Quinazolin‐4(3H)‐one‐Based Hydroxamic Acids: Design,Synthesis and Evaluation of Histone Deacetylase Inhibitory Effects and Cytotoxicity

The present article describes the synthesis and biological activity of various series of novel hydroxamic acids incorporating quinazolin‐4(3H)‐ones as novel small molecules targeting histone deacetylases. Biological evaluation showed that these hydroxamic acids were potently cytotoxic against three human cancer cell lines (SW620, colon; PC‐3, prostate; NCI?H23, lung). Most compounds displayed superior cytotoxicity than SAHA (suberoylanilide hydroxamic acid, Vorinostat) in term of cytotoxicity. Especially, N‐hydroxy‐7‐(7‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)heptanamide ( 5b ) and N‐hydroxy‐7‐(6‐methyl‐4‐oxoquinazolin‐3(4H)‐yl)heptanamide ( 5c ) (IC₅₀ values, 0.10–0.16 μm ) were found to be approximately 30‐fold more cytotoxic than SAHA (IC₅₀ values of 3.29–3.67 μm ). N‐Hydroxy‐7‐(4‐oxoquinazolin‐3(4H)‐yl)heptanamide ( 5a ; IC₅₀ values of 0.21–0.38 μm ) was approximately 10‐ to 15‐fold more potent than SAHA in cytotoxicity assay. These compounds also showed comparable HDAC inhibition potency with IC₅₀ values in sub‐micromolar ranges. Molecular docking experiments indicated that most compounds, as represented by 5b and 5c , strictly bound to HDAC2 at the active binding site with binding affinities much higher than that of SAHA.     

Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30 % and on insoluble crystalline as well as amorphous cellulose by over 50 %.     

Basis for formulating biosurfactant mixtures to achieve ultra low interfacial tension values against hydrocarbons

Biosurfactants could potentially replace or be used in conjunction with synthetic surfactants to provide for more cost-effective subsurface remediation. The design of surfactant formulations that are effective in lowering interfacial tension (IFT), which is necessary to mobilize entrapped hydrocarbons, requires information about the surface-active agent (surfactant) and the targeted non-aqueous phase liquids (NAPL). We hypothesized that biosurfactant and synthetic surfactant mixtures can be formulated to provide the appropriate hydrophobic/hydrophilic conditions necessary to produce low IFT against NAPLs, and that such mixtures will produce synergism that make them more effective than individual biosurfactants or synthetic surfactants. Our work tested the interfacial activity of biosurfactants from individual strains and mixtures of biosurfactants from different strains with and without a synthetic surfactant. Multiple regression analysis showed that, for lipopeptide biosurfactants produced by various Bacillus species, the interfacial activity against toluene depended on the relative proportions of 3-OH-C₁₄, C₁₅, C₁₆, and C₁₈ in the fatty acid tail. As the fatty acid composition became more heterogeneous the system produced lower IFT against toluene. In mixtures of lipopeptide biosurfactants with the more hydrophilic, rhamnolipid biosurfactant, the IFT against toluene decreased as the percentage of the 3-OH C₁₄ fatty acid increased in the lipopeptide. Mixtures of lipopeptide biosurfactants with the more hydrophobic synthetic surfactant, C12, C13-8PO SO₄Na, were able to produce low IFT against hexane and decane. In general, we found that lipopeptide biosurfactants with a heterogeneous fatty acid composition or mixtures of lipopeptide and rhamnolipid biosurfactants lowered the IFT against hydrophilic NAPLs. Conversely, mixtures of lipopeptide biosurfactants with a more hydrophobic synthetic surfactant lowered the IFT against hydrophobic NAPLs.     

