Search

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 (MoS 2 ) 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., Competing Interests: Declaration of competing interest All the authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: A.A., M.T., T.Y., S.Matsushita, and Y.H. are employees of PHC Corporation (formerly LSI Medience Corporation). K.C. and S.Mitarai received joint research fees from PHC Corporation (formerly LSI Medience Corporation). T.I. and T.S. are employees of LSI Medience Corporation.

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

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 Fe 3 O 4 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.

The association between PM 2.5 (particulate matter ≤ 2.5 µm) short-term exposure and its health effect is non-linear from the epidemiological studies. And this nonlinearity is suggested to be related with the PM 2.5 heterogeneity, however, the underlying biological mechanism is still unclear. Here, a total of 38 PM 2.5 filters were collected continuously for three weeks in winter Beijing, with the ambient PM 2.5 varying between 10 and 270 µg/m 3 . Human monocytes-derived macrophages (THP-1) were treated with PM 2.5 water-soluble elutes at 10 µg/mL to investigate the PM 2.5 short-term exposure effect from a proinflammatory perspective. The proinflammatory cytokine tumor necrosis factor (TNF) induced by the PM 2.5 elutes at equal concentrations were unequal, showing the heterogeneity of PM 2.5 proinflammatory potentials. Of the various chemical and biological components, lipopolysaccharide (LPS) showed a strong positive association with the TNF heterogeneity. However, some outliers were observed among the TNF-LPS association. Specifically, for PM 2.5 from relatively clean air episodes, the higher LPS amount corresponded to relatively low TNF levels. And this phenomenon was also observed in the promotion tests by treating macrophages with PM 2.5 elutes dosed with additional trace LPS. Gene expression analysis indicated the involvement of oxidative-stress related genes in the LPS signaling pathway. Therefore, a potential oxidative-stress-mediated suppression on the PM 2.5 -borne LPS proinflammatory effect was proposed to be accounted for the outliers. Overall, the results showed the differential role of LPS in the heterogeneity of PM 2.5 proinflammatory effects from a component-based perspective. Future experimental studies are needed to elucidate the signaling pathway of LPS attached on PM 2.5 from different air quality episodes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

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-1β 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-1β, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1β, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1β, 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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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-1β 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., Competing Interests: SD and RD have received grant funding from Nestlé Nutrition R&D. SD has given scientific presentations and educational webinars for Nestlé Nutrition Institute. JY was an employee of Nestlé Product Technology Center Nutrition at the time the study was conducted. JH is an employee of Nestlé Institute of Health Sciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Nestlé Nutrition. The funder provided input on the study design and reviewed the final manuscript., (Copyright © 2023 Monaco, Wang, Hauser, Yan, Dilger and Donovan.)

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., (© 2023. The Author(s).)

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., (Copyright © 2023. Published by Elsevier Inc.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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 (log 10 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-1β (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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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/m 3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10-119 EU/mL (water) and 0.13-0.64 EU/m 3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 10 4 -10 5 cells eq./mL (water) and 10 1 -10 3 cells eq./m 3 (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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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.

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., Competing Interests: Declaration of Interests None., (Copyright © 2022 The Academy of Dental Materials. All rights reserved.)

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.

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., (© 2022 British Endodontic Society. Published by John Wiley & Sons Ltd.)

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 IC 50 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.

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 Cu 2+ -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.

