Wetting/Spreading on Porous Media and on Deformable, Soluble Structured Substrates as a Model System for Studying the Effect of Morphology on Biofilms Wetting and for Assessing Anti-Biofilm Methods

Biofilm is a layer of syntrophic microorganisms stick to each other and to the surface. The importance of biofilms is enormous in various industrial applications and human everyday life. The effects of biofilm could be either positive or negative. Positive effects are encountered in industrial processes, bioremediation, and wastewater treatment. Negative effects are more common with the marine industry being one of the sectors, which confronts severe corrosion problems caused by biofouling on the surfaces of equipment and infrastructures. In space industry, microbial contamination and biofouling adversely affect both crew health and mission-related equipment, the latter including hardware, water systems, piping, and electrical tools. The capacity of biofilms to grow in space environment was confirmed already in 1991. One of the most important surface properties of biofilms is wettability, which dictates not only how a liquid spreads over the uneven external surface of biofilms but also how it penetrates into their porous and morphologically complex structure. To investigate wetting and spreading onto biofilms, model materials are often used to simulate different morphological and functional features of biofilms in a controlled way, for example, soft, deformable, soluble, structured, porous materials. Here, we review recent advances in wetting and spreading on porous and soft deformable surface together with biofilms wetting properties and its importance in space industry. We conclude with a discussion of the main directions for future research efforts regarding biofilm wetting

Analyzing the Impact of a Hub and Spoke Supply Chain Design for Long-Haul, High-Volume Transportation of Densified Biomass

This dissertation proposes a framework in support of biomass supply chain network design. This framework relies in the use of trucks for short distance biomass transportation, and relies in the use of rail for long-haul, and high-volume transportation of densified biomass. A hub and spoke network design model is proposed for the case when biomass is shipped by rail. These models are created and solved for the following problems: 1) designing a biomass supply chain to deliver densified biomass to a coal fired power plant for coiring and 2) designing biomass-to-biorefinery supply chain using rail for long-haul, and high-volume shipment of densified biomass under economic, environmental, and social criteria. The first problem is modeled as a Mixed-Integer Linear Programming (MILP). A Benders’ decomposition-based algorithm is developed to solve the MILP model because its large size makes it difficult to solve using CPLEX. The numerical analysis indicates that the total unit transportation cost from the farm to a coal plant is $36/ton. Numerical analysis also indicates that biomass cofiring is cost efficient compare to direct coal firing if the renewable energy production tax credit is applied and biomass is located within 75 miles from a coal plant. The second problem is also modeled as a MILP mode. This MILP identifies the number, capacity and location of biorefineries needed to make use of the biomass available in the region. A case study is created using data from a number of States in the Midwest USA. The numerical analysis show that 24.38$4.01 to $4.02 per gallon. The numerical analysis also reveals the tradeoffs that exist among the economics, environmental impact, and social objectives of using densified biomass for production of biofuel. Finally, this dissertation presents a detailed analysis of the rail transportation cost for products that have similar physical characteristics to densified biomass and biofuel. A numbers of regression equations are developed in order to evaluate and quantify the impact of important factors on the unit transportation cost

Nitric oxide mediates antimicrobial peptide gene expression by activating eicosanoid signaling

<div><p>Nitric oxide (NO) mediates both cellular and humoral immune responses in insects. Its mediation of cellular immune responses uses eicosanoids as a downstream signal. However, the cross-talk with two immune mediators was not known in humoral immune responses. This study focuses on cross-talk between two immune mediators in inducing gene expression of anti-microbial peptides (AMPs) of a lepidopteran insect, <i>Spodoptera exigua</i>. Up-regulation of eight AMPs was observed in <i>S</i>. <i>exigua</i> against bacterial challenge. However, the AMP induction was suppressed by injection of an NO synthase inhibitor, L-NAME, while little expressional change was observed on injecting its enantiomer, D-NAME. The functional association between NO biosynthesis and AMP gene expression was further supported by RNA interference (RNAi) against NO synthase (SeNOS), which suppressed AMP gene expression under the immune challenge. The AMP induction was also mimicked by NO alone because injecting an NO analog, SNAP, without bacterial challenge significantly induced the AMP gene expression. Interestingly, an eicosanoid biosynthesis inhibitor, dexamethasone (DEX), suppressed the NO induction of AMP expression. The inhibitory activity of DEX was reversed by the addition of arachidonic acid, a precursor of eicosanoid biosynthesis. AMP expression of <i>S</i>. <i>exigua</i> was also controlled by the Toll/IMD signal pathway. The RNAi of Toll receptors or Relish suppressed AMP gene expression by suppressing NO levels and subsequently reducing PLA₂ enzyme activity. These results suggest that eicosanoids are a downstream signal of NO mediation of AMP expression against bacterial challenge.</p></div

