Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers.

Extraction of proteins from the membrane using styrene maleic acid co-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and N-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg2+ ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.

Qualitative Assessment for Milk Adulteration: Extent, Common Adulterants, and Utility of Rapid Tests.

Background: The adulteration of milk is an important issue that needs to be addressed because of its deleterious effects on health. Detection of adulterants in milk can be done in laboratories or by purchasing adulteration detection kits. There are few home-based rapid tests to detect common milk adulterants, which are less known by our community. Objectives of this study were to estimate the proportion of milk adulteration and to identify common adulterants, to determine the association of milk adulteration with gastrointestinal and renal diseases; and also to validate rapid home-based tests by comparing its result with standard milk adulteration detection kit. Materials and Methods: A community-based study was conducted on 330 medical and paramedical staff. Collected milk samples during family visits were tested by standard milk adulteration detection tests and rapid home-based tests. Results: Among 330 tested milk samples, 233 samples were adulterated with one or more than one adulterant. The most common adulteration observed was the addition of water to milk, followed by detergent, urea, and neutralizer. The specificity of the rapid tests was found to be almost 100% in the detection of water and detergent in milk and accuracy was above 80%; with "substantial agreement" (kappa = 0.7) between rapid tests and standard tests on kappa statistics. Conclusion: The present study has identified a large extent of milk adulteration and common adulterants, which may have harmful effects on health. The utility of home-based rapid tests is clearly proven to detect common adulterants.

ZASP: A Highly Compatible and Sensitive ZnCl2 Precipitation-Assisted Sample Preparation Method for Proteomic Analysis.

Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 and methanol-induced protein precipitation at room temperature (RT). ZASP can remove trypsin digestion and LC-MS incompatible detergents such as SDS, Triton X-100, and urea at high concentrations in solution and unbiasedly recover proteins independent of the amount of protein input. We demonstrated the sensitivity and reproducibility of ZASP in an analysis of samples with 1 µg to 1000 µg of proteins. Compared to commonly used sample preparation methods such as SDC-based in-solution digestion, acetone precipitation, FASP, and SP3, ZASP has proven to be an efficient approach. Here, we present ZASP, a practical, robust, and cost-effective proteomic sample preparation method that can be applied to profile different types of samples.

Phenotypic and genotypic determination of resistance to common disinfectants among strains of Acinetobacter baumannii producing and non-producing biofilm isolated from Iran.

BACKGROUND: Nosocomial infections are a global problem in hospitals all around the world. It is considered a major health problem, especially in developing countries. The increase in the patient's stay in hospitals has increased the mortality rate, and consequently, the costs drastically increase. The main purpose of using disinfectants in the hospital environment is to reduce the risk of nosocomial infections. Ethylene diamine tetra acetic acid (EDTA) causes lysis and increases susceptibility to antimicrobial agents in the planktonic form of bacteria. This substance affects the permeability of the outer membrane of bacteria. It also prevents the formation of biofilms by bacteria. MATERIALS AND METHODS: In the current study, 120 isolates of Acinetobacter baumannii (A. baumannii) were confirmed by phenotypic and genotypic methods. Antibiogram was performed and then the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of isolates against 5% sodium hypochlorite, ethanol %70, sayasept-HP 2%, chlorhexidine 2%, dettol 4/8% were evaluated. In addition, the disinfectant effect was re-evaluated with the mixture of EDTA solution. All isolates were examined for biofilm presence by crystal violet staining method in triplicates and repeated three times for each strain. Also for all isolates detection of efflux pump genes (Qac-E, qacE-Δ1, SUG-E) by PCR technique was done. RESULTS: Antibiogram results of A. baumannii showed that 6.7% were Multi-drug-resistant (MDR), and 89.2% were Extensively drug-resistant (XDR) isolates. The highest effect of disinfectants was related to 5% sodium hypochlorite, and the least effect was 70% ethanol. EDTA increases the efficacy of selected disinfectants significantly. The highest prevalence of the efflux pump genes was related to SUG-E (95%) and Qac-E (91.7%), and, the qacE-Δ1 gene with 12.5%. The biofilm production rate was 91.3% among all isolates. CONCLUSION: The best and safest way to disinfect hospital floors and surfaces is to choose the right disinfectants, and learn how to use them properly. In this study, a mixture of disinfectants and EDTA had a significant effect on bactericidal activity. it was found that improper use of disinfectants, especially the use of sub-inhibitory dilutions, increases the resistance of bacteria to disinfectants.

Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Cleanup Techniques.

