Carnivorous Nutrition in Pitcher Plants (<i>Nepenthes</i> spp.) via an Unusual Complement of Endogenous Enzymes

Plants belonging to the genus <i>Nepenthes</i> are carnivorous, using specialized pitfall traps called “pitchers” that attract, capture, and digest insects as a primary source of nutrients. We have used RNA sequencing to generate a cDNA library from the <i>Nepenthes</i> pitchers and applied it to mass spectrometry-based identification of the enzymes secreted into the pitcher fluid using a nonspecific digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid subproteome includes enzymes across a variety of functional classes. The most abundant proteins present in the secreted fluid are proteases, nucleases, peroxidases, chitinases, a phosphatase, and a glucanase. Nitrogen recovery involves a particularly rich complement of proteases. In addition to the two expected aspartic proteases, we discovered three novel nepenthensins, two prolyl endopeptidases that we name neprosins, and a putative serine carboxypeptidase. Additional proteins identified are relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes discovered in this study suggests that carnivory in the genus <i>Nepenthes</i> can be sustained by plant-based mechanisms alone and does not absolutely require bacterial symbiosis

Additional file 2: Table S2. of Clinical characteristics associated with the onset of delirium among long-term nursing home residents

Demographic and clinical characteristics associated with the onset of delirium stratified by dementia status. The is a table summarizing the logistic regression results looking at factors associated with the onset of delirium. The study population was divided into two groups based on the presence or absence of dementia at the time of delirium onset or their last available assessment in those without delirium. (DOCX 16 kb

Additional file 1: Table S1. of Clinical characteristics associated with the onset of delirium among long-term nursing home residents

Change in medication use from baseline stratified by dementia status at baseline. This is a table summarizing the the change in the use of different medication classes described in text from baseline stratified by dementia status at baseline. (DOCX 14 kb

An <i>In Vitro</i> Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

<div><p>Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous <i>in vitro</i> assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into <i>in vivo</i> tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.</p></div

The role of endogenous growth factors in basal cord formation.

<p>(<b>A</b>) Basal cords made with ADSCs and ECFCs without the addition of growth factors were treated with IgG, or anti-VEGF or anti-HGF antibodies and stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) Basal cords treated with IgG, anti-VEGF, or anti-HGF were quantified on the ArrayScan and the total tube area (left) and SMA index (right) are shown. * p<0.0001 vs basal. n = 3 per group. Scale bars are 250 µm.</p

Induction of cord formation with a TGF-β inhibitor.

<p>(<b>A</b>) Co-cultures of ADSCs and ECFCs were treated with a TGF-β inhibitor (LY2157299) and stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) ArrayScan quantifications of total tube area following treatment with LY2157299 in basal medium (left) versus VEGF driven cords (right). n = 6 per group. (<b>C</b>) Mice harboring A549 tumor xenografts were treated with a TGF-β inhibitor (LY2157299), an inactive control (LY596144), or sunitinib (Sutent; SU11248) for 5 days. Tumors were fixed, sectioned, stained for tumor vessels (CD31; green), and quantified using Image J. * = p<0.05 vs. control. n = 4–6 per treatment group. Scale bars in A are 250 µm. Scale bars in B are 50 µm.</p

Cross-talk between angiogenic growth factors.

<p>Basal, VEGF-, bFGF-, or EGF-driven cords were treated with a VEGFR-2 antibody (IMC-1121B; Ramucirumab; Ram), a bFGF antibody (anti-bFGF), or an EGFR inhibitor (Gefitinib) and the percent inhibition of total tube area from the ArrayScan was graphed. n = 3 per group.</p

Additional file 1: Figure S1. of Membrane feeding of dengue patient’s blood as a substitute for direct skin feeding in studying Aedes-dengue virus interaction

Patient’s arm inside a glove-box during feeding experiments. (DOCX 25 kb

Characterization of growth factors present in the ADSC/ECFC co-culture system.

<p>(<b>A</b>) Basal or VEGF driven co-cultures of ADSCs and ECFCs were stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) Media was collected from ADSCs alone or ADSC/ECFC co-cultures in basal or VEGF driven conditions at 48 (blue bar) and 96 hrs (red bar). Examination of angiogenic growth factors present in the collected media was measured using Luminex. VEGF, HGF, TGF-β, Ang1, and Ang2 had detectable levels above basal media, while bFGF, EGF, and PDGF were not detected (data not shown). n = 3–4 per group with similar results found on two separate experiments. * = p<0.05 vs. all other treatment groups. † = p<0.05 vs. basal media. Scale bars are 250 µm.</p

Phenotypes of angiogenic growth factor combinations.

<p>(<b>A</b>) ADSC and ECFC co-cultures were treated with combinations of angiogenic growth factors (V = VEGF, H = HGF, F = bFGF, E = EGF) with or without anti-VEGF (Bevacizumab; Bev) and stained for cords (CD31; green), smooth muscle actin (SMA; red), and nuclei (Hoechst 33342; blue). (<b>B</b>) ArrayScan quantifications of total tube area, branching index, length to width ratio, and SMA index of samples treated with combinations of growth factors with and without bevacizumab. n = 3 per group. * = p<0.05 vs. V+H. † = p<0.05 vs. V+F. ‡ = p<0.05 vs. H+E. # = p<0.05 vs. F+E. ₊ = p<0.05 vs. H+E+F. Scale bars are 250 µm.</p