The toxic nature of bacterial endotoxins is affected by the structural details of lipid A, including the variety and position of acyl chains and phosphate group(s) on its diglucosamine backbone. Negative-ion mode tandem mass spectrometry is a primary method for the structure elucidation of lipid A, used independently or in combination with separation techniques. However, it is challenging to accurately characterize constitutional isomers of lipid A extracts by direct mass spectrometry, as the elemental composition and molecular mass of these molecules are identical. Thus, their simultaneous fragmentation leads to a composite, so-called chimera mass spectrum. The present study focuses on the phosphopositional isomers of the classical monophosphorylated, hexaacylated Escherichia coli-type lipid A. Collision-induced dissociation (CID) was performed in an HPLC-ESI-QTOF system. Energy-resolved mass spectrometry (ERMS) was applied to uncover the distinct fragmentation profiles of the phosphorylation isomers. A fragmentation strategy applying multi-levels of collision energy has been proposed and applied to reveal sample complexity, whether it contains only a 4\'-phosphorylated species or a mixture of 1- and 4\'-phosphorylated variants. This comparative fragmentation study of isomeric lipid A species demonstrates the high potential of ERMS-derived information for the successful discrimination of co-ionized phosphorylation isomers of hexaacylated lipid A.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Ultrasonic-assisted activated carbon separation (UACS) was first employed to improve product quality by regulating adsorption rate and removing bacterial endotoxin from salvia miltiorrhizae injection. The adsorption rate was related to three variables: activated carbon dosage, ultrasonic power, and pH. With the increase of activated carbon dosage from 0.05 % to 1.0 %, the adsorption rates of salvianolic acids and bacterial endotoxin increased simultaneously. The adsorption rates at which bacteria endotoxins increased from 52.52 % to 97.16 % were much higher than salvianolic acids. As the ultrasonic power increased from 0 to 700 W, the adsorption rates of salvianolic acids on activated carbon declined to less than 10 %, but bacterial endotoxin increased to more than 87 %. As the pH increased from 2.00 to 8.00, the adsorption rate of salvianolic acid dropped whereas bacterial endotoxin remained relatively stable. On the basis of response surface methodology (RSM), the optimal separation conditions were established to be activated carbon dose of 0.70 %, ultrasonic power of 600 W, and pH of 7.90. The experimental adsorption rates of bacterial endotoxin were 94.15 %, which satisfied the salvia miltiorrhizae injection quality criterion. Meanwhile, salvianolic acids\' adsorption rates were 1.92 % for tanshinol, 4.05 % for protocatechualdehyde, 2.21 % for rosmarinic acid, and 3.77 % for salvianolic acid B, all of which were much lower than conventional activated carbon adsorption (CACA). Salvianolic acids\' adsorption mechanism on activated carbon is dependent on the component\'s molecular state. Under ideal separation conditions, the molecular states of the four salvianolic acids fall between 1.13 % and 6.60 %. The quality of salvia miltiorrhizae injection can be improved while maintaining injection safety by reducing the adsorption rates of salvianolic acids to less than 5 % by the use of ultrasound to accelerate the desorption mass transfer rate on the activated carbon surface. When activated carbon adsorption was used in the process of producing salvia miltiorrhizae injection, the pH of the solution was around 5.00, and the proportion of each component\'s molecular state was tanshinol 7.05 %, protocatechualdehyde 48.93 %, rosmarinic acid 13.79 %, and salvianolic acid B 10.28 %, respectively. The loss of useful components was evident, and the corresponding activated carbon adsorption rate ranged from 20.74 % to 41.05 %. The average variation rate in plasma His and IgE was significant (P < 0.05) following injection of 0.01 % activated carbon, however the average variation rate of salvia miltiorrhizae injection was dramatically decreased with the use of UACS and CACA (P > 0.05). The ultrasonic at a power intensity of 60 W/L and the power density of 1.20 W/cm2 may resolve the separation contradiction between salvianolic acids and bacterial endotoxin, according to experiments conducted with UACS at different power intensities. According to this study, UACS has a lot of potential applications in the pharmaceutical manufacturing industry and may represent a breakthrough in the field of ultrasonic separation.

Surrogate viruses theoretically provide an opportunity to study the viral spread in an indoor environment, a highly needed understanding during the pandemic, in a safe manner to humans and the environment. However, the safety of surrogate viruses for humans as an aerosol at high concentrations has not been established. In this study, Phi6 surrogate was aerosolized at high concentration (Particulate matter2.5: ∼1018 μg m-3) in the studied indoor space. Participants were closely followed for any symptoms. We measured the bacterial endotoxin concentration of the virus solution used for aerosolization as well as the concentration in the room air containing the aerosolized viruses. In addition, we measured how the bacterial endotoxin concentration of the sample was affected by different traditional virus purification procedures. Despite the purification, bacterial endotoxin concentration of the Phi6 was high (350 EU/ml in solution used for aerosols) with both (two) purification protocols. Bacterial endotoxins were also detected in aerosolized form, but below the occupational exposure limit of 90 EU/m3. Despite these concerns, no symptoms were observed in exposed humans when they were using personal protective equipment. In the future, purification protocols should be developed to reduce associated bacterial endotoxin levels in enveloped bacterial virus specimens to ensure even safer research use of surrogate viruses.

OBJECTIVE: We evaluated bacterial endotoxin adhesion, superficial micromorphology and mechanical properties of latex and non-latex intermaxillary orthodontic elastics.
METHODS: To quantify the adhered bacterial endotoxin, elastics were divided into 5 groups: experimental (n = 12) latex and non-latex elastics, previously contaminated by an endotoxin solution, negative control (n = 6) latex and non-latex elastics without contamination, and positive control (n = 6) stainless steel specimens (metallic replicas), contaminated by an endotoxin solution. In parallel, the structural micromorphology (n = 6) and surface roughness of latex and non-latex intermaxillary orthodontic elastics were assessed using confocal laser microscopy. Force degradation (g) and deformation of the internal diameter change (mm) were also evaluated. Structural micromorphology, surface roughness (µm), force degradation (g) and internal diameter (mm) change were evaluated at time 0 and after 24 and 72 h in a deformation test. Data were analyzed by the Shapiro-Wilk, Kruskal-Wallis, Dunn, ANOVA and Bonferroni tests (α = 5%).
RESULTS: Endotoxin adhered similarly to both types of elastics with scores of 3 (> 1.0 EU/mL). The surface microstructure of both types of elastics showed irregularities and porosities at all times. Initially, the latex elastics had a higher surface roughness (p < 0.001) than the non-latex ones. After 24 h loading, surface roughness of the latex elastics was significantly reduced (p < 0.001), while after 72 h, the values were similar for both types (p > 0.05). The non-latex elastics had significantly higher force generation values (p < 0.05) at 0, 24 and 72 h compared with the latex elastics, although there was a significant reduction (p < 0.001) in force over time for both elastics. Despite similar initial values, non-latex elastics had a significantly larger internal diameter (p < 0.001) after the loading periods of 24 and 72 h compared with the latex elastics.
CONCLUSIONS: Both elastics showed high affinity with endotoxin and microstructural irregularities of their surface. The non-latex elastics generated higher force values but demonstrated greater deformation of the internal diameter after loading.
UNASSIGNED: ZIELSETZUNG: Wir untersuchten die bakterielle Endotoxinadhäsion, die oberflächliche Mikromorphologie und die mechanischen Eigenschaften von latexhaltigen und nicht-latexhaltigen intermaxillären kieferorthopädischen Elastics.
METHODS: Zur Quantifizierung des anhaftenden bakteriellen Endotoxins wurden die Elastics in 5 Gruppen eingeteilt: experimentelle (n = 12) Latex- und Nicht-Latex-Elastics, die zuvor mit einer Endotoxinlösung kontaminiert worden waren, Negativkontrolle (n = 6) Latex- und Nicht-Latex-Elastics ohne Kontamination und Positivkontrolle (n = 6) Edelstahlproben (metallische Nachbildungen), die mit einer Endotoxinlösung kontaminiert waren. Parallel dazu wurden die strukturelle Mikromorphologie (n =6 ) und die Oberflächenrauheit von intermaxillären Elastics aus Latex und aus Nicht-Latex mittels konfokaler Lasermikroskopie untersucht. Ebenfalls bewertet wurden der Kraftabbau (g) und die Verformung des Innendurchmessers (mm). Die strukturelle Mikromorphologie, die Oberflächenrauheit (µm), der Kraftabbau (g) und die Änderung des Innendurchmessers (mm) wurden zum Zeitpunkt 0 sowie nach 24 und 72 h in einem Verformungstest bewertet. Die Daten wurden mit den Tests Shapiro-Wilk, Kruskal-Wallis, Dunn, ANOVA und Bonferroni (α = 5%) analysiert.
UNASSIGNED: Das Endotoxin haftete an beiden Arten von Elastics in ähnlicher Weise mit Scores von 3 (> 1,0 EU/ml). Die Oberflächenmikrostruktur beider Arten von Elastics zeigte zu jedem Zeitpunkt Unregelmäßigkeiten und Porositäten. Zu Beginn wiesen die Latex-Elastics eine höhere Oberflächenrauheit auf (p < 0,001) als die Nicht-Latex-Elastics. Nach 24 h Belastung war die Oberflächenrauheit der Latex-Elastics deutlich geringer (p < 0,001), während nach 72 h die Werte für beide Typen ähnlich waren (p < 0,05). Die Nicht-Latex-Elastics wiesen nach 0, 24 und 72 h signifikant höhere Werte für die Krafterzeugung auf (p < 0,05) als die Latex-Elastics, auch wenn die Kraft bei beiden Elastics im Laufe der Zeit signifikant abnahm (p < 0,001). Trotz ähnlicher Ausgangswerte wiesen die Nicht-Latex-Elastics nach den Belastungszeiten von 24 und 72 h einen signifikant größeren Innendurchmesser (p < 0,001) auf als die Latex-Elastics.
UNASSIGNED: Beide Elastics zeigten eine hohe Affinität zu Endotoxin und mikrostrukturelle Unregelmäßigkeiten auf ihrer Oberfläche. Die latexfreien Elastics erzeugten höhere Kraftwerte, wiesen aber nach der Belastung eine größere Verformung des Innendurchmessers auf.

Background and aims: Periodontitis is an inflammatory-infectious disease. Identifying markers of systemic exposure of periodontitis might be of interest to study its interaction with other conditions. Soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) is upregulated during bacterial infections. Our aim was therefore to investigate whether periodontitis and its treatment are associated with bacterial endotoxin and sTREM-1. Methods: Fifty patients with severe periodontitis and 50 age-matched controls were included in a case-control study (all never smokers). A secondary analysis of a previously published intervention study was performed, in which included 69 patients with severe periodontitis were randomized to receive either intensive (IPT) or control periodontal therapy (CPT) and monitored over 6 months. Serum levels of bacterial endotoxin and sTREM-1 were determined at one time point (case-control study) and at baseline, 1 day, 1 and 6 months after periodontal treatment (intervention study). Results: Severe periodontitis was associated with elevated circulating endotoxin levels when cases (22.9 ± 2.2 EU/ml) were compared to controls (3.6 ± 0.5 EU/ml, p < 0.001) and with sTREM-1 levels (1302.6 ± 47.8 vs. 870.6 ± 62.0 pg/ml, p < 0.001). A positive correlation was observed between sTREM-1 and endotoxin levels (r = 0.4, p < 0.001). At 6 months after treatment, IPT significantly decreased serum levels of sTREM-1 compared to CPT (adjusted mean difference of 500.2 pg/ml, 95% CI: 18.9-981.4; p = 0.042). No substantial differences were noted in endotoxin levels at any time point after treatment between groups. Conclusions: Severe periodontitis is linked to increased circulating endotoxin and sTREM-1 levels and following IPT a reduction in sTREM-1 levels is observed.

UNASSIGNED: Intraamniotic infection is associated with an increased risk of multiple adverse outcomes in offspring, especially neonatal necrotizing enterocolitis (NEC), which is one of the serious gastrointestinal diseases in neonates. However, the underlying mechanism remains undefined. We hypothesize that bacterial endotoxin-induced maternal inflammation causes intestinal injury in offspring, thereby affecting the composition of the intestinal microbiome.
UNASSIGNED: Pregnant Sprague Dawley rats were received intraperitoneal injections with 700 μg/kg lipopolysaccharide (LPS, which was the same as bacterial endotoxin) or saline at 15 days of gestation. Pups were allowed to deliver naturally and euthanized at days 0, 3 and 7 after birth. Intestinal tissue and feces samples from offspring were collected to evaluate the effects of maternal inflammation on intestinal flora colonization and intestinal mucosal development.
UNASSIGNED: Significant intestinal injury of the offspring induced by prenatal LPS exposure was observed on day 0 and 3 after birth. In addition, prenatal LPS exposure also induced significant changes in the intestinal microbiome of the offspring with a significant increase in Proteobacteria (Escherichia-Shigella) and a decrease in Firmicutes at 7 days after birth.
UNASSIGNED: Thus, our findings suggest that LPS-induced maternal inflammation induces intestinal injury in offspring and subsequently leads to NEC-like changes in the composition of the intestinal microbiome.

