Rodney A. Hill

Effect of starvation on transcriptomes of brain and liver in adult female zebrafish (Danio rerio)

We used microarray and quantitative real-time PCR (qRT-PCR) analyses in adult female zebrafish (Danio rerio) to identify metabolic pathways regulated by starvation in the liver and brain. The transcriptome of whole zebrafish brain showed little response to 21 days of starvation. Only agouti-related protein 1 (agrp1) significantly responded, with increased expression in brains of starved fish. In contrast, a 21-day period of starvation significantly downregulated 466 and upregulated 108 transcripts in the liver, indicating an overall decrease in metabolic activity, reduced lipid metabolism, protein biosynthesis, proteolysis, and cellular respiration, and increased gluconeogenesis. Starvation also regulated expression of many components of the unfolded protein response, the first such report in a species other than yeast (Saccharomyces cerevisiae) and mice (Mus musculus). The response of the zebrafish hepatic transcriptome to starvation was strikingly similar to that of rainbow trout (Oncorhynchus mykiss) and less similar to mouse, while the response of common carp (Cyprinus carpio) differed considerably from the other three species.

Gold nanoparticles: the importance of physiological principles to devise strategies for targeted drug delivery

Nanotechnology and its promise for clinical translation to targeted drug delivery with limited accompanying toxicity provide exciting research opportunities that demand multidisciplinary approaches. To make rapid progress in the design of nano-platforms for drug delivery and toward their use in the clinic, basic and mechanistic studies must first be tested in vitro and then progress to in vivo studies in animal models, incorporating an understanding of body functioning. Recently, gold nanoparticles (Au NPs) have gained much attention as model drug delivery platforms because of their advantageous surface characteristics that allow easy functionalization with chemical and biological molecules and also due to their apparently low toxicity. In this study we review recent in vitro and in vivo research progress with Au NPs as drug delivery platforms and suggest experimental strategies for future studies for efficacious, targeted delivery.

Sexual dimorphism in hepatic gene expression and the response to dietary carbohydrate manipulation in the zebrafish (Danio rerio)

In this study, we tested for the presence of sexual dimorphism in the hepatic transcriptome of the adult zebrafish and examined the effect of long term manipulation of dietary carbohydrate on gene expression in both sexes. Zebrafish were fed diets comprised of 0%, 15%, 25%, or 35% carbohydrate from the larval stage through sexual maturity, then sampled for hepatic tissue, growth, proximate body composition, and retention efficiencies. Using Affymetrix microarrays and qRT-PCR, we observed substantial sexual dimorphism in the hepatic transcriptome. Males up-regulated genes associated with oxidative metabolism, carbohydrate metabolism, energy production, and amelioration of oxidative stress, while females had higher expression levels of genes associated with translation. Restriction of dietary carbohydrate (0% diet) significantly affected hepatic gene expression, growth performance, retention efficiencies of protein and energy, and percentages of moisture, lipid, and ash. The response of some genes to dietary manipulation varied by sex; with increased dietary carbohydrate, males up-regulated genes associated with oxidative metabolism (e.g. hadhbeta) while females up-regulated genes associated with glucose phosphorylation (e.g. glucokinase). Our data support the use of the zebrafish model for the study of fish nutritional genomics, but highlight the importance of accounting for sexual dimorphism in these studies.

Estimating the voluntary intake and digestibility of buffel-grass and lucerne hays offered to Brahman-cross cattle using n-alkanes

The n-alkane method was developed in temperate areas as a tool to estimate voluntary intake (VI) at pasture. The present study aimed to investigate the performance of n-alkanes as markers for estimating VI of steers (mean live weight 213 kg) offered a range of tropical grass hays and lucerne. Tropical and temperate forages have different n-alkane profiles and little is known about the issues which affect the accuracy of the method under tropical conditions. In two pen experiments (no. = 20 and no. = 24) n-alkanes were dosed using intraruminal controlled-release devices. Actual mean voluntary dry matter intakes for the diets ranged from 3·12 to 4·60 kg/day and actual mean dry-matter digestibility varied between 439 and 620 g/kg. n-Alkane profiles (C30 to C36) of the diets and the faeces for each animal were determined using gas chromatography. The recovery of each n-alkane was determined for each animal. Recoveries of n-alkanes were highly variable and generally varied between diets and between experiments. When adjacent n-alkanes were used to estimate VI (ratio method), agreement with actual VI was often poor. Despite this, where the recoveries of n-alkane pairs were similar, group mean VI were accurately estimated. From these data, it is concluded that estimation of VI in cattle offered tropical grass hays or lucerne hay, requires measured recoveries of both dosed and natural plant n-alkanes. The dosed and natural n-alkane pairs having the most similar recoveries should be used in the ratio method to estimate VI.

