Endotoxin Concentrations Research Articles

Клиническое значение интегральной регистрации маркеров эндотоксического иммунитета и колонизационной резистентности буккальных эпителиоцитов при рецидивирующем бронхите у подростков

Recurrent bronchitis (RB) occupies one of the key places in the structure of nonspecific diseases of the respiratory tract and remains a significant medical and social problem in healthcare. The purpose — to investigate and evaluate the clinical significance of integral registration of markers of endotoxic immunity and colonization resistance of buccal epithelial cells (BE) in RB in adolescents. Material and methods. The study included 117 children (12–17 years old), including 78 with RB — the main group, 29 with acute bronchitis (AB) — the comparison group and 23 conditionally healthy children — the control group. The analysis of anamnestic, clinical, paraclinical data, verification of etiologically significant viruses, registration of artificial colonization of buccal epithelial cells (BE), colonization index (CI), micro-LAL test were carried out. Statistical processing of the results was carried out using the SPSS program. Results. A significant difference in markers of colonization resistance and anti-endotoxin protection was revealed in recurrent inflammation in the respiratory tract in comparison with AB in peers. In RB, the initial degree of Candida albicans adhesion to BE was significantly higher in relation to AB (p = 0.0427) and upon discharge from the hospital did not reach normative values, being significantly different from patients with AB (p = 0.0391). The high index of artificial colonization indicates the activation of allochthonous flora and disruption of the microbiota in the oral cavity, which negatively affects the body reactivity. In patients with RB, the titer of antiglycolipid antibodies significantly decreased and in 75% the values did not return to normal, while in AB they did not differ from the control. High levels of endotoxin were noted in RB compared to AB (p = 0.0317). Such patients were diagnosed with a more severe course of the disease with symptoms of endogenous intoxication and a long hospitalization period. Using the method of correlation analysis, a high direct relationship was established between artificial colonization and endotoxin concentration (r = 0.71). The relationship between these parameters in AB was significantly less pronounced (r = 0.3). The results obtained indicate close integration and mutual modulation of the local oral immunity and antiendotoxin defense. Conclusion. Integral registration of the markers of reactivity of specific anti-endotoxin immunity and colonization resistance reflect the severe inflammatory process in the respiratory tract and predict the risk of a chronic pathological process in the bronchopulmonary system.

Upregulation of YciM expression reduces endotoxin contamination of recombinant proteins produced in <i>Escherichia coli</i> cells

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation.

Previously developed whole-cell vaccines against Bordetella pertussis, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in B. pertussis through the heterologous production of the phosphatase LpxE from Francisella novicida did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in B. pertussis. The kdtA and kdkA genes, responsible for the incorporation of a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of Escherichia coli kdtA. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the eptB gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.

Dietary crude protein time-dependently modulates the bacterial community and metabolites and changes dietary nutrient efficiency in growing pigs

The reduced nutrient digestibility of low-protein (LP) diets has been shown to be caused by the weakened fermentative capacity of the post-gut flora. The dynamic regulation of dietary protein contents on post-gut microbial population and fermentative metabolism is unclear. Twelve growing barrows (19.9 ± 0.8 kg) fitted with a T-cannula at the blind end of the cecum were randomly administered a high-protein (HP, 21.5% crude protein [CP]) diet or an LP (15.5% CP) diet for 28 d. The cecal content and feces were collected at d 1, 14, and 28 of the experiment for microflora structures and metabolite concentrations analysis. The nutrient digestibility coefficient and plasma biochemical parameters were also determined. Compared with the HP treatment, the LP treatment showed decreased plasma urea nitrogen concentration and apparent total tract digestibility of dry matter, gross energy, and CP (P < 0.01). In addition, urinary nitrogen losses, total nitrogen losses, and daily nitrogen retention in the LP treatment were lower than those in the HP treatment (P < 0.01), and the nitrogen retention-to-nitrogen intake ratio in the LP treatment was increased (P < 0.01). The HP group showed increased cecal total short-chain fatty acids (SCFA) concentration and fecal propionate, butyrate, and total SCFA concentrations (P < 0.05) on d 14 and 28, which may be mainly related to the elevated abundance of SCFA-producing bacteria, such as Ruminococcus, Lactobacillus, and Prevotella (P < 0.05). Probiotics, such as Bifidobacterium, Bacteroidales S24-7, and Rikenella, enriched in the LP treatment possibly contributed to reduced plasma endotoxin content. The differences in the abundances of almost all the above-mentioned flora appeared on d 28 but not d 14. Likewise, differences in the Simpson and Shannon indices and clustering patterns of the microbiota between treatments were also only observed on d 28. To sum up, in a time-dependent manner, the LP diet increased probiotics with gut-improving functions and decreased SCFA-producing bacteria, which may cause enhanced intestine health and reduced nutrient digestibility.

