Inhibition of FAK-Actin pathway by indoxyl sulfate suppresses arteriogenesis in a mouse model of chronic kidney disease

Chronic Kidney Disease (CKD) has been implicated in multiple conditions including cardiovascular related diseases and Type 2 Diabetes (T2D). Decreased filtration results in accumulation of urea, electrolytes, and uremic which are implicated in disease progression. Examination of the progression of CKD effect on vascular regeneration, and T2D development are necessary. First, examination of CKD progression and buildup of uremic toxin indoxyl sulfate (IS) was undertaken. In vivo and in vitro analysis implicated CKD and IS accumulation in impaired vascular regeneration increasing risk of mortality. CKD and the concomitant increase in IS resulted in impaired regeneration through alteration to vascular smooth muscle (VSMC). VSMCs exhibited alteration in actin cytoskeletal structure, and decreased motility. This was driven by loss of focal adhesion protein focal adhesion kinase (FAK). Loss of FAK resulted in decreased signal transduction for neuronal Wiskott-Aldrich syndrome protein (N-WASP) necessary for activation of actin nucleation complex Arp2/3. Secondly, examination was undertaken into T2D progression. High-fat induced T2D resulted in a significant increase in Neuropilin 1 (Nrp1) expression in vascular endothelial cells. Transmembrane Nrp1 receptor is a single-pass glycoprotein with well-established roles in vascular events, including tumor angiogenesis, hypoxia, and growth factor-mediated signal transduction. Increased expression of Nrp1 led to alteration in insulin signaling. Altered insulin signaling by Nrp1 contributed to insulin resistance. Thus, our studies demonstrate that CKD induced accumulation of IS impaired vascular regeneration through loss of VSMC motility and impaired vascular regeneration. Also, it was determined that T2D induced expression of Nrp1 resulted in altered insulin signaling and exacerbating insulin resistance

METHANOGEN METABOLIC FLEXIBILITY

Methanogens are obligately anaerobic archaea which produce methane as a byproduct of their respiration. They are found across a wide diversity of environments and play an important role in cycling carbon in anaerobic spaces and the removal of harmful fermentation byproducts which would otherwise inhibit other organisms. Methanogens subsist on low-energy substrates which requires them to utilize a highly efficient central metabolism which greatly favors respiratory byproducts over biomass. This metabolic strategy creates high substrate:product conversion ratios which is industrially relevant for the production of biomethane, but may also allow for the production of value-added commodities. Particularly of interest are terpene compounds, as methanogen membranes are composed of isoprenoid lipids resulting in a higher flux through isoprenoid biosynthetic pathways compared to Eukarya and Bacteria. To assess the metabolic plasticity of methanogens, our laboratory has engineered the methanogen Methanosarcina acetivorans to produce the hemiterpene isoprene. We hypothesized that isoprene producing strains would result in a decreased growth phenotype corresponding to a depletion of metabolic precursors needed for isoprenoid membrane production. We found that the engineered methanogens responded well to the modification, directing up to 4% of total towards isoprene production and increasing overall biomass despite the additional metabolic burden. Using flux balance analysis and RNA sequencing we investigated how the engineered strains respond to isoprene production and how production can be enhanced. Advisor: Nicole R. Bua

Conceptual Linking: Ontology-based Open Hypermedia

This paper describes the attempts of the COHSE project to define and deploy a Conceptual Open Hypermedia Service. Consisting of • an ontological reasoning service which is used to represent a sophisticated conceptual model of document terms and their relationships; • a Web-based open hypermedia link service that can offer a range of different link-providing facilities in a scalable and non-intrusive fashion; and integrated to form a conceptual hypermedia system to enable documents to be linked via metadata describing their contents and hence to improve the consistency and breadth of linking of WWW documents at retrieval time (as readers browse the documents) and authoring time (as authors create the documents)