Breastfeeding reduces the risk of necrotizing enterocolitis (NEC), one of the most common causes of morbidity and mortality in preterm infants. However, the molecular substrates by which human milk (HM) offers protection against NEC are not well known. Using fetal intestinal epithelial cells treated with known NEC aggravators, namely lipopolysaccharide (LPS) and platelet-activating factor (PAF), we mapped the time-course of changes in targeted expression analysis of 35 NEC-associated genes, so-called the NEC signature. We found, first, that HM treatment fully rescued LPS/PAF-induced fetal intestinal cell death at 12 and 24 h ( n  = 5). Differential gene expression and bioinformatics revealed that HM did not mitigate inflammatory and cell death signals, but instead promoted cell proliferation and stress response pathways to mitigate LPS/PAF-induced inflammatory cell death. From this, epidermal growth factor (EGF) synthesis emerged as the central player in rescue of the fetal intestinal cell death. Functional validation was supported by reversal of the cellular rescue by HM following EGF knockdown by small interfering RNA. In conclusion, this study suggests that HM might offer protection against NEC through enhancing intestinal EGF production to rescue the inflammatory cell death. Future studies are warranted to verify these HM molecular protective effects in NEC models in vivo . The findings reported herein also support future research avenues to discover new therapeutics to boost intrinsic EGF production in the injured intestinal tissues in neonates with NEC, for example, by bioactive components in human milk, natural compounds, or small molecules.

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein Wzy SF polymerises Oag repeat units, whilst two co-polymerase proteins, Wzz SF and Wzz pHS-2 , together interact with Wzy SF to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of Wzy SF transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of Wzy SF, or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ Wzy SF arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe Wzy SF conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including Wzz SF and Wzz pHS-2 , and extrinsic factors, such as temperature. Site-directed mutagenesis of Wzy SF cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent analyses highlighted both the structural and functional importance of Wzy SF cysteines. Substitution of Wzy SF cysteine residues significantly decreased biosynthesis of the VL-Oag modal length, without disruption to S-Oag production. This phenotype was corroborated in the absence of co-polymerase competition for Wzy SF interaction. These data suggest Wzy SF cysteine substitutions directly impair the interaction between Wzy/Wzz pHS-2, without altering the Wzy/Wzz SF interplay, and in combination with structural data, we propose that the N- and C-termini of Wzy SF are arranged in close proximity, and together may form the unique Wzz pHS-2 interaction site., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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)., (© 2022. The Author(s).)

Background: This study was conducted to compare the microbiomes, the levels of lipopolysaccharides (LPS), lipoteichoic acid (LTA), and cytokines (interleukin [IL]-1β 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-1β, 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-1β and TNF-α were detected in both sites at baseline. After CMP, LPS, LTA, IL-1β 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., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The primo vascular system can be viewed as a circulatory system that plays a therapeutic function in regenerating the body tissue. The anti-CD3 monoclonal antibody was used as an immunotherapeutic agent to treat the novel coronavirus infection (COVID-19). Objectives: In this study, we observed the effect of injecting lymph nodes with Foralumab, an anti- human CD3 epsilon therapeutic monoclonal antibody, on primo vessels., Methods: The structure and atomic stoichiometry of the antibody were determined by transmission electron microscopy and energy dispersive spectroscopy. Alcian blue dying solution was injected into the lymph nodes of the abdominal vena cava of rabbits, and the solution further flowed into the lymph vessels., Results: A primo vessel with primo nodes stained with Alcian blue was clearly visible in the lymph vessel. By injecting Foralumab into lymph nodes of rabbits with lipopolysaccharide-induced inflammation, the floating primo vessel in the lymph vessel appeared thicker and was distinctly visible., Conclusion: The observation of the primo vessel post-treated with Foralumab in the inflamed lymphatic system suggests the possibility of a functional role of the primo vascular circulatory system in pathophysiological conditions.

Mycobacterium tuberculosis (MTB) and M. avium-intracellulare complex (MAC) enter host phagocytes, such as neutrophils through lipoarabinomannan (LAM) binding to pattern-recognition receptors, inducing innate immune responses including phagocytosis. Phagocytosis of mycobacteria by human neutrophils depends on the binding of α(1 → 2)-monomannose branching α(1 → 6)-mannan core of LAM/lipomannan (LM), a common component among mycobacterial species, to lactosylceramide (LacCer)-enriched lipid microdomains. We investigated the binding specificities of several anti-LAM antibodies (Abs) to LAMs/LM and found anti-LAM monoclonal IgMs TMDU3 and LA066 were directed against mannan core. Each IgM showed different binding specificity to mannan core. Confocal and stimulated emission depletion microscopy revealed TMDU3 and LA066 strongly bind to MTB and MAC, respectively. Flow cytometric analysis revealed human neutrophils do not express Dectin-2, DC-SIGN or mannose receptor. Furthermore, neutrophil phagocytosis of mycobacteria was markedly inhibited by TMDU3 and LA066, respectively. Similarly, treatment of each mAb with neutrophils reduced the numbers of intracellular MAC. Together, our results suggest that the interaction of LacCer-enriched lipid microdomains with mannan core and its blocking are therapeutic or diagnostic targets for both TB and non-tuberculous mycobacteria infection., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021. The Author(s).)