Diagnostic significance of endotoxin and antitoxic opsonin – fibronectin in the blood flow in purulent-necrotic inflammatory of the lower limb in patients with diabetes mellitus

Introduction. In the cascade of pathogenetic mechanisms of diabetes mellitus, one of the central positions is occupied by systemic vascular lesions, including the lower extremities with the formation of diabetic foot syndrome. Aim. To investigate and evaluate the diagnostic significance of endotoxin concentration and plasma fibronectin parameters in indicating the severity of purulent-necrotic inflammation of the feet in patients with diabetes mellitus. Materials and methods. The study included 112 patients with purulent-necrotic inflammation of the feet aged 61.4 ± 12.5 years. By types of surgical intervention, patients were divided into groups: the main group (21 patients with transmetadarsal ammutation of the foot and 27 with amputation at the level of the lower leg), the comparison group – 64 patients with necrectomy and plastic closure of wounds on the foot. The concentration of endotoxin in the bloodstream was determined by the micro-LAL test (EU/ml), the content of plasma fibronectin was assessed by enzyme immunoassay (µg/ml). Results. With the progressive development of purulent inflammation of the feet in patients of the main group, the concentration of endotoxin reached 9.32 (0.91) EU/ml, in the comparison group – 5.73 (0.36) EU/ml, in which purulent-necrotic the process was limited. In the main group of examined patients, hypofibronectinemia also had a more significant character. At the same time, the lowest fibronectin in patients was observed with an extensive purulent-necrotic process, in whom an amputation was performed at the level of the lower leg – 124.7 (6.2) µg/ml, the concentration of endotoxin in them reached the maximum figures – 10.51 (0.5) EU/ml. Discussion.The main factor determining the depth of endotoxin aggression and destructive lesions of the extremities can be the enhanced utilization of fibronectin. Conclusion. The progression of endotoxin aggression is a predictor of an increase in the severity of destructive tissue damage in patients with diabetes mellitus.

Dietary strategies can increase cloacal endotoxin levels and modulate the resident microbiota in broiler chickens

Endotoxins released from poultry feces have been associated with impaired human health. Because endotoxins are released from gram-negative intestinal bacteria, it was hypothesized that dietary strategies may influence endotoxin excretion via modulation of gut microbiota. We therefore tested dietary strategies that could potentially reduce cloacal endotoxin levels in broiler chickens. One-day-old male Ross 308 (N = 1,344) broilers were housed in 48 pens (N = 8 pens/treatment, 28 chickens per pen) and fed 1 of 6 diets for 35 days (d) in a 3-phase feeding program: a basic diet (CON) that served as the reference diet, or basic diet supplemented with butyrate (BUT), inulin (INU), medium-chain fatty acids (MCFA) or Original XPC™LS (XPC), or a high-fiber-low-protein (HF-LP) diet. A significant (P < 0.05) increase in cloacal endotoxin concentration at d 35 was observed in BUT as compared to CON. Analysis of cloacal microbiota showed a trend (P < 0.07) for a higher gram-negative/gram-positive ratio and for a higher relative abundance of gram-negative bacteria at d 35 (P ≤ 0.08) in BUT and HF-LP as compared to CON. A significant (P < 0.05) increase in average daily gain (ADG) and improved feed conversion ratio (P < 0.05) were observed in MCFA during the grower phase (d 14-28), and a significant (P < 0.05) increase in average daily feed intake (ADFI) was observed in MCFA during d 0 to 28. Broilers fed HF-LP had a significantly (P < 0.05) higher FCR and lower ADG throughout the rearing period. No treatment effects were found on footpad dermatitis, but BUT had worst hock burn scores at d 35 (P < 0.01) and MCFA had worst cleanliness scores at d 21 but not at d 35 (treatment*age P < 0.05), while INU had better cleanliness as compared to CON at d 35 (P < 0.05). In conclusion, especially BUT and HF-LP were able to modulate resident microbiota and BUT also increased cloacal endotoxin levels, which was opposite to our hypothesis. The present study indicates that cloacal endotoxin release can be affected by the diet but further study is needed to find dietary treatments that can reduce cloacal endotoxin release.

