Aptasensor based on gold nanostructure-decorated 2D Cu metal-organic framework nanosheets for highly sensitive and specific electrochemical lipopolysaccharide detection.

Detecting lipopolysaccharide (LPS) using electrochemical methods is significant because of their exceptional sensitivity, simplicity, and user-friendliness. Two-dimensional metal-organic framework (2D-MOF) that merges the benefits of MOF and 2D nanostructure has exhibited remarkable performance in constructing electrochemical sensors, notably surpassing traditional 3D-MOFs. In this study, Cu[tetrakis(4-carboxylphenyl)porphyrin] (Cu-TCPP) and Cu(tetrahydroxyquinone) (Cu-THQ) 2D nanosheets were synthesized and applied on a glassy carbon electrode (GCE). The 2D-MOF nanosheets, which serve as supporting layers, exhibit improved electron transfer and electronic conductivity characteristics. Subsequently, the modified electrode was subjected to electrodeposition with Au nanostructures, resulting in the formation of Au/Cu-TCPP/GCE and Au/Cu-THQ/GCE. Notably, the Au/Cu-THQ/GCE demonstrated superior electrochemical activity because of the 2D morphology, redox ligand, dense Cu sites, and improved deposition of flower-like Au nanostructure based on Cu-THQ. The electron transfer specific surface area was increased by the improved deposition of Au nanostructures, which facilitates enriched binding of LPS aptamer and significantly improved the detection performance of Apt/Au/Cu-THQ/GCE electrochemical aptasensor. The limit of detection for LPS reached 0.15 fg/mL with a linear range of 1 fg/mL - 100 pg/mL. The proposed aptasensor demonstrated the ability to detect LPS in serum samples with satisfactory accuracy, indicating significant potential for clinical diagnosis.

Boronic functionalized aptamer macroarrays with dual-recognition and isothermal amplification of lipopolysaccharide detection.

A highly sensitive dual-recognition fluorescence amplification method is presented for lipopolysaccharide (LPS) detection based on boronic functionalized aptamer macroarrays with dual-recognition and isothermal amplification. The surface of the polystyrene microplate was firstly carboxylated, and then, 3-aminophenylboronic acid was conjugated to the carboxyl groups through EDC/NHS reaction, creating boronic acid groups as the capture moiety for LPS. A recognition DNA aptamer labeled with the fluorescent dye 6-FAM, which exhibits specificity towards LPS, was selected as the signal reporting moiety. By introducing primers and Klenow enzyme, the fluorescent-labeled aptamers are released from the microplate bottom, and double-stranded structures were formed via isothermal amplification. The addition of SYBR Green I, which strongly fluoresces upon binding to the double-stranded structures, enables signal amplification and detection. This detection method exhibits a linear range of 1-10,000 ng/mL and has a detection limit as low as 401.93 pg/mL. This analytical approach shows high selectivity and sensitivity and may serve as a universal platform in lipopolysaccharide detection.

Development and preliminary evaluation toward a new tuberculosis treatment monitoring tool: the PATHFAST TB LAM Ag assay.

The PATHFAST TB LAM Ag assay is based on a chemiluminescent enzyme immunoassay to quantify lipoarabinomannan (LAM) in sputum within 1 h, and was developed as an alternative to conventional culture methods for monitoring tuberculosis (TB) treatment. This study aimed to evaluate the analytical performance and initial clinical feasibility of using five Mycobacterium tuberculosis variants, 178 non-tuberculous mycobacteria (NTM), 34 upper respiratory and oral cavity microorganisms, 100 sputum specimens from untreated patients, and potential interfering substances, including 27 drugs. The results reveled a single-site repeatability coefficient of variation (CV) of 5.2%-7.0%, and a multi-site reproducibility CV of 7.1%-8.4%. The limit of blank, limit of detection, and limit of quantification were 3.03 pg/mL, 6.67 pg/mL, and 7.44 pg/mL, respectively. Linearity was observed over the analytical measurement range (10.0 pg/mL-50,000 pg/mL), and no hook effect was observed. The assay tended to cross-react with slow-growing NTMs, but not with common upper respiratory and oral cavity microorganisms, except Nocardia asteroides, Nocardia farcinica, and Tsukamurella paurometabola. No interference was observed in the presence of mucin, blood, or major anti-TB, anti-HIV, and anti-pneumonia drugs. Regarding clinical performance, the assay had a sensitivity of 88.8% (95% CI: 80.0%-94.0%) and specificity of 100.0% (95% CI: 83.9%-100.0%) using mycobacterial culture as the reference standard, and a correlation (Spearman's r = -0.770) was observed between LAM concentration and time to detection of culture. These findings show, for the first time, that the PATHFAST TB LAM Ag assay has potential value for monitoring TB treatment.

