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More than one million HIV-exposed, uninfected infants are born annually to HIV-positive mothers worldwide. This growing population of infants experiences twice the mortality of HIV-unexposed infants. We found that although there were very few differences seen in the microbiomes of mothers with and without HIV infection, maternal HIV infection was associated with changes in the microbiome of HIV-exposed, uninfected infants. Furthermore, we observed that human breast milk oligosaccharides were associated with the bacterial species in the infant microbiome. The disruption of the infant’s microbiome associated with maternal HIV infection may contribute to the increased morbidity and mortality of HIV-exposed, uninfected infants.

Purpose: This study evaluates the effect of dual PI3K and mTOR inhibition using NVP-BEZ235 in preclinical models of ovarian cancer as a potential novel therapeutic strategy.Experimental Design: Inhibition of PI3K/Akt/mTOR signaling by NVP-BEZ235 was demonstrated by immunoblotting. The effect on cell proliferation was assessed in 18 ovarian cancer cell lines, including four pairs of syngeneic cisplatin-sensitive and cisplatin-resistant cell lines. The in vivo effects of NVP-BEZ235 on established tumor growth were evaluated using an immunocompetent, transgenic murine ovarian cancer model (LSL-K-ras G12D/þ Pten loxP/loxP ).Results: NVP-BEZ235 decreased cell proliferation in all ovarian cancer cell lines assayed and sensitized cisplatin-resistant cells to the cytotoxic effects of cisplatin. Cell lines with PI3K-activating mutations or Pten deletions were significantly more sensitive to the effect of NVP-BEZ235 than cell lines without these mutations (P < 0.05). A statistically significant correlation was found between relative levels of p4E-BP1 and the IC 50 for NVP-BEZ235. In LSL-K-ras G12D/þ Pten loxP/loxP mice with established intraperitoneal tumor disease, oral administration of NVP-BEZ235 decreased pAkt, p4E-BP1 and Ki67 in tumor tissue, and resulted in significantly longer survival compared to control animals (P < 0.05). NVP-BEZ235 also induced cell cycle arrest, caspase 3 activity, and reduced cell migration. Conclusions: Targeting PI3K and mTOR simultaneously using NVP-BEZ235 effectively inhibits ovarian cancer cell growth even in the presence of platinum resistance and prolongs survival of mice with intraabdominal ovarian tumor disease. We propose that dual PI3K and mTOR inhibition using NVP-BEZ235 may be an effective novel therapeutic approach in patients with ovarian cancer.

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Cell mechanical phenotype or ‘mechanotype’ is emerging as a valuable label-free biomarker. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and cancer progression. Here we describe a simple and scalable technique to measure cell mechanotype: this parallel microfiltration assay enables multiple samples to be simultaneously measured by driving cell suspensions through porous membranes. To validate the method, we compare the filtration of untransformed and HRasV12-transformed murine ovary cells and find significantly increased deformability of the transformed cells. Inducing epithelial-to-mesenchymal transition (EMT) in human ovarian cancer cells by overexpression of key transcription factors (Snail, Slug, Zeb1) or by acquiring drug resistance produces a similar increase in deformability. Mechanistically, we show that EMT-mediated changes in epithelial (loss of E-Cadherin) and mesenchymal markers (vimentin induction) correlate with altered mechanotype. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application.

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with ''personalized'' driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice.

Here, we demonstrate that arginine vasopressin (AVP) induces multiple intracellular signal transduction pathways in rat intestinal epithelial IEC-18 cells via a V 1A receptor. Addition of AVP to these cells induces a rapid and transient increase in cytosolic Ca 2ϩ concentration and promotes protein kinase D (PKD) activation through a protein kinase C (PKC)-dependent pathway, as revealed by in vitro kinase assays and immunoblotting with an antibody that recognizes autophosphorylated PKD at Ser 916 . AVP also stimulates the tyrosine phosphorylation of the nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinase phosphorylation at Tyr 418 , indicative of Src activation. AVP induces extracellular signal-related kinase (ERK)-1 (p44 mapk ) and ERK-2 (p42 mapk ) activation, a response prevented by treatment with mitogen-activated protein kinase kinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors (GF-I and Ro-31-8220), depletion of Ca 2ϩ (EGTA and thapsigargin), selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or the selective Src family kinase inhibitor PP-2. Furthermore, AVP acts as a potent growth factor for IEC-18 cells, inducing DNA synthesis and cell proliferation through ERK-, Ca 2ϩ -, PKC-, EGFR tyrosine kinase-, and Src-dependent pathways. arginine vasopressin; protein kinase D; protein kinase C; Src; proline-rich tyrosine kinase 2; intestinal epithelial proliferation THE SEQUENTIAL PROLIFERATION, lineage-specific differentiation, migration, and cell death of epithelial cells of the intestinal mucosa is a tightly regulated process that is modulated by a broad spectrum of regulatory peptides (8,36,70). The nontransformed IEC-6 and IEC-18 cells, derived from rat small intestinal crypt (56), have provided an in vitro model to examine intestinal epithelial cell migration, differentiation, and proliferation (16,27,58,70). Previous studies demonstrated that the proliferation and migration of these intestinal epithelial cells is regulated by a variety of polypeptide growth factors, including epidermal growth factor (EGF), insulin-like growth factor I, and hepatocyte growth factor, which act via single-pass transmembrane tyrosine kinase receptors (3,17,51). Neuropeptides and vasoactive peptides that signal through G protein-coupled receptors (GPCRs), characterized by seven-transmembrane helices, also act as potent cellular growth factors for a variety of cell types (63,64,66,67). However, the role of GPCRs and their ligands in intestinal epithelial cell signaling and proliferation remains poorly understood.The neurohypophysial nonapeptide arginine vasopressin (AVP), also known as antidiuretic hormone, is traditionally recognized for its role as a vasoconstrictor hormone acting on vascular smooth muscle cells and its antidiuretic effect via the renal collecting system. In addition to its function in the regulation of body fluid osmolality, vascular tone, and blood pressure, AVP acts as a growth-promo...

Changes in cell stiffness (Young's modulus, E), as measured via Atomic Force Microscopy (AFM), is a newly recognized characteristic of cancer cells and may play a role in platinum drug resistance of ovarian cancers. We previously showed that, compared to their syngeneic cisplatin-sensitive counterpart, cisplatin-resistant ovarian cancer cells are stiffer, and this cell stiffness was dependent on actin polymerization and presence of stress fibers. Here, we measured the correlation between Young's modulus (via AFM measurements on live, non-apoptotic cells in physiological buffer) and cisplatin-sensitivity (IC50 as determined via the XTT cell viability assay) in a panel of nine ovarian cancer cell lines representing a range of cisplatin sensitivities. We found that cisplatin-sensitive cells had a lower Young's modulus, compared to cisplatin-resistant cells and resistant cells had a cytoskeleton composed of long actin stress fibers. As Rho GTPase mediates stress fiber formation, we examined the role of Rho GTPase in cell stiffness and platinum resistance. Rho inhibition decreased cell stiffness in cisplatin-resistant CP70 cells and increased their cisplatin sensitivity while Rho activation increased cell stiffness in cisplatin-sensitive A2780 cells and decreased their cisplatin sensitivity. Based on changes in cell stiffness, IC50 and cellular actin stress fiber organization in CP70 and A2780 cells, our findings reveal a direct role of Rho mediated actin remodeling mechanism in cisplatin resistance of ovarian cancer cells. These findings suggest the potential applicability of cell mechanical phenotyping as a model for determining sensitivity of ovarian cancer cells that could have major implications in ovarian cancer diagnosis and personalized medicine.