Lipopolysaccharide (LPS), as the major component of the outer membrane of Gram-negative bacteria, can trigger a variety of biological effects such as sepsis, septic shock, and even multiorgan failure. Herein, we developed a near-infrared-fluorescent probe for fluorescent turn-on analysis of LPS based on plasmon-enhanced fluorescence (PEF). Gold nanorods (Au NRs) modified polyethylene glycol (PEG) was used as PEF materials. Au NRs were prepared with different longitudinal surface plasmon resonance (LSPR), and their fluorescence enhancement was investigated. Three kinds of molecular weights (1000, 5000, and 10000) of polyethylene glycol (PEG) were employed to control the distance between the Au NRs and the fluorescence substances of cyanine 7 (Cy7). Experimental analysis showed that the enhancement was related to the spectral overlap between the plasmon resonance of Au NRs and the extinction/emission of fluorophore. The three-dimensional finite-difference time-domain (3D-FDTD) simulation further revealed that the enhancement was caused by local electric field enhancement. Furthermore, the probe was used for the ultrasensitive analysis of LPS with a detection limit of 3.85 ng/mL and could quickly distinguish the Gram-negative bacterium- Escherichia coli ( E. coli ) (with LPS in the membrane) from Gram-positive bacterium- Staphylococcus aureus ( S. aureus ) (without LPS), as well as quantitative determination of E. coli with a detection limit of 1.0 × 10 6 cfu/mL. These results suggested that the prepared probe has great potential for biomedical diagnosis and selective detection of LPS from different bacterial strains.

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-MS n ). 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-MS n is an attractive strategy for the structural profiling of small quantities of complex bacterial LPS mixtures in their intact form.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium ( Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

Engineered gold nanoparticles (AuNPs) find application in several fields related to human activities ( i.e. , food and cosmetic industry or water purification) including medicine, where they are employed for diagnosis, drug delivery and cancer therapy. As for any material/reagent for human use, the safety of AuNPs needs accurate evaluation. AuNPs are prone to contamination by bacterial endotoxin (lipopolysaccharide, LPS), a potent elicitor of inflammatory responses in mammals. It is therefore important, when assessing AuNP immunosafety and immune-related effects, to discriminate between inflammatory effects intrinsic to the NPs from those caused by an undeliberate and undetected LPS contamination. Detection of LPS contamination in AuNP preparations poses different problems when using the current LPS detection assays, given the general interference of NPs, similar to other particulate agents, with the assay reagents and endpoints. This leads to time-consuming search for optimal assay conditions for every NP batch, with unpredictable results, and to the use in parallel of different assays, each with its weaknesses and unpredictability. Thus, the development of highly sensitive, quantitative and accurate assays able to detect of LPS on AuNPs is very important, in view of their medical applications. Surface-enhanced Raman spectroscopy (SERS) is a label-free, sensitive, chemical-specific, nondestructive and fast technique that can be used to directly obtain molecular fingerprint information and a quantitative analysis of LPS adsorbed on AuNPs. Within this study, we describe the use of SERS for the label-free identification and quantitative evaluation - down to few attograms - of the LPS adsorbed on the surface of 50 nm AuNPs. We thus propose SERS as an efficient tool to detect LPS on the AuNP surface, and as the basis for the development of a new sensitive and specific LPS-detection sensor based on the use of AuNPs and SERS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Verde, Mangini, Managò, Tramontano, Rea, Boraschi, Italiani and De Luca.)

