Improved Cell Membrane Permeability Research Articles

Taurine-mediated gene transcription and cell membrane permeability reinforced co-production of bioethanol and Monascus azaphilone pigments for a newly isolated Monascus purpureus

BackgroundTaurine, a semi-essential micronutrient, could be utilized as a sulfur source for some bacteria; however, little is known about its effect on the accumulation of fermentation products. Here, it investigated the effect of taurine on co-production of bioethanol and Monascus azaphilone pigments (MonAzPs) for a fungus.ResultsA newly isolated fungus of 98.92% identity with Monascus purpureus co-produced 23.43 g/L bioethanol and 66.12, 78.01 and 62.37 U/mL red, yellow and orange MonAzPs for 3 d in synthetic medium (SM). Taurine enhanced bioethanol titer, ethanol productivity and ethanol yield at the maximum by 1.56, 1.58 and 1.60 times than those of the control in corn stover hydrolysates (CSH), and red, yellow and orange MonAzPs were raised by 1.24, 1.26 and 1.29 times, respectively. Taurine was consumed extremely small quantities for M. purpureus and its promotional effect was not universal for the other two biorefinery fermenting strains. Taurine intensified the gene transcription of glycolysis (glucokinase, phosphoglycerate mutase, enolase and alcohol dehydrogenase) and MonAzPs biosynthesis (serine hydrolases, C-11-ketoreductase, FAD-dependent monooxygenase, 4-O-acyltransferase, deacetylase, NAD(P)H-dependent oxidoredutase, FAD-dependent oxidoredutase, enoyl reductase and fatty acid synthase) through de novo RNA-Seq assays. Furthermore, taurine improved cell membrane permeability through changing cell membrane structure by microscopic imaging assays.ConclusionsTaurine reinforced co-production of bioethanol and MonAzPs by increasing gene transcription level and cell membrane permeability for M. purpureus. This work would offer an innovative, efficient and taurine-based co-production system for mass accumulation of the value-added biofuels and biochemicals from lignocellulosic biomass.

Cordyceps fungus-mediated enantioselective synthesis of chiral alcohol and enhanced biocatalytic efficiency in cyclodextrin/surfactant-buffer system

Asymmetric synthesis of chiral alcohols by biocatalysts holds promising prospect. In this study, a medicinal Cordyceps fungus, namely Tolypocladium inflatum, was adopted as a novel biocatalyst for the synthesis of chiral alcohols. Among the assessed prochiral ketone substrates, T. inflatum exhibited the best catalytic activity towards 1-(4-bromophenyl)−2,2,2-trifluoroethanone (BPFO), yielding a valuable pharmaceutical intermediate (S)−1-(4-bromophenyl)−2,2,2-trifluoroethanol ((S)-BPFL) with >99.9 % enantiomeric excesses (ee). For more efficient biosynthesis of (S)-BPFL, cyclodextrins or surfactants were introduced to the reaction system as additives to strengthen this bioprocess. Among which methyl β-cyclodextrin (MCD) improved cell membrane permeability and alleviated product inhibition, while Triton X-100 enhanced BPFO solubility. Furthermore, synergistic promotion for BPFO bioreduction by MCD and Triton X-100 was observed. Under the optimal reaction conditions, 50 mM BPFO was converted into (S)-BPFL with 89.1 % yield and >99.9 % ee in 12 h, showing 16-fold increase of space-time yield in the developed MCD/Triton X-100-buffer system compared to neat buffer solution. This study illustrated the potential of the enantioselective preparation of chiral alcohols mediated by Cordyceps fungus. Additionally, the biocatalytic efficiency was significantly improved in the constructed synergistic reaction system, and the underlying mechanism was also explored.

Houttuynia cordata Thunb. crude extract inactivates Cronobacter sakazakii: Antibacterial components, antibacterial mechanism, and application as a natural disinfectant

The purpose of this study was to reveal the antibacterial components of Houttuynia cordata Thunb. crude extract (HCCE), analyze the antibacterial activity and mechanism of HCCE against Cronobacter sakazakii, and evaluate the bactericidal effect of HCCE as a natural disinfectant. Using an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-QE-MS) non-target metabolomics technology, 15 kinds oforganic acids (18.65%), 14 kinds of phenols (16.17%), 10 kinds of flavonoids (12.32%), 5 kinds of terpenoids (5.87%), 3 kinds of alkaloids (1.62%), and 3 kinds of aldehydes (1.28%) were detected from HCCE. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of HCCE against C. sakazakii were 7.5 mg/mL and 15 mg/mL, respectively, and when the concentration was 2 MIC, C. sakazakii stagnated in sluggish phase. C. sakazakii exposed to HCCE showed hyperpolarization of cell membrane, increased intracellular ROS content, decreased protein and DNA content, impaired cell membrane integrity, improved cell membrane permeability, cell fluid leakage, and partial cell fragmentation. After treatments with 1 MIC and 2 MIC of HCCE for 5 min, C. sakazakii strains on 8 contact surfaces including stainless steel, tinplate, glass, polystyrene, bamboo, plank, ceramic, and tin foil paper were significantly inhibited (P < 0.05), and the reduction amounts ranged from 3.00 to 3.68 log CFU/100 cm2 and 3.97 to 4.53 log CFU/100 cm2, respectively. These findings demonstrate the potential of HCCE as a natural disinfectant and provide a new idea to reduce the C. sakazakii contamination caused by surface contact.

Surfactant aggravated the antibiotic's stress on antibiotic resistance genes proliferation by altering antibiotic solubilization and microbial traits in sludge anaerobic fermentation

The excessive application of antibiotics and surfactants resulted in their massive accumulation in waste activated sludge (WAS), but the co-occurrent impacts of antibiotics and surfactants on the antibiotic resistant genes (ARGs) fates have seldom reported. This work mainly revealed the roles and critical mechanisms of sodium dodecyl benzene sulfonate (SDBS) on the sulfadiazine (SDZ) stressing for ARGs distribution during WAS anaerobic fermentation. High-throughput qPCR and metagenomic analysis revealed that SDBS aggravated the SDZ selective pressure, and accelerated the proliferation of ARGs. The total abundance of ARGs was increased from 8.81 × 1010 in SDZ to 1.17 × 1011 copies/g TSS in the SDBS/SDZ co-occurrence system. Specifically, the absolute abundances of ermF (MLSB), mefA (MLSB), tetM-01 (tetracycline), tetX (tetracycline), sul2 (sulfonamide) and strB (aminoglycoside) were risen from 4.60 × 108–7.44 × 109 copies/g TSS in the SDZ reactor to 1.02 × 109–4.63 × 1010 copies/g TSS in SDBS/SDZ reactor. SDBS was contributed to the SDZ solubilization and simultaneously effective in disintegrating extracellular polymeric substances and improving cell membrane permeability, which would facilitate the SDZ transport and its interactions with ARGs hosts. Consequently, the microbial community structure was evidently altered, and the typical ARGs hosts (i.e., Saccharimonadales and Ahniella) were greatly enriched. Also, the cell signal transduction systems (i.e., glnL, glrK and pilG), oxidative stress response (i.e., frmA and recA) and bacterial secretion systems (i.e., VirB4), which were related with ARGs propagation, were all provoked in the co-occurred SDBS/SDZ reactor compared with that of sole SDZ. PLS-PM analysis suggested that the bacterial community was the predominant factor that determined the ARGs fates, followed by mobile genetic elements and metabolic pathway. This work demonstrated the interactive effects of surfactants and antibiotics on the ARGs fates in WAS fermentation systems and gave insightful implications on the ecological risks of different exogenous pollutants.

3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.

Te(IV) bioreduction in the sulfur autotrophic reactor: Performance, kinetics and synergistic mechanism

A laboratory-scale sulfur autotrophic reactor (SAR) was first constructed for treating tellurite [Te(IV)] wastewater. The SAR had excellent Te(IV) bioreduction efficiency (90-96%) at 5-30 mg/L and conformed to the First-order kinetic model. The Te(IV) bioreduction was elaborated deeply from extracellular polymeric substances (EPS) functions, microbial metabolic activity, key enzyme activity, microbial community succession and quorum sensing. Te(IV) stimulated the increase of redox substances in EPS and the improved cell membrane permeability led to the increase of electron transport system activity. Catalase and reduced nicotinamide adenine dinucleotide (NADH) alleviated the oxidative stress caused by Te(IV) toxicity to maintain metabolic activity. The increase of sulfur dioxygenase activity (SDO) suggested that more ATP produced by sulfur oxidation might provide energy for various physiological activities. Meanwhile, nitrate reductase (NAR), nitrite reductase (NIR) and sulfide: quinone oxidoreductase (SQR) played an active role in sulfur oxidation and Te(IV) bioreduction. Combined with the above results and dynamic succession of three functional microbial communities, a synergistic mechanism was proposed to explain the excellent performance of SAR. This work provided a promising strategy for Te(IV) wastewater treatment process and Te(IV) bioreduction mechanism.

