Microfluidic-based diagnostics for cervical cancer cells

The use of biomarkers has facilitated the detection of specific tumor cells. However, the technology to apply these markers in a clinical setting has not kept pace with their increasing availability. In this project, we use an antibody-based microfluidics platform to recognize and capture cervical cancer cells. Because HPV-16 infection of cervical cells and up-regulation of alpha6-integrin cell surface receptors are correlated, we utilized alpha6-integrin as a capture antibody bound to the channel surface. Normal human glandular epithelial cells (HGEC), human cervical stromal cells (HCSC) and cervical cancer cells (HCCC) were suspended in PBS and flowed through the system. Greater than 30% of the cancer cells were captured while the capture of the normal cell types was less than 5%. The technique is sensitive and accurate. It is potentially useful in the detection of cervical cancer at all stages, as well as other of cancers with similar characteristics of cell surface antigen expression.     

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47^phox−/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4-fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47^phox−/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47^phox−/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47^phox−/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS.     

Development of a multiplex PCR for identification of vineyard mealybugs

A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.     

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Background and Aims

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells.

Methods

Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins.

Key Results

The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls.

Conclusions

The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.     

A decision-support tool to predict spray deposition of insecticides in commercial potato fields and its implications for their performance

Approximately US $1.3 billion is spent each year on insecticide applications in major row crops. Despite this significant economic importance, there are currently no widely established decision-support tools available to assess suitability of spray application conditions or of the predicted quality or performance of a given commercial insecticide applications. We conducted a field study, involving 14 commercial spray applications with either fixed wing airplane (N=8) or ground rig (N=6), and we used environmental variables as regression fits to obtained spray deposition (coverage in percentage). We showed that (1) ground rig applications provided higher spray deposition than aerial applications, (2) spray deposition was lowest in the bottom portion of the canopy, (3) increase in plant height reduced spray deposition, (4) wind speed increased spray deposition, and (5) higher ambient temperatures and dew point increased spray deposition. Potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), mortality increased asymptotically to approximately 60% in response to abamectin spray depositions exceeding around 20%, whereas mortality of psyllid adults reached an asymptotic response approximately 40% when lambda-cyhalothrin/thiamethoxam spray deposition exceeded 30%. A spray deposition support tool was developed (http://pilcc.tamu.edu/) that may be used to make decisions regarding (1) when is the best time of day to conduct spray applications and (2) selecting which insecticide to spray based on expected spray deposition. The main conclusion from this analysis is that optimization of insecticide spray deposition should be considered a fundamental pillar of successful integrated pest management programs to increase efficiency of sprays (and therefore reduce production costs) and to reduce risk of resistance development in target pest populations.     

Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered.