The present paper is a systematic, comparative study on the reconstitution of an apocytochrome b.sub.6 purified from a heterologous system using a detergent-free method and reconstitution into ...liposomes performed using three different detergents: SDS, Triton X-100 and DM, and two methods of detergent removal by dialysis and using Bio-Beads. The product size, its distribution and zeta potential, and other parameters were monitored throughout the process. We found that zeta potential of proteoliposomes is correlated with reconstitution efficiency and, as such, can serve as a quick and convenient quality control for reconstitution experiments. We also advocate using detergent-free protein purification methods as they allow for an unfettered choice of detergent for reconstitution, which is the most crucial factor influencing the final product parameters.
Background
Epithelial barrier impairment is associated with many skin and mucosal inflammatory disorders. Laundry detergents have been demonstrated to affect epithelial barrier function in vitro ...using air–liquid interface cultures of human epithelial cells.
Methods
Back skin of C57BL/6 mice was treated with two household laundry detergents at several dilutions. Barrier function was assessed by electric impedance spectroscopy (EIS) and transepidermal water loss (TEWL) measurements after the 4 h of treatments with detergents. RNA sequencing (RNA‐seq) and targeted multiplex proteomics analyses in skin biopsy samples were performed. The 6‐h treatment effect of laundry detergent and sodium dodecyl sulfate (SDS) was investigated on ex vivo human skin.
Results
Detergent‐treated skin showed a significant EIS reduction and TEWL increase compared to untreated skin, with a relatively higher sensitivity and dose–response in EIS. The RNA‐seq showed the reduction of the expression of several genes essential for skin barrier integrity, such as tight junctions and adherens junction proteins. In contrast, keratinization, lipid metabolic processes, and epidermal cell differentiation were upregulated. Proteomics analysis showed that the detergents treatment generally downregulated cell adhesion‐related proteins, such as epithelial cell adhesion molecule and contactin‐1, and upregulated proinflammatory proteins, such as interleukin 6 and interleukin 1 beta. Both detergent and SDS led to a significant decrease in EIS values in the ex vivo human skin model.
Conclusion
The present study demonstrated that laundry detergents and its main component, SDS impaired the epidermal barrier in vivo and ex vivo human skin. Daily detergent exposure may cause skin barrier disruption and may contribute to the development of atopic diseases.
Household laundry detergents led to reduced electrical impedance, indicating impaired skin barrier. RNA‐seq and targeted multiplex protein analyses in skin biopsy demonstrated that laundry detergents disrupted several major pathways involved in the skin barrier and induced inflammation. Laundry detergents and SDS caused barrier damage in ex vivo human skin.Abbreviations: ACVRL1, activin A receptor like type; CCL, C‐C motif chemokine ligand; CLSTN2, calsyntenin‐2; CNTN1, contactin‐1; EPCAM, epithelial cell adhesion molecule; FASTL3, follistatin‐related protein 3; IL1B, interleukin 1 beta; IL6, interleukin 6; PBS, phosphate‐buffered saline; RNA‐seq, RNA sequencing; SDS, sodium dodecyl sulfate
Defects in the epithelial barrier have recently been associated with asthma and other allergies. The influence of laundry detergents on human bronchial epithelial cells (HBECs) and their barrier ...function remain unknown.
We investigated the effects of laundry detergents on cytotoxicity, barrier function, the transcriptome, and the epigenome in HBECs.
Air-liquid interface cultures of primary HBECs from healthy control subjects, patients with asthma, and patients with chronic obstructive pulmonary disease were exposed to laundry detergents and detergent residue after rinsing. Cytotoxicity and epithelial barrier function were evaluated. RNA sequencing, Assay for Transposase Accessible Chromatin with high-throughput sequencing, and DNA methylation arrays were used for checking the transcriptome and epigenome.
Laundry detergents and rinse residue showed dose-dependent toxic effects on HBECs, with irregular cell shape and leakage of lactate dehydrogenase after 24 hours of exposure. A disrupted epithelial barrier function was found with decreased transepithelial electrical resistance, increased paracellular flux, and stratified tight junction (TJ) immunostaining in HBECs exposed to laundry detergent at 1:25,000 dilutions or rinse residue at further 1:10 dilutions. RNA sequencing analysis showed that lipid metabolism, apoptosis progress, and epithelially derived alarmin-related gene expression were upregulated, whereas cell adhesion–related gene expression was downregulated by laundry detergent at 1:50,000 dilutions after 24 hours of exposure without substantially affecting chromatin accessibility and DNA methylation.
Our data demonstrate that laundry detergents, even at a very high dilution, and rinse residue show significant cell-toxic and directly disruptive effects on the TJ barrier integrity of HBECs without affecting the epigenome and TJ gene expression.
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This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS ...strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 μM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.
In the 1980s, virus inactivation steps were implemented into the manufacturing of biopharmaceuticals in response to earlier unforeseen virus transmissions. The most effective inactivation process for ...lipid‐enveloped viruses is the treatment by a combination of detergents, often including Triton X‐100 (TX‐100). Based on recent environmental concerns, the use of TX‐100 in Europe will be ultimately banned, which forces the pharmaceutical industry, among others, to switch to an environmentally friendly alternative detergent with fully equivalent virus inactivation performance such as TX‐100. In this study, a structure–activity relationship study was conducted that ultimately led to the synthesis of several new detergents. One of them, named “Nereid,” displayed inactivation activity fully equivalent to TX‐100. The synthesis of this replacement candidate has been optimized to allow for the production of several kg of detergent at lab scale, to enable the required feasibility and comparison virus inactivation studies needed to support a potential future transition. The 3‐step, chromatography‐free synthesis process described herein uses inexpensive starting materials, has a robust and simple work‐up, and allows production in a standard organic laboratory to deliver batches of several hundred grams with >99% purity.
Structure determination of membrane proteins is critical to the molecular understanding of many life processes, yet it has historically been a technically challenging endeavor. This past decade has ...given rise to a number of technological advancements, techniques, and reagents, which have facilitated membrane protein structural biology, resulting in an ever-growing number of membrane protein structures determined. To collate these advances, we have mined available literature to analyze the purification and structure determination specifics for all uniquely solved membrane protein structures from 2010 to 2019. Our analyses demonstrate the strong impact of single-particle cryo-electron microscopy on the field and illustrate how this technique has affected detergent and membrane mimetic usage. Furthermore, we detail how different structure determination methods, taxonomic domains and protein classes have unique detergent/membrane mimetic profiles, highlighting the importance of tailoring their selection. Our analyses provide a quantitative overview of where the field of membrane protein structural biology stands and how it has developed over time. We anticipate that these will serve as a useful tool to streamline future membrane protein structure determination by guiding the choice of detergent/membrane mimetic.
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•Tailored detergent selection is crucial for membrane protein structural biology.•Generation of a detergent and membrane mimetic dataset covering 2010–2019.•Protein type and structure determination method impact detergent choice.•Nanodiscs and amphipols have increased in popularity over the last decade.•Cryo-EM has significantly impacted membrane protein structural biology.
Membrane protein purification by means of detergents is key to isolating membrane‐bound therapeutic targets. The role of the detergent structure in this process, however, is not well understood. ...Detergents are optimized empirically, leading to failed preparations, and thereby raising costs. Here we evaluate the utility of the hydrophilic‐lipophilic balance (HLB) concept, which was introduced by Griffin in 1949, for guiding the optimization of the hydrophobic tail in first‐generation, dendritic oligoglycerol detergents (G1 OGDs). Our findings deliver qualitative HLB guidelines for rationalizing the optimization of detergents. Moreover, G1 OGDs exhibit strongly delipidating properties, regardless of the structure of the hydrophobic tail, which delivers a methodological enabling step for investigating binding strengths of endogenous lipids and their role for membrane protein oligomerization. Our findings will facilitate the analysis of challenging drug targets in the future.
Tailored detergents. Guidelines are introduced based on the hydrophilic‐lipophilic balance (HLB) concept, which was established by Griffin in 1949, to enable the directed structural optimization of detergents for the in vitro investigation of membrane protein‐lipid interactions.
Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, ...detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.
In this study, we present a method for isolation and propagation of Phytophthora infestans, a challenging-to-isolate phytopathogenic oomycete. The procedure initiated with naturally infected potato ...leaves, which underwent a 30-minute sanitization under running water. Subsequently, healthy potato tubers were meticulously washed with a neutral detergent, sterilized using alcohol, and then flamed. Slices 4 mm thick were carefully cut out of the potato tubers. Slices were used to cover 2 x 4 mm sterilized leaf pieces cut from the border of two-day old young P. infestans lesions, within sterilized empty Petri dishes. The sealed plates were then transferred to BOD growth chambers set at 18 degreesC in complete darkness for 5 days. At the end of this incubation period, the development of a sparse, white mycelium was observed on top of the potato slices. Using a Drigalski loop, the visible mycelium was carefully transferred to rye agar medium in Petri dishes. To avoid contamination, care was taken not no touch potato slices. After mycelium reaching the Petri dish border, and with aid of a Neubauer chamber, the inoculum density per plate was of 1.79 x 10.sup.4 sporangia mL.sup.-1. Sporangia germination rate ranged from 69 to 78%. This isolation technique simplify in vitro production of P. infestans, enhancing the possibility for research with this important pathogen. Key words: hemibiotrophic, late blight, massive inoculum production. Neste estudo, apresentamos tecnica de isolamento e multiplicacao de Phytophthora infestans, oomiceto fitopatog nico de isolamento desafiador. O procedimento teve in cio com folhas de batata naturalmente infectadas, as quais foram higienizadas em agua corrente por 30 minutos. Posteriormente, tuberculos de batata saudaveis foram lavados com detergente neutro, esterilizados em alcool e flambados. Em seguida, foram cortadas rodelas de 4 mm do tuberculo. As rodelas foram usadas para cobrir pedacos de 2 x 4 mm da borda de les es foliares de P infestans com dois dias, em placas de Petri vazias e esterilizadas. As placas foram seladas e transferidas para c maras de crescimento do tipo BOD, mantidas a 18 degreesC no escuro por cinco dias. Apos esse per odo, observou-se o crescimento de micelio ralo de cor branca sobre as rodelas de batata. Utilizando alca de Drigalski, o micelio vis vel foi transferido para meio de centeio e agar. Apos o preenchimento da placa por P infestans, estimou-se com c mara de Neubauer a concentracao de 1,79 x 10.sup.4 espor ngios mL.sup.-1, com taxa de germinacao variando entre 69 e 78%. Essa tecnica de isolamento simplifica o processo de obtencao in vitro de P infestans, contribuindo significativamente para pesquisas relacionadas a essa importante doenca. Palavras-chave: hemibiotrofico, producao massiva de inoculo, requeima.
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