Insights into the mechanism of high lipid–detergent crystallization of membrane proteins

Obtaining well diffracting crystals of membrane proteins is often challenging, but chances can be improved by crystallizing them in lipidic conditions that mimic their natural membrane environments. One approach is the high lipid–detergent (HiLiDe) method, which works by mixing the target protein with high concentrations of lipid and detergent prior to crystallization. Although this approach is convenient and flexible, understanding the effects of systematically varying lipid/detergent ratios and a characterization of the lipid phases that form during crystallization would be useful. Here, a HiLiDe phase diagram is reported for the model membrane protein MhsT, which tracks the precipitation and crystallization zones as a function of lipid and detergent concentrations, and is augmented with data on crystal sizes and diffraction properties. Additionally, the crystallization of SERCA1a solubilized directly with native lipids is characterized as a function of detergent concentration. Finally, HiLiDe crystallization drops are analysed with transmission electron microscopy, which among other features reveals liposomes, stacked lamellae that may represent crystal precursors, and mature crystals with clearly discernible packing arrangements. The results emphasize the significance of optimizing lipid/detergent ratios over broad ranges and provide insights into the mechanism of HiLiDe crystallization.

A novel approach for the purification of aggregation prone proteins

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

The effect of N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate addition on washing properties of liquid laundry detergents

The use of colorimetric analysis based on a CIELab system to determine detergency of commercial liquid laundrydetergents before and after modification with SB3C14 sulfobetaine (N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) is presented. The EMPA 101 standard cotton fabric soiled with carbon black and olive oil was usedin washing tests under the following conditions: temperature 40°C, rotational speed 200 rpm, 30 minute washing cycle,water hardness 5.35 mval/l, the concentration of liquid laundry detergents 1.25–50 g/l. The physicochemical analysis ofcolour, form, odour, pH, density, viscosity and content of anionic surfactants showed compliance with the manufacturers’declarations. The studies demonstrated that with increasing laundry detergent concentration a gradual increase indetergency occurred. At the highest tested concentrations of 50 g/L, detergency of 18.1% and 22.2% for cheaper L1 andmore expensive L2 products was achieved, respectively. Modification with the 5% addition of the zwitterionicsulfobetaine SB3C14 led to an improvement of the washing properties by 4.7% on average. At a concentration of 50 g/l,the modified L1 and L2 solutions demonstrated the highest detergency equal to 22.8% and 35.3%, respectively. Thisproves the existence of synergistic effect by the interaction of all ingredients in the solutions with higher concentrations.Microscopic analysis of EMPA 101 fabrics before and after washing processes showed no serious damage to the fibres,only the presence of slight fraying of individual ones. The results suggest that the SB3C14 sulfobetaine can besuccessfully used in liquid laundry detergents due to its very favourable surface properties

The effect of N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate addition on washing properties of liquid laundry detergents

The use of colorimetric analysis based on a CIELab system to determine detergency of commercial liquid laundrydetergents before and after modification with SB3C14 sulfobetaine (N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) is presented. The EMPA 101 standard cotton fabric soiled with carbon black and olive oil was usedin washing tests under the following conditions: temperature 40°C, rotational speed 200 rpm, 30 minute washing cycle,water hardness 5.35 mval/l, the concentration of liquid laundry detergents 1.25–50 g/l. The physicochemical analysis ofcolour, form, odour, pH, density, viscosity and content of anionic surfactants showed compliance with the manufacturers’declarations. The studies demonstrated that with increasing laundry detergent concentration a gradual increase indetergency occurred. At the highest tested concentrations of 50 g/L, detergency of 18.1% and 22.2% for cheaper L1 andmore expensive L2 products was achieved, respectively. Modification with the 5% addition of the zwitterionicsulfobetaine SB3C14 led to an improvement of the washing properties by 4.7% on average. At a concentration of 50 g/l,the modified L1 and L2 solutions demonstrated the highest detergency equal to 22.8% and 35.3%, respectively. Thisproves the existence of synergistic effect by the interaction of all ingredients in the solutions with higher concentrations.Microscopic analysis of EMPA 101 fabrics before and after washing processes showed no serious damage to the fibres,only the presence of slight fraying of individual ones. The results suggest that the SB3C14 sulfobetaine can besuccessfully used in liquid laundry detergents due to its very favourable surface properties

Analysis of Detergent Waste Absorption Using Water Spinach (Ipomoea aquatica)

This study aims to determine the absorption of water detergent waste using water spinach (ipomoea aquatica). This experimental study used a randomized block design. Water samples were planted with water plants according to the research design. Detergent concentrations were observed using the MBAS (Methylene Blue Active Substance) method. The results showed that water spinach was able to reduce the detergent concentration and improve the concentration of water quality parameters (DO, BOD, and COD). After the treatment of water kangkong, the ability to absorb detergent was seen with a decrease in detergent levels until day 28 of 54.17%. The highest detergent absorption on days 7 and 14.