Novel aptasensor based on polyaniline functionalized carboxylated dobby carbon nanotubes and molybdenum disulfide for endotoxin detection.

Endotoxins, also known as lipopolysaccharides (LPS), are present within the cell walls of Gram-negative bacteria and are released upon cellular death, which can pose a significant risk to human and animal health. Due to the minimal amount of endotoxin required to trigger an inflammatory response in human body, the demand for sensitive methods with low endotoxin detection limits is essential necessary. This paper presents a straightforward aptamer sensor which can enhance the conductivity and specific surface area of molybdenum disulfide (MoS2) by incorporating carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) and polyaniline (PANI). Doping with gold nanoparticles (AuNPs) improves biocompatibility and sensitivity while providing binding sites for thiolated endotoxin-binding aptamers (LBA). This biosensor achieved a remarkable detection limit as low as 0.5 fg mL-1, enabling trace-level identification of LPS. It also exhibits excellent repeatability, selectivity, and stability, facilitating rapid and accurate LPS detection. Moreover, this method demonstrates high recovery rates and specificity for LPS analysis in food samples, showcasing its promising application prospects in trace-level LPS detection within the food industry.

Novel automated AIMLAM for diagnosis of Mycobacterium tuberculosis.

Aim: A rapid and precise diagnostic method is crucial for timely intervention and management of tuberculosis. The present study compared the diagnostic accuracy of a novel lipoarabinomannan (LAM) antigen test, AIMLAM, for tuberculosis in urine samples. Methodology: The study subjected 106 TB suspects to smear microscopy, MGIT, GeneXpert and AIMLAM. Results: Among 106, smear microscopy identified 36 as positive (33%) (sensitivity; 70.93%, 95% CI (60.14-80.22%), while MGIT showed 38 positive (36.8%). GeneXpert detected 59 positives (sensitivity; 96.83, 95% CI (89.00-99.61%)). AIMLAM declared 61 as positive (57.5%) (sensitivity; 100.00, 95% CI (94.13-100.00%) and 45 as negative (42.5%). Conclusion: Overall, AIMLAM demonstrated better diagnostic accuracy than GeneXpert Assay, smear microscopy and MGIT liquid culture in urine samples.

This study describes a new way to detect tuberculosis, called AIMLAM. Unlike traditional methods that use sputum or blood, AIMLAM tests urine samples and bodily fluids. It is automated and uses easily accessible samples to identify a tuberculosis infection, so may be a convenient and noninvasive option for healthcare providers. The test shows promising results in terms of accuracy and sensitivity.

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

Systemic and local responses of cytokines and tissue histology following intramammary lipopolysaccharide challenge in dairy cows.

During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1ß showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1ß, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1ß, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1ß, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue.

Exhaled breath particles as a novel tool to study lipid composition of epithelial lining fluid from the distal lung.

