Slow Hydrolysis Rate Research Articles

Abstract 1361: Characterization of TpeL, a RAS specific clostridial toxin

Abstract Clostridium perfringes lethal toxin (TpeL) belongs to the family of large clostridial glycosylating cytotoxins. Clostridial toxins are glycosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glycosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. Glycosylation of RAS at Thr35 prevents binding to the primary effector, RAF kinase and results in a blockade RAS signal transduction. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop tools to disrupt the RAS signaling pathway. In order to assess the effect of TpeL in vivo, we use RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E. We expect that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected by any toxin. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells treated with TpeL, suggesting that KRAS Q61R is resistant to glycosylation. This may be due in part to the relatively slow intrinsic rate of GTP hydrolysis and very high levels of KRAS-GTP in KRAS Q61R cells. We also developed and adapted biochemical assays to study TpeL-KRAS interaction in vitro. Based on Alpha Assay and UDP Glow, TpeL activity does not appear to be nucleotide specific, in contrast to previously reported findings. We are currently testing if TpeL RAS glycosylation is competitive with or is inhibited by other molecules that bind to switch I, such as the RBD domain of RAF1 or GEF/GAP proteins. These data may indicate that the lack of activity in KRAS Q61R expressing MEF cells is due not to the high levels of RAS-GTP, but to the constitutive binding to effector molecules, which prevent TpeL from accessing KRAS. We also hope to identify the critical residues required for TpeL binding and catalysis. By elucidating the mechanism of substrate binding and substrate specificity we may gain insight into novel binding pockets that could be exploited therapeutically. Citation Format: Maria T. Abreu-Blanco. Characterization of TpeL, a RAS specific clostridial toxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2017-1361

An efficient method to improve the production of methane from anaerobic digestion of waste activated sludge.

Methane production from waste activated sludge (WAS) anaerobic digestion is always low due to slow hydrolysis rate and inappropriate ratio of carbon to nitrogen (C/N). In this work, a novel approach, i.e., co-digestion of WAS and tobacco waste (TW) using ozone pretreatment, to greatly enhance the production of methane is reported. Experimental results showed the optimal C/N and ozone dosage for methane production was 24:1 and 90 mg/g suspended solids, and the corresponding methane production was 203.6 mL/g volatile suspended solids, which was 1.3-fold that in mono-WAS digestion. Further investigation showed the co-digestion of WAS and TW was beneficial to the consumptions of protein and cellulose; also, the presence of ozone enhanced the disruption of organic substrates and production of short chain fatty acids, which provided sufficient digestion substrates for methane generation. Analysis of microbial community structure suggested that members of the phyla Bacteroidetes and Firmicutes were the dominant species when ozone pretreatment was applied. The findings obtained in this work might be of great importance for the treatment of WAS and TW.

Synergistic effects of anaerobic digestion from sewage sludge with lime mud

Waste reuse and renewable energy generation are encouraged in many countries. Anaerobic digestion of sewage sludge is usually limited by the slow hydrolysis rate of the sludge. To resolve the problem the lime mud was used in this biomethane production. Batch tests were performed at 55 °C with lime mud concentrations ranging from 1.25 to 5.00 g/L. The results showed that the highest methane yield per gram of volatile solid and volatile solid reduction were 146 ml and 42.4% at lime mud concentration of 2.50 g/L, which were 1.77 and 3.01 times higher than that of the control tests without lime mud, respectively. However, high levels of lime mud reduced the amount of available substrate and inhibited the anaerobes, thereby decreasing the methane production. The modified Gompertz model showed a better fit for the results than the first-order model. It was also found that the eco-friendly treatment of two types of solid waste had the potential to synergistically contribute to waste reuse and energy recovery.

Effect of CaO on the reaction process of MgO-SiO2-H2O cement pastes

As a potential cementitious material for nuclear waste immobilization magnesium silicate hydrate (M-S-H) phase was prepared by MgO and silica fume at room temperature. Attributable to the slow hydrolysis rate of MgO, the strength of M-S-H paste is lower than Portland cement in 3days. This work has demonstrated that CaO content has a direct influence on the chemical environment in pore solution and phases transformation in CaO-MgO-SiO2-H2O (C/M/S/H) system. Appropriate CaO content (with optimal Ca/Si molar ratio at approximately 0.02) can accelerate M-S-H gel formation in high W/C paste contributing to early age strength improvement. Ca/Si molar ratio should be controlled at a range between 0.02 and 0.1, because the formation of the calcium silicate hydrate (C-S-H) phase is much is easier, which results in the inhibition of the M-S-H formation.