Production and Characterisation Partial Lipase of Bacillus halodurans Cml Mutant for Biodetergen

This study aims to produce lipase of the Bacillus halodurans CM1 mutant and its assess partial characteristics, performed in Bora and Bora modified medium. The purification was conducted using Ultrafiltration (UF), ammonium sulfate (AS) and polyethylene glycol (PEG). Results revealed that the highest purity lipase of B. halodurans CM1 mutant was 1.49-fold from the UF-AS-dyalisis, with a molecular weight of 35.7-37.4 KDa. The optimum condition of lipase enzyme was achieved at pH 7 and temperature 50 °C, relatively stable at pH 7-8 and temperature 30-70 °C. Mg2+, Ca2+, Zn2+, Mn2+, Fe2+ and K+ ions of concentrations, 1 mM to 10 mM increased enzyme lipase activity. The Km value was 0.23 mg/mL and Vmax 4.07 U/mL. Lipase was stable with the addition of a detergent concentration of 1-2% (69.60-57.10%), and with the washing test, the enzyme capable of hydrolyzing oil on cloth is 8.40%.

Ex-vivo recellularisation and stem cell differentiation of a decellularised rat dental pulp matrix

Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

A Novel Way of Addressing Eminent Food Insecurity Challenges Caused by American Fall Army Worms Spodoptera Frugiperda)

American Fall Army Worms Spodoptera frugiperda (J.E. Smith) has been a great nuisance to farmers in West, South, East Africa and Asia imparting significant losses ranging from 80 to 100% to maize crops while in the fields. This paper presents and describes the results of an investigative study carried out to examine the efficacy of a simple detergent water mixture as an effective remedy for controlling American Fall Army Worms. This should be good news and great relief to governments and farmers at large with regards to time and financial resources that have been used to control these worms. The experiments were carried out at a laboratory in Kilimanjaro street, Kihonda ward, Morogoro region, Tanzania. Various concentrations of 0.1 graduation (0.1% - 1.5%) of detergents (Trade names: KLEESOFT, OMO, FOMA, ARIEL and FAMILY) water mixtures were prepared. These were applied to the worms to detect; which concentration was effective in killing them in the shortest time possible. Five worms were subjected to each concentration of detergent:water mixture and the time was recorded to detect the duration it took to kill the worms. Simple detergent:water mixtures have shown to be very effective in controlling the American Fall Army Worms. For each concentration prepared from 0.1% to 1.5% we observed a 100% mortality of the worms, with only difference being the time to kill. On average at 0.1%, the worms were killed in 689 seconds (≈ 12 minutes), while at 1.5%, the worms were killed in 32 seconds (≈ 0.5 minutes). The duration decreased with level of concentration of the detergent. As the detergent concentration increased less and less time was required to kill the worms completely. From the lowest concentration 0.1% to the highest 1.5%, there was a 100% mortality of the worms, with the only difference being time to kill. A 0.5% detergent:water mixture has been shown to be very effective in killing the fall army worms within 60 seconds as the difference in time to kill was negligible after this concentration. The findings of this study call for urgent need to introduce this remedy to all farmers in order to protect maize and other crops in the fields during production season and to reduce financial burden incurred by farmers in purchasing costly pesticides. Further research is needed to explore the specific content of the detergents that causes abrupt death and the mechanism in which the worms are killed

Effects of Detergent on α-Synuclein Structure: A Native MS-Ion Mobility Study

The intrinsically disordered protein α-synuclein plays a major role in Parkinson’s disease. The protein can oligomerize resulting in the formation of various aggregated species in neuronal cells, leading to neurodegeneration. The interaction of α-synuclein with biological cell membranes plays an important role for specific functions of α-synuclein monomers, e.g., in neurotransmitter release. Using different types of detergents to mimic lipid molecules present in biological membranes, including the presence of Ca2+ ions as an important structural factor, we aimed to gain an understanding of how α-synuclein interacts with membrane models and how this affects the protein conformation and potential oligomerization. We investigated detergent binding stoichiometry, affinity and conformational changes of α-synuclein taking detergent concentration, different detergent structures and charges into account. With native nano-electrospray ionization ion mobility-mass spectrometry, we were able to detect unique conformational patterns resulting from binding of specific detergents to α-synuclein. Our data demonstrate that α-synuclein monomers can interact with detergent molecules irrespective of their charge, that protein-micelle interactions occur and that micelle properties are an important factor.