Identification and Characterization of the Corazonin Receptor and Possible Physiological Roles of the Corazonin-Signaling Pathway in Rhodnius prolixus.

Neuropeptides control many physiological and endocrinological processes in animals, acting as neuroactive chemicals within the central and peripheral nervous systems. Corazonin (CRZ) is one such neuropeptide that has a variety of physiological roles associated with control of heartbeat, ecdysis behavior initiation, and cuticle coloration. These physiological effects are mediated by the CRZ receptor (CRZR). In order to understand the role of the CRZ-signaling pathway in Rhodnius prolixus, the cDNA sequence encoding the Rhopr-CRZR was isolated and cloned revealing two splice variants (Rhopr-CRZR-α and β). Sequence analysis revealed characteristics of rhodopsin-like GPCRs. Rhopr-CRZR-α and β were dose-dependently activated by Rhopr-CRZ with EC50 values of 2.7 and 1 nM, respectively, when tested in a functional receptor assay using CHOKI-aeq cells. Neither receptors were activated by the evolutionarily-related peptides, Rhopr-AKH, or Rhopr-ACP. For 5th instars, qPCR revealed expression of Rhopr-CRZR transcript in the CNS, the dorsal vessel, abdominal dorsal epidermis, and prothoracic glands with associated fat body. Interestingly, transcript expression was also found in the female and male reproductive tissues. Rhopr-CRZR transcript was reduced after injection of dsCRZR into adult R. prolixus. In these insects, the basal heartbeat rate was reduced in vivo, and the increase in heartbeat frequency normally produced by CRZ on dorsal vessel in vitro was much reduced. No effect of dsCRZR injection was seen on ecdysis or coloration of the cuticle

The Proctolin Gene and Biological Effects of Proctolin in the Blood-Feeding Bug, Rhodnius prolixus

We have reinvestigated the possible presence or absence of the pentapeptide proctolin in Rhodnius prolixus and report here the cloning of the proctolin cDNA. The transcript is expressed in the central nervous system (CNS) and some peripheral tissues. The proctolin prepropeptide encodes a single copy of proctolin along with a possible proctolin-precursor-associated peptide. We have biochemically identified proctolin in CNS extracts and shown its distribution using proctolin-like immunoreactivity. Immunostained processes are found on the salivary glands, female and male reproductive tissues, and heart and associated alary muscles. Proctolin-like immunoreactive bipolar neurons are found on the lateral margins of the common oviduct and bursa. Proctolin is biologically active on R. prolixus tissues, stimulating increases in contraction of anterior midgut and hindgut muscles, and increasing heartbeat frequency. Contrary to the previous suggestion that proctolin is absent from R. prolixus, proctolin is indeed present and biologically active in this medically important bug

Corticotropin-releasing factor-like diuretic hormone acts as a gonad-inhibiting hormone in adult female, Rhodnius prolixus

Within insects, corticotropin-releasing factor/diuretic hormones (CRF/DHs) are responsible for the modulation of a range of physiological and behavioural processes such as feeding, diuresis, and reproduction. Rhopr-CRF/DH plays a key role in feeding and diuresis in Rhodnius prolixus, a blood-gorging insect and a vector for human Chagas disease. Here, we extend our understanding on the role of this neurohormone in reproduction in adult female R. prolixus. Double-label immunohistochemistry displays co-localized staining of CRF-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in the same neurosecretory cells (NSCs) in the mesothoracic ganglionic mass (MTGM) and in their neurohemal sites in adult female R. prolixus, suggesting these peptides could work together to regulate physiological processes. qPCR analysis reveals that the transcript for Rhopr-CRF/DH receptor 2 (Rhopr-CRF/DH-R2) is expressed in reproductive tissues and fat body (FB) in adult female R. prolixus, and its expression increases post blood meal (PBM), a stimulus that triggers diuresis and reproduction. Using RNA interference, transcript expression of Rhopr-CRF/DH-R2 was knocked down, and egg production monitored by examining the major yolk protein, vitellogenin (Vg), the number and quality of eggs laid, and their hatching ratio. Injection of dsCRFR2 into adult females reduces Rhopr-CRF/DH-R2 transcript expression, accelerates oogenesis, increases the number of eggs produced, and reduces hatching rate in female R. prolixus. Downregulation of Rhopr-CRF/DH-R2 leads to an increase in the transcript expression of RhoprVg1 in the fat body and ovaries, and increases the transcript level for the Vg receptor, RhoprVgR, in the ovaries. A significant increase in Vg content in the fat body and in the hemolymph is also observed. Incubation of isolated tissues with Rhopr-CRF/DH leads to a significant decrease in transcript expression of RhoprVg1 in the fat body and RhoprVg1 in the ovaries. In addition, Rhopr-CRF/DH reduces transcript expression of the ecdysteroid biosynthetic enzymes and reduces ecdysteroid titer in the culture medium containing isolated ovaries. These results suggest the involvement of the CRF-signaling pathway in reproduction, and that Rhopr-CRF/DH acts as a gonad-inhibiting hormone in the adult female R. prolixus, as previously shown for the colocalized glycoprotein, GPA2/GPB5

