Presence Of High Salt Concentrations Research Articles

Wax Ester and Triacylglycerol Production in Acinetobacter baumannii: Role in Osmostress Protection, Reactive Oxygen Species, and Antibiotic Sensitivity.

Wax esters (WEs) are neutral lipids that are produced by many different bacteria as potential carbon and energy storage compounds. Comparatively little is known about the role of WE in pathogenic bacteria. The opportunistic pathogen Acinetobacter baumannii is a major cause of hospital-acquired infections worldwide. Salt and desiccation resistance foster A. baumannii infections such as urinary tract infections and allow for reinfection when bacteria are taken up from dry surfaces in the hospital environment. Here we report on WE and triacylglycerol (TAG) production in A. baumannii as a response to nitrogen limitation and high salt stress. Fatty acids and fatty alcohols with chain lengths of C16 and C18 were identified as the most prominent WE constituents. We identified the terminal key enzyme of WE biosynthesis, the bifunctional wax ester synthase/acylCoA:diacylglycerol acyltransferase (WS/DGAT) encoded by the wax/dgat gene, and demonstrated that transcription of wax/dgat and production of WS/DGAT are independent of the nitrogen concentration. A Δwax/dgat mutant was impaired in growth in the presence of high salt concentration and was more sensitive to imipenem and reactive oxygen species.

Assessing Listeria monocytogenes growth during Spanish-style green table olive fermentation

Table olives are one of the most important fermented vegetables in the Mediterranean countries. Although this fermented vegetable has a long record of microbial safety, various studies have shown the presence of Listeria monocytogenes during table olive processing. This study focused on evaluating the behaviour of a cocktail of L. monocytogenes strains during fermentation of Spanish-style green Manzanilla olives. For this purpose, a challenge test consisting of artificial inoculation of olive fermentation with high levels of the food-borne pathogen (7.0 log10 CFU/ml) was performed. The Fermentation was carried out at the laboratory scale in sealed glass bottles (n = 168) under controlled conditions to avoid the initiation of lactic acid fermentation intentionally. This point was confirmed by the evolution of pH, which ranged from an initial 9.0 to final 6.0 units (decrease kinetic constant of −0.0016 h−1), as well as by the behaviour of lactic acid and mesophilic bacteria during the study (55 days). The fate of L. monocytogenes during fermentation was described using mathematical models, including the two-term Gompertz model. During this time, the food-borne pathogen showed an initial growth phase which lasted until 250 h, reaching a maximum population level close to 9.0 log10 CFU/ml, even in the presence of high salt concentrations (>6.0% NaCl). Then, a rapid decline phase was observed but continuously detecting a residual tail of around 2 log10 CFU/ml. Data obtained in this work shows the importance of the control of olive fermentation, especially during the first weeks when pH is alkaline-neutral and environmental conditions could favour L. monocytogenes growth.

Investigation into the distinct structural features of the internal fusion loop within the LASV fusion domain.

Infection with Lassa virus (LASV), an Old-World arenavirus that is endemic to West Africa, causes Lassa fever – a lethal hemorrhagic fever. Delivery of LASV's genetic material into the host cell is an integral component of its lifecycle. This is accomplished via membrane fusion – a process widely accepted to be initiated by a hydrophobic sequence known as the fusion domain (FD).The LASV FD (G260–N295) consists of two structurally distinct regions: an N-terminal fusion peptide (FP: G260–P275) and an internal fusion loop (FL: G276–N295). Here, we identified a conserved disulfide bond (C279 and C292) and salt bridge (R282 and E289) within the FL that are pertinent to fusion. Through a FRET-based fusion assay, we witnessed a significant reduction in fusion when the disulfide bond was broken. Additionally, we found that fusion is reduced in the presence of high salt concentrations. When single point mutations were made such that each residue was the same charge (i.e., R282E and E289R) to ablate the salt bridge, we observed reduced fusion, which was partially returned when a double mutation was introduced (R282E/E289R). This was corroborated by ITC where the binding affinity to the lipid bilayer was decreased in comparison to wildtype for each salt bridge mutant and without the disulfide bond. While it could be suggested aggregation is causing this loss in function, CD spectroscopy and gel filtration revealed that both the global secondary structure and oligomeric state, respectively, of the FD remains unchanged, even after mutagenesis or disruption of the disulfide bond. In conclusion, our findings suggest that both the salt bridge and disulfide bond within the FL are critical for the proper association of the complete FD with the membrane to elicit efficient fusion.