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identifications. Single-pot, solid-phase-enhanced sample preparation (SP3) is a cleanup technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. Recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer cleanup technique that employs protein aggregation to capture proteins without modified particles. We hypothesize that differences in capture mechanisms of SP3 and SP4 affect which proteins are identified by each cleanup technique. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. Ultimately, we demonstrate that protein-capture mechanisms are distinct, and the selection of a cleanup technique that yields high proteome coverage is dependent on protein-sample hydrophobicity. Data has been deposited into MassIVE (MSV000094130) and ProteomeXchange (PXD049965).

In silico and experimental characterization of a new polyextremophilic subtilisin-like protease from Microbacterium metallidurans and its application as a laundry detergent additive.

Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy's ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390 U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60 °C and 1 M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box-Behnken Design (BBD) with four variables, namely, time (15-75 min), temperature (30-60 °C), MmSP enzyme concentration (5-10 U/mL) and pH (7-11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5 U/mL of MmSP during 30 min at 32 °C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency.

Probabilistic Risk Assessment of Polioencephalomalacia from Texas Beef Consuming Rations Containing Multiple Sources of Dietary Sulfur.

The purpose of this probabilistic assessment was to estimate the risk of sulfur-induced polioencephalomalacia (S-PEM) for beef raised across Texas, from a dietary perspective. Ruminant nutritionists in Amarillo, TX, formulated two typical nutritional regimens based on cattle production stages, each containing six feed ingredients and well water. The Office of the Texas State Chemist (OTSC), National Research Council (NRC), and the published literature provided S data for feed ingredients. The Texas Water Development Board provided data for S content in Texas well water, categorized into twelve districts established by the Texas A&M AgriLife Research Extension Service. The S-PEM risk was estimated at five different eNDF levels ranging from 0% to 8% in 2% increments, using rumen degradable S (RDS) as an input value. Findings identified cattle raised in the South Plains district as the most susceptible population to S toxicity, with beef in the finishing production stage experiencing increased sensitivity. The most potential (MP) risk scenario suggested that the S-PEM risk could reach 28.5% for growers and 100% for finishers. Results further revealed that when S concentrations in well water exceeded 14.5 mg/L, water became the greatest contributor to RDS content for Texas beef, suggesting that high S content in well water is the most prominent concern for Texas beef.

Characterization of a thermostable protease from Bacillus subtilis BSP strain.

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Pediatric injuries and poisonings associated with detergent packets: results from the Canadian Hospitals Injury Reporting and Prevention Program (CHIRPP), 2011-2023.

BACKGROUND: Detergent packets are common household products; however, they pose a risk of injuries and poisonings, especially among children. This study examined the epidemiological characteristics of pediatric injuries and poisonings related to all types of detergent packets in Canada using emergency department (ED) data from the Canadian Hospitals Injury Reporting and Prevention Program (CHIRPP) database. METHODS: The CHIRPP database was searched for ED visit records for injuries and poisonings related to all types of detergent packets between April 1, 2011 and October 12, 2023 (N = 2,021,814) using variable codes and narratives. Data for individuals aged 17 years and younger were analyzed descriptively. Temporal trends in the number of detergent packet-related injuries and poisonings per 100,000 CHIRPP cases were assessed using Joinpoint regression and annual percent change (APC). A proportion ratio and 95% confidence intervals (CI) were calculated to compare the proportion of detergent packet-related cases in CHIRPP during two 34-months periods, pre-COVID-19 pandemic and after the beginning of the pandemic. RESULTS: There were 904 detergent packet-related cases among children and youth aged 17 years and younger identified in CHIRPP between April 1, 2011 and October 12, 2023, representing 59.9 cases per 100,000 CHIRPP cases. The majority (86.5%) of cases were among children aged 4 years and younger. Poisonings (58.8%) and eye injuries (30.6%) were the most frequent primary diagnoses. Unintentional ingestion (56.9%) and squeezing/breaking a detergent packet (32.3%) were the most frequent exposure mechanisms. Sixty-five patients (7.2%) were admitted to hospital. The number of detergent packet-related cases per 100,000 CHIRPP cases increased by 5.0% (95% CI 0.8, 10.2) annually between 2012 and 2022. The number of detergent packet-related poisonings per 100,000 CHIRPP cases decreased by 15.3% (95% CI - 22.3, - 10.6) annually between 2015 and 2022, whereas eye injuries showed an average annual percent increase of 16.6% (95% CI 11.2, 23.0) between 2012 and 2022. The proportion of detergent packet-related cases in CHIRPP after the beginning of the pandemic (79.9/100,000 CHIRPP cases) was 1.43 (95% CI 1.20, 1.71) times greater than pre-pandemic (55.7/100,000 CHIRPP cases). CONCLUSIONS: Detergent packet-related injuries and poisonings are a persisting issue. Continued surveillance and prevention efforts are needed to reduce detergent packet-related injuries and poisonings in Canada, particularly among children and youth.