Pulsed electric field promotes the growth metabolism of aerobic denitrifying bacteria Pseudomonas putida W207-14 by improving cell membrane permeability

ABSTRACT The purpose of this study was to explore the stimulation mechanism of low pulsed electric field (PEF) strength treatment to promote the growth metabolism of aerobic denitrifying bacteria Pseudomonas putida W207-14. The results indicated that compared with the control group, the strain W207-14 treated with PEF entered the logarithmic growth phase 5 h earlier, the growth time to reached the maximum cell optical density at 600 nm (OD600) of 1.935 ± 0.04 was only 24 h, which shortened by half. With the reduction of growth time, the metabolic rate of the strain increased significantly, in which the removal efficiency of COD, NO3 −–N and TN was 97.67 ± 1.12%, 90.34 ± 0.73% and 90.13 ± 0.10% in 24 h, respectively. The maximum nitrate removal rate increased from 3.49 mg/L/h to 7.53 mg/L/h. A large number of cells with simultaneous cell membrane damage and high physiological activity were observed by flow cytometry (FCM) in combination with fluorescence staining analysis, which confirmed the reversible electroporation on the cell membrane of strain W207-14 treated with PEF. Transcriptomic analysis indicated that PEF activated the highly significant differential expression of membrane porin (opdB, opdC, and oprB) and cytochrome oxidoreductase related genes (ccoP, ccoN, cioA and cioB) on the cell membrane, which promoted the transport of nutrients through the cell membrane and electron transfer during aerobic respiration and provided an explanation for the possible mechanism of PEF promoting the growth metabolism of strain W207-14 at the micro level. These results lay a foundation for the practical application of PEF enhanced aerobic denitrification technology.

Chemical profiling and anti-breast cancer potential of hexane fraction of Sphaeranthus indicus flowers

Purpose: The current study aimed to determine the phytochemicals and anti-breast cancer potential of Sphaeranthus indicus.Methods: S. indicus flowers were extracted with methanol followed by fractionation using n-hexane. For the chemical composition of n-hexane fraction, qualitative phytochemical and GC-MS analysis were performed. The anti-proliferative activity was measured by MTT assay, whereas, cytotoxic and proapoptotic effects in MCF-7 (breast cancer) cells were determined using propidium iodide, 4′,6-diamidino- 2-phenylindole, dichlorofluorescin diacetate, and JC-1 staining through fluorescent microscopy.Results: The phytochemical analysis indicated presence of phytosterols, oils and resins in the nhexane fraction. GC-MS analysis showed that n-hexane fraction comprises of 11 compounds including methyl esters of caprylic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid and behanic acid. The tested fraction showed remarkable cytotoxic activity against breast cancer (MCF-7) cells while it was found less toxic towards non-cancerous (BHK-21) cells. Furthermore, morphological assessment through fluorescent microscopy revealed cytotoxic and apoptotic effects by improved cell membrane permeability, increased reactive oxygen species level, compromised mitochondrial activity and condensation of chromatin network.Conclusion: The n-hexane fraction of S. indicus contains phytosterols, oils and fatty acid methyl esters and produced apoptotic effect against breast cancer cells.

Integration of natural deep-eutectic solvent and surfactant for efficient synthesis of chiral aromatic alcohol mediated by Cyberlindnera saturnus whole cells