Influence of Toll/IMD signaling on immune mediation by NO/eicosanoid in <i>S</i>. <i>exigua</i>.

<p>Specific RNA interference (RNAi) against Toll and IMD signal pathways was performed by injecting 800 ng of dsRNA (dsToll or dsRelish) specific to Toll (contig 06215) or Relish (contig 00977) of <i>S</i>. <i>exigua</i> transcriptome (SRX259774) to fifth instar larva. At 48 h after dsRNA injection, immune challenge was initiated by injecting <i>E</i>. <i>coli</i> for Gram-negative (G-) and <i>P</i>. <i>polymyxa</i> for Gram-positive (G+) at a dose of 1 × 10⁵ cells per larva. (A) Cross-talk between Toll/IMD and NO signaling. NO signal was quantified by measuring nitrate amount from a whole body after 8 h of bacterial challenge. (B) Cross-talk between Toll/IMD and eicosanoid signaling. Eicosanoid signal was quantified by measuring PLA₂ enzyme activity after 8 h of bacterial challenge. Each treatment was conducted three times. Different letters above the error bars indicate significant differences between means at Type I error = 0.5 (LSD).</p

Nitric oxide mediates antimicrobial peptide gene expression by activating eicosanoid signaling - Fig 9

<p>Influence of Toll/IMD signaling on gene expression of (A) NO synthase (SeNOS) and (B) calcium-independent PLA₂ (SeiPLA₂) under bacterial challenge in <i>S</i>. <i>exigua</i>. Specific RNA interference (RNAi) against Toll and IMD signal pathways was initiated by injecting 800 ng of dsRNA (dsToll or dsRelish) specific to Toll (contig 06215) or Relish (contig 00977) of <i>S</i>. <i>exigua</i> transcriptome (SRX259774) into fifth instar larva. At 48 h after dsRNA injection, immune challenge was initiated by injecting <i>E</i>. <i>coli</i> for Gram-negative (‘G-’) and <i>P</i>. <i>polymyxa</i> for Gram-positive (G+) at a dose of 1 × 10⁵ cells per larva. After 8 h of bacterial challenge, fat bodies were collected for cDNA preparation. For RNAi control (dsCON), larvae were injected with dsRNA that was specific to a viral gene, CpBV-ORF302, in same doses. Each treatment was conducted three times. Target gene (SeNOS, SeiPLA₂) expressions were quantified by RT-qPCR. RL32, a ribosomal protein, was used as a reference gene for qPCR. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).</p

Inducing NO and PLA₂ activity by bacterial challenge in <i>S</i>. <i>exigua</i> fifth instar larvae.

<p>Bacterial challenge used <i>E</i>. <i>coli</i> for Gram-negative (G-) and <i>P</i>. <i>polymyxa</i> for Gram-positive (G+) at a dose of 1 × 10⁵ cells per larva. For control (CON), larvae were injected with a phosphate buffer used for diluting bacterial cells. After 8 h of bacterial infection, the fat bodies were collected and used to assess NO amounts and for PLA₂ enzyme assay. NO concentration was indirectly measured by quantifying nitrate amount using Griess reagent. PLA₂ activity was measured using a pyrene-labeled fluorescence substrate. Each treatment was conducted three times. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).</p

Rescue effect of arachidonic acid (AA, a PLA₂ catalytic product) on suppressing AMP expression of <i>S</i>. <i>exigua</i> fifth instar larvae under blocking NO biosynthesis.

