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PDF file - 44K, Supplementary Table S2. Primer sequences for RT-PCR.

Obesity is associated with increased liver cancer risks and mortality. We recently showed that apo-10′-lycopenoic acid, a lycopene metabolite generated by beta-carotene-9′,10′-oxygenase (BCO2), inhibited carcinogen-initiated, high-fat diet (HFD)–promoted liver inflammation, and hepatic tumorigenesis development. The present investigation examined the outstanding question of whether lycopene could suppress HFD-promoted hepatocellular carcinoma (HCC) progression, and if BCO2 expression is important using BCO2-knockout (BCO2-KO) and wild-type male mice. Results showed that lycopene supplementation (100 mg/kg diet) for 24 weeks resulted in comparable accumulation of hepatic lycopene (19.4 vs. 18.2 nmol/g) and had similar effects on suppressing HFD-promoted HCC incidence (19% vs. 20%) and multiplicity (58% vs. 62%) in wild-type and BCO2-KO mice, respectively. Intriguingly, lycopene chemopreventive effects in wild-type mice were associated with reduced hepatic proinflammatory signaling (phosphorylation of NK-κB p65 and STAT3; IL6 protein) and inflammatory foci. In contrast, the protective effects of lycopene in BCO2-KO but not in wild-type mice were associated with reduced hepatic endoplasmic reticulum stress–mediated unfolded protein response (ERUPR), through decreasing ERUPR-mediated protein kinase RNA-activated like kinase–eukaryotic initiation factor 2α activation, and inositol requiring 1α–X-box–binding protein 1 signaling. Lycopene supplementation in BCO2-KO mice suppressed oncogenic signals, including Met mRNA, β-catenin protein, and mTOR complex 1 activation, which was associated with increased hepatic microRNA (miR)-199a/b and miR214 levels. These results provided novel experimental evidence that dietary lycopene can prevent HFD-promoted HCC incidence and multiplicity in mice, and may elicit different mechanisms depending on BCO2 expression. Cancer Prev Res; 7(12); 1219–27. ©2014 AACR.

PDF file - 61K, Supplementary Figure S2. Inhibition of carcinogen-initiated (DEN), HFD-promoted glucose intolerance by APO10LA. Glucose tolerance test was conducted on 6-8 mice/group and calculated for AUC.

PDF file - 30K, Supplementary Table S1. High-fat diet composition.

Supplementary Table S1: Primers sequences for RT-PCR analysis of liver tissue.

Obesity is associated with increased risk in hepatocellular carcinoma (HCC) development and mortality. An important disease control strategy is the prevention of obesity-related hepatic inflammation and tumorigenesis by dietary means. Here, we report that apo-10′-lycopenoic acid (APO10LA), a cleavage metabolite of lycopene at its 9′,10′-double bond by carotene-9′,10′-oxygenase, functions as an effective chemopreventative agent against hepatic tumorigenesis and inflammation. APO10LA treatment on human liver THLE-2 and HuH7 cells dose dependently inhibited cell growth and upregulated sirtuin 1 (SIRT1), a NAD+-dependent protein deacetylase that may suppress hepatic carcinogenesis. This observed SIRT1 induction was associated with decreased cyclin D1 protein, increased cyclin-dependent kinase inhibitor p21 protein expression, and induced apoptosis. APO10LA supplementation (10 mg/kg diet) for 24 weeks significantly reduced diethylnitrosamine-initiated, high fat diet (HFD)–promoted hepatic tumorigenesis (50% reduction in tumor multiplicity; 65% in volume) and lung tumor incidence (85% reduction) in C57Bl/6J mice. The chemopreventative effects of APO10LA were associated with increased hepatic SIRT1 protein and deacetylation of SIRT1 targets, as well as with decreased caspase-1 activation and SIRT1 protein cleavage. APO10LA supplementation in diet improved glucose intolerance and reduced hepatic inflammation [decreased inflammatory foci, TNFα, interleukin (IL)-6, NF-κB p65 protein expression, and STAT3 activation] in HFD-fed mice. Furthermore, APO10LA suppressed Akt activation, cyclin D1 gene, and protein expression and promoted PARP protein cleavage in transformed cells within liver tumors. Taken together, these data indicate that APO10LA can effectively inhibit HFD-promoted hepatic tumorigenesis by stimulating SIRT1 signaling while reducing hepatic inflammation. Cancer Prev Res; 6(12); 1304–16. ©2013 AACR.

PDF file - 48K, Supplementary Figure S1. Chemical structure of lycopene, APO10LA, acyclic retinoic acid and all-trans retinoic acid.

PDF file - 54K

Humans are consistently exposed to thousands of untested chemicals that have been detected in the follicular fluid of the ovaries, and can disrupt reproductive health. Human granulosa cells (GCs) are the functional unit of the ovarian follicle with steroidogenic and signaling activities, and play a pivotal role in oocyte development. During follicle progression, GCs multiply to form a 3D avascular structure, and establish gap junction intercellular communication (GJIC) that is critical to maintaining optimal viability and function. We developed a high-throughput in vitro platform of human GCs for the screening of chemicals that can impact GJIC and estradiol (E2) production of human granulosa. Our granulosa 3D microtissues fabricated with human ovarian granulosa-like tumor KGN cells are multicell-layered structures that mimic the avascular granulosa layers surrounding the oocyte. These microtissues robustly expressed the steroidogenic CYP19 aromatase enzyme and GJIC intercellular membrane channel, connexin 43. Granulosa microtissues produced E2 at rates comparable to primary human GCs as previously reported. E2 production was suppressed by the CYP19 inhibitor, letrozole, and induced by CYP19 activators, bisphenol A at 100 µM, and genistein at 100 µM. Granulosa microtissues displayed active GJIC function, as demonstrated by the connexin 43-dependent diffusion of calcein fluorescent dye from microtissue surface to the core using high-throughput confocal microscopy in conjunction with our open-sourced automated image analysis tool. Overall, our 3D human granulosa screening platform is highly promising for predictive and efficient in vitro toxicity testing to screen for chemicals that contaminate follicular fluid and may affect fertility.

Provided are the scope and sequence of mathematics topics for Grade 6 to be implemented by teachers and supervisors. The guide is presented in the form of 62 units, each being organized around the themes of (1) sets, numbers, and numeration; (2) operations; (3) geometry and measurement; and (4) algebraic concepts, graphs, probability, and statistics. The sequence is structured to provide a spiral or cyclic approach for presentation of the concepts and skills. (Author/JG)

Provided are the scope and sequence of mathematics topics for Grade 1 to be implemented by teachers and supervisors. The guide is presented in the form of 66 units, each being organized around the themes of (1) sets, numbers, and numeration; (2) operations; (3) geometry and measurement; and (4) algebraic concepts, graphs, probability, and statistics. The sequence is structured to provide a spiral or cyclic approach for presentation of the concepts and skills. (Author/JG)

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechan-ics and biochemistry, since these are both dependent on ECM protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and microstructure can be systematically tuned over a wide range. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, first increasing as the hydrogel stiffness increases, then decreasing as the pore size of silk fibroin decreases. Indeed, mesenchymal morphology exhibits a similar biphasic depen-dence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.