Introduction: Patients with community-acquired pneumonia display enhanced levels of lipopolysaccharides (LPS) compared with controls, suggesting that low-grade endotoxemia may be implicated in vascular disturbances. It is unknown whether this occurs in patients with coronavirus 2019 (COVID-19) and its impact on thrombotic complications., Methods: We measured serum levels of zonulin, a marker of gut permeability, LPS, and D-dimer in 81 patients with COVID-19 and 81 healthy subjects; the occurrence of thrombotic events in COVID-19 during the intrahospital stay was registered., Results: Serum LPS and zonulin were higher in patients with COVID-19 than in control subjects and, in COVID-19, significantly correlated (R = 0.513; P < 0.001). Among the 81 patients with COVID-19, 11 (14%) experienced thrombotic events in the arterial (n = 5) and venous circulation (n = 6) during a median follow-up of 18 days (interquartile range 11-27 days). A logistic regression analysis showed that LPS (P = 0.024) and D-dimer (P = 0.041) independently predicted thrombotic events., Discussion: The study reports that low-grade endotoxemia is detectable in patients with COVID-19 and is associated with thrombotic events. The coexistence of low-grade endotoxemia with enhanced levels of zonulin may suggest enhanced gut permeability as an underlying mechanism., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: This study aimed to investigate the relationship between lipopolysaccharide (LPS) and zonulin levels and also to show the effect of acute hyperglycemic stress induced by oral glucose tolerance testing (OGTT) on zonulin levels in pre-diabetic patients., Methods: Four groups were constituted according to the criteria of the American Diabetes Association (ADA), based on OGTT results: control group (n:40); prediabetic group (n:56), divided into two subgroups: impaired fasting glucose group (IFG) (n:36), and impaired glucose tolerance (IGT) + IFG group (n:20) and type-2 diabetes mellitus (T2DM) group (n:45)., Results: Zonulin and LPS did not significantly differ between the prediabetes and control groups, but were significantly higher in the T2DM group compared to both the prediabetic and the control group (P<0.001). After OGTT, zonulin and LPS were significantly higher in the prediabetes group compared to the control group (P<0.01 and P<0.05, respectively), and significantly lower in the IFG and IFG+IGT groups compared to the T2DM group (P<0.001, P<0.001 and P<0.001, P<0.001, respectively). A positive correlation was detected between fasting zonulin and 2-hour zonulin (r=0.727, P<0.001) and between fasting LPS (r=0.555, P<0.001) and 2-hour LPS (r=0.567, P<0.001) in the prediabetic group. Increased zonulin and LPS levels and the positive correlation between these levels during the prediabetic period although non significant suggests onset of intestinal permeability., Conclusions: During acute hyperglycemia in prediabetic patients, up-regulation of zonulin and LPS may affect intestinal function. The intestines may play a key role in up-regulation of glucose and the pathogenesis of diabetes., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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.

The very high content of structurally diverse and biologically active lipids of exotic structures is the hallmark of Mycobacteria. As such the lipid composition is commonly used to characterize mycobacterial strains at the species and type-species levels. The present chapter describes the methods that allow the purification of the most commonly isolated biologically active lipids and those used for analyzing extractable lipids and their constituents, cell wall-linked mycolic acids (MA), and lipoarabinomannan (LAM). These involve various chromatographic techniques and analytical procedures necessary for structural and metabolic studies of mycobacterial lipids. In addition, as the use of physical methods has brought important overhang on chemical structures of the very-long-chain MA, which typify mycobacteria, NMR and mass spectrometry data of these specific fatty acids are included.