Stromal cells promote anti-estrogen resistance of breast cancer cells through an insulin-like growth factor binding protein 5 (IGFBP5)/B-cell leukemia/lymphoma 3 (Bcl-3) axis

There is strong evidence that stromal cells promote drug resistance of cancer. Here, we show that mesenchymal stem cells (MSCs) and carcinoma-associated fibroblasts (CAFs) desensitize ERa-positive breast cancer cells to the anti-estrogen fulvestrant. In search for the mechanism, we found that MSCs and CAFs similarly increased the activity of the PI3K/AKT and the JAK/STAT3 pathways and upregulated the expression of integrin ß1, IGF1R, HIF1α, CAIX and Bcl-3 in MCF-7 cells. Further analyses revealed that MSCs and CAFs coordinately induce these changes by triggering the downregulation of IGFBP5. Loss of IGFBP5 in MCF-7 cells was an early and long-lasting event in response to MSCs and CAFs and was accompanied by growth stimulation both in the absence and presence of fulvestrant. The growth-stimulatory effect in the absence of fulvestrant could be attributed to PI3K/AKT pathway activation and could be mimicked by insulin. The growth-promoting effect in the presence of fulvestrant depended upon the upregulation of Bcl-3. By cRNA microarray analysis we identified additional IGFBP5 targets, of which two (KLHL4 and SEPP1) were inversely regulated by IGFBP5 and Bcl-3. BT474 cells also responded to stromal cells by downregulating IGFBP5 and upregulating the P-AKT, Bcl-3 and IGF1R levels, whereas T47D cells did not show any of these responses. In conclusion, our data suggest that, by targeting IGFBP5 expression in ERa-positive breast cancer cells, such as MCF-7 cells, MSCs and CAFs are able to orchestrate a variety of events, particularly activation of the PI3K/AKT pathway, upregulation of Bcl-3 expression and desensitization to anti-estrogen

A Rhodnius prolixus Insulin Receptor and Its Conserved Intracellular Signaling Pathway and Regulation of Metabolism

The insulin signaling pathway is a modulator of metabolism in insects and can regulate functions associated with growth and development, as well as lipid and carbohydrate balance. We have previously reported the presence of an insulin-like peptide and an insulin-like growth factor in Rhodnius prolixus, which are involved in the homeostasis of lipids and carbohydrates in post-feeding and non-feeding periods. In the present study, we have characterized the first insulin receptor (IR) to be discovered in R. prolixus, Rhopr-IR, and investigated its intracellular signaling cascade and its role in nutrient control. We identified a candidate protein sequence within R. prolixus putative peptidome and predicted its conserved features using bioinformatics. Tissue-specific expression analyses indicated that the Rhopr-IR transcript is differentially-expressed in all tissues tested, with the highest values observed in the central nervous system (CNS). Treatment of insects with the IR kinase activator BpV(phen), glucose, or porcine insulin resulted in the activation of protein phosphorylation in the fat body, and stimulated the phosphorylation of protein kinase Akt, an evolutionarily conserved key regulator of the intracellular insulin signaling cascade. We also observed activation of Akt and phosphorylation of its downstream targets glycogen synthase kinase 3 β (GSK3β) and the transcription factor FOXO for several days following a blood meal. We used dsRNA to knockdown transcript expression and examined the resulting effects on metabolism and intracellular signaling. Furthermore, knockdown of the Rhopr-IR transcript increased lipid levels in the hemolymph, while reducing lipid content in the fat body. Interestingly, the levels of carbohydrates in the hemolymph and in the fat body did not show any alterations. The activation of Akt and phosphorylation of FOXO were also reduced in knockdown insects, while the phosphorylation pattern of GSK3β did not change. Our results support the identification of the first IR in R. prolixus and suggest that Rhopr-IR signaling is involved in hemolymph nutrient homeostasis and fat body storage both in post-feeding and in non-feeding stages. These metabolic effects are likely regulated by the activation of Akt and downstream cascades similar to mammalian insulin signaling pathways