Cell-Penetrating Cross-β Peptide Assemblies with Controlled Biodegradable Properties.

Although self-assembled peptide nanostructures (SPNs) have shown potential as promising biomaterials, there is a potential problem associated with the extremely slow hydrolysis rate of amide bonds. Here, we report the development of cell-penetrating cross-β SPNs with a controllable biodegradation rate. The designed self-assembling β-sheet peptide incorporating a hydrolyzable ester bond (self-assembling depsipeptide; SADP) can be assembled into bilayer β-sandwich one-dimensional (1D) fibers similarly to conventional β-sheet peptides. The rate of hydrolysis can be controlled by the pH, temperature, and structural characteristics of the ester unit. The 1D fiber of the SADP transforms into vesicle-like 3D structures when the hydrophilic cell-penetrating peptide segment is attached to the SADP segment. Efficient cell internalization of the 3D nanostructures was observed, and we verified the intracellular degradation and disassembly of the biodegradable nanostructures. This study illustrates the potential of biodegradable cross-β SPNs and provides a valuable toolkit that can be used with self-assembling peptides.

Anaerobic digestion of recalcitrant textile dyeing sludge with alternative pretreatment strategies

Abundant organic compounds in textile dyeing sludge (TDS) provide possibility for its anaerobic digestion (AD) treatment. However, preliminary test showed little biogas generation in direct AD of the TDS during 20days. In order to improve the AD availability of TDS, alkaline, acid, thermal and thermal alkaline pretreatments were performed. Color and aromatic amines were specifically measured as extra characteristics for the AD of TDS. The rate-limiting steps of AD of TDS were slow hydrolysis rate and inhibited acidogenesis, which were somewhat overcome by pretreatments. Thermal alkaline pretreated TDS performed best enhancement on solubilisation. The biochemical methane potential tests revealed that thermal pretreated TDS showed highest total methane production of 55.9mL/gVSfed compared to the control with little methane generation. However, thermal alkaline pretreated TDS did not perform well in BMP test as expected. Moreover, the hydrophilicity of reactive dyes in TDS could seriously affect dewaterability of TDS.

Formation of organic–inorganic nanomatrix structure with nanosilica networks and its effect on properties of rubber

Abstract Graft-copolymerization of vinyl monomer containing silica precursor, p -styryltrimethoxysilane, onto natural rubber microparticles was performed in latex stage to form three-dimensional networks of nanosilica. After the latex was cast into a film, morphology observation by transmission electron microscopy showed that the natural rubber microparticles were dispersed in organic–inorganic nanomatrix consisting of vinyl polymer and silica nanoparticles. In particular, generation of large silica particles was prevented by slow rate of hydrolysis and condensation of silicon alkoxides for p -styryltrimethoxysilane and the nanosilica networks were formed. The nanomatrix structure with the nanosilica networks increased tensile strength of the rubber, and destruction of the structure decreased the strength. Master curves of dynamic moduli of the rubber showed that the value of storage modulus at rubbery plateau region was high and it was almost flat at low-frequency region because of the nanosilica networks.

Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1)

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.

Polyhydroxyalkanoates in waste activated sludge enhances anaerobic methane production through improving biochemical methane potential instead of hydrolysis rate.

Anaerobic sludge digestion is the main technology for sludge reduction and stabilization prior to sludge disposal. Nevertheless, methane production from anaerobic digestion of waste activated sludge (WAS) is often restricted by the poor biochemical methane potential and slow hydrolysis rate of WAS. This work systematically investigated the effect of PHA levels of WAS on anaerobic methane production, using both experimental and mathematical modeling approaches. Biochemical methane potential tests showed that methane production increased with increased PHA levels in WAS. Model-based analysis suggested that the PHA-based method enhanced methane production by improving biochemical methane potential of WAS, with the highest enhancement being around 40% (from 192 to 274 L CH4/kg VS added; VS: volatile solid) when the PHA levels increased from 21 to 143 mg/g VS. In contrast, the hydrolysis rate (approximately 0.10 d−1) was not significantly affected by the PHA levels. Economic analysis suggested that the PHA-based method could save $1.2/PE/y (PE: population equivalent) in a typical wastewater treatment plant (WWTP). The PHA-based method can be easily integrated into the current WWTP to enhance methane production, thereby providing a strong support to the on-going paradigm shift in wastewater management from pollutant removal to resource recovery.