Efficiency of whole-cell biocatalysis in aqueous buffer solution is mainly limited by the low cellular membrane permeability and poor solubility of hydrophobic non-natural substrate. In this study, highly efficient bioreduction of 3,5-bis(trifluoromethyl)acetophenone (BTAP) to (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-BTPE) with excellent enantioselectivity (> 99.9 % ee) by a novel yeast isolate Cyberlindnera saturnus ZJPH1807 was achieved via a integrated strategy of natural deep-eutectic solvent (NADES) and surfactant. The introduction of L-carnitine: lysine (C:Lys, molar ratio 1:2) in the reaction medium improved cell membrane permeability effectively and relieved BTAP toxicity to the cells. The Tween-80 addition further accelerated catalytic yield by increasing solubility of BTAP for 7.6-fold compared with neat aqueous buffer solution. After the optimization of key reaction parameters, a 81.0 % yield within 24 h under 500 mM substrate loading was achieved in the established C:Lys/Tween-80-containing system, compared with a 76.4 % yield for 30 h in aqueous buffer system under 200 mM BTAP concentration. This biocatalytic process was also feasible at 500 mL preparation scale with a 79.7 % (S)-BTPE yield under 500 mM BTAP. The developed combinational strategy of NADES with surfactant is an effective approach for enhancing bioreductive efficiency of hydrophobic substrates mediated by whole-cell catalyst, and has great potential in biocatalysis.

Trichoderma biofertilizer (mixTroTha) mediates Malus sieversii resistance to Alternaria alternata

As a biocontrol agent, Trichoderma can ameliorate fungal phytopathogen infection of plants by inducing plant resistance, promoting plant growth, and improving the soil environment. Trichoderma rossicum T7 (TroT7) and Trichoderma harzianum T2 (ThaT2) can significantly inhibit the growth of Alternaria alternata, Fusarium spp., and Cytospora mandshurica, with inhibition rates ranging from 34% to 58%. Therefore, spores of TroT7 and ThaT2 were mixed as a biofertilizer (mixTroTha). Soil irrigated with mixTroTha had a decreased population of phytopathogens. Following pretreatment with mixTroTha, the leaves of two-month-old Malus sieversii seedlings infected by A. alternata had improved cell membrane permeability and decreased numbers of diseasespots (by 16.67%, in the laboratory). The leaves of three-year-old M. sieversii seedlings also had 6.71% fewer diseasespots (in the field). The mechanism might involve the increased jasmonic acid content, which upregulated the expression of PDF1.2 (encoding plant defensin 1.2), by 12.4-fold at 72 h compared with the control. In addition, following Trichoderma colonization of the apple tree rhizosphere, the fungal communities changed significantly: 12 of 14 pathogenic genera showed a lower species abundance, and 4 of 5 biocontrol genera had a higher species abundance. However, the bacterial communities did not change significantly. Moreover, exposure to mixTroTha promoted the growth of lateral roots of three-year-old M. sieversii seedlings, and also accelerated soil metabolic processes, further increasing the contents of available nutrients in soil. Thus, mixTroTha could mediate M. sieversii seedling resistance to A. alternata via diverse methods.

Metal-surfactant hybridize living cells to form micro-urchin with high catalytic activity

The cell membrane, as the supporting structure of cells, maintains the stability of the internal microenvironment of cells for whole-cell catalysis, but is also a barrier in transmembrane transport of substrate and product. However, methods for improving cell membrane permeability are usually accompanied with reduced membrane structural strength. Here, we show that a self-assembled biocomposite formed by Mg2+ ions and sodium deoxycholate (NaDC) can form sea urchin-shaped 3D complexes, incorporating living microbial cells. Cell membrane permeability was significantly improved by a fused structure between cell and Mg(DC)2 nanoparticles without membrane damage. Due to with hydrophobic mesopores, hydrophobic core and disseminated structure, the biocomposite greatly improved catalytic activity (up to 427.3%) of an environment-sensitive P450 enzyme expressed in Escherichia coli, which catalyzed hydroxylation with aromatic hydrocarbon. We also show that the biocomposite successfully improved the membrane permeability of live Bacillus cereus and Saccharomyces cerevisiae, making it a promising application platform for improving membrane permeability in whole-cell catalysis.

Synergistic Anti-tumor Mechanisms of Low-frequency Ultrasound-targeted Microbubble Destruction:Mechanisms and Application

Ultrasound contrast agent microbubbles combined with low frequency ultrasound named as low-frequency ultrasound-targeted microbubble destruction technology has become an effective and non-invasive anti-tumor therapy for deep tumors.It can enhance the efficacies of chemotherapy,gene therapy,immunotherapy,and anti-angiogenic therapy by improving cell membrane permeability and destroying tumor neovasculature.It can be applied to sonodynamic therapy and realize multimodal synergistic therapy on the basis of nanoparticles,which increases the anti-tumor efficiency and offers a promising target therapy for tumors.