Intestinal immunoglobulins (Ig) are abundantly secreted antibodies that bind bacteria and bacterial components in the gut. This binding is considered to accelerate bacterial transit time and prevent the interaction of potentially immunogenic compounds with intestinal immune cells. Ig secretion is regulated by alterations in gut microbiome composition, an event rarely mapped in an intervention setting in humans. Here, we determined the intestinal and systemic Ig response to a major intervention in gut microbiome composition. Healthy humans and humans with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Coinciding with a vancomycin-induced increase in Gram-negative bacteria, fecal levels of the immunogenic bacterial components lipopolysaccharide (LPS) and flagellin drastically increased. Intestinal antibodies (IgA and IgM) significantly increased, whereas peripheral antibodies (IgG, IgA, and IgM) were mostly unaffected by vancomycin treatment. Bacterial cell sorting followed by 16S rRNA sequencing revealed that the majority of Gram-negative bacteria, including opportunistic pathogens, were IgA-coated after the intervention. We suggest that the intestinal Ig response after vancomycin treatment prevents the intrusion of pathogens and bacterial components into systemic sites.

This study aimed to compare the effects of three food-grade particles (micro-TiO 2 , nano-TiO 2 , and nano-SiO 2 ) on the murine intestinal tract and to investigate their potential mechanisms of action. A 28-day oral exposure murine model was established. Samples of blood, intestinal tissues and colon contents were collected for detection. The results showed that all three particles could cause inflammatory damage to the intestine, with nano-TiO 2 showing the strongest effects. Exposure also led to changes in gut microbiota, especially mucus-associated bacteria. Our results suggest that the toxic effects on the intestine were due to reduced intestinal mucus barrier function and an increase in metabolite lipopolysaccharides which activated the expression of inflammatory factors downstream. In mice exposed to nano-TiO 2 , the intestinal PKC/TLR4/NF-κB signalling pathway was activated. These findings will raise awareness of toxicities associated with the use of food-grade TiO 2 and SiO 2 ., (Copyright © 2020. Published by Elsevier B.V.)

Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis , the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2- N -seryl, 3- N -acetylglucuronamide in P. gingivalis and 2- N -glycyl, 3- N -acetylmannuronic acid in T. forsythia The structures indicated the involvement of the Wbp pathway to produce 2,3-di- N -acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis The wbpS gene was identified as PGN&#95;1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalis vimA mutant which lacks A-LPS was successfully complemented by the T. forsythia vimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway. IMPORTANCE Porphyromonas gingivalis and Tannerella forsythia , two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies., (Copyright © 2020 Veith et al.)

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.

Miso, a fermented paste made from soybeans, is used traditionally for seasoning of food. It has been a protein and nitrogen source since ancient times in Japan because of its high nutritional value. Furthermore, it has important health functions such as the estrogen-like activity of isoflavones, anti-oxidation, and angiotensin-converting-enzyme inhibition activity. Moreover, it has activity for neutralization of lipopolysaccharide (LPS) from Escherichia coli. Nevertheless, the mechanisms of that activity remain unclear. For this study, we purified and identified the proteins responsible for LPS-neutralization. After proteins were isolated from a miso extract using Blue native polyacrylamide gel electrophoresis, a protein found at 10 to 30 kDa on the polyacrylamide gel was identified using nano LC-MS/MS as 2S albumin in soybean (Glycine max). The protein had two LPS binding motifs: SKWQHQ (22 amino acid residues) and EKQKKKMEKE (131 amino acid residues). The protein in miso was found to have LPS neutralization activity, as assayed by prostaglandin D 2 (PGD 2 ) production from macrophage cells. The PGD 2 production by macrophage cells was inhibited by LPS-neutralizing protein (LNP) from miso. Particularly, 50 mg/mL of LNP solution and LPS (10 µg/mL) inhibited production of PGD 2 from the cells. The data were inferred as significantly different (P < 0.05) from statistical analyses by analysis of variance testing and Tukey tests. The 2S albumin in soybean is LNP, an LPS-neutralizing protein, produced in miso. PRACTICAL APPLICATION: A protein from miso fermented soy paste neutralizes an Escherichia coli intestinal bacterial product, lipopolysaccharide (LPS), which causes intestinal inflammation. Miso and its protein are anticipated for use as a probiotic agent to prevent intestinal inflammation in humans and domestic animals. Miso is useful not only as a seasoning for food, but also as a health-functional food because it is an LPS-neutralizing agent., (© 2020 Institute of Food Technologists®.)