In vitro proliferating cell models to study cytotoxicity of silica nanowires

UNLABELLED Proliferating cells representing two disease models (HeLa and Panc 10.05 cells) and a more physiologically relevant cell model (3T3-L1 cells) were used to study the acute toxicologic effects of silica nanowires (NWs). Cellular responses to NW effects were determined over a 4- to 20-hour exposure time-course. Proliferation, viability, metabolic activity, and toxicologic mechanism (apoptosis vs. necrosis) data showed the following: 3 x 10(4) NWs per cell inhibited cell proliferation. The effect was rapid in HeLa cells, but 3T3-L1 and Panc 10.05 cells appeared to be more tolerant to NWs, effects being significant only after 20 or 16 hours, respectively. Cells of all three cell lines showed a significant reduction in cellular metabolic activity after 20 hours of treatment with NWs. Assay of NW-invoked mechanism of cell death (caspase 3/7 activity) indicated that apoptosis was minimally induced. Small but significant effects of NWs were detected in 3T3-L1 and HeLa cells after 20-hour treatment. No NW-induction of caspase 3/7 activity was detected for Panc 10.05 cells. Proliferating cells provide a sensitive model to study treatment with silica NWs. Silica NWs appeared to be well tolerated by these three cell lines at the doses tested. When effects were detected, cell necrosis and not apoptosis was the main mechanism of silica NW-induced cell death. FROM THE CLINICAL EDITOR In this study, three relevant cell culture models were used to study the acute toxicological effects of silica nanowires (NW). All cell lines cells showed a significant reduction in cellular metabolic activity after 20 h of treatment with NW. Overall, silica NW appeared to be well-tolerated by these cell lines at the tested doses. Cell necrosis as opposed to apoptosis was the main mechanism of silica NW-induced cell death.

Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells

The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)2D3) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100nM) of 1, 25-(OH)2D3 were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)2D3 were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)2D3. In response to 1, 25-(OH)2D3 (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)2D3 tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)2D3 was also tested, with no effect detected. These results indicate that 1, 25-(OH)2D3 inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)2D3 in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.

Dietary carbohydrate level affects transcription factor expression that regulates skeletal muscle myogenesis in rainbow trout.

Understanding the effects of dietary carbohydrates on transcription factors that regulate myogenesis provides insight into the role of nutrient sensing by satellite cells towards myocyte differentiation. We evaluated the influence of dietary carbohydrate level (0, 15, 25 or 35%) on the temporal mRNA expression patterns (4, 8 or 12 weeks) of transcription factors that regulate satellite cell myocyte addition (MA) in rainbow trout (Oncorhynchus mykiss), a vertebrate with indeterminate growth. Relative to the 0% carbohydrate (NC) diet, 15 (IC-15) and 25% (IC-25) carbohydrate containing diets significantly up-regulate MyoD and Myf5, but not Pax7, after 12 weeks of feeding. Simultaneously, the Pax7/MyoD mRNA expression ratio declined significantly with both the IC diets. Myogenin mRNA expression also increased in rainbow trout (RBT) fed the IC-15 diet. The high carbohydrate (HC) diet (35%) attenuated the increased mRNA expression of these transcription factors. It is of note that the 4 and 8 week samples lacked the promyogenic expression patterns. The myogenic gene expression in fish fed the IC-15 diet for 12 weeks indicate a transcriptional signature that reflects increased satellite cell myogenesis. Our results suggest a potential role for satellite cells in the nutrient sensing ability of a vertebrate with indeterminate skeletal muscle growth.