Berberine derivatives with a long alkyl chain branched by hydroxyl group and methoxycarbonyl group at 9-position show improved anti-proliferation activity and membrane permeability in A549 cells.

Berberine (BBR) exhibits diverse bioactivities, including anticancer activity; but its poor druggability limits its applications. In this study, we designed and synthesized a series of 9-O position modified BBR derivatives aiming to improve its cell permeability and anticancer activity, utilizing a long alkyl chain branched by hydroxyl group and methoxycarbonyl group. Among these compounds, B10 showed 3.6-fold higher intracellular concentration than BBR, as well as 60-fold increased anti-proliferation activity against human lung cancer A549 cells compared with BBR. Treatment with B10 (1, 2 μM) induced apoptosis of A549 cells. Further investigations showed that B10 treatment dose-dependently affected mitochondrial functions, including oxygen consumption rate (OCR), mitochondrial membrane potential (MMP) and the morphology of mitochondria in A549 cells. Therefore, this work offers a new way for BBR structural modification through improving cell membrane permeability to affect mitochondrial functions and potential anti-tumor therapy in the future.

A novel biosensor for zinc detection based on microbial fuel cell system

Microbial fuel cell (MFC) biosensors are self-sustainable device for monitoring of various substrates; however, for heavy metals detection are still scarce. In this study, E. coli BL21 was engineered to express the zntR, ribB, and oprF genes with PzntA promoter, which could sense zinc (Zn2+) for riboflavin and porin production. The engineered strain produced high levels of riboflavin (2.4–3.6 μM) and improved cell membrane permeability, with a positive correlation of Zn2+ (0–400 μM). The strain was then employed in MFC biosensor under the following operational parameters: external resistance 1000 Ω, pH 9, and temperature 37 °C for Zn2+ sensing. The maximum voltages (160, 183, 260, 292, and 342 mV) of the constructed MFC biosensor have a linear relationship with Zn2+ concentrations (0, 100, 200, 300, and 400 μM, respectively) (R2 = 0.9777). An Android App was developed for the biosensor system that could sense Zn2+ in real-time and in situ. The biosensor was applied to wastewater with different Zn2+ concentrations and the results showed that the detection range for Zn2+ was 20–100 μM, which covers common Zn2+ safety standards. The results obtained with developed MFC biosensor were comparable to conventional methods such as colorimetric, flame atomic absorption spectroscopy (FAAS), and inductively coupled plasma optical emission spectroscopy (ICP-OES). In summary, MFC biosensor with biosynthetic strain is an efficient and affordable system for real-time monitoring and sensing of heavy metals.

Synthesis and characterization of 4′-C-guanidinomethyl-2′-O-methyl-modified RNA oligomers

This study investigated the synthesis and properties of 4′-C-guanidinomethyl-2′-O-methyluridine and RNAs containing the analog. Thermal and thermodynamic stabilities of double-stranded RNAs (dsRNAs) containing the nucleoside analog were examined. It was found that although the analog decreased the thermal and thermodynamic stabilities of dsRNA, it had base-discrimination ability. The 4′-C-guanidinomethyl modification increased stability of RNAs in a buffer containing serum. Furthermore, small interference RNAs incorporating one analog at the passenger strand still preserved their RNA interference activities. It was suggested that the 4′-guanidinomethyl modification significantly improved cell membrane permeability of RNA. Thus, 4′-C-guanidinomethyl-2′-O-methyl analogs may be useful in improving the properties of therapeutic siRNA molecules.

Protection of rat liver against hepatic ischemia-reperfusion injury by a novel selenocysteine-containing 7-mer peptide.