Simultaneous high resolution meausurement of phonons and ionization created by particle interactions in a 60 g germanium crystal at 25 mK

We demonstrate simultaneous high energy resolution (rms≊800 eV) measurements of ionization and phonons created by particle interactions in a semiconductor crystal of macroscopic size (60 g germanium) at 25 mK. We present first studies of charge collection at biases below 1 V/cm, and find that, contrary to commonly held opinion, the full recoil energy of particle interactions is recovered as phonons when charge trapping is negligible. We also report an unanticipated correlation between charge collection and phonon energy at very low bias, and discuss this effect in terms of charge trapping

Measurement of ionization and phonon production by nuclear recoils in a 60 g crystal of germanium at 25 mK

We report on the first measurement of the absolute phonon energy and the amount of ionization produced by the recoil of nuclei and electrons in a 60 g germanium cyrstal at a temperature of ≊25 mK. We find good agreement between our results and previous measurements of ionization yield from nuclear recoils in germanium. Our device achieves 10:1 discrimination between neutrons and photons in the few keV energy range, demonstrating the feasibility of this technique for large reductions of background in searches for direct interactions of weakly interacting massive particle dark matter

NOTCH3 Expression Is Linked to Breast Cancer Seeding and Distant Metastasis

Background: Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. Methods: We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database. Results: In this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens. Conclusions: These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer

Evaluation of polygenic risk scores for breast and ovarian cancer risk prediction in BRCA1 and BRCA2 mutation carriers

Background: Genome-wide association studies (GWAS) have identified 94 common single-nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk and 18 associated with ovarian cancer (OC) risk. Several of these are also associated with risk of BC or OC for women who carry a pathogenic mutation in the high-risk BC and OC genes BRCA1 or BRCA2. The combined effects of these variants on BC or OC risk for BRCA1 and BRCA2 mutation carriers have not yet been assessed while their clinical management could benefit from improved personalized risk estimates. Methods: We constructed polygenic risk scores (PRS) using BC and OC susceptibility SNPs identified through population-based GWAS: for BC (overall, estrogen receptor [ER]-positive, and ER-negative) and for OC. Using data from 15 252 female BRCA1 and 8211 BRCA2 carriers, the association of each PRS with BC or OC risk was evaluated using a weighted cohort approach, with time to diagnosis as the outcome and estimation of the hazard ratios (HRs) per standard deviation increase in the PRS. Results: The PRS for ER-negative BC displayed the strongest association with BC risk in BRCA1 carriers (HR = 1.27, 95% confidence interval [CI] = 1.23 to 1.31, P = 8.2 x 10(53)). In BRCA2 carriers, the strongest association with BC risk was seen for the overall BC PRS (HR = 1.22, 95% CI = 1.17 to 1.28, P = 7.2 x 10(-20)). The OC PRS was strongly associated with OC risk for both BRCA1 and BRCA2 carriers. These translate to differences in absolute risks (more than 10% in each case) between the top and bottom deciles of the PRS distribution; for example, the OC risk was 6% by age 80 years for BRCA2 carriers at the 10th percentile of the OC PRS compared with 19% risk for those at the 90th percentile of PRS. Conclusions: BC and OC PRS are predictive of cancer risk in BRCA1 and BRCA2 carriers. Incorporation of the PRS into risk prediction models has promise to better inform decisions on cancer risk management

Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature