High Salt Solution Research Articles

English

Extraction of non-degraded and contaminant-free DNA from field specimen requires collection under liquid nitrogen which is not readily available in resource constrained laboratories in low and middle income countries (LMICs). A method of extracting DNA from silica gel-preserved common bean (Proteus vulgaris L.) leaves is presented. The method, which does not involve the use of phenol, chloroform or isoamyl alcohol also obviates the need for low temperature incubation during the DNA extraction steps and the grinding of desiccated leaf tissue in liquid nitrogen. It relies on inactivating proteins using SDS and proteinase K along with precipitation of polysaccharides using a high salt solution (0.8 M NaCl). DNA is further purified by exploiting its insolubility in aqueous media. High quality pure DNA (mean concentration 2.84 ± 0.013 µg/g of dry leaf tissue) with mean DNA purity values of 2.1 ± 0.1 was extracted. The DNA was also found to be free of protein and polysaccharide contamination. This method enables DNA amplification using molecular markers routinely used in molecular biology laboratories like random amplified polymorphic (RAPD) markers, inter simple sequence repeat (ISSR) markers, sequence-characterized amplified region (SCAR) markers and simple sequence repeat (SSR) markers. The findings of this study show that it is possible to obtain high quality DNA from leaf tissue preserved in silica gel. The method used in this research will be invaluable to resource constrained laboratories in low and middle income countries (LMICs) that cannot afford to buy or access liquid nitrogen in order to extract high quality DNA and for research groups undertaking field surveys that require several days or weeks off station without laboratory freezers to maintain the integrity of the tissues which is crucial for obtaining high quality DNA. Key words: Random amplified polymorphic (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-characterized amplified region (SCAR), deoxyribonucleic acid (DNA), low and middle income countries (LMICs).

Alginate-graphene oxide hydrogels with enhanced ionic tunability and chemomechanical stability for light-directed 3D printing

Nanocomposite hydrogels that incorporate 2D carbon nanomaterials could enable augmented and responsive behaviors not observed with polymeric matrices alone. In particular, non-covalent interactions could facilitate enhanced mechanical performance that can be self-recovered with external stimuli. Here, we demonstrate alginate-graphene oxide (GO) hydrogels using a non-covalent, ionic crosslinking mechanism compatible with light-directed 3D printing. We show that alginate-GO hydrogels exhibit improved mechanical performance in shear, compression, and tension, including a two-fold increase in shear modulus, a three-fold decrease in inelastic deformation, and a nine-fold increase in fracture energy relative to alginate-only hydrogels. Moreover, alginate-GO hydrogels are stabilized by hydrogen bonding between nanosheets and remain intact after removal of ionic crosslinkers by chelation. As a consequence, the shear modulus of these nanocomposite hydrogels can be tuned by over 500-fold via external ion concentration. We demonstrate that alginate-GO can be stereolithographically printed into robust, freestanding and overhanging 3D structures. These designer material architectures exhibit outstanding stability and superoleophobicity in high salt solution, which can be used to repel and manipulate a variety of oils. Overall, such nanocomposite hydrogels with engineered non-covalent interactions could enable “smart” multiresponsive and multifunctional devices for aqueous and marine environments.

Alterations in vascular function by syncytiotrophoblast extracellular vesicles via lectin-like oxidized low-density lipoprotein receptor-1 in mouse uterine arteries.

Syncytiotrophoblast extracellular vesicles (STBEVs), released into the maternal circulation during pregnancy, have been shown to affect vascular function; however, the mechanism remains unknown. In rats, STBEVs were shown to reduce endothelium-mediated vasodilation via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a multi-ligand scavenger receptor that has been associated with vascular dysfunction. Recently, LOX-1 was shown to interact with the angiotensin II type 1 receptor (AT-1). We hypothesized that, in pregnant mice, STBEVs would impair vascular function via LOX-1 and would specifically affect angiotensin II responses. Uterine arteries from pregnant control (C57BL/6) and LOX-1 knockout (LOX-1KO) mice were isolated on gestational day (GD) 18.5. Endothelium-dependent (methylcholine (MCh); ± N(G)-Nitro-L-arginine methyl ester to assess nitric oxide (NO) contribution), and -independent (sodium nitroprusside) vasodilation, and vasoconstriction (angiotensin II; ± AT-1 [candesartan] or angiotensin II type 2 receptor (AT-2) [PD123.319] receptor antagonists; high potassium salt solution) responses were assessed using wire myography. AT-1 and AT-2 expression was analyzed using fluorescence microscopy. Human umbilical vein endothelial cells (HUVECs) were stimulated with STBEVs ± LOX-1 blocking antibody, and superoxide and peroxynitrite production were analyzed. Although MCh-induced vasodilation was decreased (P=0.0012), NO contribution to vasodilation was greater in LOX-1KO mice (P=0.0055). STBEVs delayed angiotensin II tachyphylaxis in arteries from control but not LOX-1KO mice (P<0.0001), while AT-1 and AT-2 expression was unchanged. STBEVs increased peroxynitrite production in HUVECs via LOX-1 (P=0.0091). In summary, LOX-1 deletion altered endothelium-mediated vasodilation, suggesting that LOX-1 contributes to vascular adaptations in pregnancy. STBEVs increased angiotensin II responsiveness and oxidative stress levels via LOX-1, suggesting that increased LOX-1 expression/activation or STBEVs could adversely affect vascular function and contribute to vascular complications of pregnancy.

Dynamic mass spectrometry probefor electrospray ionization mass spectrometry monitoring of bioreactors for therapeutic cell manufacturing.

Large-scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). Electrospray ionization mass spectrometry (ESI-MS) is a highly sensitive label-free method to detect and identify biomolecules, but requires extensive sample preparation before analysis, making online application of ESI-MS challenging. We present a microfabricated, monolithically integrated device capable of continuous sample collection, treatment, and direct infusion for ESI-MS detection of biomolecules in high-salt solutions. The dynamic mass spectrometry probe (DMSP) uses a microfluidic mass exchanger to rapidly condition samples for online MS analysis by removing interfering salts, while concurrently introducing MS signal enhancers to the sample for sensitive biomolecular detection. Exploiting this active conditioning capability increases MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low-concentration biomolecules is significantly improved, and multiple proteins can be detected from chemically complex samples. Thus, the DMSP has significant potential to serve as an enabling portion of a novel analytical tool for discovery and monitoring of CQAs relevant to therapeutic cell manufacturing.

Unique ion rectification in hypersaline environment: A high-performance and sustainable power generator system.

The development of membrane science plays a fundamental role in harvesting osmotic power, which is considered a future clean and renewable energy source. However, the existing designs of the membrane cannot handle the low conversion efficiency and power density. Theory has predicted that the Janus membrane with ionic diode-type current would be the most efficient material. Therefore, rectified ionic transportation in a hypersaline environment (the salt concentration is at least 0.5 M in sea) is highly desired, but it still remains a challenge. Here, we demonstrate a versatile strategy for creating a scale-up Janus three-dimensional (3D) porous membrane-based osmotic power generator system. Janus membranes with tunable surface charge density and porosity were obtained by compounding two kinds of ionomers. Under electric fields or chemical gradients, the Janus membrane has ionic current rectification properties and anion selectivities in a hypersaline environment. Experiments and theoretical calculation demonstrate that abundant surface charge and narrow pore size distribution benefit this unique ionic transport behavior in high salt solution. Thus, the output power density of this membrane-based generator reaches 2.66 W/m2 (mixing seawater and river water) and up to 5.10 W/m2 at a 500-fold salinity gradient (i.e., flowing salt lake into river water). Furthermore, a generator, built by connecting a series of membranes, could power a calculator for 120 hours without obvious current decline, proving the excellent physical and chemical stabilities. Therefore, we believe that this work advances the fundamental understanding of fluid transport and materials design as a paradigm for a high-performance energy conversion generator.

Purification, characterization and potential applications of a novel halotolerant metalloprotease from marine bacterium Vibrio sp. LA‐05

AbstractBACKGROUNDMany reported proteases showed low activity and stability under harsh operating conditions, e.g. high pH, salinity and surfactants, which hinders expansion of their applications in industry. Here, a novel halotolerant metalloprotease from marine bacterium Vibrio sp. LA‐05 endowed with superior properties was purified and characterized, and its industrial application also evaluated.RESULTSThe protease was active and stable at 25–40°C and pH 6.0–10.0. Moreover, it was remarkably stable in high salt solution, tolerant to denaturing agents, organic solvents, surfactants and bleaching agents, and quite compatible and stable with many commercial detergents. Its residual activities were above 22% even after co‐incubation with 20% NaCl for 480 h. Except Ariel and Tide, the residual activities were all above 55% after co‐incubation with five other liquid detergents for 240 h. The corresponding theoretical analyses show that it is a metalloprotease containing 1 mol of zinc ion per mole of protease, the mature active protease predominantly in α‐helix form consists of 321 amino acids, and the central zinc ion along with 11 other conserved and crucial amino acid residues forms the catalytic domain.CONCLUSIONThese excellent properties indicated that the protease has considerable potential as a bio‐additive in the application of detergent formulations. © 2018 Society of Chemical Industry

Facile Assembly of a Large‐Area BNNSs Film for Oxidation/Corrosion‐Resistant Coatings

AbstractCopper is one of the most widely used materials in industrial applications, such as electric power, electronics, and mechanical engineering fields. However, under high humidity, high temperature environment, acid, base, and salt solutions, the copper is vulnerable to destruction, which induces the oxidation and corrosion of copper surface. For circumventing these problems, a robust hydrophobic hexagonal boron nitride nanosheets (BNNSs) coating is facilely prepared through integrating the methods of liquid exfoliation, interface self‐assembly, and physics vapor deposition. The coating can prevent the copper to be oxidized at temperature as high as 500 °C. The corrosive droplets on the coating surface keep in Cassie's state and cannot penetrate into the valley of the topography, which effectively prevents the electrochemical corrosion at the interface between copper and the coating materials even under the DC voltage of 10 V. This finding sheds light on large‐scale constructing of oxidation/corrosion‐resistant coating for copper base materials toward practical industrial application.

Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples.

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS2 and pseudo-MS3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

Recovery of lithium from spent lithium-ion batteries using precipitation and electrodialysis techniques

At present, the projected demand for lithium calls for processing all viable resources especially secondary resources. This paper presents a promising approach for recovering lithium from low lithium high-salt solution which is usually produced in the spent LIBs recycling process. The solution was firstly purified, and then lithium was precipitated by phosphate, the effects of operating conditions on the Li3PO4 precipitation behaviors were evaluated. The results indicated that temperature is a more important factor than seed crystal or flocculant. After that, Li3PO4 was dissolved by acid as the anolyte, and electrodialysis with cation-exchange membranes was used to investigate the separation performance of Li and P. The results showed that Li and P was effectively separated by electrodialysis, and the P/Li mass ratio of the catholyte was reduced to 0.23 (6.5 times lower compared to a feed P/Li ratio of 1.48). Deep separation of lithium and phosphorus in the catholyte was achieved by raising pH to make the lithium precipitated with the permeated phosphorus. The lithium concentration of purified catholyte was 22.5 g/L and was used to prepare lithium carbonate. The Li2CO3 precipitation rate reached 88.3% at 80 °C under CO33−/Li+ molar ratio of 1.1:2. The final product of lithium carbonate was in accord with the standard specification (Li2CO3-0, GB/T 11075-2013). The results of this study provide an efficient and green cyclic process for recovering lithium from spent LIBs.

Technetium-99m-based simple and convenient radiolabeling of Escherichia coli for in vivo tracking of microorganisms

We herein report a convenient radiolabeling method capable of real-time in vivo imaging and biodistribution of bacteria in a living subject. Escherichia coli (E. coli), a model microorganism in this study, was incubated with technetium-99m (99mTc) tricarbonyl under physiological conditions provided radiolabeled bacterial cells. 99mTc-labeled bacterial cells were obtained in high yield (42%, n = 5) and purity (> 99%) within 30 min. In vitro radiolabeling study using cellular components revealed that 99mTc could be incorporated with cytosolic proteins and it was maintained stably in E. coli under several media including mouse serum, high salt solutions with varying pH. SPECT/CT images and organ biodistribution of 99mTc-labeled bacterial cells showed quantitative in vivo behavior and accumulation of E. coli in mice. The radiolabeling method demonstrated here will be a promising platform tool for in vivo tracking and biodistribution of infectious microorganism.

