Forward genetic analysis of cellulose biosynthesis inhibitor resistance and wall hydrolysis sensitivity.

The functional analysis of components involved in cellulose biosynthesis is central in understanding cell wall assembly and structure in plants. We conducted screens using the herbicides, isoxaben and flupoxam which inhibit cellulose biosynthesis in higher plants. Mutations resulting in a high degree of resistance to isoxaben (ixr) or flupoxam (fxr) were attributed to single amino acid substitutions in primary wall CESAs. Twelve novel resistance alleles were isolated and no cross-resistance was observed. Point mutations were mostly clustered around the C-terminal regions of CESA1 and CESA3, and CESA3 and CESA6 for fxr and ixr respectively. Resistance to isoxaben was also conferred by modification to the putative catalytic regions of CESA3. This resulted in cellulose deficient phenotypes characterized by reduced crystallinity and dwarfism. These results provide genetic evidence supporting CESA1-CESA3, and CESA3-CESA6 association with flupoxam and isoxaben respectively targeting and disrupting these interactions. The ixr and fxr mutants also exhibited enhanced saccharification under enzymatic degradation schemes which is consistent with the observed reduction in cellulose crystallinity. A second forward genetic screen was performed using mild acid hydrolysis to isolate mutants with enhanced saccharification. This screen identified sixty-three responsive to acid hydrolysis (rah) lines. Unconventional strategies to increase sugar yields from plant biomass where highlighted. These included starch hyper-accumulators such as starch excess 4 (sex4) loss-of-function mutants and the perturbation of polar auxin transport. Disruption of the serine/threonine kinase positive regulator of auxin efflux, PINOID (PID) was found to significantly enhance sugar release in Arabidopsis and similar effects were observed in the maize orthologue, BARREN INFLORESENCE 2 (BIF2). Furthermore, the application of N-1-naphthylphthalamic acid (NPA) in Arabidopsis, maize, Miscanthus and switchgrass phenocopied the enhanced wall saccharification effects of PID. This study attempted to elucidate some of the interactions of seemingly unrelated pathways in the context of wall biosynthesis and saccharification enhancement

Decreased DBC1 Expression Is Associated With Poor Prognosis in Patients With Non-Muscle-Invasive Bladder Cancer

Purpose The deleted in bladder cancer 1 (DBC1) gene is located within chromosome 9 (9q32-33), a chromosomal region that frequently shows loss of heterozygosity in bladder cancer (BC). It is suspected that it acts as a tumor suppressor gene, but its prognostic value remains unclear. The aim of the present study was to investigate the value of DBC1 as a prognostic marker in BC. Materials and Methods The expression of DBC1 was determined by real-time polymerase chain reaction analysis in 344 patients with BC (220 non-muscle-invasive BC [NMIBC] and 124 muscle-invasive BC [MIBC]) and in 34 patients with normal bladder mucosa. The results were compared with clinicopathologic parameters, and the prognostic value of DBC1 was evaluated by Kaplan-Meier analysis and a multivariate Cox regression model. Results: DBC1 expression was significantly decreased in patients with MIBC compared with those diagnosed with NMIBC (p=0.010). Patients with aggressive tumor characteristics had lower DBC1 expression levels in NMIBC (each, p<0.05). By multivariate Cox regression analysis, low DBC1 expression was a predictor of progression to MIBC (hazard ratio, 7.104; p=0.013). Kaplan-Meier estimates revealed a significant difference in tumor recurrence, progression to MIBC, and cancer-specific survival depending on the level of DBC1 expression in NMIBC (log-rank test, each, p<0.05). Conclusions: The expression of DBC1 was associated with tumor aggressiveness, progression to MIBC, and survival in NMIBC. Our results suggest that DBC1 expression can be a useful prognostic marker for patients with NMIBC

Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen.

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus

Bioelectric stimulation controls tissue shape and size

Abstract Epithelial tissues sheath organs and electro-mechanically regulate ion and water transport to regulate development, homeostasis, and hydrostatic organ pressure. Here, we demonstrate how external electrical stimulation allows us to control these processes in living tissues. Specifically, we electrically stimulate hollow, 3D kidneyoids and gut organoids and find that physiological-strength electrical stimulation of ∼ 5 - 10 V/cm powerfully inflates hollow tissues; a process we call electro-inflation. Electro-inflation is mediated by increased ion flux through ion channels/transporters and triggers subsequent osmotic water flow into the lumen, generating hydrostatic pressure that competes against cytoskeletal tension. Our computational studies suggest that electro-inflation is strongly driven by field-induced ion crowding on the outer surface of the tissue. Electrically stimulated tissues also break symmetry in 3D resulting from electrotaxis and affecting tissue shape. The ability of electrical cues to regulate tissue size and shape emphasizes the role and importance of the electrical micro-environment for living tissues

Effect of pellicin on cellulose production in liquid culture and solid medium.

<p><i>G. xylinus was</i> grown at 30°C in liquid culture under agitated (A, B) and static conditions (C, D); (A, C) are DMSO controls, (B, D) grown in the presence of 10 µM pellicin. Arrows in (A, C) indicate either the large aggregates or pellicle that form in the absence of pellicin. Colony morphology of <i>G. xylinus</i> grown on SH agar plates that were supplemented with (E, G) DMSO or (F, H) 10 µM pellicin. Photographs of (E, F) were taken with illumination from above and (G, H) were taken with illumination from below of the same colonies. Note the larger, undulate, raised colonies forming on pellicin supplemented plates (F). Arrows in (G) indicate the filiform projections emerging from colony, which are absent in (H). Scale bars equal 0.5 mm.</p

Pellicin affects the crystallization of cellulose produced by <i>G. xylinus</i>.

<p><i>In vitro</i> cellulose synthase assays of crude enzyme prepared from <i>G. xylinus</i> grown SH medium and incubated with DMSO or pellicin for 1 hour prior to UDP-[³H]glucose addition. Reactions treated with 0.5 M NaOH contain both cellulose I and cellulose II. Acetic-nitric acid treatment of reactions removes cellulose II while retaining cellulose I. Data show the mean ± SE of three experimental determinations.</p

<i>In vitro</i> cellulose synthase assay using membrane preparations of <i>G. xylinus</i> grown in the presence and absence of pellicin using UDP-[³H]glucose as substrate.

<p>1) Boiled control 2) Untreated 3) Pellicin pretreated. Cells grown in the presence of pellicin show an increased cellulose synthase activity. Data show the mean ± SE of three experimental determinations.</p

The influence of pellicin on <i>G. xylinus</i> growth.

<p>Pellicin does not affect the viability of <i>G. xylinus</i>. <i>G. xylinus</i> was grown at 30°C in Schramm-Hestrin broth containing 0.1% cellulase and either 10 µM pellicin (▪) or DMSO (▴) as a control. Data show the mean ± SD of four experimental determinations.</p

Pellicin remains in the extracellular environment.

<p>High performance liquid chromatography of organic extracts of A) <i>G. xylinus</i> in the absence and B) presence of pellicin. Data shown is representative of triplicate experiments: (red dashed line) pellicin standard, (black line) extracellular supernatant, (blue line) pellet wash prior to cellulase treatment, (green line) supernatant from cellulase treated cells, (orange line) cell free lysate, (purple line) cellular debris analyzed at 220 nm, the optimum wavelength for pellicin detection.</p