Cell-cell transfer of adaptation traits benefits kin and actor in a cooperative microbe.

Microbes face many physical, chemical, and biological insults from their environments. In response, cells adapt, but whether they do so cooperatively is poorly understood. Here, we use a model social bacterium, Myxococcus xanthus, to ask whether adapted traits are transferable to naïve kin. To do so we isolated cells adapted to detergent stresses and tested for trait transfer. In some cases, strain-mixing experiments increased sibling fitness by transferring adaptation traits. This cooperative behavior depended on a kin recognition system called outer membrane exchange (OME) because mutants defective in OME could not transfer adaptation traits. Strikingly, in mixed stressed populations, the transferred trait also benefited the adapted (actor) cells. This apparently occurred by alleviating a detergent-induced stress response in kin that otherwise killed actor cells. Additionally, this adaptation trait when transferred also conferred resistance against a lipoprotein toxin delivered to targeted kin. Based on these and other findings, we propose a model for stress adaptation and how OME in myxobacteria promotes cellular cooperation in response to environmental stresses.

Extending the Lifetime of Frying Oil through Optimization of Fryer Cleaning.

With increases in consumer demand for fried foods in Japan over the last several decades, the consumption of frying oil has also steadily increased. Fryers used in restaurants to cook large quantities of food are typically cleaned using neutral kitchen detergents at the end of the day after removing the oil from the tank. However, significant amounts of debris can remain in the fryer after cleaning, possibly accelerating oil deterioration and thus reducing the quality of the fried foods. In this study, debris obtained from fryer tanks used in actual restaurants was assessed using scanning electron microscopy-energy dispersive X-ray spectroscopy together with Fourier transform infrared spectroscopy, and were determined to comprise polymerized oil and carbonized organic matter. Experiments using artificially prepared debris confirmed that these materials increased the acid value (AV) of frying oil. Trials in two restaurants serving similar amounts of fried chicken, French fries and doughnuts examined the effects of cleaning the fryer with either an alkaline detergent or a neutral kitchen detergent on debris removal and oil life. The alkaline detergent was found to completely remove debris while the neutral detergent left significant amounts of debris. After cleaning, the fryers were operated with new oil as usual and the deterioration of this oil was monitored by assessing the color difference, AV, carbonyl value and peroxide value. These indices increased 1.3 to 2.0 times faster in the case that the neutral kitchen detergent was used, suggesting that cleaning fryer tanks with an alkaline detergent could contribute to extending the lifetime of frying oil, reducing food losses and thus achieving sustainable development goals.

Synthesis of Low-Silicon X-Type Zeolite from Lithium Slag and Its Fast Exchange Performance of Calcium and Magnesium Ions.

Without the addition of silicon and aluminum sources, a pure-phase KNaLSX zeolite was successfully synthesized from the residue (lithium slag), which was produced from spodumene in the production process of lithium carbonate. The KNaLSX samples were characterized by an X-ray Diffractometer (XRD), Scanning Electron Microscope (SEM), X-ray Fluorescence Spectrometer (XRF), Thermogravimetric Differential Thermal Analysis (TG-DTA), Fourier Transform Infrared Spectrometer (FT-IR), and N2 adsorption measurement. The ion exchange capacity and the ion exchange rate of calcium and magnesium ions were measured as used for a detergent builder, and the results were compared with the standard zeolites (KNaLSX and 4A). The experimental results show that the pure-phase KNaLSX synthSynthesis and characterization of co-crystalline zeolite composite of LSX/esized from lithium slag has a SiO2/Al2O3 ratio of 2.01 with a grain size of 3~4 µm, which is close to the commercial KNaLSX sample of a SiO2/Al2O3 ratio of 2.0. The BET-specific surface area of KNaLSX is 715 m2/g, which is larger than the low-silicon X-type zeolite (LSX) synthesized from waste residue reported in the literature. The ion exchange rate constant of calcium and magnesium ions in KNaLSX is 5 times and 3 times that of 4A zeolite, respectively. KNaLSX also has a high ion exchange capacity for magnesium ion of 191 mgMgCO3/g, which is 2 times than that of 4A zeolite, and a high ion exchange capacity for calcium ion of 302 mgCaCO3/g, which meets the first-grade standard of zeolite for detergent builders in China. The work provides the basis for high-value resource utilization of lithium slag and the development of a detergent builder for rapid washing.