BACKGROUND: Surfactant phospholipid (PL) composition plays an important role in lung diseases. We compared the PL composition of non-invasively collected exhaled breath particles (PEx) with bronchoalveolar lavage (BAL) and induced sputum (ISP) at baseline and following endotoxin (LPS) challenges. METHODS: PEx and BAL were collected from ten healthy nonsmoking participants before and after segmental LPS challenge. Four weeks later, PEx and ISP were sampled in the week before and after a whole lung LPS inhalation challenge. PL composition was analysed using mass spectrometry. RESULTS: The overall PL composition of BAL, ISP and PEx was similar, with PC(32:0) and PC(34:1) representing the largest fractions in all three sample types (baseline PC(32:0) geometric mean mol%: 52.1, 56.9, and 51.7, PC(34:1) mol%: 11.7, 11.9 and 11.4, respectively). Despite this similarity, PEx PL composition was more closely related to BAL than to ISP. For most lipids comparable inter-individual differences in BAL, ISP, and PEx were found. PL composition of PEx was repeatable. The most pronounced increase following segmental LPS challenge was detected for SM(d34:1) in BAL (0.24 to 0.52 mol%) and following inhalation LPS challenge in ISP (0.45 to 0.68 mol%). An increase of SM(d34:1) following segmental LPS challenge was also detectable in PEx (0.099 to 0.103 mol%). The inhalation challenge did not change PL composition of PEx. CONCLUSION: Our data supports the peripheral origin of PEx. The lack of PL changes in PEx after inhalation challenge might to be due to the overall weaker response of inhaled LPS which primarily affects the larger airways. Compared with BAL, which always contains lining fluid from both peripheral lung and central airways, PEx analysis might add value as a selective and non-invasive method to investigate peripheral airway PL composition. TRIAL REGISTRATION: NCT03044327, first posted 07/02/2017.

Pharmacological blockade of the interleukin-1 receptor suppressed Escherichia coli lipopolysaccharide-induced neuroinflammation in preterm fetal sheep.

BACKGROUND: Intraamniotic inflammation is associated with preterm birth, especially in cases occurring before 32 weeks' gestation, and is causally linked with an increased risk for neonatal mortality and morbidity. Targeted anti-inflammatory interventions may assist in improving the outcomes for pregnancies impacted by intrauterine inflammation. Interleukin-1 is a central upstream mediator of inflammation. Accordingly, interleukin-1 is a promising candidate target for intervention therapies and has been targeted previously using the interleukin-1 receptor antagonist, anakinra. Recent studies have shown that the novel, noncompetitive, allosteric interleukin-1 receptor inhibitor, rytvela, partially resolved inflammation associated with preterm birth and fetal injury. In this study, we used a preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela and anakinra, administered in the amniotic fluid in the setting of intraamniotic Escherichia coli lipopolysaccharide exposure. OBJECTIVE: We hypothesized that both rytvela and anakinra would reduce lipopolysaccharide-induced intrauterine inflammation and protect the fetal brain. STUDY DESIGN: Ewes with a singleton fetus at 105 days of gestation (term is ∼150 days) were randomized to one of the following groups: (1) intraamniotic injections of 2 mL saline at time=0 and time=24 hours as a negative control group (saline group, n=12); (2) intraamniotic injection of 10 mg Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2 mL saline at time=0 hours and time=24 hours as an inflammation positive control group (lipopolysaccharide group, n=11); (3) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2.5 mg rytvela at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of rytvela (lipopolysaccharide + rytvela group, n=10); or (4) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 100 mg anakinra at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of anakinra (lipopolysaccharide + anakinra group, n=12). Amniotic fluid was sampled at time 0, 24, and 48 hours (ie, at each intervention and at delivery). Fetal umbilical cord blood was collected at delivery for differential blood counts and chemical studies. Inflammation was characterized by the analysis of fetal tissue cytokine and chemokine levels using quantitative polymerase chain reaction, enzyme-linked inmmunosorbent assay, and histology. The primary study outcome of interest was the assessment of anakinra and rytvela brain-protective effects in the setting of Escherichia coli lipopolysaccharide-induced intrauterine inflammation. Secondary outcomes of interest were to assess protection from fetal and intrauterine (ie, amniotic fluid, chorioamnion) inflammation. RESULTS: Intraamniotic administration of lipopolysaccharide caused inflammation of the fetal lung, brain, and chorioamnionitis in preterm fetal sheep. Relative to treatment with saline only in the setting of lipopolysaccharide exposure, intraamniotic administration of both rytvela and anakinra both significantly prevented periventricular white matter injury, microglial activation, and histologic chorioamnionitis. Anakinra showed additional efficacy in inhibiting fetal lung myeloperoxidase activity, but its use was associated with metabolic acidaemia and reduced fetal plasma insulin-like growth factor-1 levels at delivery. CONCLUSION: Intraamniotic administration of rytvela or anakinra significantly inhibited fetal brain inflammation and chorioamnionitis in preterm fetal sheep exposed to intraamniotic lipopolysaccharide. In addition, anakinra treatment was associated with potential negative impacts on the developing fetus.

