Field evaluation and system improvement of a semi-automated mechanical intra-row weeder for vegetable crops

This was the original project description: This is an expansion of a previous Leopold Center competitive grant (M2009-23), which supported the development of a basic semi-automated mechanical intra-row weed removal system for vegetable crops. The investigators will conduct field trials to evaluate and improve the prototype

Aldose Reductase, Oxidative Stress, and Diabetic Mellitus

Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications

Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP)

AbstractProcessing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP

Transferrin-Liposome-Mediated Systemic p53 Gene Therapy in Combination with Radiation Results in Regression of Human Head and Neck Cancer Xenografts

This is the published version, also available here: http://dx.doi.org/10.1089/10430349950016357.The use of cationic liposomes as nonviral vehicles for the delivery of therapeutic molecules is becoming increasingly prevalent in the field of gene therapy. We have previously demonstrated that the use of the transferrin ligand (Tf) to target a cationic liposome delivery system resulted in a significant increase in the transfection efficiency of the complex [Xu, L., Pirollo, K.F., and Chang, E.H. (1997). Hum. Gene Ther. 8, 467-475]. Delivery of wild-type (wt) p53 to a radiation-resistant squamous cell carcinoma of the head and neck (SCCHN) cell line via this ligand-targeted, liposome complex was also able to revert the radiation resistant phenotype of these cells in vitro. Here we optimized the Tf/liposome/DNA ratio of the complex (LipT) for maximum tumor cell targeting, even in the presence of serum. The efficient reestablishment of wtp53 function in these SCCHN tumor cells in vitro, via the LipT complex, restored the apoptotic pathway, resulting in a significant increase in radiation-induced apoptosis that was directly proportional to the level of exogenous wtp53 in the tumor cells. More significantly, intravenous administration of LipT-p53 markedly sensitized established SCCHN nude mouse xenograft tumors to radiotherapy. The combination of systemic LipT-p53 gene therapy and radiation resulted in complete tumor regression and inhibition of their recurrence even 6 months after the end of all treatment. These results indicate that this tumor-specific, ligand-liposome delivery system for p53 gene therapy, when used in concert with conventional radiotherapy, can provide a new and more effective means of cancer treatment

Internalization of the Active Subunit of the Aggregatibacter Actinomycetemcomitans Cytolethal Distending Toxin Is Dependent upon Cellugyrin (Synaptogyrin 2), a Host Cell Non-Neuronal Paralog of the Synaptic Vesicle Protein, Synaptogyrin 1

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is a heterotrimeric AB2 toxin capable of inducing lymphocytes, and other cell types, to undergo cell cycle arrest and apoptosis. Exposure to Cdt results in binding to the cell surface followed by internalization and translocation of the active subunit, CdtB, to intracellular compartments. These events are dependent upon toxin binding to cholesterol in the context of lipid rich membrane microdomains often referred to as lipid rafts. We now demonstrate that, in addition to binding to the plasma membrane of lymphocytes, another early and critical event initiated by Cdt is the translocation of the host cell protein, cellugyrin (synaptogyrin-2) to the same cholesterol-rich microdomains. Furthermore, we demonstrate that cellugyrin is an intracellular binding partner for CdtB as demonstrated by immunoprecipitation. Using CRISPR/cas9 gene editing we established a Jurkat cell line deficient in cellugyrin expression (JurkatCg−); these cells were capable of binding Cdt, but unable to internalize CdtB. Furthermore, JurkatCg− cells were not susceptible to Cdt-induced toxicity; these cells failed to exhibit blockade of the PI-3K signaling pathway, cell cycle arrest or cell death. We propose that cellugyrin plays a critical role in the internalization and translocation of CdtB to critical intracellular target sites. These studies provide critical new insight into the mechanism by which Cdt, and in particular, CdtB is able to induce toxicity

MiRNA-200C expression in Fanconi anemia pathway functionally deficient lung cancers

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy

Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells

Membrane Localization of the Repeats-in-Toxin (RTX) Leukotoxin (LtxA) Produced by Aggregatibacter Actinomycetemcomitans

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane. © 2018 Brown et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Self-Assembly of a Virus-Mimicking Nanostructure System for Efficient Tumor-Targeted Gene Delivery

This is the published version, also available here: http://dx.doi.org/10.1089/10430340252792594.Molecular therapy, including gene therapy, is a promising strategy for the treatment of human disease. However, delivery of molecular therapeutics efficiently and specifically to the target tissue remains a significant challenge. A human transferrin (Tf)-targeted cationic liposome-DNA complex, Tf-lipoplex, has shown high gene transfer efficiency and efficacy with human head and neck cancer in vitro and in vivo (Xu, L., Pirollo, K.F., Tang, W.H., Rait, A., and Chang, E.H. Hum. Gene Ther. 1999;10:2941-2952). Here we explore the structure, size, formation process, and structure-function relationships of Tf-lipoplex. We have observed Tf-lipoplex to have a highly compact structure, with a relatively uniform size of 50-90 nm. This nanostructure is novel in that it resembles a virus particle with a dense core enveloped by a membrane coated with Tf molecules spiking the surface. More importantly, compared with unliganded lipoplex, Tf-lipoplex shows enhanced stability, improved in vivo gene transfer efficiency, and long-term efficacy for systemic p53 gene therapy of human prostate cancer when used in combination with conventional radiotherapy. On the basis of our observations, we propose a multistep self-assembly process and Tf-facilitated DNA cocondensation model that may provide an explanation for the resultant small size and effectiveness of our nanostructural Tf-lipoplex system

Transient Tumor-Fibroblast Interactions Increase Tumor Cell Malignancy by a TGF-β Mediated Mechanism in a Mouse Xenograft Model of Breast Cancer

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-β1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-β appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-β pathway