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RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4 + helper T cells and increased CD8 + cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Context: During pregnancy, the immune system is altered, putting women at increased risk of complications from respiratory viruses. There is limited information about the effects of COVID-19 on pregnant women and obstetrics programs, particularly in rural regions., Aims: Most published reports have been from large urban hospitals. This study sought to gain insight into how the COVID-19 pandemic is impacting rural obstetrics programs., Materials and Methods: This qualitative study surveyed participants who worked in rural obstetrics programs in New Mexico using a free response questionnaire., Results: The pandemic has changed the obstetrics experience in rural New Mexico by impacting the relationship between patients and providers and altering the family-centered model of birth. Difficulties unique to rural obstetrics included lack of transportation, limited hospital rooms and limited staff, including OB providers, nurses and housekeeping. Wellness has been impacted for providers, manifested in increased anxiety, stress and burnout. Patients reported increased anxiety, decreased family support, and potential disruptions in maternal-infant bonding., Conclusions: Rural hospitals and clinics in New Mexico and across the United States operate with challenges at baseline, with many clinics across the nation closing, leading to increasing shortages of healthcare services in rural areas. This study showed that COVID-19 has increased the challenges that rural obstetrics providers face, altering the way they practice and creating uncertainty for the future due to potentially permanent changes to their practice. Future research will explore the lasting effects of the pandemic on rural obstetrics providers specifically, and rural hospitals generally., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Family Medicine and Primary Care.)

Background: Luminescent semiconductor nanocrystals, also known as quantum dots (QD), possess highly desirable optical properties that account for development of a variety of exciting biomedical techniques. These properties include long-term stability, brightness, narrow emission spectra, size tunable properties and resistance to photobleaching. QD have many promising applications in biology and the list is constantly growing. These applications include DNA or protein tagging for in vitro assays, deep-tissue imaging, fluorescence resonance energy transfer (FRET), and studying dynamics of cell surface receptors, among others. Here we explored the potential of QD-mediated labeling for the purpose of tracking an intracellular protein inside live cells., Results: We manufactured dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QD, not available commercially, and coupled them to the cell cycle regulatory protein Cyclin E. We then utilized the QD fluorescence capabilities for visualization of Cyclin E trafficking within cells of Xenopus laevis embryos in real time., Conclusions: These studies provide "proof-of-concept" for this approach by tracking QD-tagged Cyclin E within cells of developing embryos, before and during an important developmental period, the midblastula transition. Importantly, we show that the attachment of QD to Cyclin E did not disrupt its proper intracellular distribution prior to and during the midblastula transition. The fate of the QD after cyclin E degradation following the midblastula transition remains unknown.

RNA binding protein (RBPs) and microRNAs (miRNAs or miRs) are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR) containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC). Despite this, miR-16 and HuR do not affect the other's expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

Cyclins are regulatory subunits that bind to and activate catalytic Cdks. Cyclin E associates with Cdk2 to mediate the G1/S transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how cyclin E is normally downregulated during development in order to determine if disruption of similar mechanisms could either contribute to its overexpression in cancer, or be exploited to decrease its expression. In Xenopus laevis embryos, cyclin E protein level is high and constant until its abrupt destabilization by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT) and remodeling of the embryonic to the adult cell cycle. Since degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent, we examined the role of phosphorylation in Xenopus cyclin E turnover. We show that similarly to human cyclin E, phosphorylation of serine 398 and threonine 394 plays a role in cyclin E turnover at the MBT. Immunofluorescence analysis shows that cyclin E relocalizes from the cytoplasm to the nucleus preceding its degradation. When nuclear import is inhibited, cyclin E stability is markedly increased after the MBT. To investigate whether degradation of Xenopus cyclin E is mediated by the proteasomal pathway, we used proteasome inhibitors and observed a progressive accumulation of cyclin E in the cytoplasm after the MBT. Ubiquitination of cyclin E precedes its proteasomal degradation at the MBT. These results show that cyclin E destruction at the MBT requires both phosphorylation and nuclear import, as well as proteasomal activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this., Results: Here we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4alpha and xCdc4beta. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail., Conclusions: We demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation.

We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after the midblastula transition (MBT) of Xenopus laevis embryogenesis. By MBT, the stage when the embryonic cell cycle is remodeled and zygotic transcription of mRNAs is initiated, each embryo has accumulated approximately 10(9) molecules of miR-427 processed from multimeric pri-miR-427 transcripts synthesized after fertilization. We demonstrate that the maternal mRNAs for cyclins A1 and B2 each contain a single miR-427 target sequence, spanning less than 30 nucleotides, that is both necessary and sufficient for deadenylation, and that inactivation of miR-427 leads to stabilization of the mRNAs. Although this deadenylation normally takes place after MBT, exogenous miRNAs produced prematurely in vivo can promote deadenylation prior to MBT, indicating that turnover of the maternal mRNAs is limited by the amount of accumulated miR-427. Injected transcripts comprised solely of the cyclin mRNA 3' untranslated regions or bearing a 5' ApppG cap undergo deadenylation, showing that translation of the targeted RNA is not required. miR-427 is not unique in promoting deadenylation, as an unrelated miRNA, let-7, can substitute for miR-427 if the reporter RNA contains an appropriate let-7 target site. We propose that miR-427, like the orthologous miR-430 of zebrafish, functions to down-regulate expression of maternal mRNAs early in development.