Endotoxic Septic Shock: Diagnosis and Treatment.

Endotoxin, also referred to as lipopolysaccharide (LPS), is a potent stimulator of the inflammatory cascade which may progress to sepsis and septic shock. The term endotoxic septic shock has been used for patients who have a clinical phenotype that is characterized by high endotoxin activity in addition to a high burden of organ failure; especially a pattern of organ failure including hepatic dysfunction, acute kidney injury, and various forms of endothelial dysfunction. Endotoxic septic shock has been a target for drug therapy for decades with no success. A likely barrier to their success was the inability to quantify endotoxin in the bloodstream. The Endotoxin Activity Assay (EAA) is positioned to change this landscape. In addition, medical devices using adsorptive technology in an extra-corporeal circulation has been shown to remove large quantities of endotoxin from the bloodstream. Focusing on the use of EAA to determine high concentrations of endotoxin will allow patients with endotoxic septic shock to be identified quickly and these patients may benefit most from removal of endotoxin using extracorporeal methods.

Endotoxin reduction from biotech silk material inhibits the production of anti-silk antibodies in mice.

Eliminating endotoxins is a common problem in the development of biotechnologically produced pharmaceuticals or biomaterials. Residual endotoxins in the final sample may hamper the properties of the product or induce severe adverse effects. Developing an effective downstream purification protocol that ensures a lack of minimal endotoxin content in the final product can be a challenging task. In our previous studies, we developed nanospheres produced from bioengineered silks. Despite their good overall biocompatibility, in vivo characterization of spheres showed mild activation of the immune system (mainly in terms of anti-silk antibody production). Herein, we examined, if the endotoxins delivered with the silk spheres might have contributed to activating the adaptive immune response. We investigated various commercially available methods for endotoxin removal that can be applied as an extra step in downstream endotoxin removal from MS1-type silk proteins. We selected a method that allowed for a 10-fold reduction of endotoxin content in soluble silk and 2-fold in the final product (silk spheres). The reduced level of endotoxins improved the biocompatibility of the silk spheres as these particles induced negligible titers of anti-silk antibodies in an in vivo immune study. Since endotoxins can enhance life-threatening immune responses, it is crucial to optimize the method of their removal before clinical use not only of silk-based products but also of other biomolecules produced biotechnologically.

Respiratory exposure to agricultural dust extract promotes increased intestinal Tnfα expression, gut barrier dysfunction, and endotoxemia in mice.

Concentrated animal feeding operations (CAFOs) are responsible for the production of global greenhouse gases and harmful environmental pollutants including hydrogen sulfide, ammonia, and particulate matter. Swine farmers are frequently exposed to organic dust that is proinflammatory in the lung and are thus at greater risk of developing pneumonia, asthma, and other respiratory conditions. In addition to respiratory disease, air pollutants are directly associated with altered gastrointestinal (GI) physiology and the development of GI diseases, thereby highlighting the gut-lung axis in disease progression. Instillation of hog dust extract (HDE) for 3 wk has been reported to promote the development of chronic airway inflammation in mice, however, the impact of HDE exposure on intestinal homeostasis is poorly understood. We report that 3-wk intranasal exposure of HDE is associated with increased intestinal macromolecule permeability and elevated serum endotoxin concentrations in C57BL/6J mice. In vivo studies also indicated mislocalization of the epithelial cell adhesion protein, E-cadherin, in the colon as well as an increase in the proinflammatory cytokine, Tnfα, in the proximal colon. Moreover, mRNA expression of the Paneth cell-associated marker, Lyz1, was increased the proximal colon, whereas the expression of the goblet cell marker, Muc2, was unchanged in the epithelial cells of the ileum, cecum, and distal colon. These results demonstrate that airway exposure to CAFOs dusts promote airway inflammation and modify the gastrointestinal tract to increase intestinal permeability, induce systemic endotoxemia, and promote intestinal inflammation. Therefore, this study identifies complex physiological consequences of chronic exposure to organic dusts derived from CAFOs on the gut-lung axis.NEW & NOTEWORTHY Agricultural workers have a higher prevalence of occupational respiratory symptoms and are at greater risk of developing respiratory diseases. However, gastrointestinal complications have also been reported, yet the intestinal pathophysiology is understudied. This work is novel because it emphasizes the role of an inhaled environmental pollutant on the development of intestinal pathophysiological outcomes. This work will provide foundation for other studies evaluating how agricultural dusts disrupts host physiology and promotes debilitating gastrointestinal and systemic disorders.

