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The delta opioid receptor (DOR), a physiologically relevant prototype for G protein–coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

Cancer is considered one of the leading causes of death in the United States. Although preventive strategies, early detection, and improved treatment options have been developed, novel targets and therapeutics are still needed. Since concluding that cancer is mediated by genetic and epigenetic alterations of the cell, many research groups are now focusing on other means of prevention and therapy via nutrition, epigenetic mechanisms, and non-coding RNAs which have been shown to control gene expression and have many different functions at the cellular level. With the advent of high-throughput sequencing in human cancer, the potential to identify novel biomarkers and therapeutic targets of disease has increased tremendously and led to the identification of many non-coding RNAs that are dysregulated in various cancers. Gene expression and regulation is important in maintaining the homeostasis of normal tissues and cells. Not uncommonly, up- or down-regulation of particular genes are associated with cancer as a result of increased or decreased expression of transcriptional targets. This review focuses on the role of nutrition in cancer and the dysregulation of non-coding RNAs with particular emphasis on long non-coding RNAs and microRNAs in different cancer types.

Alternative splicing (AS), a tightly regulated process, contributes to proteome diversification essential for tissue development and identity. During brain development, splicing targets include genes involved in several cellular processes like endocytosis, cytoskeleton dynamics and vesicle-mediated transport. Deregulation of AS in cancers enables tumor cells to alter the transcriptome in favor of isoforms that drive tumor progression. Annexin A7 (ANXA7) is a hydrophilic protein that binds membranes in a calcium-dependent manner; it is important for membrane scaffolding and vesicle trafficking. ANXA7 is alternatively spliced by PTBP1 and expressed as two isoforms - unspliced ANXA7 Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). We determined that this splicing of ANXA7 is lineage-specific in the brain - I1 is highly expressed in post-mitotic mature neurons while I2 is expressed in neural and glial precursors. In order to understand the functional impact of these isoforms, we generated GBM cell lines that express endogenous I2 (I2 cells), or endogenous I2 + exogenous I1 (I1 cells). We evaluated the influence of I1 and/or I2 expression on RTK levels, activation and downstream events using western blot and co-immunoprecipitation assays. Receptor trafficking was observed using flow cytometry and immunofluorescence assays tagging different components of the endocytic pathway. In GBM, we observed high levels of PTBP1 with a subsequent increase in I2 levels indicative of dysregulated AS. Consequently, we observed upregulated activation of several receptor tyrosine kinases (RTKs) such as EGFR, MET, PDGFRα and EGFR vIII in the I2 cells. In I1 cells, however, we observed reduced levels and activation of all aforementioned RTKs along with the diminished activation of downstream pathways. Using EGFR signaling as a model, we found that in I1 cells EGFR co-localized with markers of lysosomal degradation indicating efficient trafficking of activated EGFR for endosomal degradation; co-localization was absent in I2 cells indicative of impaired trafficking. We propose that I1, by virtue of the amino acids encoded by cassette exon 6, binds to conserved motifs within multiple RTKs and targets them for endosomal degradation thereby attenuating tumorigenic signaling in GBM. Our results show that ANXA7 I1 is tumor suppressive in GBM and that loss of I1 promotes tumorigenicity by perpetuating RTK signaling. We present a mechanism that explains, in part, how I1 mediates the degradation of multiple tumorigenic RTKs by spatio-temporally regulating their endosomal trafficking. A refined understanding of how I1 functions will provide the foundation for future, selective therapies that target dysregulated AS in GBM. Citation Format: Sindhu Nair, Rajani Rajbhandari, Centdrika Dates-Hurt, Markus Bredel. Dysregulated splicing of Annexin A7 impairs receptor tyrosine kinase trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3747.