MicroRNA-132 controls water homeostasis through regulating MECP2-mediated vasopressin synthesis.

Fine-tuning of the body's water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.

Reprocessable porous poly(ionic liquid) membranes derived from main-chain polyimidazolium

A main-chain polyimidazolium poly(ionic liquid) was obtained via one-step Debus-Radziszewski (DB) reaction, which was followed by anion exchange to afford the hydrophobic analogue. After confirmation of their chemical structures by nuclear magnetic resonance (NMR) spectroscopy and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, their thermal behavior and solubility were fully characterized. Via interpolyelectrolyte complexation with poly(acrylic acid), a new main-chain poly(ionic liquid)-based porous membrane was constructed. Upon tuning molecular weight of poly(acrylic acid), the average pore size of membranes was found to vary from 100 to 500 nm. Eventually, this porous membrane was proven to be reprocessable through treatment with a high ionic salt solution, which offers a simple but efficient method to reuse porous polymer membranes.

Fabrication of three functionalized silica adsorbents: Impact of co-immobilization of imidazole, phenyl and long-chain alkyl groups on bisphenol A adsorption from high salt aqueous solutions

Abstract Based on the physicochemical properties of bisphenol A (BPA), chemical co-immobilizations of imidazole, phenyl and hexadecyl were done onto silica to obtain three functionalized silica adsorbents. Accordingly, the combinations of hexadecyl and imidazole, hexadecyl and phenyl, and phenyl and imidazole were named as Sil-(HDTMS-MPS)-VIm, Sil-(HDTMS-MPS)-S and Sil-MPS-(S-VIm), respectively. The composites were characterized by FTIR, TGA and SEM. The aim of this work was to explore how these typical functional groups affect the adsorption behavior of BPA. Experiments demonstrated that alkaline imidazole would play a key role for the BPA adsorption, and the synergy of imidazole and hexadecyl exhibited the highest adsorption affinities for BPA in the pH range of 4.0–9.0 due to the high Kow value of BPA. The maximum adsorption capacity of Sil-(HDTMS-MPS)-VIm for BPA obtained from Langmuir model was 89.6 mg/g at 293 K. Furthermore, the prepared three functionalized silicas retained their good adsorption capacities of BPA in high salt solutions, and the adsorption behaviors of BPA for these adsorbents were well described by the pseudo-second-order kinetic model and the Freundlich model. This work suggests that the synergy of hydrogen bond-forming alkaline functional group and hydrophobic long-chain alkyl group benefits BPA removal from aqueous solutions.

Gold nanoparticle aggregation: Colorimetric detection of the interactions between avidin and biotin

This paper reported a novel colorimetric assay strategy for avidin and biotin interactions based on terminal protection of the biotinylated single-stranded DNA and the surface plasmon resonance adsorption of gold nanoparticles (AuNPs). In this assay, it was firstly found that biotin-ssDNA specifically bound to the target protein avidin with strong affinity could be protected from hydrolysis by exonuclease I (Exo I). Furthermore, a colorimetric strategy was designed for the detection of avidin and biotin interactions. In the process, in the presence of avidin, the interaction of avidin and biotin protected the digestion of Exo I towards the biotin-ssDNA. The biotin-ssDNA with negatively charged would attach to the surface of AuNPs with positively charge in high salt solution through electrostatic interactions, which prevented AuNPs to aggregate. With the increased addition of avidin, the absorbance of AuNPs in 520 nm increased gradually and the color showed gradually wine red. By taking advantage of terminal protection, the developed strategy could offer high sensitivity for detecting small molecule-protein interactions. The results revealed that the developed strategy was highly sensitive for detecting avidin in the concentration ranging from 0.01 to 0.2 μg/mL with the detection limit of 4 × 10−3 μg/mL.The developed assay also showed highly specific, cost-efficient and convenient. Moreover, this strategy only required labeling the small molecule on a single-stranded DNA, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new colorimetric method could have potential to become a universal, sensitive, and selective platform for detection of small molecule−protein interactions.