Hexagonal CeO2 nanostructures: an efficient electrode material for supercapacitors.

Cerium oxide (CeO2) has emerged as a new and promising pseudocapacitive material due to its prominent valance states and extensive applications in various fields. In the present study, hexagonal CeO2 nanostructures have been prepared via the hydrothermal method employing cationic surfactant cetyl trimethyl ammonium bromide (CTAB). CTAB ensures a slow rate of hydrolysis to form small sized CeO2 nanostructures. The role of calcination temperature on the morphological, structural, electrochemical properties and cyclic stability has been assessed for supercapacitor applications. The mesoscopic hexagonal architecture endows the CeO2 with not only a higher specific capacity, but also with an excellent rate capability and cyclability. When the charge/discharge current density is increased from 2 to 10 A g(-1) the reversible charge capacity decreased from 927 F g(-1) to 475 F g(-1) while 100% capacity retention at a high current density of 20 A g(-1) even after 1500 cycles could be achieved. Furthermore, the asymmetric supercapacitor based on CeO2 exhibited a significantly higher energy density of 45.6 W h kg(-1) at a power density of 187.5 W kg(-1) with good cyclic stability. The electrochemical richness of the CeO2 nanostructure makes it a suitable electrode material for supercapacitor applications.

Engineered Hematite Mesoporous Single Crystals Drive Drastic Enhancement in Solar Water Splitting.

Mesoporous single crystals (MSCs) rendering highly accessible surface area and long-range electron conductivity are extremely significant in many fields, including catalyst, solar fuel, and electrical energy storage technologies. Hematite semiconductor, whose performance has been crucially limited by its pristine poor charge separation efficiency in solar water splitting, should benefit from this strategy. Despite successful synthesis of many metal oxide MSCs, the fabrication of hematite MSCs remains to be a great challenge due to its quite slow hydrolysis rate in water. Herein, for the first time, we have developed a synthetic strategy to prepare hematite MSCs and systematically investigated their growth mechanism. The electrode fabricated with these crystals is able to achieve a photocurrent density of 0.61 mA/cm(2) at 1.23 V vs RHE under AM 1.5G simulated sunlight, which is 20 times higher than that of electrodes made of solid single crystals. The enhancement is ascribed to the superior light absorption and enhanced charges separation. Our results demonstrate the advantage of incorporation of nanopores into the large-sized hematite single crystals and provide a valuable insight for the development of high performance photoelectrodes in PEC application.

Nanoparticles Containing High Loads of Paclitaxel-Silicate Prodrugs: Formulation, Drug Release, and Anticancer Efficacy.

We have investigated particle size, interior structure, drug release kinetics, and anticancer efficacy of PEG-b-PLGA-based nanoparticles loaded with a series of paclitaxel (PTX)-silicate prodrugs [PTX-Si(OR)3]. Silicate derivatization enabled us to adjust the hydrophobicity and hydrolytic lability of the prodrugs by the choice of the alkyl group (R) in the silicate derivatives. The greater hydrophobicity of these prodrugs allows for the preparation of nanoparticles that are stable in aqueous dispersion even when loaded with up to ca. 75 wt % of the prodrug. The hydrolytic lability of silicates allows for facile conversion of prodrugs back to the parent drug, PTX. A suite of eight PTX-silicate prodrugs was investigated; nanoparticles were made by flash nanoprecipitation (FNP) using a confined impingement jet mixer with a dilution step (CIJ-D). The resulting nanoparticles were 80-150 nm in size with a loading level of 47-74 wt % (wt %) of a PTX-silicate, which corresponds to 36-59 effective wt % of free PTX. Cryogenic transmission electron microscopy images show that particles are typically spherical with a core-shell structure. Prodrug/drug release profiles were measured. Release tended to be slower for prodrugs having greater hydrophobicity and slower hydrolysis rate. Nanoparticles loaded with PTX-silicate prodrugs that hydrolyze most rapidly showed in vitro cytotoxicity similar to that of the parent PTX. Nanoparticles loaded with more labile silicates also tended to show greater in vivo efficacy.

An efficient and green pretreatment to stimulate short-chain fatty acids production from waste activated sludge anaerobic fermentation using free nitrous acid.