3D culture of cancer cells in alginate hydrogel beads as an effective technique for emergency cell storage and transportation in the pandemic era

Due to the restrictions in accessing research laboratories and the challenges in providing proper storage and transportation of cells during the COVID‐19 pandemic, having an effective and feasible mean to solve these challenges would be of immense help. Therefore, we developed a 3D culture setting of cancer cells using alginate beads and tested its effectiveness in different storage and transportation conditions. The viability and proliferation of cancer cells were assessed using trypan blue staining and quantitative CCK‐8 kit, respectively. The developed beads allowed cancer cells survival up to 4 weeks with less frequent maintenance measures such as change of the culture media or subculture of cells. In addition, the recovery of cancer cells and proliferation pattern were significantly faster with better outcomes in the developed 3D alginate beads compared to the standard cryopreservation of cells or the 2D culture conditions. The 3D alginate beads also supported the viability of cells while the shipment at room temperature for a duration of up to 5 days with no humidity or CO₂ support. Therefore, 3D culture in alginate beads can be used to store or ship biological cells with ease at room temperature with minimal preparations.     

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)     

Photosystem I light-harvesting proteins regulate photosynthetic electron transfer and hydrogen production

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H₂) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H₂ production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H₂ photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H₂ photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H₂ photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

Alteration of the light-harvesting composition of photosystem I impacts photosynthetic electron transfer and hydrogen production.     

Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives

Methylated cytosines appear as sequence variations following bisulfite treatment and polymerase chain reaction (PCR) amplification. By using methylation-specific PCR (MSP), it is possible to detect methylated sequences in a background of unmethylated DNA with a high level of sensitivity. MSP is frequently used to identify methylated alleles in carcinogenesis, and may be combined with the TaqMan real-time PCR system, which uses fluorescence-based detection of amplification products during the amplification phase of the PCR and increases the sensitivity of detection (MethyLight). Sequences that have been incompletely converted during the bisulfite treatment are frequently coamplified during MSP, resulting in an overestimation of DNA methylation. The presence of amplified sequences originating from partially unconverted material may be determined by sequencing or by restriction digests or Southern blots of MSPs. Alternately, we have developed a method where the PCR and conversion assay are combined within a single TaqMan reaction by using an additional fluorescent probe directed against unconverted DNA (ConLight-MSP). We recommend that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.     

Life cycle cost analysis of fuel ethanol produced from cassava in Thailand

Background, aim, and scope

As a net oil importer, Thailand has a special interest in the development of biofuels, especially ethanol. At present, ethanol in the country is mainly a fermentation/distillery product of cane molasses, but cassava holds superior potential for the fuel. This study aims to assess the economics of cassava-based ethanol as an alternative transportation fuel in Thailand. The scope of the study includes the cassava cultivation/processing, the conversion to ethanol, the distribution of the fuel, and all transportation activities taking place within the system boundary.

Materials and methods

The life cycle cost assessment carried out follows three interrelated phases: data inventory, data analysis, and interpretation. The functional unit for the comparison between ethanol and gasoline is the specific distance that a car can travel on 1 L ethanol in the form of E10, a 10% ethanol blend in gasoline.

Results

The results of the analysis show, despite low raw material cost compared to molasses and cane-based ethanol, that cassava ethanol is still more costly than gasoline. This high cost has put an economic barrier to commercial application, leading to different opinions about government support for ethanol in the forms of tax incentives and subsidies.

Discussion

Overall, feedstock cost tends to govern ethanol’s production cost, thus, making itself and its 10% blend in gasoline less competitive than gasoline for the specific conditions considered. However, this situation can also be improved by appropriate measures, as discussed later.

Conclusions

To make ethanol cost-competitive with gasoline, the first possible measure is a combination of increasing crop yield and decreasing farming costs (chemical purchase and application, planting, and land preparation) so as to make a 47% reduction in the cost per tonne of cassava. This is modeled by a sensitivity analysis for the cost in the farming phase. In the industrial phase of the fuel production cycle, utilization of co-products and substitution of rice husk for bunker oil as process energy tend to reduce 62% of the price gap between ethanol and gasoline. The remaining 38% price gap can be eliminated with a 16% cut of raw material (cassava) cost, which is more practical than a 47% where no savings options in ethanol conversion phase are taken into account.

Recommendations and perspectives

The life cycle cost analysis helps identify the key areas in the ethanol production cycle where changes are required to improve cost performance. Including social aspects in an LCC analysis may make the results more favorable for ethanol.