Hepatic ischemia-reperfusion (I-R) injury causes acute organ damage or dysfunction, and remains a problem for liver transplantation. In the I-R phase, the generation of reactive oxygen species aggravates the injury. In the current study, a novel selenocysteine-containing 7-mer peptide (H-Arg-Sec-Gly-Arg-Asn-Ala-Gln-OH) was constructed to imitate the active site of an antioxidant enzyme, glutathione peroxidase (GPX). The 7-mer peptide which has a lower molecular weight, and improved water-solubility, higher stability and improved cell membrane permeability compared with other GPX mimics. Its GPX activity reached 13 U/µmol, which was 13 times that of ebselen (a representative GPX mimic). The effect of this GPX mimic on I-R injury of the liver was assessed in rats. The 7-mer peptide significantly inhibited the increase in serum hepatic amino-transferases, tissue malondialdehyde, nitric oxide contents, myeloperoxidase activity and decrease of GPX activity compared with I-R tissue. Following treatment with the 7-mer peptide, the expression of B-cell CLL/lymphoma-2 (Bcl-2) was significantly upregulated at the mRNA and protein level compared with the I-R group, as determined by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. By contrast, Bcl-2 associated X protein (Bax) was downregulated by the 7-mer peptide compared the I-R group. Histological and ultrastructural changes of the rat liver tissue were also compared among the experimental groups. The results of the current study suggest that the 7-mer peptide protected the liver against hepatic I-R injury via suppression of oxygen-derived free radicals and regulation of Bcl-2 and Bax expression, which are involved in the apoptosis of liver cells. The findings of the present study will further the investigation of the 7-mer peptide as an effective therapeutic agent in hepatic I-R injury.

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability

Fluorescence imaging of calcium ions (Ca2+) has become an essential technique for investigation of signaling pathways involving Ca2+ as a second messenger. But, Ca2+ signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca2+ and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca2+ that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca2+ concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca2+ red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.

Focused library with a core structure extracted from natural products and modified: application to phosphatase inhibitors and several biochemical findings.

Synthesis of a focused library is an important strategy to create novel modulators of specific classes of proteins. Compounds in a focused library are composed of a common core structure and different diversity structures. In this Account, we describe our design and synthesis of libraries focused on selective inhibitors of protein phosphatases (PPases). We considered that core structures having structural and electronic features similar to those of PPase substrates, phosphate esters, would be a reasonable choice. Therefore, we extracted core structures from natural products already identified as PPase inhibitors. Since many PPases share similar active-site structures, such phosphate-mimicking core structures should interact with many enzymes in the same family, and therefore the choice of diversity structures is pivotal both to increase the binding affinity and to achieve specificity for individual enzymes. Here we present case studies of application of focused libraries to obtain PPase inhibitors, covering the overall process from selection of core structures to identification and evaluation of candidates in the focused libraries. To synthesize a library focused on protein serine-threonine phosphatases (PPs), we chose norcantharidin as a core structure, because norcantharidin dicarboxylate shows a broad inhibition profile toward several PPs. From the resulting focused library, we identified a highly selective PP2B inhibitor, NCA-01. On the other hand, to find inhibitors of dual-specificity protein phosphatases (DSPs), we chose 3-acyltetronic acid extracted from natural product RK-682 as a core structure, because its structure resembles the transition state in the dephosphorylation reaction of DSPs. However, a highly selective inhibitor was not found in the resulting focused library. Furthermore, an inherent drawback of compounds having the highly acidic 3-acyltetronic acid as a core structure is very weak potency in cellulo, probably due to poor cell membrane permeability. Therefore, we next modified the core structure from acidic to neutral by transformation to the enamine derivative and constructed a second-generation focused library (RE derivatives). The resulting compounds showed dramatically improved cell membrane permeability and inhibitory selectivity and included VHR (vaccinia VH1-related)-selective RE12 and CDC25A/B (cell division cycle 25A/B)-selective RE44. These inhibitors act on target enzymes in cellulo and do not generate reactive oxygen species, which is a potential problem with quinoid-type inhibitors of CDC25s. The cellular activity of RE12 was further improved by replacement of the side chain to afford RE176, which showed more potent antiproliferative activity than RE12 against HeLa cells. The dramatic change of inhibitory selectivity obtained by core structure modification from 3-acyltetronic acid to its enamine derivative was associated with a change in the mode of action. Namely, RE derivatives were found to be noncompetitive inhibitors with respect to a small-molecular substrate of CDC25A/B, whereas RK-682 was a competitive inhibitor of VHR. We identified the binding site of RE derivatives on the CDC25A as a pocket adjacent to the active site; this appears to be a promising target site for development of further novel inhibitors of CDC25s.

Enhanced biotransformation of dehydroepiandrosterone to 3β,7α,15α-trihydroxy-5-androsten-17-one with Gibberella intermedia CA3-1 by natural oils addition.

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3β,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2%, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8% compared with that of the control at DHEA concentration of 8.0g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates.

Advances in Ultrasound Mediated Gene Therapy Using Microbubble Contrast Agents

Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.