Short-chain fatty acid (SCFA) production from waste activated sludge (WAS) anaerobic fermentation is often limited by the slow hydrolysis rate and poor substrate availability, thus a long fermentation time is required. This paper reports a new pretreatment approach, i.e., using free nitrous acid (FNA) to pretreat sludge, for significantly enhanced SCFA production. Experimental results showed the highest SCFA production occurred at 1.8 mg FNA/L with time of day 6, which was 3.7-fold of the blank at fermentation time of day 12. Mechanism studies revealed that FNA pretreatment accelerated disruption of both extracellular polymeric substances and cell envelope. It was also found that FNA pretreatment benefited hydrolysis and acidification processes but inhibited the activities of methanogens, thereby promoting the yield of SCFA. In addition, the FNA pretreatment substantially stimulated the activities of key enzymes responsible for hydrolysis and acidification, which were consistent with the improvement of solubilization, hydrolysis and acidification of WAS anaerobic fermentation.

The effect of rate of hydrolysis on structural and optical properties of the TiO2 nanoparticles prepared by a sol–gel method

The nanocrystalline TiO2 powders with different crystallinity and phase structures were obtained by controlling hydrolysis rate and annealing temperature. Rutile phase TiO2 nanoparticles with crystallite sizes of about 80nm, were obtained by annealing the TiO2 powders at 700°C. The crystallite sizes of anatase phase TiO2 increases from 8 to 11nm and band gap energy ranges from 2.49 and 3.26eV, calculated using XRD pattern and UV–vis reflectance spectra, respectively. In agreement with change of the TiO2 band gap after different rate of hydrolysis the PL emission peak in the UV region shift slightly from 335 to 339nm, which corresponds to the direct recombination between electrons in the conduction band and holes in the valence band. The defect related emission shifted from 376 to 385nm with the change in the rate of hydrolysis. TiO2 nanoparticles synthesized at slow hydrolysis rate (6ml of H2O) shows the attainment of best luminescence and maximum reflectance in the visible range. Therefore, these TiO2 nanoparticles can also be employed as coating material to develop reflectors with maximum diffuse reflectance.

In vitro starch digestibility of bread with banana (Musa acuminata X balbisiana ABB cv. Awak) pseudo-stem flour and hydrocolloids

Novel composite breads were prepared by using banana pseudo-stem flour (BPF). BPF was substituted for commercial wheat flour (CWF) (at 10%, w/w) to prepare B10BPF and the B10BPF with added hydrocolloids [xanthan gum (XG) or sodium carboxy-methyl cellulose (Na CMC)] to prepare B10BPFXG and B10BPFCMC, respectively. The prepared composite breads had high level of minerals (calcium, phosphorus, potassium, magnesium, iron, and manganese), total sugars, and resistant starch contents. Except for the composite bread prepared by adding XG, all other samples showed non-significant decrease in the total and digestible starch. Slow starch hydrolysis rate (corresponding to increase in time) was recorded with a subsequent reduction of hydrolysis index and estimated glycemic index in the composite breads. As this is a novel composite bread developed with health promoting properties, it is expected to benefit consumers, especially for those requiring high fiber diets.

Enhancing methane production from waste activated sludge using a novel indigenous iron activated peroxidation pre-treatment process

Methane production from anaerobic digestion of waste activated sludge (WAS) is limited by the slow hydrolysis rate and/or poor methane potential of WAS. This study presents a novel pre-treatment strategy based on indigenous iron (in WAS) activated peroxidation to enhance methane production from WAS. Pre-treatment of WAS for 30min at 50mg H2O2/g total solids (dry weight) and pH 2.0 (iron concentration in WAS was 7mg/g TS) substantially enhanced WAS solubilization. Biochemical methane potential tests demonstrated that methane production was improved by 10% at a digestion time of 16d after incorporating the indigenous iron activated peroxidation pre-treatment. Model-based analysis indicated that indigenous iron activated peroxidation pre-treatment improved the methane potential by 13%, whereas the hydrolysis rate was not significantly affected. The economic analysis showed that the proposed pre-treatment method can save the cost by $112,000 per year in a treatment plant with a population equivalent of 300,000.

Zero valent iron significantly enhances methane production from waste activated sludge by improving biochemical methane potential rather than hydrolysis rate.

