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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Sputum lipoarabinomannan (LAM) as a biomarker to determine sputum mycobacterial load: exploratory and model-based analyses of integrated data from four cohorts.

BACKGROUND: Despite the high global disease burden of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb) infection, novel treatments remain an urgent medical need. Development efforts continue to be hampered by the reliance on culture-based methods, which often take weeks to obtain due to the slow growth rate of Mtb. The availability of a "real-time" measure of treatment efficacy could accelerate TB drug development. Sputum lipoarabinomannan (LAM; an Mtb cell wall glycolipid) has promise as a pharmacodynamic biomarker of mycobacterial sputum load. METHODS: The present analysis evaluates LAM as a surrogate for Mtb burden in the sputum samples from 4 cohorts of a total of 776 participants. These include those from 2 cohorts of 558 non-TB and TB participants prior to the initiation of treatment (558 sputum samples), 1 cohort of 178 TB patients under a 14-day bactericidal activity trial with various mono- or multi-TB drug therapies, and 1 cohort of 40 TB patients with data from the first 56-day treatment of a standard 4-drug regimen. RESULTS: Regression analysis demonstrated that LAM was a predictor of colony-forming unit (CFU)/mL values obtained from the 14-day treatment cohort, with well-estimated model parameters (relative standard error ≤ 22.2%). Moreover, no changes in the relationship between LAM and CFU/mL were observed across the different treatments, suggesting that sputum LAM can be used to reasonably estimate the CFU/mL in the presence of treatment. The integrated analysis showed that sputum LAM also appears to be as good a predictor of time to Mycobacteria Growth Incubator Tube (MGIT) positivity as CFU/mL. As a binary readout, sputum LAM positivity is a strong predictor of solid media or MGIT culture positivity with an area-under-the-curve value of 0.979 and 0.976, respectively, from receiver-operator curve analysis. CONCLUSIONS: Our results indicate that sputum LAM performs as a pharmacodynamic biomarker for rapid measurement of Mtb burden in sputum, and thereby may enable more efficient early phase clinical trial designs (e.g., adaptive designs) to compare candidate anti-TB regimens and streamline dose selection for use in pivotal trials. Trial registration NexGen EBA study (NCT02371681).

Chemomechanical preparation influences the microbial community and the levels of LPS, LTA and cytokines in combined endodontic-periodontal lesions: A clinical study.

BACKGROUND: This study was conducted to compare the microbiomes, the levels of lipopolysaccharides (LPS), lipoteichoic acid (LTA), and cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]), before and after chemomechanical preparation (CMP) of the root canals (RC) and their associated periodontal pockets (PP) in teeth with combined EPL. MATERIALS: Samples were taken from 10 RC and PP, before and after CMP. The microbiomes (next-generation sequencing, V3-V4 hypervariable region of the 16S rRNA gene), microbiome diversity (bioinformatics analyses), LPS (limulus amebocyte lysate), LTA, IL-1ß, and TNF-α (ELISA) were evaluated. A statistical analysis was performed with significance level set at 5%. RESULTS: The most abundant phyla in both sites were Firmicutes and Proteobacteria. Comparative studies of bacterial genera species revealed that some increased and others decreased after CMP at both sites. A 3% reduction in Gram-negative bacteria (RC) and a 4% increase in Gram-positive bacteria (PP) were detected. LPS levels were 4.4 times higher in PP than in the RC. LTA was detected in all samples investigated. Higher levels of IL-1ß and TNF-α were detected in both sites at baseline. After CMP, LPS, LTA, IL-1ß and TNF-α were reduced in both sites. CONCLUSION: The microbial community in the RC and PP in teeth with combined EPL indicated a similarity between both sites. CMP effectively reduced the microbial load and the LPS levels from teeth with EPL, and consequently diminished the cytokine levels. The reduction in LTA levels in the RC and PP proved challenging.

A modified dentine infection model with fluorescent lipopolysaccharide and lipopolysaccharides sampling technique to compare XP-Endo finisher and passive ultrasonic irrigation.

AIM: The lipopolysaccharides-dentine-infection (LPS-dentine-infection) models and sampling techniques frequently used to evaluate LPS disinfection have limitations. In this study, a LPS-dentine-infection model was devised using fluorescent conjugate LPS. Secondly, a sampling technique using cryogenic grinding for intraradicular LPS analysis was evaluated. Thirdly, the effectiveness of the XP-endo Finisher (XP-EF) was compared with passive ultrasonic irrigation (PUI) in removing LPS from root canal system. METHODOLOGY: Sixty-nine mandibular premolars were submitted to dentine pretreatment and inoculated with fluorescent LPS conjugate (Alexa Fluor® 594). Twenty-three teeth were analysed under confocal laser scanning microscopy (CLSM) to validate this modified LPS-dentine-infection model. Forty-six teeth were randomly divided into two experimental groups: XP-EF (n = 23) and PUI (n = 23). All teeth were instrumented with XP-endo shaper (XPS; FKG Dentaire) and 2.5% NaOCl. The root canals were sampled with paper points before (s1) and after (s2) instrumentation and after supplemental treatment (s3) with XP-EF and PUI. After s3, all roots were cryogenically ground for intraradicular LPS analysis (s4). Limulus amebocyte lysate assay was used for LPS quantification. The Friedman test was used for differences in LPS among four time-points (s1, s2, s3, and s4). Dunn's test was used for pairwise testing of time-points. The significance level was set at 5% (p < .05). RESULTS: Fluorescent LPS conjugate was detected in 100% of the samples under CLSM with a penetration depth of approximately 400 µm into dentine. Chemo-mechanical preparation using XPS files significantly reduced LPS levels (p < .05). Both the XPS and PUI improved the LPS disinfection (p < .05), with no difference between them (p > .05). LPS was recovered from all samples after cryogenic grinding. The residual amount of LPS detected using the cryogenically sampling technique at s4 was approximately three times greater than with the paper-point sampling technique at s3. CONCLUSION: This study established a modified LPS-dentine-infection model using fluorescent conjugate LPS, and validated a LPS sampling technique for using cryopulverization intraradicular LPS analysis. Moreover, both the XP-EF and PUI further improved LPS disinfection from the root canals, and the innovative XP-EF was as effective as PUI.