Ionic Conductivity of Polyelectrolyte Hydrogels.

Polyelectrolytes have many important functions in both living organisms and man-made applications. One key property of polyelectrolytes is the ionic conductivity due to their porous networks that allow the transport of water and small molecular solutes. Among polyelectrolytes, zwitterionic polymers have attracted huge attention for applications that involve ion transport in a polyelectrolyte matrix; however, it is still unclear how the functional groups of zwitterionic polymer side chains affect their ion transport and swelling properties. In this study, zwitterionic poly(carboxybetaine acrylamide), poly(2-methacryloyloxyethyl phosphorylcholine), and poly(sulfobetaine methacrylate) hydrogels were synthesized and their ionic conductivity was studied and compared to cationic, anionic, and nonionic hydrogels. The change of the ionic conductivity of zwitterionic and nonionic hydrogels in different saline solutions was investigated in detail. Zwitterionic hydrogels showed much higher ionic conductivity than that of the widely used nonionic poly(ethylene glycol) methyl ether methacrylate hydrogel in all tested solutions. For both cationic and anionic hydrogels, the presence of mobile counterions led to high ionic conductivity in low salt solutions; however, the ionic conductivity of zwitterionic hydrogels surpassed that of cationic and ionic hydrogels in high salt solutions. Cationic and anionic hydrogels showed much higher water content than that of zwitterionic hydrogels in deionized water; however, the cationic hydrogels shrank significantly with increasing saline concentration. This work provides insight into the effects of polyelectrolyte side chains on ion transport. This can guide us in choosing better polyelectrolytes for a broad spectrum of applications, including bioelectronics, neural implants, battery, and so on.

DNA Extraction from silica gel preserved common bean (Phaseolus vulgaris L.) leaves

Extraction of high-quality DNA from field specimen requires collection under liquid nitrogen which is not readily available in resource constrained laboratories in low- and middle-income countries (LMICs). A method of extracting DNA from silica gel preserved common bean (Phaseolus vulgaris L.) leaves is presented. Our method which does not involve the use of phenol, chloroform or isoamyl alcohol also obviates the need for low temperature incubation during the DNA extraction steps and the grinding of desiccated leaf tissue in liquid nitrogen. It relies on inactivating proteins using SDS and proteinase K and precipitation of polysaccharides using a high salt solution. DNA is further purified by exploiting its insolubility in aqueous media. We were able to extract high quality pure DNA (mean concentration 2.98 ± 0.84 μg/g of leaf tissue) with mean A260/280 of 2.1 ± 0.1 and A260/230 of 2.4 ± 0.15. The DNA was also found to be amenable to amplification using molecular marker types routinely used in molecular biology laboratories like random amplified polymorphic (RAPD) markers, inter simple sequence repeat (ISSR) markers, sequence-characterized amplified region (SCAR) markers and simple sequence repeat (SSR) markers. Our findings show that it is possible to obtain high quality DNA from leaf tissue preserved in silica gel. Our method will be invaluable to resource constrained laboratories especially in LMICs that cannot afford to buy or access liquid nitrogen in order to extract high quality DNA and to research groups undertaking field surveys that require several days or weeks off station without laboratory freezers to maintain the integrity of the tissues which is crucial for obtaining high quality DNA.