Proteinase K Pretreatment for the Quantitative Recovery and Sensitive Detection of the Tuberculosis Biomarker Mannose-Capped Lipoarabinomannan Spiked into Human Serum.

This paper reports on an investigation of an enzymatic pretreatment protocol using proteinase K (ProK) for the analysis of human serum samples spiked with mannose-capped lipoarabinomannan (ManLAM). ManLAM is an antigenic biomarker found in the serum, urine, and other body fluids of individuals infected with tuberculosis (TB). Immunometric measurements of ManLAM are compromised by steric effects due to its complexation with high-molecular-weight components in these matrices that interfere with its capture and/or labeling. Recent work has shown that deproteinization of these types of samples by perchloric acid acidification or ProK digestion releases ManLAM from complexation. Releasing ManLAM greatly improves its detectability and, as a result, its utility as a TB biomarker. The work detailed herein examined how different ProK reaction conditions (e.g., enzyme concentration and digestion time and temperature) affect the recovery and detectability of ManLAM in human serum. As measured by enzyme-linked immunosorbent assay (ELISA), we show that using the optimal set of digestion conditions to free ManLAM, which also yield a small, quantitatively reproducible level of sample concentration, it is possible to achieve a spiked ManLAM recovery of 98 ± 13% and a limit of detection of 10 pg/mL (0.6 pM). Experiments also demonstrated that the ELISA responses measured for a given ManLAM concentration in serum after pretreatment were statistically indistinguishable from those directly determined for the same amounts of ManLAM added to an innocuous buffered solution. Possible adaptations of the digestion protocol for use in point-of-care TB testing are also briefly discussed.

Foodomics revealed the effects of ultrasonic extraction on the composition and nutrition of cactus fruit (Opuntia ficus-indica) seed oil.

Cactus is a tropical fruit with a high nutritional value; however, little information is available regarding the comprehensive utilization of its byproducts. This study aimed to explore the composition and nutritional value of cactus fruit seed oil (CFO) and reveal the effects of ultrasound-assisted extraction and traditional solvent extraction on oil quality. Foodomics analysis showed that CFO extracted using a traditional solvent is rich in linolenic acid (9c12cC18:2, 57.46 ± 0.84 %), α-tocopherol (20.01 ± 1.86 mg/100 g oil), and canolol (200.10 ± 1.21 µg/g). Compared to traditional solvent extraction, ultrasound-assisted extraction can significantly increase the content of lipid concomitants in CFO, whereas excessive ultrasound intensity may lead to the oxidation of oils and the formation of free radicals. Analysis of the thermal properties showed that ultrasound had no effect on the crystallization or melting behavior of CFO. To further demonstrate the nutritional value of CFO, a lipopolysaccharide (LPS)-induced lipid metabolism imbalance model was used. Lipidomics analysis showed that CFO significantly reduced the content of oxidized phospholipids stimulated by LPS and increased the content of highly bioactive metabolites such as ceramides, thus alleviating LPS-induced damage in C. elegans. Hence, CFO is a functional oil with high value, and ultrasound-assisted extraction is advocated. These findings provide new insights into the comprehensive utilization of cactus fruits.

Effects of replacing inorganic salts of trace minerals with organic trace minerals in the diet of prepartum cows on quality of colostrum and immunity of newborn calves.