Mechanisms for initiation of food allergy by skin pre-disposed to atopic dermatitis.

Food allergy can be life-threatening and often develops early in life. In infants and children, loss-of-function mutations in skin barrier genes associate with food allergy. In a mouse model with skin barrier mutations (Flakey Tail, FT+/- mice), topical epicutaneous sensitization to a food allergen peanut extract (PNE), an environmental allergen Alternaria alternata (Alt) and a detergent induce food allergy and then an oral PNE-challenge induces anaphylaxis. Exposures to these allergens and detergents can occur for infants and children in a household setting. From the clinical and preclinical studies of neonates and children with skin barrier mutations, early oral exposure to allergenic foods before skin sensitization may induce tolerance to food allergens and thus protect against development of food allergy. In the FT+/- mice, oral food allergen prior to skin sensitization induce tolerance to food allergens. However, when the skin of FT+/- pups are exposed to a ubiquitous environmental allergen at the time of oral consumption of food allergens, this blocks the induction of tolerance to the food allergen and the mice can then be skin sensitized with the food allergen. The development of food allergy in neonatal FT+/- mice is mediated by altered skin responses to allergens with increases in skin expression of interleukin 33, oncostatin M and amphiregulin. The development of neonate food allergy is enhanced when born to an allergic mother, but it is inhibited by maternal supplementation with α-tocopherol. Moreover, preclinical studies suggest that food allergen skin sensitization can occur before manifestation of clinical features of atopic dermatitis. Thus, these parameters may impact design of clinical studies for food allergy, when stratifying individuals by loss of skin barrier function or maternal atopy before offspring development of atopic dermatitis.

An essential host dietary fatty acid promotes TcpH inhibition of TcpP proteolysis promoting virulence gene expression in Vibrio cholerae.

Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated directly by ToxT and indirectly by two transmembrane transcription regulators (TTRs), ToxR and TcpP, that promote the expression of toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH-mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. The absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that α-linolenic acid, a dietary fatty acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs) in a mechanism requiring the TcpH transmembrane domain. Taken together, our data support a model where V. cholerae cells use exogenous α-linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity. IMPORTANCE: Vibrio cholerae continues to pose a significant global burden on health and an alternative therapeutic approach is needed, due to evolving multidrug resistance strains. Transcription of toxT, stimulated by TcpP and ToxR, is essential for V. cholerae pathogenesis. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction. Furthermore, we identified a gut metabolite, α-linolenic acid, that stimulates the co-association of TcpP and TcpH within detergent-resistant membranes (also known as lipid-ordered membrane domains), thereby supporting TcpH-dependent antagonism of TcpP proteolysis. Data presented here extend our knowledge of RIP, virulence gene regulation in V. cholerae, and, to the best of our knowledge, provides the first evidence that lipid-ordered membranes exist within V. cholerae. The model presented here also suggests that TTRs, common among bacteria and archaea, and co-component signal transduction systems present in Enterobacteria, could also be influenced similarly.

Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target. OBJECTIVE: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment. METHODS: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied. RESULTS: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ. CONCLUSION: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.

Laboratory features in acute paediatric poisoning with liquid laundry detergent capsules: a seven-year retrospective study in Romania.

INTRODUCTION: The epidemiological and clinical characteristics of acute poisoning with liquid laundry detergent capsules have been comprehensively reported. However, studies of laboratory test results in these exposures are uncommon. This study analyzed the impact of the ingestion of liquid laundry detergent capsules on admission laboratory tests in paediatric patients. METHODS: This retrospective study was conducted in the clinical toxicology unit of a paediatric poison centre between 2015 and 2021. Paediatric patients (less than 18 years of age) who ingested liquid laundry detergent capsules were included. The relationship between the European Association of Poisons Centers and Clinical Toxicologists/European Commission/International Programme on Chemical Safety Poisoning Severity Score and admission laboratory test results was assessed using Fisher's exact test or analysis of variance. RESULTS: A total of 156 patients were included in the study. A considerable proportion of patients presented with leucocytosis, acidosis, hyperlactataemia or base deficit. The median values of white blood cell count (P = 0.042), pH (P = 0.022), and base excess (P = 0.013) were significantly different among the Poisoning Severity Score groups. Hyperlactataemia was strongly associated with the Poisoning Severity Score (P = 0.003). DISCUSSION: Leucocytosis is a non-specific marker of severity following ingestion of liquid laundry detergent capsules. The incidence of metabolic acidosis and hyperlactataemia was higher in this study than in previous reports, but these metabolic features were not related to the severity of exposure. The exact mechanisms of toxicity are not yet known, but the high concentration of non-ionic and anionic surfactants, as well as propylene glycol and ethanol, in the capsule are likely contributing factors. CONCLUSIONS: Pediatric patients who ingest liquid laundry detergent capsules may develop leucocytosis, metabolic acidosis, hyperlactataemia, and a base deficit.

Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association.

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

NDT-C11 as a Viable Novel Detergent for Single Particle Cryo-EM.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Evaluation of biofilm formation and expression of psl, pel, alg genes of Pseudomonas aeruginosa in exposure to detergents.

Pseudomonas aeruginosa has been in the center of attention for several years as an opportunistic human pathogen implicated in many severe acute and chronic infections particularly in immunocompromised patients. Its high persistence and resistance against many antimicrobial agents are mostly attributed to biofilm formation. Biofilms are microbial communities mainly consisting of extracellular polymeric substances that encapsulate bacteria together and protect them from extracellular stresses. This cell aggregation is a stress response that P. aeruginosa employes as a survival strategy during growth with the toxic detergents. This process has shown to involve several operons such as psl, pel, and alg. Here we used P. aeruginosa strain PAO1 in control group, 40 P. aeruginosa strains from sink and 40 strains from surface of public places. Biofilm formation and gene expression were measured before and after exposure to sub minimum inhibitory concentration (sub-MIC) of biocides chlorhexidine diacetate and benzalkonium chloride. The qRT-PCR and biofilm formation results demonstrated an increase in biofilm formation ability and gene expression of pslA/B and pelA/B in two groups collected from sink and surface in contrast to the control group. A remarkable increase was observed in the biofilm formation and expression of pslA in the bacterial strain collected from the sink after exposure to biocides chlorhexidine diacetate. Both Pel and Psl appeared to have redundant functions as structural scaffolds in biofilms. Sub-MIC levels of detergents can improve biofilm formation ability of P. aeruginosa and therefore trigger resistance.

Unraveling the potential of uninvestigated thermoalkaliphilic lipases by molecular docking and molecular dynamic simulation: an in silico characterization study.

Thermoalkaliphilic lipase enzymes are mostly favored for use in the detergent industry. While there has been considerable research on Geobacillus lipases, a significant portion of these enzymes remains unexplored or undocumented in the scientific literature. This work performed in silico phylogeny, sequence alignment, structural and enzyme-substrate interaction analyses of the five thermoalkaliphilic lipases belonging to different Geobacillus species (Geobacillus stearothermophilus lipase = GsLip, Geobacillus sp. B4113_201601 lipase = Gb4Lip, Geobacillus kaustophilus HTA426 lipase = GkLip, Geobacillus sp. SP22 lipase = GspLip, Geobacillus sp. NTU 03 lipase = GntLip). For this purpose, unreviewed enzyme sequences of five Geobacillus thermoalkaliphilic lipases were analyzed at sequence and phylogeny levels. 3D homology enzyme models were built, validated, and investigated by different bioinformatics tools. The ligand interactions screening using seven para-nitrophenyl (pNP) esters and enzyme-ligand interactions were analyzed on Gb4Lip:pNP-C12 and BTL2:pNP-C12 by MD simulation. Biophysicochemical characteristic analysis showed that Gb4Lip had a theoretical T m value of above 65 ºC, and a higher aliphatic index indicating greater thermal stability. Sequence alignment showed a hydrophilic threonine in the α6 helix of Gb4Lip, indicating high enzymatic activity. A normalized temperature factor B (B'-factor) analysis showed that the lid domains of five lipases significantly possessed lower B'-factor values, compared to G. thermocatenulatus lipase 2 (BTL2), indicating that they had higher rigidity. Molecular docking results indicated that the five lipases had the highest binding affinity toward pNP-C12. The RMSF investigation revealed that the thermostability of Gb4Lip is influenced by specific molecular elements: D202-S203 within the αB region of the lid domain, and E274-Q275 within the b3 strand, as well as W278 in the b3-b4 loop, and H282 in the b4 strand of the Ca2+-binding region. MD simulation analysis showed that catalytic residue S114 and at least one oxyanion hole residue (F17 and/or Q114) in Gb4Lip frequently formed hydrogen bonds with the pNP-C12 ligand at 343 K and 348 K throughout the simulation process, indicating that Gb4Lip might catalyze relatively long-chain ligand pNP-C12 with high performance. In conclusion, Gb4Lip might be a more suitable candidate as the detergent additive. In addition, this investigation can offer valuable perspectives on Family I.5 lipases such as Gb4Lip for future exploration in the field of protein engineering. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04023-5.