Purpose/Objective(s): Glioblastomas (GBM) are highly malignant tumors that arise from astrocytes. GBMs rely on signaling through receptor tyrosine kinases (RTK) to ensure tumorigenicity. EGFR, PDGFRA and MET are distinct RTKs; each is capable of binding a ligand, which promotes receptor dimerization and kinase activation. Annexin A7 (ANXA7), a protein involved in membrane binding and vesicle trafficking, is spliced into 2 isoforms - isoforms 1 (I1) and isoform 2 (I2). A normal adult brain will express both isoforms, however, in GBM, I1 expression is prevented. We determined that I1, but not I2, is tumor suppressive, in part, because it promotes the endocytic degradation of EGFR. Because I1 is typically not expressed in GBM, EGFR signaling persists unabated and tumors thrive. Recently, we extended these observations and determined that I1 also impacts additional RTKs, including PDGFRA and MET. By understanding how ANXA7 I1 impacts PDGFRA and MET signaling, we hope to target multiple tumorigenic RTKs and improve therapy for patients with GBM. Materials/Methods: To understand how and when I1 downregulates PDGFRα and MET, U251-malignant glioma (MG) cells, which only express I2 (P), were modified to express I1. Cells were serum starved overnight, and then stimulated with PDGFAA and HGF(20 ng/ml), which activate the aforementioned receptors respectively. As a positive control, cells were stimulated with EGF to activate EGFR. Total protein was then collected and analyzed by western blot analyses. Next, we sought to determine whether I1 was competent to down-regulate several RTKs coincidentally. To do this, P and I1 cells were serum starved, stimulated with EGF, PDGFAA or both, and total protein levels were evaluated. Results: The levels of activated EGFR, PDGFRα and MET are dramatically reduced in those cells that express I1 as compared to cells expressing I2 (P). Upon activation with both PDGFAA and EGF, in cells expressing I1 (I1), levels of PDGFRα and EGFR were markedly reduced 30 minutes post-stimulation as compared to cells expressing I2. Conclusion: Our results suggest I1 may be a master regulator of RTK endocytosis and degradation. Further investigations of the impact of I1 on endocytic trafficking patterns using immunofluorescence and confocal microscopy are underway. Collectively, understanding how I1 functions and targets multiple tumorigenic RTKs will provide the foundation for future selective therapies that restore I1 expression in GBM and reduce tumorigenicity. Citation Format: Zachary B. White, Centdrika Dates Hurt, Rajani Rajbhandari, Sindhu Nair, Susan Nozell, Markus Bredel. Impact of ANXA7 I1 expression on PDGFRA and MET endosomal trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3758.

Dysregulation of receptor tyrosine kinase genes is a frequent genetic abnormality in GBM. A common genetic alteration is amplification and/or overexpression of the epidermal growth factor receptor (EGFR) gene. Annexin A7 (ANXA7) is a hydrophilic protein that binds membranes in a calcium dependent manner; it is important for membrane scaffolding and vesicle trafficking. ANXA7 is alternatively spliced and expressed as two isoforms – unspliced ANXA7 Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). Our lab has determined that I1 is tumor suppressive in GBM and that loss of I1 promotes tumorigenicity by perpetuating EGFR signaling. However, if restored, I1 but not I2, reduces EGFR levels, activation and downstream pathways and diminishes tumorigenicity. In patients, I1 levels directly correlate with survival and prognosis. We propose that I1 inhibits EGFR signalling by binding to and promoting degradation of EGFR in a calcium dependent manner. In GBM, I1 is absent, preventing endosomal degradation of EGFR and leading to perpetual signalling. Our approach is novel; rather than address how/whether EGFR is activated, we focus on why EGFR fails to be downregulated afterwards. Herein, we present a mechanism that explains, in part, how I1 mediates endosomal degradation of EGFR. Further studies are underway to identify exactly where and how I1 modifies endosomal trafficking and degradation of EGFR. We are also exploring the role of I1 in regulating the degradation of other receptor tyrosine kinases (RTKs), such as MET and PDGFRA. Collectively, understanding how I1 functions will provide the foundation for future, selective therapies that restore I1 expression and/or function to reduce GBM tumorigenicity.

Increased aerobic glycolysis and de novo lipid biosynthesis are common characteristics of invasive cancers. UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that in normal cells possess the ability to glucuronidate these lipids and speed their excretion; however, de-regulation of these enzymes in cancer cells can lead to an accumulation of bioactive lipids, which further fuels cancer progression. We hypothesize that UGT2B isoform expression is down-regulated in cancer cells and that exogenous re-introduction of these enzymes will reduce lipid content, change the cellular phenotype, and inhibit cancer cell proliferation. In this study, steady-state mRNA levels of UGT isoforms from the 2B family were measured using qPCR in 4 breast cancer and 5 pancreatic cancer cell lines. Expression plasmids for UGT2B isoforms known to glucuronidate cellular lipids, UGT2B4, 2B7, and 2B15 were transfected into MCF-7 and Panc-1 cells, and the cytotoxic effects of these enzymes were analyzed using trypan blue exclusion, annexin V/PI staining, TUNEL assays, and caspase-3 immunohistochemistry. There was a significant decrease in cell proliferation and a significant increase in the number of dead cells after transfection with each of the 3 UGT isoforms in both cell lines. Cellular lipids were also found to be significantly decreased after transfection. The results presented here support our hypothesis and emphasize the important role UGTs can play in cellular proliferation and lipid homeostasis. Evaluating the effect of UGT expression on the lipid levels in cancer cell lines can be relevant to understanding the complex regulation of cancer cells, identifying the roles of UGTs as “lipid-controllers” in cellular homeostasis, and illustrating their suitability as targets for future clinical therapy development.

