami T Azar, Hala Kfoury Kassouf, and Assaad A. Eid

NETosis, a novel form of neutrophil-related cell death, acts as a major regulator of diabetes and diabetes-associated complications. In this review, we show that the extrusion of neutrophil extracellular traps, termed NETs, plays an important role in the pathogenesis of type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and diabetes-induced complications. In T1DM, β-cell death induces the sequestration of neutrophils in the pancreas and seems to be correlated with increased NETosis. In T2DM patients, products of NETs release are significantly elevated. Increased levels of dsDNA are correlated with the presence of cardiovascular disease and diabetic kidney disease, further supporting the role of NETosis in the pathogenesis of other diabetes-induced complications such as impaired wound healing and diabetic retinopathy. NETosis is induced by high glucose through incompletely understood mechanisms, but it also appears to be elevated in patients with diabetes who have tightly controlled glucose levels. We hypothesize that hyperglycemia worsens the already elevated baseline of NETosis in diabetic patients to further increase its detrimental effects.

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Ruiqi Ma, Lu Gan, Hui Ren, Andrew Harrison, and Jiang Qian

The study aimed to investigate the role of pyruvate dehydrogenase kinase (PDK) in regulating glycolysis and proliferation of perimysial orbital fibroblasts (pOFs) during the pathogenesis of thyroid-associated ophthalmopathy (TAO). EdU and BrdU incorporation assays were performed to examine cell proliferation. Lactate production and oxygen consumption assays were conducted to evaluate glycolysis. Real-time PCR was adapted to quantify PDK mRNA levels. Capillary western immunoassay was adapted to quantify PDK2, Akt, pAkt308 and GAPDH in protein samples. The TAO pOFs exhibited stronger proliferation activity, higher intracellular lactate concentration, and lower oxygen consumption rate than the control pOFs. The PDK inhibitor dichloroacetic acid (DCA) dose-dependently suppressed proliferation of both TAO and control pOFs. DCA reduced lactate production and promoted oxygen consumption in the TAO pOFs but showed no significant effects on glycolysis in the control pOFs. Among four PDK isotypes, PDK2 was overexpressed in the TAO pOFs. The potential PDK signaling mediator, cytoplasmic Akt, was more abundant in TAO pOFs than control pOFs. Knockdown of PDK2 resulted in lower lactate production, stronger oxygen consumption, weaker proliferation activity, and less cytoplasmic Akt in the TAO pOFs but showed no significant effects in the control pOFs. The Akt inhibitor MK2206 suppressed proliferation in both TAO and control pOFs, and lactate production was inhibited by MK2206 in the TAO OFs but not the control pOFs. To conclude, PDK2 overexpression enhances glycolysis and promotes proliferation via Akt signaling in the TAO pOFs. These findings yield insights that PDK2 is a potential therapeutic target for TAO treatment.

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Juan Carlos Arapa-Diaz, Wender Nascimento Rouver, Jéssyca Aparecida Soares Giesen, Marcela Daruge Grando, Lusiane Maria Bendhack, and Roger Lyrio dos Santos

Physiological or supraphysiological levels of testosterone appear to be associated with the development of risk factors for cardiovascular diseases such as hypertension, as this hormone modulates the release of endothelial factors. However, its actions are still controversial, especially in the coronary circulation of hypertensive animals. This study was designed to assess the effects of testosterone treatment (T) on endothelium-dependent coronary vascular reactivity in orchiectomized SHR. The animals were divided into SHAM, orchiectomized (ORX), ORX+T and ORX+T+aromatase inhibitor (AI). All treatments lasted 15 days. Blood pressure (BP) was measured. Dose-response curves to bradykinin (BK) were constructed using the Langendorff technique, followed by inhibition of endothelium mediators (NO, prostanoids, EETs) and potassium channels. The intensity of eNOS, COX-1, COX-2, Akt, and gp91phox protein expression was quantified by Western blotting. BP was elevated in SHAM, ORX+T, and ORX+T+AI groups. However, we did not observe differences in the ORX group. Baseline coronary perfusion pressure (CPP) remained unaffected. Orchiectomy did not change the BK-induced relaxation compared to the SHAM group, whereas testosterone treatment increased it. This response was diminished in the absence of NO, prostanoids, and EETs in the SHAM and ORX groups, while in ORX+T group the relaxation was diminished only in absence of NO and EETs. There was no difference in eNOS, COX-1, COX-2, and gp91phox protein expression, though Akt expression was increased in ORX and ORX+T groups. These results show that testosterone treatment can modulate endothelial function, especially in the coronary circulation under hypertension conditions, via NO and EETs pathways.

