The U.S. Must Embrace Partner-led Global Health Engagement.

Direct-care Medical Readiness Training Exercises (MEDRETEs) are a popular method for conducting global health engagement. Unfortunately, direct-care MEDRETEs build partnerships without building partner nation capacity. This article proposes that global health engagement should instead focus on partner-led health engagements to accomplish both of these goals.

Early caregiver burden in owners of pets with suspected cancer: Owner psychosocial outcomes, communication behavior, and treatment factors.

BACKGROUND: Owners of companion animals with serious illnesses are likely to experience "caregiver burden." This topic has not been fully evaluated in veterinary oncology. OBJECTIVES: To examine owners of a dog or cat with suspected cancer for relationships between early caregiver burden and (a) psychosocial factors: depression, stress, and quality of life; (b) owner communication behavior; and (c) specific pet treatment plan factors. ANIMALS: None. METHODS: This cross-sectional, observational study recruited 164 owners of a cat or dog presenting for evaluation by a veterinary oncology service at a single referral institution. Measures of caregiver burden, psychosocial function, treatment plan elements, and demographics were collected online via owner self-report. Medical records were reviewed to identify factors including diagnosis, medications, treatment schedules, and owner communications. RESULTS: Caregiver burden correlated with higher stress (rs = 0.40, P < .001), greater symptoms of depression (rs = 0.50, P < .001), and lower quality of life (rs = 0.39, P < .001). Pet treatment plan factors related to caregiver burden included changes in care routines, perception that compliance with new routines was challenging, and difficulty adhering to medication routines. There was low correlation between caregiver burden and owner-driven communications (rs = 0.15, P = .07). CONCLUSIONS AND CLINICAL IMPORTANCE: Findings suggest caregiver burden is similar in owners of pets with cancer and owners of pets with other diseases. Caregiver burden is present in the earliest stages of disease. Major correlates of burden including life-disruptive treatments and schedules provide key areas for potential intervention by veterinary teams.

Anemia in Older Adults.

Anemia is associated with increased morbidity and mortality in older adults. Diagnostic cutoff values for defining anemia vary with age, sex, and possibly race. Anemia is often asymptomatic and discovered incidentally on laboratory testing. Patients may present with symptoms related to associated conditions, such as blood loss, or related to decreased oxygen-carrying capacity, such as weakness, fatigue, and shortness of breath. Causes of anemia in older adults include nutritional deficiency, chronic kidney disease, chronic inflammation, and occult blood loss from gastrointestinal malignancy, although in many patients the etiology is unknown. The evaluation includes a detailed history and physical examination, assessment of risk factors for underlying conditions, and assessment of mean corpuscular volume. A serum ferritin level should be obtained for patients with normocytic or microcytic anemia. A low serum ferritin level in a patient with normocytic or microcytic anemia is associated with iron deficiency anemia. In older patients with suspected iron deficiency anemia, endoscopy is warranted to evaluate for gastrointestinal malignancy. Patients with an elevated serum ferritin level or macrocytic anemia should be evaluated for underlying conditions, including vitamin B12 or folate deficiency, myelodysplastic syndrome, and malignancy. Treatment is directed at the underlying cause. Symptomatic patients with serum hemoglobin levels of 8 g per dL or less may require blood transfusion. Patients with suspected iron deficiency anemia should be given a trial of oral iron replacement. Lower-dose formulations may be as effective and have a lower risk of adverse effects. Normalization of hemoglobin typically occurs by eight weeks after treatment in most patients. Parenteral iron infusion is reserved for patients who have not responded to or cannot tolerate oral iron therapy.

The ANK repeats of Notch-4/Int3 activate NF-κB canonical pathway in the absence of Rbpj and causes mammary tumorigenesis.