Our objectives were to evaluate the impact of supplementary trace mineral (TM) form-inorganic salts (STM; Co, Cu, Mn, Zn sulfates, and Na selenite) or organic (OTM; Co, Cu, Mn, Zn proteinates, and selenized yeast)-in the prepartum diet on quantity and quality of colostrum, passive immunity, antioxidant biomarkers, cytokine responses to lipopolysaccharide (LPS), health, and growth of newborn calves. Pregnant heifers (n = 100) and cows (n = 173) were enrolled at 45 d before calving, blocked by parity and body condition score, and allocated randomly to STM (50 heifers; 86 cows) or OTM (50 heifers; 87 cows) supplementation. Cows in both treatments were fed the same diet, except for the source of supplementary TM. Within 2 h of calving, dams and calves were separated, colostrum was harvested, the yield was measured, and a sample was saved for posterior analyses of colostrum quality. A subgroup of calves (n = 68) had a blood sample collected before colostrum feeding. After colostrum feeding, all samples and data collection were limited to 163 calves (STM = 82; OTM = 81) fed 3 L of good quality (Brix% >22) maternal colostrum via nipple bottle minutes after harvesting. Concentration of IgG in colostrum and serum was determined 24 h after colostrum feeding using radial immunodiffusion. Concentration of TM in colostrum and serum were performed by inductively coupled plasma mass spectrometry. Activity of glutathione peroxidase, ferric reducing ability of plasma, and concentration of superoxide dismutase were evaluated in plasma by colorimetric assays. Ex vivo whole blood stimulation with LPS was performed on d 7 of life to evaluate cytokine responses in a subgroup of 66 calves. Health events were recorded from birth to weaning, and body weight was recorded at birth (all calves) and on d 30 and 60 (heifers only). Continuous variables were analyzed by ANOVA and binary responses were analyzed by logistic regression. Complete replacement of STM by OTM in prepartum diet resulted in greater concentration of Se (461 vs. 543 ± 7 µg/g; ± SEM) but did not alter the concentration or total mass of other TM and IgG in colostrum. Female calves of the OTM group had greater concentration of Se in serum at birth (0.23 vs. 0.37 ± 0.05 µg/mL), were lighter in weight at birth (40.9 vs. 38.8 ± 0.6 kg) and weaning (93.2 vs. 89.7 ± 1.6 kg) than those of the STM group. Maternal treatments did not affect passive immunity or antioxidant biomarkers. On d 7, basal concentrations (log10 of concentration in pg/mL) of IFNγ (0.70 vs. 0.95 ± 0.083) and LPS-stimulated concentrations of CC chemokine ligand 2 (CCL2; 2.45 vs. 2.54 ± 0.026), CC chemokine ligand 3 (CCL3; 2.63 vs. 2.76 ± 0.038), IL-1α (2.32 vs. 2.49 ± 0.054), and IL-1ß (3.62 vs. 3.86 ± 0.067) were greater in OTM than in STM. Supplementation with OTM in pregnant heifers, but not in pregnant cows, reduced the incidence of preweaning health problems in their calves (36.4 vs. 11.5%). Complete replacement of STM by OTM in the prepartum diet did not cause major changes in colostrum quality, passive immunity, and antioxidant capacity, but increased cytokine and chemokine responses to LPS on d 7 of life and benefited preweaning health of calves born to primiparous cows.

Performance of Tuberculosis Molecular Bacterial Load Assay Compared to Alere TB-LAM in Urine of Pulmonary Tuberculosis Patients with HIV Co-Infections.

Alternative tools are needed to improve the detection of M. tuberculosis (M. tb) in HIV co-infections. We evaluated the utility of Tuberculosis Molecular Bacterial Load Assay (TB-MBLA) compared to lipoarabinomannan (LAM) to detect M. tb in urine. Sputum Xpert MTB/RIF-positive patients were consented to provide urine at baseline, weeks 2, 8, 16, and 24 of treatment for TB-MBLA, culture, and LAM. Results were compared with sputum cultures and microscopy. Initial M. tb. H37Rv spiking experiments were performed to validate the tests. A total of 63 urine samples from 47 patients were analyzed. The median age (IQR) was 38 (30-41) years; 25 (53.2%) were male, 3 (6.5%) had urine for all visits, 45 (95.7%) were HIV positive, of whom 18 (40%) had CD4 cell counts below 200 cells/µL, and 33 (73.3%) were on ART at enrollment. Overall urine LAM positivity was 14.3% compared to 4.8% with TB-MBLA. Culture and microscopy of their sputum counterparts were positive in 20.6% and 12.7% of patients, respectively. Of the three patients with urine and sputum at baseline, one (33.33%) had urine TB-MBLA and LAM positive compared to 100% with sputum MGIT culture positive. Spearman's rank correction coefficient (r) between TB-MBLA and MGIT was -0.85 and 0.89 with a solid culture, p > 0.05. TB-MBLA has the promising potential to improve M. tb detection in urine of HIV-co-infected patients and complement current TB diagnostics.