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Maya Elena Lee, Aisha Aderayo Tepede, Adel Mandl, Lee Scott Weinstein, Jaydira del Rivero, Sunita K Agarwal, and Jenny E Blau

Gastroenteropancreatic neuroendocrine tumors (GEP NETs) comprise a heterogenous and diverse group of neoplasms arising from a common neuroendocrine cell origin. The majority of these tumors occur sporadically while ~20% manifest within the context of hereditary syndromes. Germline MEN1 mutations cause a syndrome with an increased susceptibility to multifocal primary GEP NETs. In addition, somatic MEN1 mutations also occur in these sporadic lesions. MEN1 alterations are the most frequent somatic mutation found in pancreatic neuroendocrine tumors. In this review, we explore the implication of the loss of the MEN1-encoded protein menin as a key pathogenic driver in subsets of GEP NETs with downstream consequences including upregulation of the oncogenic receptor c-MET (hepatocyte growth factor receptor). Furthermore, the review will summarize the data related to the clinical presentation, therapeutic standards, and outcomes of these tumors in both sporadic and germline MEN1 mutation-associated contexts. Finally, we present the data on c-MET expression in GEP NETs, clinical trials using c-MET inhibitors and provide an overview of the molecular mechanisms by which c-MET inhibition in these lesions represents a potential precision-medicine targeted approach.

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Leonard Y M Cheung and Karine Rizzoti

In the last 15 years, single-cell technologies have become robust and indispensable tools to investigate cell heterogeneity. Beyond transcriptomic, genomic and epigenome analyses, technologies are constantly evolving, in particular toward multi-omics, where analyses of different source materials from a single cell are combined, and spatial transcriptomics, where resolution of cellular heterogeneity can be detected in situ. While some of these techniques are still being optimized, single-cell RNAseq has commonly been used because the examination of transcriptomes allows characterization of cell identity and, therefore, unravel previously uncharacterized diversity within cell populations. Most endocrine organs have now been investigated using this technique, and this has given new insights into organ embryonic development, characterization of rare cell types, and disease mechanisms. Here, we highlight recent studies, particularly on the hypothalamus and pituitary, and examine recent findings on the pancreas and reproductive organs where many single-cell experiments have been performed.

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Yujia Pan, Weikang Yun, Bingshuai Shi, Rongjun Cui, Chi Liu, Zhong Ding, Jialin Fan, Wenqian Jiang, Jiebing Tang, Tianhu Zheng, Xiaoguang Yu, and Ying Liu

miR-146b-5p is overexpressed in papillary thyroid carcinoma (PTC) and is thought to be a related diagnostic marker. Previous studies have indicated the effects of iodine on oncogenic activation. However, the effect of iodine on the proliferation of PTC cells and the associated underlying mechanisms remain unclear. We found that miR-146b-5p was downregulated and smad4 was upregulated in patients exposed to high iodine concentration by in situ hybridisation (ISH) and immunohistochemical (IHC). NaI (10−3 M) treatment downregulated miR-146b-5p and upregulated Smad4 in PTC cell lines. Luciferase assay was used to confirm that Smad4 is a target of miR-146b-5p. Furthermore, MTT assay and cell cycle analysis indicated that 10−3 M NaI suppressed cell proliferation and caused G0/G1 phase arrest. Real-time PCR and Western blotting demonstrated that 10−3 M NaI increased p21, p27, and p57 levels and reduced cyclin D1 levels in PTC cells. Our findings suggest that 10−3 M NaI increases Smad4 levels through repression of miR-146b-5p expression, curbing the proliferation in PTC.

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Kai Huang, Gezi Chen, Wenqian Fan, and Linli Hu

A receptive endometrium is required in a successful embryo implantation. The ubiquitination-induced β-catenin degradation is related to the implantation failure.This study aimed to elucidate whether miR-23a-3p regulates endometrial receptivity via the modulation of β-catenin ubiquitination.The expressions of miR-23a-3p and CUL3 were detected in endometrial epithelial cells (EECs) isolated from pregnant mice and in hormone-induced EEC-like Ishikawa cells. The ubiquitination experiment was performed to explore the effect of CUL3 and miR-23a-3p on β-catenin ubiquitination level. The trophoblast attachment was detected by co-culturing JAR (choriocarcinoma cell line) spheroids with Ishikawa cell monolayers. miR-23a-3p was upregulated while CUL3 was downregulated in EECs at day 4 after pregnancy compared with day 1, as well as in hormone-induced Ishikawa cells. miR-23a-3p positively regulated the protein level of β-catenin without affecting the mRNA level. The ubiquitination and degradation of β-catenin was suppressed by miR-23a-3p, while it was promoted by CUL3. Immunoprecipitation confirmed the binding between CUL3 and β-catenin. Luciferase reporter assay confirmed the target relationship between miR-23a-3p and CUL3. The ubiquitination of β-catenin was modulated by the miR-23a-3p/CUL3 pathway. The overexpression of miR-23a-3p promoted JAR spheroid attachments in Ishikawa cells. miR-23a-3p is beneficial for the endometrial receptivity and embryo implantation, whose mechanism is partly through the modulation of CUL3/β-catenin.