Introduction: Lipopolysaccharides (LPSs) of Gram-negative bacteria (GNB) are highly toxic and induce inflammation. Therefore, we investigated both the LPS activity and composition of GNB in the gastric fluid (GF) to assess the potential toxicity of them accumulated in the stomach., Methods: GF and saliva samples were obtained from 158 outpatients who were undergoing upper gastrointestinal endoscopy and 36 volunteers using a nasogastric tube. The LPS activity was measured by assay kits including recombinant Factor C or Limulus amebocyte lysate. To assess the bacterial composition in the samples, a 16S ribosomal DNA-based operational taxonomic unit analysis was performed. We focused on the genera representing >0.1% of the whole microbiota., Results: We found a high LPS activity in the GF samples with weak acidity (approximately > pH 4), whereas little/no activity in those with strong acidity (approximately < pH 2). Spearman test also demonstrated a close correlation between pH and LPS in those samples (r = 0.872). The relative abundance of GNB in the saliva showed no significant difference between the subject groups with weak- and strong-acidity GF. In addition, in the subjects whose GF acidity was weak, the GNB abundance in the GF was almost the same as that in the saliva. By contrast, in the subjects whose GF acidity was strong, the GNB abundance in the GF was significantly lower than that in the saliva., Discussion: GNB that have recently moved from the oral cavity might account for the prominent LPS activity in a stomach with weak acidity.

We present two types of two-dimensional (2D) photonic crystals (PC) hydrogel sensors based on glycated albumin (G-alb) as a proof-of-concept for utilizing recognition between G-alb and bacterial cell surface lipopolysaccharides (LPS) to detect and discriminate Gram-negative bacteria. The G-alb functionalized PC-G-alb hydrogel provides recognition of different LPS via hydrogen bonding and can discriminate different Gram-negative bacteria based on their LPS types. The hydrogel delivered LOD of 0.87 ng mL -1 for E.coli LPS, 153 CFU mL -1 for E.coli, 1.22 ng mL -1 for P.aeruginosa LPS and 225 CFU mL -1 for P.aeruginosa. On the other hand, LPS bioimprinted hydrogel (PC-G-alb-LPS imp ) provides selective recognition of E.coli LPS with LOD 0.76 ng mL -1 and for E.coli 58 CFU mL -1 , via generation of flexible specific cavities for E.coli and its LPS. The two hydrogels showed remarkable recoveries for both LPS and Gram-negative bacteria in the relevant samples of milk, orange juice, river water, and serum with a short response time of 6-12 min. In the binding process, the hydrogels shrink, and 2D PC particle spacing decreases with diffraction shift from green to blue. The diffraction shifts can be visually observed, measured through Debye's diffraction ring diameter by a laser pointer or determined from a spectrometer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Endotoxins or lipopolysaccharides (LPS) are pathogens released from the outer membrane of gram-negative bacteria which produce toxic effects on humans. The sensitive and selective detection of LPS is in high demand, especially in the field of medical supplies, therapeutics and in the food industry. Herein we report a new nano-probe based on a gold nanoparticle loaded, water-soluble layered molybdenum disulfide-polyacrylic acid (Au/MoS 2 -PAA) nanocomposite as a label-free voltammetric aptasensor for ultrasensitive LPS detection. MoS 2 nanosheets were obtained through one-step sonication assisted exfoliation of bulk MoS 2 with polyacrylic acid (PAA). Au nanoparticles were incorporated into the MoS 2 -PAA nanocomposite and thiol terminated LPS binding aptamers (LBA) were immobilized on this. The specific binding of LPS with LBA is investigated electrochemically by differential pulse voltammetry. The apparent binding constant (K b ) of LPS with LBA has been calculated to be 1.53 × 10 2 mL g -1 . The aptasensor demonstrated LPS detection down to the ag mL -1 level without incorporating any redox mediator and showed wide linearity from 100 ag mL -1 to 100 pg mL -1 with a low limit of detection of 29 ag mL -1 . The sensor showed excellent recovery upon spiking LPS in clinical grade insulin, suggesting that LBA/Au/MoS 2 -PAA/GCE has promising application for the trace analysis of LPS in the field of pharmaceutical products.