MicroRNAs (miRNAs) are a class of naturally occurring, small, non-coding RNAs that control gene expression during development,normal cell function and disease. Although there is emerging evidence that some miRNAs can function as oncogenes or tumor suppressors, there is limited understanding of the role of miRNAs in cancer. In this study, we observed that the expression of miR-125a was inversely correlated with HuR expression in several different breast carcinoma cell lines. HuR is a stress-induced RNA binding protein whose expression is elevated or localization perturbed in several different cancers. Increased cytoplasmic localization of HuR is a prognostic marker in breast cancer. Real time PCR and gene reporter assays indicated that HuR was translationally repressed by miR-125a. Re-establishing miR-125a expression in breast cancer cells decreased HuR protein level and inhibited cell growth. Using MCF-7 breast cancer cells, we further clarified that miR-125a inhibited cell growth via a dramatic suppression of cell proliferation and promotion of apoptosis.In addition, cell migration was also inhibited by miR-125a overexpression. Importantly, the repression of cell proliferation and migration engendered by miR-125a was partly rescued by HuR re-expression. Our results suggest that miR-125a may function as a tumor suppressor for breast cancer, with HuR as a direct and functional target.

The early cell divisions of Xenopus laevis and other metazoan embryos occur in the presence of constitutively high levels of the cell cycle regulator cyclin E1. Upon completion of the 12th cell division, a time at which many maternal proteins are downregulated by deadenylation and destabilization of their encoding mRNAs, maternal cyclin E1 protein is downregulated while its mRNA is polyadenylated and stable. We report here that stable polyadenylation of cyclin E1 mRNA requires three cis-acting elements in the 3' untranslated region; the nuclear polyadenylation sequence, a contiguous cytoplasmic polyadenylation element and an upstream AU-rich element. ElrA, the Xenopus homolog of HuR and a member of the ELAV gene family binds the cyclin E1 3'UTR with high affinity. Deletion of these elements dramatically reduces the affinity of ElrA for the cyclin E1 3'UTR, abolishes polyadenylation and destabilizes the mRNA. Together, these findings provide compelling evidence that ElrA functions in polyadenylation and stabilization of cyclin E1 mRNA via binding these elements.

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.

At the midblastula transition, the Xenopus laevis embryonic cell cycle is remodeled from rapid alternations between S and M phases to become the complex adult cell cycle. Cell cycle remodeling occurs after zygotic transcription initiates and is accompanied by terminal downregulation of maternal cyclins A1 and B2. We report here that the disappearance of both cyclin A1 and B2 proteins is preceded by the rapid deadenylation of their mRNAs. A specific mechanism triggers this deadenylation. This mechanism depends upon discrete regions of the 3' untranslated regions and requires zygotic transcription. Together, these results strongly suggest that zygote-dependent deadenylation of cyclin A1 and cyclin B2 mRNAs is responsible for the downregulation of these proteins. These studies also raise the possibility that zygotic control of maternal cyclins plays a role in establishing the adult cell cycle.

A subset of adult peripheral blood leukocytes functions as endothelial cell progenitors called angioblasts. They can incorporate into the vasculature in animal models of neovascularization and accelerate the restoration of blood flow to mouse ischemic limbs. Earlier reports suggested that CD34-expressing (CD34+) but not CD34+ cell-depleted (CD34-) leukocytes can differentiate into endothelial cells (EC) in vitro and in vivo. Recent findings suggest that CD14+ cells, which are typically CD34-, also have angioblast-like properties in vitro. To determine the identity of angioblasts, the potential of CD34+, CD34-, CD34- CD14+, and CD34- CD14- cells to produce EC was compared. We show that a subset of monocyte (CD34- CD14+)-enriched cells can take on an EC-like phenotype in culture, but that the EC-like cells also express dendritic cell antigens. These findings suggest that monocytes differentiate into macrophages, dendritic cells, or EC depending on environmental cues. The data also demonstrate that angioblasts are more abundant in the blood than previously thought. Finally, we demonstrate that CD34- and CD34- CD14+ cells incorporate into the endothelium of blood vessels in mouse ischemic limbs. However, incorporation of these cells requires co-injection with CD34+ cells, indicating that leukocyte-leukocyte interactions may play a critical role in governing angioblast behavior in vivo.

During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1). Delta34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins. However, DNA synthesis was not inhibited. Several hallmarks of the MBT were delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Delta34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.

We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.

Mitogen-activated protein kinase (MAPK) is one of the protein kinases activated during meiotic maturation of Xenopus laevis oocytes. The c-Mosxe protein kinase, which has been shown to be sufficient to promote germinal vesicle breakdown (GVBD) in meiosis I, can directly activate MAP kinase kinase in vitro and leads to the activation of MAPK in vivo. Recently we have shown that constitutively activated MAPK induces metaphase arrest when injected into one blastomere of a two-cell embryo. This arrest mimics the natural arrest of vertebrate unfertilized eggs in second meiotic metaphase due to cytostatic factor and c-Mosxe activity. We show here that microinjection of constitutively activated thiophosphorylated MAPK into resting oocytes is able to activate maturation-promoting factor (MPF) and promote GVBD. These results strongly support the hypothesis that MAPK plays an important role in the pathway that links c-Mosxe to the activation of MPF.

We describe a simple technique for maintaining highly contractile long-term chicken myogenic cultures on Matrigel, a gel composed of basement membrane components extracted from the Engelbreth-Holm-Swarm mouse tumor. Cultures grown on Matrigel consist of three-dimensional multilayers of cylindrical, contracting myotubes which endure for at least 60 d without myotube detachment. A Matrigel substrate increases the initial plating efficiency but does not effect cell proliferation. Large-scale differentiation in cultures maintained on Matrigel is delayed by 1 to 2 d, compared to cultures grown on gelatin-coated dishes. Long-term maintenance on Matrigel also results in increased expression of the neonatal and adult fast myosin heavy chain isoforms. Culturing of cells on a Matrigel substrate could thus facilitate the study of later events of in vitro myogenesis.