Biotechnological potential of microorganisms isolated from the salar del hombre muerto, Argentina.

Bacterial strains were isolated from soil and aqueous solution samples from the Salar del Hombre Muerto, Argentina. A total of 141 strains were characterized and the tolerance to sodium chloride was evaluated. We performed a screening to search for molecules of biotechnological interest: carotenoids (11%), emulsifiers (95%), and exopolysaccharides (6%), and to assess the production of enzymes, including proteolytic (39%), lipolytic (26%), hemolytic (50%), and catalase activities (99%); 25 bacterial strains were selected for further studies. Some of them produced biofilms, but only Bacillus sp. HA120b showed that ability in all the conditions assayed. Although 21 strains were able to form emulsions, the emulsifying index Kocuria sp. M9 and Bacillus sp. V3a cultures were greater than 50% and, emulsions were more stable when the bacteria grew in higher salt concentrations. Only pigmented Kocuria sp. M9 showed lipolytic activity on olive oil medium and was able to produce biofilms when cultured without and with 4 M of NaCl. Yellow pigments, lipase activity, and biosurfactant production were observed for Micrococcus sp. SX120. Summarizing, we found that the selected bacteria produced highly interesting molecules with diverse industrial applications and, many of them are functional in the presence of high salt concentrations.

Deciphering Structure-Function Relationship Unveils Salt-Resistant Mode of Action of a Potent MRSA-Inhibiting Antimicrobial Peptide, RR14.

Multidrug-resistant (MDR) bacteria lead to considerable morbidity and mortality, threatening public health worldwide. In particular, infections of methicillin-resistant Staphylococcus aureus (MRSA) in hospital and community settings are becoming a serious health problem. Antimicrobial peptides (AMPs) are considered novel therapeutic targets against MDR bacteria. However, salt sensitivity reduces the bactericidal potency of AMPs, posing a major obstacle for their development as antibiotics. Thus, the design and development of salt-insensitive peptides with potent antibacterial activity is imperative. Here, we employed biochemical and biophysical examinations coupled with molecular modeling to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. The secondary structure of RR14 was characterized as an apparent α-helix, a structure that confers strong membrane-permeabilizing ability targeting bacterial-mimetic membranes. Additionally, the bioactive structure of RR14 was determined in complex with dodecylphosphocholine (DPC) micelles, where it possesses a central α-helical segment comprising residues R4 to K13 (R4-K13). RR14 was observed to orient itself into the DPC micelle with its N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of positively charged residues of RR14 on its amphipathic helical conformation has great potential to render its strong salt resistance ability. Our study explored the structure-function relationship of RR14, explaining its possible mode of action against MRSA and other microbes. The insights obtained are of great applicability for the development of new antibacterial agents. IMPORTANCE Many antimicrobial peptides have been observed to become inactive in the presence of high salt concentrations. To further develop new and novel AMPs with potent bactericidal activity and salt insensitivity, understanding the structural basis for salt resistance is important. Here, we employed biochemical and biophysical examinations to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. RR14 was observed to orient itself into DPC micelles with the N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of cationic residues of RR14 on its amphipathic helical conformation renders its strong salt resistance ability. The insights obtained are of great applicability for developing new antibacterial agents.

Salt- and pH-Dependent Thermal Stability of Photocomplexes from Extremophilic Bacteriochlorophyll b-Containing Halorhodospira Species.

Halorhodospira (Hlr.) species are the most halophilic and alkaliphilic of all purple bacteria. Hlr. halochloris exhibits the lowest LH1 Qy transition energy among phototrophic organisms and is the only known triply extremophilic anoxygenic phototroph, displaying a thermophilic, halophilic, and alkaliphilic phenotype. Recently, we reported that electrostatic charges are responsible for the unusual spectroscopic properties of the Hlr. halochloris LH1 complex. In the present work, we examined the effects of salt and pH on the spectroscopic properties and thermal stability of LH1-RCs from Hlr. halochloris compared with its mesophilic counterpart, Hlr. abdelmalekii. Experiments in which the photocomplexes were subjected to different levels of salt or variable pH revealed that the thermal stability of LH1-RCs from both species was largely retained in the presence of high salt concentrations and/or at alkaline pH but was markedly reduced by lowering the salt concentration and/or pH. Based on the amino acid sequences of LH1 polypeptides and their composition of acidic/basic residues and the Hofmeister series for cation/anion species, we discuss the importance of electrostatic charge in stabilizing the Hlr. halochloris LH1-RC complex to allow it to perform photosynthesis in its warm, hypersaline, and alkaline habitat.

Electrochemical evaluation of the grafting density of self-assembled monolayers of polyethylene glycol of different chain lengths formed by the grafting to approach under conditions close to the cloud point

• The grafting density of the SAMs of polymer films is a useful parameter that is very difficult to obtain. • SAMs of polyethylene glycol built from good solvents like water, maintain their solution conformation upon adsorption. • The presence of high amount of water in the polyethylene glycol SAMs is responsible of the high charge density obtained in the reductive desorption process. • The SAMs of polyethylene glycol formed under cloud point conditions exclude most of the water giving more realistic grafting density values. • The proposed methodology to get grafting densities of polyethylene glycol SAMs can be easily extended to other polymer SAMs. Grafting densities of polymers on gold surfaces are important parameters that inform about the chain conformation adopted by the chains, either mushroom or brush conformation. Most of the literature reports on this topic are based on ellipsometry measurements with other few results obtained from surface plasmon resonance or quartz crystal microbalance. In this work, we report the use of cyclic voltammetry of the reductive desorption (RD) process of mercapto-polyethylene glycol self-assembled monolayers (EGn-SAMs) to get the grafting densities of these polymers with different chain lengths (c.a. EG136, EG45 and EG18). When the EGn-SAMs are formed from aqueous solutions where the polymer chains are highly hydrated, an excess of charge density is obtained that apart from the capacitive contribution found in these processes, include an extra charge due to the hydrogen evolution reaction (HER) produced by the high ratio of water contained in the film that reduces concomitantly with the SAM RD process. However, preparing the EGn-SAM in the presence of high salt concentrations that lowers the water content within the chains, that is, using the cloud point strategy, very realistic grafting density values are obtained. The RD profiles of the EGn-SAMs completely change from a broad peak with high charge density to a structured peak that allows us the determination of grafting density values. Capacitance curves and the behavior of the electrochemical process of the Fe(CN) 6 3-/4- redox pair in the presence of the films are also in agreement with the CV results. The grafting density values obtained by this methodology are higher than those reported for these SAMs by using other techniques but agree with the footprints expected for these chains when organized in a brush conformation. Moreover, they also are close to the values obtained by thermogravimetric analysis of the same films built on gold nanoparticles that behave as planar surfaces.

Isolation and Characterization of Halophilic Kocuria salsicia Strains from Cheese Brine.

Kocuria salsicia can survive in extreme environments and cause infections, including catheter-related bacteremia, in humans. Here, we investigated and evaluated the characteristics of nine K. salsicia strains (KS1–KS9) isolated from cheese brine from a farmstead cheese-manufacturing plant in Korea from June to December, 2020. Staphylococcus aureus American Type Culture Collection (ATCC) 29213 was used as a positive control in the growth curve analysis and biofilm-formation assays. All K. salsicia isolates showed growth at 15% salt concentration and temperatures of 15°C, 25°C, 30°C, 37°C, and 42°C. KS6 and KS8 showed growth at 5°C, suggesting that they are potential psychrotrophs. In the biofilm-formation analysis via crystal violet staining, KS6 exhibited the highest biofilm-forming ability at various temperatures and media [phosphate buffered saline, nutrient broth (NB), and NB containing 15% sodium chloride]. At 25°C and 30°C, KS3, KS6, and KS8 showed higher biofilm-forming ability than S. aureus ATCC 29213. The antimicrobial resistance of the isolates was evaluated using the VITEK® 2 system; most isolates were resistant to marbofloxacin and nitrofurantoin (both 9/9, 100%), followed by enrofloxacin (7/9, 77.8%). Five of the nine isolates (5/9, 55.6%) showed multidrug resistance. Our study reports the abilities of K. salsicia to grow in the presence of high salt concentrations and at relatively low temperatures, along with its multidrug resistance and tendency to form biofilms.

Isolation and characterization of halophilic strains from beef jerky

Beef jerky is a typically Brazilian food product that consists of adding salt (sodium chloride, NaCl) to bovine meat. Salt is used as main preservative when preparing jerked meat, however, certain species of microorganisms, known as halophiles, optimally develop in the presence of high salt concentrations, thus salty food may contain halophiles and cause undesirable alterations in the products. In this context, this study aims to isolate, characterize, and assess the growth parameters of halophilic strains isolated from beef jerky. Two halophilic strains were isolated: C11 strain, with cocci-shaped morphological features from white cream color, and V22 strain, with red pleomorphism. Both the lineages were gran-negative, catalase, and oxidase positive, with optimum growth time after 15 incubation days at 37 °C. The strains were neutrophilic, growing only at pH 7.0 and subjected to a 2x3 factorial experimental design to assess and optimize the growth of strains at different concentrations of NaCl, MgCl2, and yeast extract. We found that for both the optimal growth conditions, the studied variables ranged from 25 to 35 g L-1 of MgCl2, 5 g L-1 of yeast extract, but for NaCl, the C11 strain did not grow in medium containing 300 g L-1 (optimally at 200), while the V22 showed no growth in medium containing 100 g L-1 (optimally at 300). Therefore, the features of the isolates suggest that they belong to the Halobacteriaceae family.

Biosurfactant production using Egyptian oil fields indigenous bacteria for microbial enhanced oil recovery

Biosurfactant production is one of the most efficient mechanisms in microbial enhanced oil recovery (MEOR) processes. This work investigates the production of biosurfactants by indigenous bacteria isolated from Egyptian oil fields, and how to optimize these produced biosurfactants for MEOR. 59 Egyptian oil reservoirs were screened to evaluate the potential applicability of MEOR processes, based on their rock and fluid properties. Results showed that 8 reservoirs from the Gulf of Suez and 3 reservoirs from the Western Desert had the potential to MEOR. Combined analysis of morphological, and biochemical characterization was performed on the 11 bacterial strains isolated from different crude oil samples collected from the reservoirs that have the potential to MEOR process to identify their types. Bacillus spp, a bacilli species that can produce biosurfactants, was selected for further studies. To optimize the surface activity of the produced biosurfactant, ten different reported nutrient media, and a new proposed nutrient media were examined. Bacillus spp has shown the ability to produce a very active biosurfactant that reduced the surface tension of water from 71.8 ± 1.9 mN/m to 25.7 ± 1.2 mN/m, and the interfacial tension of water against kerosene from 48.4 ± 2.1 mN/m to 0.38 ± 0.07 mN/m at Critical Micelle Concentration (CMC) of 0.04 ± 0.01 g/l, in a medium supplemented by the new proposed nutrient medium H. The growth rate of Bacillus spp was studied, and it was found it reached its maximum (OD600nm 2.59 ± 0.16) after 24 h of incubation. Biosurfactant production has no significant change in its surface activity over a wide range of temperature range up to 120 °C, which means the studied species Bacillus spp is a thermophilic bacterium. Bacillus spp grew well in the presence of high salt concentration up to 20% (w/v) NaCl, the optimal surface activity was obtained in the range of 0–2% (w/v) NaCl, and at pH 7. The emulsification activity of the produced biosurfactant was examined, and it reached the maximum (69.6 ± 1.5%) against kerosene at temperature 25 °C, Salinity 0% (w/v) NaCl (distilled water), and pH 7. The produced biosurfactant was purified and extracted by acid precipitation method, and the biosurfactant yield of the purified compound was found to be 2.8 ± 0.3 g/l. Finally, the core-flooding experiments were conducted to investigate the effect of produced biosurfactants by Bacillus subtilis in oil recovery. The obtained results reveal the potential of Bacillus spp to grow in the new proposed medium H and produce effective and efficient biosurfactants that enhanced oil recovery by 25.19–39.35% of additional oil over the water flooding residual oil saturation in the studied cores and retain more than 60% of its surface activity under harsh conditions and that are relevant to Microbial Enhanced Oil Recovery, MEOR.

Effectiveness of dual-detergent strategy using Triton X-100 in membrane protein purification

Membrane solubilization by detergents is a critical step for successful membrane protein purification. Alkyl maltoside detergents such as DDM and DM are very expensive and are commonly used to produce most of the high-quality proteins in stable and functional form. Recently, dual-detergent strategy using inexpensive detergents for membrane solubilization step has been shown to be highly effective in purifying different classes of membrane proteins in a cost-effective manner. In this work, we have monitored the effectiveness of ‘dual-detergent strategy’ towards successful purification of the isolated voltage sensing domain (VSD) of KvAP and the inward rectifying K+ channel, KirBac1.1. We demonstrate that the inexpensive detergent Triton X-100 extracts the activated conformation of the KvAP-VSD well without compromising the structural integrity of the sensor, and also retains its proper structural dynamics. Importantly, the cost associated with solubilizing the KvAP sensor can be reduced by ∼2000 fold. To the best of our knowledge, our results constitute the first report characterizing the purification of KvAP voltage sensor using an inexpensive detergent. However, the dual-detergent strategy using Triton X-100 for membrane solubilization is not effective for the purification of inward rectifying K+ channel, KirBac1.1 even in presence of high salt concentration during solubilization. We propose that the dual-detergent strategy will be useful for extracting stable and functional proteins that are both DDM- and DM-extractable, but will be ineffective if the protein is only DM-extractable. The relevance of the effectiveness of dual-detergent strategy with respect to the hydrophobic thickness of proteins is discussed.

Distribution of Denitrification among Haloarchaea: A Comprehensive Study.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

Elabela Protects Spontaneously Hypertensive Rats From Hypertension and Cardiorenal Dysfunctions Exacerbated by Dietary High-Salt Intake.

Objectives: Arterial hypertension, when exacerbated by excessive dietary salt intake, worsens the morbidity and mortality rates associated with cardiovascular and renal diseases. Stimulation of the apelinergic system appears to protect against several circulatory system diseases, but it remains unknown if such beneficial effects are conserved in severe hypertension. Therefore, we aimed at determining whether continuous infusion of apelinergic ligands (i.e., Apelin-13 and Elabela) exerted cardiorenal protective effects in spontaneously hypertensive (SHR) rats receiving high-salt diet.Methods: A combination of echocardiography, binding assay, histology, and biochemical approaches were used to investigate the cardiovascular and renal effects of Apelin-13 or Elabela infusion over 6 weeks in SHR fed with normal-salt or high-salt chow.Results: High-salt intake upregulated the cardiac and renal expression of APJ receptor in SHR. Importantly, Elabela was more effective than Apelin-13 in reducing high blood pressure, cardiovascular and renal dysfunctions, fibrosis and hypertrophy in high-salt fed SHR. Unlike Apelin-13, the beneficial effects of Elabela were associated with a counter-regulatory role of the ACE/ACE2/neprilysin axis of the renin-angiotensin-aldosterone system (RAAS) in heart and kidneys of salt-loaded SHR. Interestingly, Elabela also displayed higher affinity for APJ in the presence of high salt concentration and better resistance to RAAS enzymes known to cleave Apelin-13.Conclusion: These findings highlight the protective action of the apelinergic system against salt-induced severe hypertension and cardiorenal failure. As compared with Apelin-13, Elabela displays superior pharmacodynamic and pharmacokinetic properties that warrant further investigation of its therapeutic use in cardiovascular and kidney diseases.

Analysis of drugs and pesticides for forensic purposes using noble metal-modified silica monolith as SALDI-MS substrate

In this study, a 3D SiO2 (M−SiO2) monolith was modified with four noble metals, namely, Au, Ag, Pt, and Pd nanoparticles (NPs), and was explored as a surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) substrate for forensic detection of drugs and pesticides. The loading of the NPs, reducing agent concentration, and reaction time were optimized using UV–vis spectroscopy. The prepared M−SiO2 monoliths were extensively characterized using different analytical techniques. The surface area of the pristine SiO2 monolith increased slightly upon introduction of the NPs, while the morphology of the SiO2 surface did not change after deposition. The effect of the NP loading and deposition method on SALDI-MS analysis of drugs and pesticides was investigated, and the results proved that layer-by-layer deposition provided the best SALDI performance. Among the prepared M−SiO2 monoliths, Au-SiO2 exhibited superior SALDI performance in terms of the signal-to-noise (S/N) ratio. Furthermore, the effect of NaCl salt was scrutinized using Au-SiO2 monolith, which enabled sensitive detection of the drugs and pesticides even in the presence of high salt concentrations. Finally, Au-SiO2 substrate was applied to the analyses of non-biological and biological samples. Specifically, drug-spiked carbonated malt drink and pesticide-spiked blood serum were analyzed using minute amounts of sample. The prepared substrate offered sensitive detection of the analytes with a limit of detection (LOD) of 100–1000 ng/mL and 1–100 ng/mL with a relative standard deviation (RSD) of 7.21–22.22% and 2.68–26.29% for the drugs and pesticides, respectively, indicating good spot-to-spot repeatability. Thus, the proposed method can be used as an efficient approach for forensic analysis of trace evidence owing to its simplicity and minimum sample volume.

Dynamic Surface Tension of Surfactants in the Presence of High Salt Concentrations

We study the influence of high NaCl concentrations on the equilibrium and dynamic surface tensions of ionic (CTAB) and nonionic (Tween 80) surfactant solutions. Equilibrium surface tension measurements show that NaCl significantly reduces the critical micellar concentration (CMC) of CTAB but has no effect on the CMC of Tween 80. Dynamic surface tension measurements allow comparing the surface tension as a function of time for pure surfactant solutions and in the presence of NaCl. For the ionic surfactant, the dynamics agree with the usual diffusion-limited interfacial adsorption kinetics; however, the kinetics become orders of magnitude slower when NaCl is present. Sum-frequency generation spectroscopy experiments and the equilibrium adsorption measurements show that the presence of NaCl in CTAB solution leads to the formation of ion pairs at the surface, thereby neutralizing the charge of the head group of CTAB. This change, however, is not able to account for the slowing down of adsorption dynamics; we find that it is rather the decreases in the monomer concentration (CMC) in the presence of salt which has the major influence. For the nonionic surfactant, the kinetics of interfacial tension is seen to be already very slow, and the addition of salt does not influence it further. This also correlates very well to the very low CMC of Tween 80.

Influence of ionic strength and thermal pretreatment on the freeze-thaw stability of Pickering emulsion gels

Salt addition and thermal pretreatment were used to improve the freeze-thaw stability of Pickering emulsion gels (PEGs) stabilized by compound proteins. Thermal pretreatment with the presence of salt could promote the formation of gel-like structure and alter the interactions between the emulsion droplets of PEGs, sequentially increase the resistance of the PEGs to water separation, creaming, and oiling-off during freeze-thaw cycles (freeze at −20 °C for 22 h and thawing at 37 °C for 2 h), especially at higher salt levels (200 and 500 mM). Microstructures indicated that the presence of high salt concentration and heat pretreatment could help to maintain the gel-like structures of PEGs during freeze-thaw cycles. Overall, our results showed that novel viscoelastic food materials with good freeze-thaw stability can be produced by controlling the electrostatic interactions between the emulsion droplets and the gelation of emulsion gels. These materials may be useful for application in frozen food products.

Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR).

Chromatin immunoprecipitation (ChIP) is widely used to measure protein-DNA interactions. This protocol outlines a ChIP method used to identify the association of a protein or protein modification (such as a specific histone modification-methylation, acetylation, etc.) of interest with a specific DNA sequence in a target gene in fetal mouse brains on gestational day (GD) 17. Briefly, DNA and proteins are cross-linked (via formaldehyde), and chromatin is sonicated into fragments between 200 and 1000 base pair (bp) long, with an average length of 500bp. The DNA-protein complexes are captured using antibodies directed toward the protein or protein modification of interest. These immunoprecipitated complexes are retrieved using agarose beads. The DNA-protein cross-links are reversed (via heat and via presence of high salt concentrations), and the ChIP DNA is purified and measured via a quantitative polymerase chain (qPCR) reaction. The results show the association of histone modifications at unknown sites of specific genes of interest, indicating which epigenetic modifications of specific genes may be responsible for the outcome of interest.

A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization

Due to the need for calf rennet alternatives, many attempts have been made to find new proteases. A novel cysteine protease with milk-clotting activity was purified from Ficus johannis by cation exchange chromatography. The protease was stable in various pH (3.0–10.5) with the optimum at 6.5 and showed its maximum activity at 60 °C. The Km and Vmax values of the enzyme were obtained to be 0.604 mg/ml and 0.0273 μmol Tyr/min, respectively. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and β-casein. The enzyme was almost completely active in the presence of high salt concentrations. Besides, it had high stability against autodigestion. The content of free amino acids was determined by HPLC, where leucine, lysine, valine, γ-aminobutyric acid and tyrosine were the most abundant amino acids. The cheese manufactured by using the purified protease showed similar textural properties and physico-chemical compositions to cheese produced using commercial rennet. Considering the special characteristics, including high milk-clotting activity, considerable stability over wide ranges of pH and temperature, resistance towards solvents, salts, and surfactants, the new protease might be the promising candidate for the dairy industry as well as other food and biotechnological industries.

Levansucrase from Halomonas smyrnensis AAD6T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37°C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

Chemotherapeutic Drug-Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA.

Plasmid DNA (pDNA) is an attractive therapeutic biomolecule in several diseases including cancer, AIDS, cystic fibrosis, Parkinson's disease, and Alzheimer's disease. Increasing demand for plasmid DNA as a therapeutic biomolecule for transgene expression or vaccine applications necessitate novel approaches to bioprocessing. The synthesis, characterization and evaluation of aminoglycoside-derived hydrogel microbeads (Amikabeads) for pDNA binding is described previously. Here, the generation and evaluation of novel chemotherapeutic drug-conjugated microbeads for application in pDNA binding and recovery is described. Chemotherapeutic drug-conjugated Amikabeads demonstrate higher binding of methylated pDNA compared to unmethylated pDNA in presence of high salt concentrations. Desorption of plasmids from drug-conjugated microbeads is facilitated by the use of organic modifiers. The observed differences in binding methylated versus unmethylated DNA can make drug-conjugated microbeads useful in diagnostic as well as therapeutic applications. These results demonstrate that anti-cancer drugs represent a diverse set of ligands that may be exploited for molecular engineering of novel DNA binding materials for applications in delivery, diagnostics, and biomanufacturing.