Tamoxifen (Tam) is a selective estrogen receptor modulator used to inhibit breast tumor growth. Tam can be directly N-glucuronidated via the tertiary amine group or O-glucuronidated after cytochrome P450–mediated hydroxylation. In this study, the glucuronidation of Tam and its hydroxylated and/or chlorinated derivatives [4-hydroxytamoxifen (4OHTam), toremifene (Tor), and 4-hydroxytoremifene (4OHTor)] was examined using recombinant human UDP-glucuronosyltransferases (UGTs) from the 1A subfamily and human hepatic microsomes. Recombinant UGT1A4 catalyzed the formation of N-glucuronides of Tam and its derivatives and was the most active UGT enzyme toward these compounds. Therefore, it was hypothesized that single nucleotide polymorphisms (SNPs) in the promoter region of UGT1A4 have the ability to significantly decrease the glucuronidation rates of Tam metabolites in the human liver. In vitro activity of 64 genotyped human liver microsomes was used to determine the association between the UGT1A4 promoter and coding region SNPs and the glucuronidation rates of Tam, 4OHTam, Tor, and 4OHTor. Significant decreases in enzymatic activity were observed in microsomes for individuals heterozygous for −163G/A and −217T/G. These alterations in glucuronidation may lead to prolonged circulating half-lives and may potentially modify the effectiveness of these drugs in the treatment of breast cancer.

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator used to inhibit breast tumor growth. Tam can be directly N-glucuronidated via the tertiary amine group or O-glucuronidated after cytochrome P450 mediated hydroxylation. In this study, the glucuronidation of Tam and its hydroxylated and/or chlorinated derivatives [4OHTamoxifen (4OHTam), Toremifene (Tor), and 4OHTor] was examined using recombinant human UGTs from the 1A subfamily and human hepatic microsomes. Recombinant UGT1A4 catalyzed the formation of N-glucuronides of Tam and its derivatives and was the most active UGT enzyme toward these compounds. Therefore, it was hypothesized that single nucleotide polymorphisms (SNPs) in the promoter region of UGT1A4 have the ability to significantly decrease the glucuronidation rates of Tam metabolites in the human liver. In vitro activity of 64 genotyped human liver microsomes were used to determine the association between the UGT1A4 promoter and coding region SNPs and the glucuronidation rates of Tam, 4OHTam, Tor, and 4OHTor. Significant decreases in enzymatic activity were observed in microsomes for individuals heterozygous for -163G/A and -217T/G. These alterations in glucuronidation may lead to prolonged circulating half-lives and potentially modify the effectiveness of these drugs in the treatment of breast cancer.

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.

Objetivos. Determinar la seroprevalencia de la infeccion por Trypanosoma cruzi en Ushuaia, la ciudad mas austral del mundo. Metodos. Se analizaron muestras de suero de 2 991 personas, obtenidas entre enero de 1995 y diciembre de 1996. Las muestras fueron procesadas por hemaglutinacion indirecta (HAI) e inmunoensayo enzimatico (ELISA) o inmunofluorescencia indirecta (IFI). Resultados. La seroprevalencia general de la infeccion por T. cruzi fue de 6,8%. La prevalencia segun el pais de origen fue de 41,1% en los bolivianos, 5,0% en los argentinos y 0,9% en los chilenos; en embarazadas fue de 5,9%, en examenes obligatorios de 6,3% y en consultas dirigidas de 30,8%. Conclusiones. Se destaca la magnitud de la infeccion por T. cruzi en una zona donde no existe el insecto vector. Debido al riesgo de la transmision congenita y transfusional, es necesario mantener el control de la sangre a transfundir y reforzar el seguimiento de los hijos de mujeres infectadas para un diagnostico precoz y tratamiento oportuno de la infeccion.

The Phoenix Mars Scout Lander will launch in August 2007 and land on the northern plains of Mars in May of 2008. In a departure from traditional planetary surface mission operations, it will have no direct-to-Earth communications capability and will rely entirely on Mars-orbiting relays in order to facilitate command and control as well as the return of science and engineering data. The Mars Exploration Rover missions have demonstrated the robust data-return capability using this architecture, and also have demonstrated the capability of using this method for command and control. The Phoenix mission will take the next step and incorporate this as the sole communications link. Operations for 90 Sols will need to work within the constraints of Odyssey and Mars Reconnaissance Orbiter communications availability, anomalies must be diagnosed and responded to through an intermediary and on-board fault responses must be tolerant to loss of a relay. These and other issues pose interesting challenges and changes in paradigm for traditional space operations and spacecraft architecture, and the approach proposed for the Phoenix mission is detailed herein.