Dihydroxanthene-Based Near-infrared Fluorescent Probes for Monitoring Mitochondrial Viscosity in Living Cells and Mice.

Aberrant mitochondrial viscosity is closely associated with many diseases and cellular malfunctions. Thus, the development of reliable methods for monitoring mitochondrial viscosity variations has attracted considerable attention. Herein, through stepwise structural modulation of the dihydroxanthene fluorophore (DHX), we developed three NIR fluorescent probes, named DHX-V-1-3, for detecting mitochondrial viscosity. Among them, DHX-V-3 displayed the highest signal-to-noise ratio (67-fold) for viscosity with outstanding selectivity and showed excellent mitochondria targeting and immobilization ability. At the cellular level, the DHX-V-3 probe was successfully applied to image the mitochondrial viscosity in live cells upon treatment with lipopolysaccharide (LPS) or nystatin. Moreover, benefiting from its NIR emission and the increased depth of tissue imaging, DHX-V-3 demonstrated the ability to visualize the increased viscosity in LPS-treated mice.

Cyanobacteria, cyanotoxins and lipopolysaccharides in aerosols from inland freshwater bodies and their effects on human bronchial cells.

Components of cyanobacterial water blooms were quantified in aerosols above agitated water surfaces of five freshwater bodies. The thoracic and respirable aerosol fraction (0.1-10 µm) was sampled using a high-volume sampler. Cyanotoxins microcystins were detected by LC-MS/MS at levels 0.3-13.5 ng/mL (water) and < 35-415 fg/m3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10-119 EU/mL (water) and 0.13-0.64 EU/m3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 104-105 cells eq./mL (water) and 101-103 cells eq./m3 (aerosol). Lipopolysaccharides isolated from bloom samples induced IL-6 and IL-8 cytokine release in human bronchial epithelial cells Beas-2B, while extracted cyanobacterial metabolites induced both pro-inflammatory and cytotoxic effects. Bloom components detected in aerosols and their bioactivities observed in upper respiratory airway epithelial cells together indicate that aerosols formed during cyanobacterial water blooms could induce respiratory irritation and inflammatory injuries, and thus present an inhalation health risk.

Effect of Odanacatib on the release of NTX (Amino Terminal Telopeptide) from LPS contaminated type I dentin collagen.

OBJECTIVE: To evaluated the Odanacatib inhibitor treatment on lipopolysaccharide (LPS) contamination effect on cathepsin-K mediated dentin degradation by analysis of type I collagen C- and N-termini telopeptides. METHODS: Pulverized and disks of human dentin were demineralized and LPS contaminated, or stored in deionized water (DW) for 12 h. Samples were challenged with lactic acid (LA). Aliquots of dentin powder were treated with 1 mL Odanacatib or stored in DW for 30 min. Dentin collagen degradation was determined by sub-product release of C-terminal (ICTP and CTX) and N-terminal (NTX) telopeptides, normalized to total protein (tp) concentration (n = 3). Dentin matrix was evaluated for gravimetric (n = 8) and ultrastructural changes. Data were analyzed by Student t-test, one-way ANOVA and Tukey's test (α = 5 %). RESULTS: LA incubation significantly increased telopeptide release compared with DW (p < 0.05). In untreated groups, significantly higher CTXtp, NTXtp telopeptide rates were observed for LA+LPS samples compared with DW (p < 0.01). Odanacatib significantly reduced ICTPtp, CTXtp, and NTXtp telopeptide release for LPS, LA, and LA+LPS conditions. In untreated groups, LPS and LA+LPS challenge significantly increased dentin weight loss (p = 0.02). Within each storage condition, Odanacatib treatment did not affect weight change (p > 0.05) of dentin disks. SIGNIFICANCE: This study showed that LPS contamination resulted in significantly higher rates of NTX than CTX from dentin matrix. Odanacatib significantly reduced telopeptide release rates of LPS contaminated dentin matrix.

Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography.

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

Formula supplementation with human and bovine milk oligosaccharides modulates blood IgG and T-helper cell populations, and ex vivo LPS-stimulated cytokine production in a neonatal preclinical model.

Introduction: Human milk contains structurally diverse oligosaccharides (HMO), which are multifunctional modulators of neonatal immune development. Our objective was to investigate formula supplemented with fucosylated (2'FL) + neutral (lacto-N-neotetraose, LNnt) oligosaccharides and/or sialylated bovine milk oligosaccharides (BMOS) on immunological outcomes. Methods: Pigs (n=46) were randomized at 48h of age to four diets: sow milk replacer formula (CON), BMOS (CON + 6.5 g/L BMOS), HMO (CON + 1.0 g/L 2'FL + 0.5 g/L LNnT), or BMOS+HMO (CON + 6.5 g/L BMOS + 1.0 g/L 2'FL + 0.5 g/L LNnT). Blood and tissues were collected on postnatal day 33 for measurement of cytokines and IgG, phenotypic identification of immune cells, and ex vivo lipopolysaccharide (LPS)-stimulation of immune cells. Results: Serum IgG was significantly lower in the HMO group than BMOS+HMO but did not differ from CON or BMOS. The percentage of PBMC T-helper cells was lower in BMOS+HMO than the other groups. Splenocytes from the BMOS group secreted more IL-1ß when stimulated ex vivo with LPS compared to CON or HMO groups. For PBMCs, a statistical interaction of BMOS*HMO was observed for IL-10 secretion (p=0.037), with BMOS+HMO and HMO groups differing at p=0.1. Discussion: The addition of a mix of fucosylated and sialylated oligosaccharides to infant formula provides specific activities in the immune system that differ from formulations supplemented with one oligosaccharide structure.

Structures and Anti-Inflammatory Evaluation of Phenylpropanoid Derivatives from the Aerial Parts of Dioscorea polystachya.

Seven undescribed phenylpropanoid constituents, including three new bibenzyl derivatives (1-3) along with four new benzofuran stilbene derivatives (4-7), were isolated from the aerial parts of Dioscorea polystachya. The structures of these compounds were elucidated using a combination of spectroscopic analyses, including UV, IR, HRESIMS, 1D, and 2D NMR. Further, all the compounds were evaluated on the anti-inflammatory activity for their inhibition of nitric oxide (NO) production by RAW 264.7 macrophages cells, and some of them (1-3 and 6) displayed inhibitory activity with IC50 values in the range of 9.3-32.3 µM. Moreover, compound 3 decreased the expression of iNOS in Western blot analysis, suggesting compound 3 is mediated via the suppression of an LPS-induced NF-κB inflammasome pathway.

Cu2+-Mediated Aggregation of Gold Nanoparticles as an Optical Probe for the Detection of Endotoxin.

Endotoxins or lipopolysaccharides (LPS) present in the outer layer of Gram-negative bacteria (GNB) are responsible for bacterial toxicity. It is an environmental hazard that everyone is exposed to daily to various extents. Due to its potent toxicity, quantitative detection with very high sensitivity is essential in the food, medical, and pharmaceutical industries. Herein, we report an optical nanosensor for the rapid and sensitive detection of LPS and GNB based on the Cu2+-mediated aggregation of gold nanoparticles (Cu@AuNPs). The sensor detects LPS within a linear range of 20 ag/mL to 20 ng/mL with a lower detection limit of 0.2 ag/mL. The sensor could successfully recover spiked endotoxin in grape juice with a percentage error of ±0.2, confirming its application in the food industry. The sensor could also distinguish Gram-negative bacteria from Gram-positive bacteria, and the selectivity of the Cu@AuNP sensor toward GNB is utilized to detect Escherichia coli in wastewater. The rapid detection of E. coli without any pretreatment is a promising strategy in water analysis.