Transgenic mice expressing the Notch-4 intracellular domain (designated Int3) in the mammary gland have two phenotypes exhibited with 100% penetrance: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch-4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. Interestingly, WAP-Int3/Rbpj knockout mice have normal mammary gland development but still developed mammary tumors with a slightly longer latency than the WAP-Int3 mice. Thus, Notch-induced mammary tumor development is Rbpj-independent. Here, we show that Int3 activates NF-κB in HC11 cells in absence of Rbpj through an association with the IKK signalosome. Int3 induced the canonical NF-κB activity and P50 phosphorylation in HC11 cells without altering the NF-κB2 pathway. The minimal domain within the Int3 protein required to activate NF-κB consists of the CDC10/Ankyrin (ANK) repeats domain. Treatment of WAP-Int3 tumor bearing mice with an IKK inhibitor resulted in tumor regression. In a soft agar assay, treatment of HC11-Int3 cells with P50-siRNA caused a significant decrease in colony formation. In addition, Wap-Int3/P50 knockout mice did not develop mammary tumors. This data indicates that the activation of NF-κB canonical signaling by Notch-4/Int3 is ANK repeats dependent, Rbpj-independent, and is mediated by IKK activation and P50 phosphorylation causing mammary tumorigenesis.

Original Research: Featured Article: Imatinib mesylate (Gleevec) inhibits Notch and c-Myc signaling: Five-day treatment permanently rescues mammary development.

Wap-Int3 transgenic females expressing the Notch4 intracellular domain (designated Int3) from the whey acidic protein promoter exhibit two phenotypes in the mammary gland: blockage of lobuloalveolar development and lactation, and tumor development with 100% penetrance. Previously, we have shown that treatment of Wap-Int3 tumor bearing mice with Imatinib mesylate (Gleevec) is associated with complete regression of the tumor. In the present study, we show that treatment of Wap-Int3 mice during day 1 through day 6 of pregnancy with Gleevec leads to the restoration of their lobuloalveolar development and ability to lactate in subsequent pregnancies in absence of Gleevec treatment. In addition, these mice do not develop mammary tumors. We investigated the mechanism for Gleevec regulation of Notch signaling and found that Gleevec treatment results in a loss of Int3 protein but not of Int3 mRNA in HC11 mouse mammary epithelial cells expressing Int3. The addition of MG-132, a proteasome inhibitor, shows increased ubiquitination of Int3 in the presence of Gleevec. Thus, Gleevec affects the stability of Int3 by promoting the degradation of Int3 via E3 ubiquitin ligases targeting it for the proteasome degradation. Gleevec is a tyrosine kinase inhibitor that acts on c-Kit and PDGFR. Therefore, we investigated the downstream substrate kinase GSK3ß to ascertain the possible role that this kinase might play in the stability of Int3. Data show that Gleevec degradation of Int3 is GSK3ß dependent. We have expanded our study of the effects Gleevec has on tumorigenesis of other oncogenes. We have found that anchorage-independent growth of HC11-c-Myc cells as well as tumor growth in nude mice is inhibited by Gleevec treatment. As with Int3, Gleevec treatment appears to destabilize the c-Myc protein but not mRNA. These results indicate that Gleevec could be a potential therapeutic drug for patients bearing Notch4 and/or c-Myc positive breast carcinomas.

Cripto-1 ablation disrupts alveolar development in the mouse mammary gland through a progesterone receptor-mediated pathway.

Cripto-1, a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways.

Lipid-enhancement of activated sludges obtained from conventional activated sludge and oxidation ditch processes.

Lipid-enhancement of activated sludges was conducted to increase the amount of saponifiable lipids in the sludges. The sludges were obtained from a conventional activated sludge (CAS) and an oxidation ditch process (ODP). Results showed 59-222% and 150-250% increase in saponifiable lipid content of the sludges from CAS and ODP, respectively. The fatty acid methyl ester (FAMEs) obtained from triacylglycerides was 57-67% (of total FAMEs) for enhanced CAS and 55-73% for enhanced ODP, a very significant improvement from 6% to 10% (CAS) and 4% to 8% (ODP). Regardless of the source, the enhancement resulted in sludges with similar fatty acid profile indicating homogenization of the lipids in the sludges. This study provides a potential strategy to utilize existing wastewater treatment facilities as source of significant amount of lipids for biofuel applications.

Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors.