Purpose of Review: Tuberculous meningitis (TBM) is associated with significant mortality and morbidity yet is difficult to diagnose and treat. We reviewed original research published in the last 2 years, since 1 January 2018, which we considered to have a major impact in advancing diagnosis, treatment and understanding of the pathophysiology of TBM meningitis in children and adults., Recent Findings: Studies have sought to identify a high sensitivity diagnostic test for TBM, with new data on modified Ziehl--Neelsen staining, urinary and cerebrospinal fluid (CSF) lipoarabinomannan and GeneXpert Ultra. Recent studies on CSF biomarkers provide a better understanding of the detrimental inflammatory cascade and neuromarkers of brain damage and suggest potential for novel host-directed therapy. Tryptophan metabolism appears to affect outcome and requires further study. Increased clinical trials activity in TBM focuses on optimizing antituberculosis drug regimens and adjuvant therapy; however, there are few planned paediatric trials., Summary: Tuberculous meningitis still kills or disables around half of sufferers. Although some progress has been made, there remains a need for more sensitive diagnostic tests, better drug therapy, improved management of complications and understanding of host-directed therapy if outcomes are to improve.

Background: Tuberculosis (TB) is the most common cause of death in people living with HIV (PLHIV), yet TB often goes undiagnosed since many patients are not able to produce a sputum specimen, and traditional diagnostics are costly or unavailable. A novel, rapid lateral flow assay, Fujifilm SILVAMP TB LAM (SILVAMP-LAM), detects the presence of TB lipoarabinomannan (LAM) in urine, and is substantially more sensitive for diagnosing TB in PLHIV than an earlier LAM assay (Alere Determine TB LAM lateral flow assay [LF-LAM]). Here, we present an individual participant data meta-analysis of the diagnostic accuracy of SILVAMP-LAM in adult PLHIV, including both published and unpublished data., Methods and Findings: Adult PLHIV (≥18 years) were assessed in 5 prospective cohort studies in South Africa (3 cohorts), Vietnam, and Ghana, carried out during 2012 to 2017. Of the 1,595 PLHIV who met eligibility criteria, the majority (61%) were inpatients, median age was 37 years (IQR 30-43), 43% had a CD4 count ≤ 100 cells/μl, and 35% were receiving antiretroviral therapy. Most participants (94%) had a positive WHO symptom screen for TB on enrollment, and 45% were diagnosed with microbiologically confirmed TB, using mycobacterial culture or Xpert MTB/RIF testing of sputum, urine, or blood. Previously published data from inpatients were combined with unpublished data from outpatients. Biobanked urine samples were tested, using blinded double reading, with SILVAMP-LAM and LF-LAM. Applying a microbiological reference standard for assessment of sensitivity, the overall sensitivity for TB detection was 70.7% (95% CI 59.0%-80.8%) for SILVAMP-LAM compared to 34.9% (95% CI 19.5%-50.9%) for LF-LAM. Using a composite reference standard (which included patients with both microbiologically confirmed as well as clinically diagnosed TB), SILVAMP-LAM sensitivity was 65.8% (95% CI 55.9%-74.6%), and that of LF-LAM 31.4% (95% CI 19.1%-43.7%). In patients with CD4 count ≤ 100 cells/μl, SILVAMP-LAM sensitivity was 87.1% (95% CI 79.3%-93.6%), compared to 56.0% (95% CI 43.9%-64.9%) for LF-LAM. In patients with CD4 count 101-200 cells/μl, SILVAMP-LAM sensitivity was 62.7% (95% CI 52.4%-71.9%), compared to 25.3% (95% CI 15.8%-34.9%) for LF-LAM. In those with CD4 count > 200 cells/μl, SILVAMP-LAM sensitivity was 43.9% (95% CI 34.3%-53.9%), compared to 10.9% (95% CI 5.2%-18.4%) for LF-LAM. Using a microbiological reference standard, the specificity of SILVAMP-LAM was 90.9% (95% CI 87.2%-93.7%), and that of LF-LAM 95.3% (95% CI 92.2%-97.7%). Limitations of this study include the use of biobanked, rather than fresh urine samples, and testing by skilled laboratory technicians in research laboratories, rather