The association of early-life household endotoxin exposure with asthma, recurrent wheeze, and allergic sensitization in Canadian children 3 years of age

Asthma and allergies are the leading chronic illnesses among children in Canada, causing a significant burden on healthcare systems and negatively impacting the quality of life of children and their families. Currently, the association between asthma, wheeze, and atopy development and early-life exposure to endotoxin is not fully understood. Data from the CHILD Cohort Study were analyzed using multivariate logistic regression modelling to determine whether an association exists between household early-life endotoxin exposure measured in house dust and asthma, wheeze, and allergy development at 3 years of age. The models were adjusted for covariates relating to the child’s home environment, demographics and socioeconomic status. Those with higher household endotoxin concentrations showed lower odds of allergic sensitization at 3 years of age (OR 0.49, p=0.07 and OR 0.54, p=0.11) than those with the lowest household exposure. Sex stratification found that this relationship was specific to boys. No relationship was found between endotoxin exposure and recurrent wheeze at 3 years of age. Girls in homes with the highest exposure had lower odds of developing asthma by age 3 (p=0.10). These findings suggest endotoxin exposure in early life may protect against allergy at age 3 in Canadian children, particularly boys. Endotoxin is a measure of gram-negative bacteria but may be associated with the presence of ‘good’ microbes in the home environment as well. These findings are consistent with the hygiene hypothesis and encourage more research on early-life microbiome abundance and diversity.

Effects of combined application of benzoic acid and 1-monolaurin on growth performance, nutrient digestibility, gut microbiome and inflammatory factor levels in weaned piglets

BackgroundOur previous study observed that benzoic acid and 1-monolaurin have a synergistic bactericidal effect. Moreover, their improvement effect of benzoic acid and 1-monolaurin on the growth performance and diarrhea of weaned piglets was better than the two feedings alone. However, it is not clear how the combination of benzoic acid and 1-monolaurin affects the growth performance of weaned piglets. Therefore, 100 weaned piglets (mean weight 7.03 ± 1.04 kg, mean weaning age 26 d) were randomly divided into two groups: (1) basal diet control (CON); (2) basal diet supplemented with 0.6% benzoic acid and 0.1% 1-monolaurin (CA). The experiment lasted 28 days after weaning. The effects of benzoic acid and 1-monolaurin supplementation on growth performance, apparent nutrient digestibility, intestinal flora composition and function, and inflammatory factor levels of weaned piglets were investigated.ResultsThe feed conversion efficiency of piglets in the CA group between 15 and 28 d and 1 and 28 d after weaning was significantly higher than that in the CON group (P < 0.05). Additionally, the diarrhea proportion and frequency of piglets in the CA group 1–14 days post-weaning were significantly decreased (P < 0.05). The apparent digestibility of dry matter, organic matter and crude protein of piglets in the CA group was significantly higher than the CON group on days 14 and 28 (P < 0.05). The microbial composition in the cecal digesta of piglets was detected. The results indicated that the CA group piglets were significantly supplemented with g_YRC22 at day 14 and g_Treponema, g_Pseudomonas, and g_Lachnobacterium at day 28 (P < 0.05; log LDA > 2). No significant difference was observed between the CON and CA groups in the content of short-chain fatty acids. In addition, serum IL−1β level significantly decreased at day 28 in the CA group compared with the CON group, while serum endotoxin content was significantly reduced at day 14.ConclusionTherefore, dietary supplementation of 0.6% benzoic acid and 0.1% 1-monolaurin enhanced growth performance and nutrient digestibility, affected gut microflora composition, and decreased systemic inflammatory response and intestinal permeability of weaned piglets. These outcomes provide a theoretical basis for applying of benzoic acid and 1-monolaurin over weaned piglets.

IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction.

Microbial resistance to antibiotics poses a tremendous challenge. Bacteriophages may provide a useful alternative or adjunct to traditional antibiotics. To be used in therapy, bacteriophages need to be purified from endotoxins and tested for their effects on human immune cells. Interleukin-1 Receptor Associated Kinase-3 (IRAK3) is a negative regulator of inflammation and may play a role in the modulation of immune signalling upon bacteriophage exposure to immune cells. This study aimed to investigate the immune effects of crude and purified bacteriophage FNU1, a bacteriophage that targets the oral pathobiont Fusobacterium nucleatum, on wildtype and IRAK3 knockout THP-1 monocytic cell lines. The IRAK3 knockout cell line was also used to develop a novel endotoxin detection assay. Exposure to crude FNU1 increased the production of pro-inflammatory cytokines (Tumour necrosis factor - alpha (TNF-α) and Interleukin 6 (IL-6)) compared to purified FNU1 in wildtype and IRAK3 knockout THP-1 monocytes. In the IRAK3 knockout THP-1 cells, exposure to crude FNU1 induced a higher immune response than the wildtype monocytes, supporting the suggestion that the inhibitory protein IRAK3 regulates reactions to endotoxins and impurities in bacteriophage preparations. Finally, the novel endotoxin detection assay generated here provides a robust and accurate method for determining endotoxin concentrations.