Conceptual Linking: Ontology-based Open Hypermedia

This paper describes the attempts of the COHSE project to define and deploy a Conceptual Open Hypermedia Service. Consisting of • an ontological reasoning service which is used to represent a sophisticated conceptual model of document terms and their relationships; • a Web-based open hypermedia link service that can offer a range of different link-providing facilities in a scalable and non-intrusive fashion; and integrated to form a conceptual hypermedia system to enable documents to be linked via metadata describing their contents and hence to improve the consistency and breadth of linking of WWW documents at retrieval time (as readers browse the documents) and authoring time (as authors create the documents)

Insights into the biotechnology potential of Methanosarcina

Methanogens are anaerobic archaea which conserve energy by producing methane. Found in nearly every anaerobic environment on earth, methanogens serve important roles in ecology as key organisms of the global carbon cycle, and in industry as a source of renewable biofuels. Environmentally, methanogenic archaea play an essential role in the reintroducing unavailable carbon to the carbon cycle by anaerobically converting low-energy, terminal metabolic degradation products such as one and two-carbon molecules into methane which then returns to the aerobic portion of the carbon cycle. In industry, methanogens are commonly used as an inexpensive source of renewable biofuels as well as serving as a vital component in the treatment of wastewater though this is only the tip of the iceberg with respect to their metabolic potential. In this review we will discuss how the efficient central metabolism of methanoarchaea could be harnessed for future biotechnology applications

Robust Quantification of Polymerase Chain Reactions Using Global Fitting

BACKGROUND: Quantitative polymerase chain reactions (qPCR) are used to monitor relative changes in very small amounts of DNA. One drawback to qPCR is reproducibility: measuring the same sample multiple times can yield data that is so noisy that important differences can be dismissed. Numerous analytical methods have been employed that can extract the relative template abundance between samples. However, each method is sensitive to baseline assignment and to the unique shape profiles of individual reactions, which gives rise to increased variance stemming from the analytical procedure itself. PRINCIPAL FINDINGS: We developed a simple mathematical model that accurately describes the entire PCR reaction profile using only two reaction variables that depict the maximum capacity of the reaction and feedback inhibition. This model allows quantification that is more accurate than existing methods and takes advantage of the brighter fluorescence signals from later cycles. Because the model describes the entire reaction, the influences of baseline adjustment errors, reaction efficiencies, template abundance, and signal loss per cycle could be formalized. We determined that the common cycle-threshold method of data analysis introduces unnecessary variance because of inappropriate baseline adjustments, a dynamic reaction efficiency, and also a reliance on data with a low signal-to-noise ratio. SIGNIFICANCE: Using our model, fits to raw data can be used to determine template abundance with high precision, even when the data contains baseline and signal loss defects. This improvement reduces the time and cost associated with qPCR and should be applicable in a variety of academic, clinical, and biotechnological settings

Isoprene Production from Municipal Wastewater Biosolids by Engineered Archaeon \u3ci\u3eMethanosarcina acetivorans\u3c/i\u3e

Wastewater biosolids are a promising feedstock for production of value-added renewable chemicals. Methane-producing archaea (methanogens) are already used to produce renewable biogas via the anaerobic treatment of wastewater. The ability of methanogens to efficiently convert dissolved organic carbon into methane makes them an appealing potential platform for biorefining using metabolic engineering. We have engineered a strain of the methanogen Methanosarcina acetivorans to produce the volatile hemiterpene isoprene in addition to methane. The engineered strain was adapted to grow in municipal wastewater through cultivation in a synthetic wastewater medium. When introduced to municipal wastewater the engineered methanogens were able to compete with the indigenous microorganisms and produce 0.97 mM of isoprene (65.9 ± 21.3 g per m3 of effluent). The production of isoprene in wastewater appears to be dependent on the quantity of available methanogenic substrate produced during upstream digestion by heterotrophic fermenters. This shows that with minimal adaptation it is possible to drop-in engineered methanogens to existing wastewater environments and attain value-added products in addition to the processing of wastewater. This shows the potential for utilizing methanogens as a platform for low-cost production of renewable materials without expensive feedstocks or the need to build or adapt existing facilities