The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene expression in primary human breast tumors was interrogated. Twenty of the human orthologues of MMTV CIS associated genes are deregulated and/or mutated in human breast tumors.

Effects of age and parity on mammary gland lesions and progenitor cells in the FVB/N-RC mice.

The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3-4 pregnancies) females were euthanized at 16-22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer.

Recombinant R-spondin2 and Wnt3a up- and down-regulate novel target genes in C57MG mouse mammary epithelial cells.

R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/ß-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. We observed the up- and down-regulation of several previously unidentified target genes, including ones that encode proteins involved in immune responses, effectors of other growth factor signaling pathways and transcription factors. Dozens of these changes were validated by quantitative real time RT-PCR. Time course experiments showed that Rspo2 typically had little or no effect on Wnt-dependent gene expression at 3 or 6 h, but enhanced expression at 24 h, consistent with biochemical data indicating that Rspo2 acts primarily to sustain rather than acutely increase Wnt pathway activation. Up-regulation of gene expression was inhibited by pre-treatment with Dickkopf1, a Wnt/ß-catenin pathway antagonist, and by siRNA knockdown of ß-catenin expression. While Dickkopf1 blocked Rspo2/Wnt3a-dependent down-regulation, a number of down-regulated genes were not affected by ß-catenin knockdown, suggesting that in these instances down-regulation was mediated by a ß-catenin-independent mechanism.

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/ß-catenin pathway, or short hairpin RNA targeting ß-catenin expression demonstrated that the invasive activity was not mediated by ß-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the ß-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

Expression of truncated eukaryotic initiation factor 3e (eIF3e) resulting from integration of mouse mammary tumor virus (MMTV) causes a shift from cap-dependent to cap-independent translation.

Integration of mouse mammary tumor virus (MMTV) at the common integration site Int6 occurs in the gene encoding eIF3e, the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation, but by an unknown mechanism. Because eIF3e makes up at least part of the binding site for eIF4G, we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G, signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43 S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP, c-Myc, CYR61, and Pim-1, which are translated in a cap-independent manner, were shifted to heavier polysomes whereas mRNAs encoding GAPDH, actin, L32, and L34, which are translated in a cap-dependent manner, were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between up-regulation of cap-independent translation and MMTV-induced tumorigenesis contrasts with the well established model for malignant transformation involving up-regulation of highly cap-dependent translation.

Expression of Notch receptors, ligands, and target genes during development of the mouse mammary gland.

Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.

Trp53 regulates Notch 4 signaling through Mdm2.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Transforming acidic coiled-coil protein-3 (Tacc3) acts as a negative regulator of Notch signaling through binding to CDC10/Ankyrin repeats.

We have identified the transforming acidic coiled-coil protein-3 (Tacc3) as a binding partner for Notch4/Int3 and were able to show that it binds to the intracellular domain (ICD) of all members of the Notch receptor family. Members of the Tacc family reside at the centrosomes and associates with microtubules. Recent studies suggest that Tacc3 also contributes to the regulation of gene transcription. Tacc3 specifically interacts with the Notch4/Int3 CDC10/Ankyrin repeats and to a lesser extent, with residues C-terminal to these repeats in the ICD. Dual label immunofluorescence of mouse mammary tissue shows Tacc3 co-localizes with the Notch3 ICD. Co-immunoprecipitation of endogenous Notch and Tacc3 proteins from NIH3T3 cell extracts, lung and mammary gland confirms that these two proteins interact under physiological conditions. In addition, knock down of Tacc3 in NIH3T3 cells leads to the up-regulation of Hey2, a target gene for Notch signaling. The affinity of Tacc3 binding to Notch4/Int3 ICD is similar to that between Rbpj and Notch4/Int3 ICD. Notch4/Int3 ICD-Tacc3 interaction results in the inhibition of transcription from a Hes1-Luciferase reporter vector in COS-1 cells. The inhibition was reversed in these cells by increasing the levels of Rbpj. Taken together, these results suggest that Tacc3 is a negative regulator of the Notch signaling pathway.

Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1.

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.