than at the point of care., Conclusions: In this study, we found that SILVAMP-LAM identified a substantially higher proportion of TB patients in PLHIV than LF-LAM. The sensitivity of SILVAMP-LAM was highest in patients with CD4 count ≤ 100 cells/μl. Further work is needed to demonstrate accuracy when implemented as a point-of-care test., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: TB, SGS, AM, SO, RS and CMD were previously or are currently employed by FIND. TB reports a patent in the field of lipoarabinomannan detection. CMD is a member of PLOS Medicine's Editorial Board. The rest of the authors declare no competing interests associated with this manuscript. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Weaning stresses often induce markedly structural and functional changes in the small intestine. However, little data are available on the changes of the morphology, function, and health in the cecum of piglets post-weaning. This study was conducted to measure the effects of weaning on the cecum in piglets. Forty piglets were weaned at 21 d and samples were collected at different time points (days 0, 1, 3, 7, and 14) post-weaning. The crypt depth, crypt width, and total epithelial cell numbers increased on days 7 and 14, compared with days 0, 1, and 3 (P < 0.001). The value of Ki67/total epithelial cells was highest on day 0 compared with all other days (P < 0.05). Besides, a higher concentration of malondialdehyde was observed on day 3 (P < 0.001). The activity of CuZn-superoxide dismutase (P < 0.05) enhanced from day 0 to 1 and the activity of catalase increased from day 1 to 3 (P < 0.001). In addition, days 3, 7, and 14 had greater acetic, propanoic, and butyric acid contents than on day 1 (P < 0.001). The pH and monocarboxylate transporter 1 (MCT1) expression increased from day 0 to 1 and from day 3 to 7 and decreased between days 1 and 3 (P < 0.001). The mRNA expression of solute carrier family 9 member A3 (SLC9A3) decreased on day 1 compared to all other postweaning days (P < 0.05). The abundance of toll-like receptor 4 (TLR4) and interferon-γ (IFN-γ) mRNA expression increased (P < 0.05) during the first 24 h after weaning. The concentration of lipopolysaccharide increased from day 3 to 7, then decreased on day 14 (P < 0.05). The mRNA expression of tumor necrosis factor-α (TNF-α) enhanced from day 7 to 14 (P < 0.05). The abundance of phosphorylated mammalian target of rapamycin protein was lower on day 14 than day 0 (P < 0.05). Taken together, these results show that weaning in piglets influences intestinal morphology, function, and health in the cecum., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An electrochemical aptasensor for the lipopolysaccharide (LPS) detection was constructed by using copper-based metal-organic framework (Cu-MOF) as a label and the LPS aptamer of specific single-stranded DNA as a probe. The carboxyl-functionalized polypyrrole nanowires (PPy NWs) were synthesized by electrochemical polymerization method, and the amino-terminated aptamer covalently coupling with the carboxyl group of the PPy NWs was immobilized onto the modified electrode. The aptamer can specifically combine with the target LPS molecules, and Cu-MOF was labeled by adsorption based on specific interactions of LPS carbohydrate portions with anionic groups. The fabrication processes of the aptasensor were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS). Differential pulse voltammetry (DPV) was used to measure electrochemical performance of the aptasensor, and the electrochemical signal can be directly measured by the electrochemical redox reaction of Cu(II)/Cu(I) existed in the Cu-MOF. The electrochemical aptasensor exhibited a high sensitivity toward LPS ranging from 1.0 pg/mL to 1.0 ng/mL with a detection limit of 0.29 pg/mL. Moreover, the developed sensor was found to have good selectivity, stability and regeneration properties, and the sensor also successfully detected LPS in real tap water samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)