Effects of Different Container Types on (1→3)-β-D-glucan Recovery.

It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1→3)-β-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1→3)-β-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1→3)-β-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1→3)-β-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1→3)-β-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1→3)-β-D-glucans.

Cry toxin expression in different plant parts of Bt cotton at different phenological stages

BackgroundCompared to Bollgard-I, the utilization of Cry2Ab protein in Bollgard-II cotton cultivars enhances insect control. Field and laboratory studies demonstrated reduction in the numbers of bollworm-infested terminals, squares, and bolls in Bollgard-II cotton lines when compared to both Bollgard-I and non-Bollgard cotton cultivars. This indicates that the combination of Cry2Ab with Cry1Ac increased the overall expression of proteins. The expression of Cry protein varied across different plant parts, such as leaves, bracts, squares, and bolls. As the season progresses, the expression of Cry protein decreased in these plant parts. Leaves exhibited the highest levels of Cry protein expression, followed by squares, flowers, and bolls. Variation in the expression levels of delta endotoxins in different plant parts was one of the contributing factors to the survival of pest populations on Bt cotton.ResultsUsing a commercially available QL 96 ELISA plate kit, the concentration of delta endotoxin in various plant parts at different phenological stages was determined in twelve BG-II cotton hybrids, namely Ajeet-155, JKCH-2245, RCH-3863, NCS-866, MRC-7373, JKCH-99, MRC-7387, NCEH-21, ANKR-3324, NCSI-1904, and NCHB 9902. Cry1Ac and Cry2Ab protein levels were determined from samples of flowers and fruiting parts (Rind, locule, seed, locule wall and seed) at 40, 75, 100 and 125 days after sowing (DAS) over two consecutive years 2018–2019 and 2019–2020. Cry1Ac protein content and expression was the highest at 100DAS in locule, seed and rind; followed by rind and locule wall and seed in green bolls at 125 DAS; followed by seed, locule, rind and flowers at 75 DAS; followed by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry2Ab protein content and expression was the highest at 125DAS in locule wall and seed and rind, followed by seed, locule and rind in green bolls at 100 DAS, followed by seed, locule, rind and flowers at 75 DAS, then by flowers at 40 DAS in all the tested twelve Bollgard-II hybrids. Cry1Ac protein expression was less in comparison with Cry2Ab.ConclusionsThe research findings indicate that the locule of BG-II cotton plants exhibited the highest expression levels of Cry1Ac and Cry2Ab proteins, followed by the seeds, rind, locule wall, and flowers. These results provide valuable insights into the distribution of Cry protein expression in different plant parts, which can contribute to a better understanding of insect control in Bt cotton cultivars.

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

Metagenomic signatures reveal the key role of phloretin in amelioration of gut dysbiosis attributed to metabolic dysfunction-associated fatty liver disease by time-dependent modulation of gut microbiome.

The importance of gut-liver axis in the pathophysiology of metabolic dysfunction-associated fatty liver disease (MAFLD) is being investigated more closely in recent times. However, the inevitable changes in gut microbiota during progression of the disease merits closer look. The present work intends to assess the time-dependent gut dysbiosis in MAFLD, its implications in disease progression and role of plant-derived prebiotics in its attenuation. Male C57BL/6J mice were given western diet (WD) for up to 16 weeks and phloretin was administered orally. The fecal samples of mice were collected every fourth week for 16 weeks. The animals were sacrificed at the end of the study and biochemical and histological analyses were performed. Further, 16S rRNA amplicon sequencing analysis was performed to investigate longitudinal modification of gut microbiome at different time points. Findings of our study corroborate that phloretin alleviated the metabolic changes and mitigated circulating inflammatory cytokines levels. Phloretin treatment resists WD induced changes in microbial diversity of mice and decreased endotoxin content. Prolonged exposure of WD changed dynamics of gut microbiota abundance and distribution. Increased abundance of pathogenic taxa like Desulfovibrionaceae, Peptostreptococcus, Clostridium, and Terrisporobacter was noted. Phloretin treatment not only reversed this dysbiosis but also modulated taxonomic signatures of beneficial microbes like Ruminococcus, Lactobacillus, and Alloprevotella. Therefore, the potential of phloretin to restore gut eubiosis could be utilized as an intervention strategy for the prevention of MAFLD and related metabolic disorders.

Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells.

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

P14-07: Renal injury from inhalation exposure to atmospheric endotoxin concentrations in a rat model

P14-07: Renal injury from inhalation exposure to atmospheric endotoxin concentrations in a rat model

Spatial and temporal distribution of endotoxins, antibiotic resistance genes and mobile genetic elements in the air of a dairy farm in Germany

Antimicrobial resistance (AMR) is a serious issue that is continuously growing and spreading, leading to a dwindling number of effective treatments for infections that were easily treatable with antibiotics in the past. Animal farms are a major hotspot for AMR, where antimicrobials are often overused, misused, and abused, in addition to overcrowding of animals.In this study, we investigated the risk of AMR transmission from a farm to nearby residential areas by examining the overall occurrence of endotoxins, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) in the air of a cattle farm. We assessed various factors, including the season and year, day and nighttime, and different locations within the farm building and its vicinity.The most abundant ARGs detected were tetW, aadA1, and sul2, genes that encode for resistances towards antibiotics commonly used in veterinary medicine. While there was a clear concentration gradient for endotoxin from the middle of the farm building to the outside areas, the abundance of ARGs and MGEs was relatively uniform among all locations within the farm and its vicinity. This suggests that endotoxins preferentially accumulated in the coarse particle fraction, which deposited quickly, as opposed to the ARGs and MGEs, which might concentrate in the fine particle fraction and remain longer in the aerosol phase. The occurrence of the same genes found in the air samples and in the manure indicated that ARGs and MGEs in the air mostly originated from the cows, continuously being released from the manure to the air.Although our atmospheric dispersion model indicated a relatively low risk for nearby residential areas, farm workers might be at greater risk of getting infected with resistant bacteria and experiencing overall respiratory tract issues due to continuous exposure to elevated concentrations of endotoxins, ARGs and MGEs in the air of the farm.

Dietary High Glycinin Reduces Growth Performance and Impairs Liver and Intestinal Health Status of Orange-Spotted Grouper (Epinephelus coioides).

The aim of the study was to investigate whether the negative effects of dietary glycinin are linked to the structural integrity damage, apoptosis promotion and microbiota alteration in the intestine of orange-spotted grouper (Epinephelus coioides). The basal diet (FM diet) was formulated to contain 48% protein and 11% lipid. Fish meal was replaced by soybean meal (SBM) in FM diets to prepare the SBM diet. Two experimental diets were prepared, containing 4.5% and 10% glycinin in the FM diets (G-4.5 and G-10, respectively). Triplicate groups of 20 fish in each tank (initial weight: 8.01 ± 0.10 g) were fed the four diets across an 8 week growth trial period. Fish fed SBM diets had reduced growth rate, hepatosomatic index, liver total antioxidant capacity and GSH-Px activity, but elevated liver MDA content vs. FM diets. The G-4.5 exhibited maximum growth and the G-10 exhibited a comparable growth with that of the FM diet group. The SBM and G-10 diets down-regulated intestinal tight junction function genes (occludin, claudin-3 and ZO-1) and intestinal apoptosis genes (caspase-3, caspase-8, caspase-9, bcl-2 and bcl-xL), but elevated blood diamine oxidase activity, D-lactic acid and endotoxin contents related to intestinal mucosal permeability, as well as the number of intestinal apoptosis vs FM diets. The intestinal abundance of phylum Proteobacteria and genus Vibrio in SBM diets were higher than those in groups receiving other diets. As for the expression of intestinal inflammatory factor genes, in SBM and G-10 diets vs. FM diets, pro-inflammatory genes (TNF-α, IL-1β and IL-8) were up-regulated, but anti-inflammatory genes (TGF-β1 and IL-10) were down-regulated. The results indicate that dietary 10% glycinin rather than 4.5% glycinin could decrease hepatic antioxidant ability and destroy both the intestinal microbiota profile and morphological integrity through disrupting the tight junction structure of the intestine, increasing intestinal mucosal permeability and apoptosis. These results further trigger intestinal inflammatory reactions and even enteritis, ultimately leading to the poor growth of fish.