Anaerobic digestion has been widely applied for waste activated sludge (WAS) treatment. However, methane production from anaerobic digestion of WAS is usually limited by the slow hydrolysis rate and/or poor biochemical methane potential of WAS. This work systematically studied the effects of three different types of zero valent iron (i.e., iron powder, clean scrap and rusty scrap) on methane production from WAS in anaerobic digestion, by using both experimental and mathematical approaches. The results demonstrated that both the clean and the rusty iron scrap were more effective than the iron powder for improving methane production from WAS. Model-based analysis showed that ZVI addition significantly enhanced methane production from WAS through improving the biochemical methane potential of WAS rather than its hydrolysis rate. Economic analysis indicated that the ZVI-based technology for enhancing methane production from WAS is economically attractive, particularly considering that iron scrap can be freely acquired from industrial waste. Based on these results, the ZVI-based anaerobic digestion process of this work could be easily integrated with the conventional chemical phosphorus removal process in wastewater treatment plant to form a cost-effective and environment-friendly approach, enabling maximum resource recovery/reuse while achieving enhanced methane production in wastewater treatment system.

Relative extents of activity loss between enzyme–substrate interactions and combined environmental mechanisms

Enzymatic hydrolysis of biomass undergoes a significant decrease in rate, which is often attributed to activity loss of enzyme during the incubation. Activity loss due to both interaction with substrate (for example inactivation of adsorbed enzyme) and all combined environmental mechanisms in a substrate free buffer solution were compared in this study. Enzyme-substrate interactions contributed more towards the overall activity loss than did the combined environmental sources as evidenced from three independent metrics. (1) Relative extents of inactivation were higher for enzyme-substrate interactions than for environmental mechanisms. (2) Apparent half-lives (1.37-11.01 h) following interaction with substrate were relatively small compared to environmental inactivation, which was 21.5h. (3) The inactivation rate constant for enzyme-substrate interactions (0.56 h(-1)) was 46 times higher than that of environmental inactivation (0.0123 h(-1)). These results suggest enzyme-substrate interaction is the main cause of cellulase activity loss and contributes significantly to the slow rate of hydrolysis.

Free Nitrous Acid (FNA)-Based Pretreatment Enhances Methane Production from Waste Activated Sludge

Anaerobic digestion of waste activated sludge (WAS) is currently enjoying renewed interest due to the potential for methane production. However, methane production is often limited by the slow hydrolysis rate and/or poor methane potential of WAS. This study presents a novel pretreatment strategy based on free nitrous acid (FNA or HNO2) to enhance methane production from WAS. Pretreatment of WAS for 24 h at FNA concentrations up to 2.13 mg N/L substantially enhanced WAS solubilization, with the highest solubilization (0.16 mg chemical oxygen demand (COD)/mg volatile solids (VS), at 2.13 mg HNO2-N/L) being six times that without FNA pretreatment (0.025 mg COD/mg VS, at 0 mg HNO2-N/L). Biochemical methane potential tests demonstrated methane production increased with increased FNA concentration used in the pretreatment step. Model-based analysis indicated FNA pretreatment improved both hydrolysis rate and methane potential, with the highest improvement being approximately 50% (from 0.16 to 0.25 d(-1)) and 27% (from 201 to 255 L CH4/kg VS added), respectively, achieved at 1.78-2.13 mg HNO2-N/L. Further analysis indicated that increased hydrolysis rate and methane potential were related to an increase in rapidly biodegradable substrates, which increased with increased FNA dose, while the slowly biodegradable substrates remained relatively static.

Antitumor and antiangiogenic active dendrimer/5‐fluorouracil conjugates

Antitumor and antiangiogenic active dendritic compounds conjugated with 5-fluorouracil (5-FU) were synthesized and characterized. First, the core structure based on ascorbic acid and dicarboxylic acid was synthesized, amidated with ethylenediamine. For the synthesis of higher generated dendrimer, it was reacted further with methyl acrylate to increase molecular weight through Michael addition reaction, followed by the continuous repeating amidation reaction, finally, used to synthesize covalently bound dendrimer/5-FU conjugates. The in vitro cytotoxicities of the conjugates was evaluated with mouse mammary carcinoma, mouse leukemia, and human histiocytic lymphoma as cancer cell lines and mouse liver cells as a normal cell line, and showed lower values than 5-FU. The in vivo antitumor activities of the conjugates against mice bearing sarcoma 180 tumor cell line were better than 5-FU. Especially, Second generated dendrimer (G-2)/5-FU conjugates showed excellent in vivo antitumor as well as antiangiogenic activities evaluated by the embryo chorioallantoic membrane assay due to slow hydrolysis rate and the amount of 5-FU released.