<p>RNA interference (RNAi) applied to SeNOS using its specific dsRNA at a dose of 800 ng per larva. (A) RNAi effect on <i>SeNOS</i> expression. After 24, 48, and 72 h of dsNOS injection, whole bodies were collected to extract RNA and used for cDNA preparation. For RNAi control (dsCON), larvae were injected with dsRNA that were specific to a viral gene, <i>CpBV-ORF302</i>, in same doses. (B) Effects of SeNOS RNAi on <i>defensin</i> (<i>Def</i>) expression. For bacterial challenge (BAC), <i>E</i>. <i>coli</i> was injected at a dose of 1 × 10⁵ cells per larva after 48 h of dsNOS injection. AA injection used 10 μg per larva. After 8 h of injection, each whole body per replication was used for total RNA extraction to prepare cDNA. Each treatment was conducted three times. <i>Def</i> expression was quantified by RT-qPCR. RL32, a ribosomal protein, was used as a reference gene for qPCR. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).</p

Influence of NO synthase activity on AMP expression of <i>S</i>. <i>exigua</i> fifth instar larvae.

<p>An NO synthase inhibitor, L-NAME, was injected at a dose of 50 μg per larva. D-NAME is its enantiomer and used the same dose. For bacterial challenge (BAC), <i>E</i>. <i>coli</i> was injected at a dose of 1 × 10⁵ cells per larva. For control (CON), larvae were injected with a solvent used for dissolving SNAP. After 8 h of injection, each whole body per replication was used for total RNA extraction to prepare cDNA; each treatment was conducted three times. Expression of eight AMP genes—attacin-1 (Att 1), attacin-2 (Att 2), defensin (Def), gloverin (Glv), hemolin (Hem), lysozyme (Lys), transferrin-1 (Trf 1), and transferrin-2 (Trf 2), was quantified by RT-qPCR. RL32, a ribosomal protein, was used as a reference gene for qPCR. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).</p

Toll/IMD signaling of <i>S</i>. <i>exigua</i> and specific AMPs.

<p>(A) Specific RNA interference (RNAi) against Toll and IMD signal pathways by injecting 800 ng of dsRNA (dsToll or dsRelish) specific to Toll (contig 06215) or Relish (contig 00977) of <i>S</i>. <i>exigua</i> transcriptome (SRX259774) to fifth instar larva. Each time point was tested three times. (B) Specific expressional control of Toll/IMD against two AMPs of lysozyme (Lys) and transferrin 2 (Trf 2). After 48 h of dsRNA injection, fat bodies were collected for preparing cDNA. For RNAi control (dsCON), larvae were injected with dsRNA that was specific to a viral gene, <i>CpBV-ORF302</i>, in same doses. Each treatment was conducted three times. Target gene (Toll, Relish, Lys, Trf 2) expressions were quantified by RT-qPCR. RL32, a ribosomal protein, was used as a reference gene for qPCR. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).</p

Interaction of NO and eicosanoids in AMP expression of <i>S</i>. <i>exigua</i> fifth instar larvae.

<p>For bacterial challenge (BAC), <i>E</i>. <i>coli</i> was injected in a dose of 1 × 10⁵ cells per larva. For control (CON), larvae were injected with solvent used for dissolving chemicals. SNAP (an NO donor) injection used 50 μg per larva. Dexamethasone (DEX, a PLA₂ inhibitor) injection used 10 μg per larva. Arachidonic acid (AA, a PLA₂ catalytic product) injection used 10 μg per larva. After 8 h of injection, each whole body per replication was used for total RNA extraction to prepare cDNA. Each treatment was conducted three times. Expression of eight AMP genes—attacin-1 (Att 1), attacin-2 (Att 2), defensin (Def), gloverin (Glv), hemolin (Hem), lysozyme (Lys), transferrin-1 (Trf 1), and transferrin-2 (Trf 2), was quantified by RT-qPCR. RL32, a ribosomal protein, was used as a reference gene for qPCR. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).</p