Towards an Effective Management on the Teaching and Learning of Accounting in Simple and Rapid Colorimetric Bio-sensing of Alpha-fetoprotein Using Aptamer and Unmodified Gold Nanoparticles

A simple and rapid colorimetric method for determination of alpha-fetoprotein (AFP) has been developed here using specific aptamers and free gold nanoparticles (AuNPs). In the absence of AFP, the free aptamers prevent aggregation of AuNPs in a high salt environment, which cause the mixed solution remain red. Conversely, when there are existing AFP in colorimetric system, the aptamers is consumed to decrease, forming aptamer-AFP complex. AuNPs were aggregated due to losting the protection from aptamers in the high salt solution. The color of the solution turns to blue at this time. Subsequently, a color change of red-to-blue can be observed by the naked eye with the increasing of AFP. The linear response range for AFP was 10 to 2000 ng/ml and the detection limit was 5 ng/ml. This colorimetric biosenser does not require any modification, and its composition is cheap and easy to obtain. In addition, this proposed colorimetric method is also used in simulative hepatocellular carcinoma (HCC) serum samples and showed good feasibility, holding great potential for preliminary screening of serum of HCC in the future.

The Effect of Concentration on the Pressure of a Sodium Chloride Solution Inside Dialysis Tubing

High blood pressure causes significant medical problems around the world such as strokes, blindness and even death. Many people with high blood pressure are asked to lower their intake of salt in their diet which led us to wonder how blood pressure changes with more salt in the body and how the kidney diffuses water in response to salt concentration in the blood. Chemically, the salt outside of the blood vessels affects the pressure inside of the blood vessels due to the movement of water across the vessel walls. We used dialysis tubing containing a 0.9% salt-water solution to simulate the standard concentration of sodium chloride in the blood vessels. These simulated blood vessels were then placed in high and low salt solutions and a gas pressure sensor was used to monitor pressure changes in the dialysis tubing. The results showed that when the dialysis tubing was immersed in a solution that contained less salt, the pressure increased, and when it was immersed in a solution that contained more salt, the pressure decreased. Almost no change was recorded in the 0.9% solution. These findings suggest that salt concentration in the blood would impact the rate of osmosis, changing the pressure within the blood vessels.

The importance of an efficient extraction protocol for the use of fish muscle cholinesterases as biomarkers

Esterase activity found in muscle extracts is useful to evaluate harmful effects of anticholinesterase pollutants. Yet, most procedures applied in the extraction of fish muscle esterases in order to investigate their activity as a biomarker of environmental exposure comprise the homogenization of muscle tissue in low-salt solutions, followed by centrifugation to separate the supernatant as the enzyme source. However, acetylcholinesterase (AChE), the main target in these monitoring efforts, is a membrane-bound protein and is only present in muscle extracts if homogenization is carried out using chaotropic high-salt solutions. In this context, four extraction procedures using muscle tissue from six fish species were evaluated in order to establish a reproducible and reliable AChE assay for the determination of this biomarker. Results indicate that over 80% of AChE activity might be lacking in low-salt supernatants, and that the highest activities are obtained after extraction with solutions containing either 1molL−1 NaCl or 1molL−1 NaCl plus 3% Triton X-100, preserving almost 100% esterase activity over acetylthiocholine as substrate after centrifugation. Thus, many studies in the literature suffer from theoretical flaws and report erroneous AChE activity, since typical muscle AChE activity, the end-point biomarker for anticholinesterase pollutants, may have not been consistently assayed.

Metagenomic DNA Extraction of Natural Cellulose-Degrading Consortia

A natural cellulose-degrading consortium is able to efficiently convert various types of lignocellulose to organic products, which is considered a potential method for the production of biofuels and biochemicals from lignocellulose materials. Metagenomics is an efficient method for determining the complex microbiota structure and performing metabolic mechanism analysis. However, natural cellulose-degrading consortia not only have complicated microbiome compositions and metabolites but also have tight adsorption between cells and cellulose fibers, which could have a significant influence on metagenomic DNA extraction, leading to a reduction of the DNA concentration, integrity, and diversity. Here, we found that bacterial cells that bind to cellulose fibers were efficiently washed off by a high-salt solution treatment and that the wall-breaking ratios of the biological enzyme and mechanical homogenization method were significantly higher than those of grinding and the freeze-thaw method. These results helped to establish a new method for extraction of the consortia metagenome that efficiently decreased absorption between cells and cellulose fibers, reduced the adverse impact of metabolites on DNA extraction, and improved the quality of DNA extraction.