Gold-peptide nanoconjugate cellular uptake is modulated by serum proteins

Gold nanoparticles (Au NPs, 20 nm) were conjugated with two different cysteine-terminated peptides. Radio-ligand binding studies were conducted to characterize Au NP-peptide binding, suggesting both covalent and noncovalent interactions. The interactions of serum proteins with Au NP-peptide nanoconjugates were determined using gel electrophoresis and dynamic light scattering. Serum proteins rapidly bound the nanoconjugates (15 minutes). The cellular uptake of free peptides and nanoconjugates into mouse myogenic (Sol8) cells was investigated in the absence or presence of serum. In the absence of serum, peptides presented as nanoconjugates showed significantly higher intracellular fluorescence signals compared to those in the presence of serum (P < 0.05), suggesting that serum proteins inhibit Au NP-mediated peptide delivery. The cellular uptake of nanoconjugates was also confirmed using transmission electron microscopy. These data suggest that Au NP-peptide nanoconjugates are a useful platform for intracellular delivery of therapeutics. However, a deeper understanding of the mechanisms regulating their uptake and intracellular trafficking is needed.

Effect of preslaughter feeding and ractopamine hydrochloride supplementation on growth performance, carcass characteristics, and end product quality in market dairy cows

An experiment was conducted in market dairy cows to determine the effect of feeding time and ractopamine hydrochloride (RAC) on growth performance, carcass characteristics, and end product quality. In 3 replicates, 9 Holstein cows per replicate (n = 27; 659 +/- 25.3 kg initial BW) culled from 3 dairies were randomly assigned to 3 treatments: 1) slaughter immediately (control), 2) feed for 90 d (NoR), or 3) feed for 90 d with RAC (312 mg.cow(-1).d(-1)) for the final 32 d (RAC). On d 0, NoR and RAC cows were placed in individual pens and fed a high concentrate diet (86% concentrate, DM basis) for 90 d before slaughter. All cows were subjectively scored for BCS and locomotion score on d 0, and NoR and RAC cows were evaluated again after 90 d. Individual DMI was recorded daily throughout the trial, and BW was collected every 14 d. Age and age x treatment did not affect (P > 0.05) any of the traits evaluated in this study. When cows fed for 90 d (NoR and RAC combined) were compared with nonfed controls, fed cows had greater (P < 0.001) final BCS, BW and HCW, lower (P < 0.001) final locomotion score, and greater (P < 0.03) dressing percentage, external fat thickness, and marbling score. Fed cows also tended to have more desirable yield grade (P = 0.08), ribeye area (P = 0.11), fat color (P = 0.09), lean maturity (P = 0.06), and quality grade (P = 0.09) compared with control cows. Warner-Bratzler shear force was not affected (P = 0.23) by feeding. However, a 12-member trained sensory panel revealed that fed cow carcasses had more desirable (P < 0.04) tenderness, juiciness, and overall acceptability than control cow carcasses. Flavor intensity also tended (P = 0.10) to be more desirable for fed vs. control cows. No difference (P > 0.10) in off-flavor was detected among treatments. Finally, there was no effect (P > 0.10) of RAC on growth performance, carcass characteristics, or end product quality. In conclusion, feeding a high concentrate diet for 90 d improved important live animal, carcass, and end product characteristics related to the quality and palatability of beef from market dairy cows; however, no effect of RAC supplementation was observed.

Utilization of monoclonal antibody-targeted nanomaterials in the treatment of cancer

Due to their excellent specificity for a single epitope, monoclonal antibodies (mAbs) present a means of influencing the function of cells at the molecular level. In particular they show great promise in the treatment of cancer because they can inhibit cancer cell proliferation, tumor angiogenesis, invasiveness and malignant spread of cancerous cells. Many mAbs are in various stages of testing and 11 are currently marketed in the US or Europe for the treatment of cancers that express particular antigens such as human epidermal growth factor receptor-2, CD20, epidermal growth factor receptor and vascular endothelial growth factor. Strategies to conjugate mAbs to toxins, radioactive isotopes and chemotherapeutic drugs to improve efficacy are under intense investigation and numerous immunoconjugates have been studied in the clinical setting. However, the molecules have limitations, and so nanomaterials (NMs), which potentially offer more flexibility of design and functionality in providing platforms for binding of multiple therapeutic agents in a single structure, are being examined as an alternative. Studies utilizing mAb-targeted NMs have shown that they exhibit focused targeting, improved pharmacokinetics and improved “passive” drug delivery via leaky vasculature. Nevertheless, before they can be utilized to treat cancer, potential NM toxicity must be thoroughly investigated. Thus, rigorous testing of NM-mAb conjugates in both in vitro and in vivo systems is underway to determine how NM-mAb conjugates will interact with cells and tissues of the body. In this review, we discuss the broad range of nanomaterials that are under investigation as potential platforms for the presentation of mAbs either as single therapeutics or in combination with other drugs and their advantages and limitations in specifically targeting cancer.