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

The consequences of aging of the immune system include impaired T-lymphocyte responsiveness and aberrant immunoglobulin production. Although T cells from elderly individuals have a well-described defect in lymphoblastic transformation in response to some polyclonal mitogens, immunoglobulin abnormalities have lacked a clear in vitro model. Peripheral blood mononuclear cells from 13 young and 13 old healthy donors were cultured with phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Old-donor-cell phytohemagglutinin (PHA), but not PWM, cultures had significantly lower lymphoblastic transformation compared with young donor cultures. IgG, IgA, and IgM production tended to be lower in old- versus young-donor PWM cell cultures. By contrast, despite lower lymphoblastic transformation in old-donor PHA cell cultures, immunoglobulin production was higher for old- versus young-donor cell cultures. No significant age differences were present in initial lymphocyte counts, percent B cells, T cells or monocytes, or helper/suppressor ratios to explain this enhancement in immunoglobulin production. PHA-stimulated mononuclear cell cultures in the aged demonstrate not only a defect in proliferation but also increased immunoglobulin production. This in vitro system may be useful to characterize further the pathogenesis of altered immunoglobulin production in the elderly.

Drosophila melanogaster larvae require choline for growth but when fed dl-carnitine in place of choline, carnitine is decarboxylated to β-methylcholine and phosphatidyl-β-methylcholine is synthesized to replace phosphatidylcholine. Feeding (−)-carnitine in place of choline results in a moderate deficiency of lecithin-type phospholipid and a corresponding reduction in growth. The capacity of larvae to employ (+)-carnitine for phosphatidyl-β-methylcholine synthesis and subsequently for growth is very limited. Only 41% as much phosphatidyl-β-methylcholine is synthesized by larvae fed 5.7 X 10−4 M (+)-carnitine as is formed when the same concentration of (−)-carnitine is fed. This suggests that (−)-carnitine is the preferred substrate for carnitine decarboxylase, the enzyme that degrades carnitine to form β-methylcholine. Utilization of carnitine for phospholipid synthesis is clearly dependent upon a choline-deficient state. The presence of small quantities of choline, either in body tissues or in the diet, inhibits the incorporation of carnitine derivatives into phospholipid. Intact carnitine is needed for maximum larval growth and adult activity, indicating that D. melanogaster larvae synthesize a suboptimal quantity of carnitine. Thus, dietary carnitine supplements in vivo carnitine synthesis for physiological purposes.

Several questions remain regarding the timing and nature of the Neanderthal-anatomically modern human (AMH) transition in Europe. The situation in Eastern Europe is generally less clear due to the relatively few sites and a dearth of reliable radiocarbon dates. Claims have been made for both notably early AMH and notably late Neanderthal presence, as well as for early AMH (Aurignacian) dispersal into the region from Central/Western Europe. The Kostenki-Borshchevo complex (European Russia) of Early Upper Paleolithic (EUP) sites offers high-quality data to address these questions. Here we revise the chronology and cultural status of the key sites of Kostenki 17 and Kostenki 14. The Kostenki 17/II lithic assemblage shares important features with Proto-Aurignacian material, strengthening an association with AMHs. New radiocarbon dates for Kostenki 17/II of ∼41–40 ka cal BP agree with new dates for the recently excavated Kostenki 14/IVw, which shows some similarities to Kostenki 17/II. Dates of ≥41 ka cal BP from other Kostenki sites cannot be linked to diagnostic archaeological material, and therefore cannot be argued to date AMH occupation. Kostenki 14's Layer in Volcanic Ash assemblage, on the other hand, compares to Early Aurignacian material. New radiocarbon dates targeting diagnostic lithics date to 39–37 ka cal BP. Overall, Kostenki's early EUP is in good agreement with the archaeological record further west. Our results are therefore consistent with models predicting interregional penecontemporaneity of diagnostic EUP assemblages. Most importantly, our work highlights ongoing challenges for reliably radiocarbon dating the period. Dates for Kostenki 14 agreed with the samples' chronostratigraphic positions, but standard pre-treatment methods consistently produced incorrect ages for Kostenki 17/II. Extraction of hydroxyproline from bone collagen using preparative high-performance liquid chromatography, however, yielded results consistent with the samples' chronostratigraphic position and with the layer's archaeological contents. This suggests that for some sites compound-specific techniques are required to build reliable radiocarbon chronologies.

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.