The impact of sandstorm dust events affects local air quality and public health. These issues are becoming of greater concern in Saudi Arabia. There is a significant lack of research on airborne endotoxin exposure and analysis in the Middle East countries and no coherent body of research exists focusing on sandstorm dust in worldwide. In this study, we used a novel design of an aluminum foil plate (AFP) electrostatic dust cloth (EDC) for the passive air sampling of sandstorm dust. A total of 38 sandstorm dust samples were collected during sandstorm episodes occurring between January and April 2020 in both indoor (7 days, n = 20) and outdoor environments (24 h, n = 18). After exposure, and following an extraction procedure, bacterial endotoxin levels were measured using the Limulus Amoebocyte Lysate (LAL) gel clot method. The study highlights that the airborne endotoxin level observed was between 10 and 200 EU/m2 in both indoor and outdoor environments, during a sandstorm event. Agricultural activities and farmhouses observed higher airborne endotoxin levels. In general, increased endotoxin levels were related to the severity of the sandstorms. Given that the observed values were high as per existing guidelines for respiratory health, we recommend the setting an occupational airborne exposure limit for bacterial endotoxin. This is the first report and further studies across various sandstorm-hit regions will need to be undertaken, together with various sampling methods, in order to assess for seasonal and geographic trends.

Non-alcoholic fatty liver disease (NAFLD) is by now the most prevalent liver disease worldwide. The non-proteogenic amino acid l-citrulline (L-Cit) has been shown to protect mice from the development of NAFLD. Here, we aimed to further assess if L-Cit also attenuates the progression of a pre-existing diet-induced NAFLD and to determine molecular mechanisms involved. Female C57BL/6J mice were either fed a liquid fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C) for 8 weeks to induce early stages of NASH followed by 5 more weeks with either FFC-feeding +/- 2.5 g L-Cit/kg bw or C-feeding. In addition, female C57BL/6J mice were either pair-fed a FFC +/- 2.5 g L-Cit/kg bw +/- 0.01 g/kg bw i.p. N(ω)-hydroxy-nor-l-arginine (NOHA) or C diet for 8 weeks. The protective effects of supplementing L-Cit on the progression of a pre-existing NAFLD were associated with an attenuation of 1) the increased translocation of bacterial endotoxin and 2) the loss of tight junction proteins as well as 3) arginase activity in small intestinal tissue, while no marked changes in intestinal microbiota composition were prevalent in small intestine. Treatment of mice with the arginase inhibitor NOHA abolished the protective effects of L-Cit on diet-induced NAFLD. Our results suggest that the protective effects of L-Cit on the development and progression of NAFLD are related to alterations of intestinal arginase activity and intestinal permeability.

Ultrasonic-assisted ultrafiltration (UAU) removing bacterial endotoxin from diammonium glycyrrhizinate, was firstly applied to surfactant separation. Separation efficiency was related with four variables, including ultrafiltration molecular weight cut off (MWCO), ultrasonic power, concentration and pH. The SCQ-9200E ultrasonic system was provided for the study with adjustable ultrasonic power 80 W to 800 W, and the ultrasonic frequency was 40 KHz. On the basis of response surface methodology (RSM), the optimal separation conditions were determined to be the ultrafiltration MWCO as 10 kDa, the ultrasonic power as 570 W, diammonium glycyrrhizinate concentration as 150.00 μg/mL and the pH as 4.70. The experimental rejection of bacterial endotoxin was 94.08%, meanwhile the transmittance of diammonium glycyrrhizinate was 93.65%. Based on the ultrasonic power, solution volume, and ultrasonic container size, the experiments with UAU at different power intensities showed that ultrasonic at a power intensity of 57 W/L and the power density of 0.32 W/cm2 could solve the separation contradiction between diammonium glycyrrhizinate and bacterial endotoxin. This study indicated that UAU could be an innovation in ultrasonic separation fields, and had a vast range of prospects for making use in pharmaceutical preparation area.