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.

Nanoflow LC-MS Method Allowing In-Depth Characterization of Natural Heterogeneity of Complex Bacterial Lipopolysaccharides.

The variable modification of the outer membrane lipopolysaccharide (LPS) in Gram-negative bacteria contributes to bacterial pathogenesis through various mechanisms, including the development of antibiotic resistance and evasion of the immune response of the host. Characterizing the natural structural repertoire of LPS is challenging due to the high heterogeneity, branched architecture, and strong amphipathic character of these glycolipids. To address this problem, we have developed a method enabling the separation and structural profiling of complex intact LPS mixtures by using nanoflow reversed-phase high-performance liquid chromatography (nLC) coupled to electrospray ionization Fourier transform mass spectrometry (ESI-FT-MSn). Nanogram quantities of rough-type LPS mixtures from Neisseria meningitidis could be separated and analyzed by nLC-ESI-FT-MS. Furthermore, the method enabled the analysis of highly heterogeneous smooth (S)-type LPS from pathogenic enteric bacteria such as Salmonella enterica serotype Typhimurium and Escherichia coli serotype O111:B4. High-resolution, accurate mass spectra of intact LPS containing various lengths of the O-specific polysaccharide in the range of 3 and 15 kDa were obtained. In addition, MS/MS experiments with collision-induced dissociation of intact LPS provided detailed information on the composition of oligo/polysaccharides and lipid A domains of single S-type LPS species. The structural heterogeneity of S-type LPS was characterized by unprecedented details. Our results demonstrate that nLC-ESI-FT-MSn is an attractive strategy for the structural profiling of small quantities of complex bacterial LPS mixtures in their intact form.

λ-carrageenan exacerbates Citrobacter rodentium-induced infectious colitis in mice by targeting gut microbiota and intestinal barrier integrity.

For nearly half a century, the scientific community has been unable to agree upon the safety profile of carrageenan (CGN), a ubiquitous food additive. Little is known about the mechanisms by which consumption of CGN aggravates the etiopathogenesis of murine colitis. However, analyses of gut microbiota and intestinal barrier integrity have provided a breakthrough in explaining the synergistic effect of CGN upon colitis. In Citrobacter rodentium-induced infectious murine colitis, inflammation and the clinical severity of gut tissue were aggravated in the presence of λ-CGN. Using fecal transplantation and germ-free mice experiments, we evaluated the role of intestinal microbiota on the pro-inflammatory effect of λ-CGN. Mice with high dietary λ-CGN consumption showed altered colonic microbiota composition that resulted in degradation of the colonic mucus layer, a raised fecal LPS level, and a decrease in the presence of bacterially derived short-chain fatty acids (SCFAs). Mucus layer defects and altered fecal LPS and SCFA levels could be reproduced in germ-free mice by fecal transplantation from CGN-H-fed mice, but not from germ-free CGN-H-fed mice. Our results confirm that λ-CGN may create an environment that favors inflammation by altering gut microbiota composition and gut bacterial metabolism. The present study provides evidence that the "gut microbiota-barrier axis" could be an alternative target for ameliorating the colitis promoting effect of λ-CGN.

Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells.

BACKGROUND: The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. RESULTS: Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci-lpxM, lpxP, lpxL, eptA, gutQ and kdsD-were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. CONCLUSIONS: Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Electrochemical aptasensor for ultrasensitive detection of lipopolysaccharide using silver nanoparticles decorated titanium dioxide nanotube/functionalized reduced graphene oxide as a new redox nanoprobe.

A novel and relatively simple signal-off electrochemical aptasensor was constructed for highly sensitive detection of lipopolysaccharide (LPS). For the first time, silver nanoparticles (AgNPs) decorated titanium dioxide nanotube (TNT) was conjugated with polydiallyldimethylammonium chloride (PDDA) functionalized reduced graphene oxide (rGO) to form a new nanohybrid of Ag-TNT/P-rGO. This nanohybrid with a large specific surface area exhibited excellent electrochemical activity, which not only served as the sensing platform to immobilize LPS binding aptamer (LBA) but was also employed as the redox probe to monitor the change of the electrochemical signal. The electrochemical signal responses were measured by cyclic voltammetry (CV) in the potential range -0.3 to 0.5 V at a scan rate of 0.1 V/s. The proposed aptasensor exhibited acceptable stability, reproducibility, and specificity for LPS detection with a wide linear range from 17 fg/mL to 100 ng/mL. The limit of detection (LOD) was 5 fg/mL. Furthermore, the prepared aptasensor showed acceptable recovery ranging from 96% to 103%, and the RSD varied between 1.4% and 8.5% for determining LPS in real samples.Graphical abstract.