Extracellular Protein Concentration Research Articles

Abstract LB034: Analysis of PD-L1 IHC tests using NIST SRM 1934-traceable reference materials: A new paradigm for development of predictive IHC biomarkers

Abstract BACKGROUND. The challenges in accurate patient stratification for immune checkpoint inhibitors have been compounded by the fact that the FDA-cleared PD-L1 IHC tests are analytic ‘black boxes'. Relatively basic analytic parameters such as lower limit of detection and analytic dynamic range are unknown to both developers of assays as well as end users. The recent development of standardized PD-L1 immunohistochemistry (IHC) reference materials enables quantitative test characterizations that were not previously possible. METHODS. We surveyed 41 PD-L1 testing laboratories in North America and Europe, quantitatively defining each of the PD-L1 tests' analytic performance in terms of lower limit of detection and dynamic range. All four commercial PD-L1 kits were assessed by multiple laboratories. A variety of laboratory-developed tests (LDTs) we also assessed. The reference materials incorporated defined concentrations of PD-L1 peptide (intracellular domain) or recombinant extracellular domain protein, traceable to NIST Standard Reference Material 1934. Each laboratory received a slide with 10 separate PD-L1 calibrator concentrations: 2,200 - 600,000 molecules of PD-L1 extracellular domain or 34,000 - 2,200,000 molecules of PD-L1 intracellular domain. The calibrator concentrations ranged from those that are below the lower limit of detection to others that yield maximal staining. RESULTS. The data obtained with the four PD-L1 kits (VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, DAKO PD-L1 IHC 28-8 pharmDx and DAKO PD-L1 IHC 22C3 pharmDx assays) revealed that the lower limits of detection (PD-L1 molecules per cell equivalent) are approximately: 50,000 - 180,000 (SP263), 800,000 - 1,200,000 (SP142), 220,000 - 360,000 (28-8), and 200,000 - 400,000 (22C3). The dynamic ranges for all of these tests are generally narrow, spanning less than a log concentration of PD-L1. The SP263 and SP142 assays showed no overlap of their analytic response curves. This means that a maximal stain intensity with SP263 kit can be associated with zero staining with the SP142 kit. Consequently, it is not possible to compensate for the variability in analytic sensitivity between these two tests by adjusting the percent positive cell cutoff. The 28-8 was more sensitive than, but statistically indistinguishable from the 22C3 assay. Laboratory-developed tests (LDTs) using these and other primary antibodies have their own unique analytic performance characteristics. CONCLUSIONS. The PD-L1 reference materials enable precise definitions of analytic test performance and linking them with clinical management thresholds. Therefore, this tool finds its most important implementation at the stage of development of new IHC predictive biomarkers in clinical trials as well as at the stage of methodology transfer to clinical IHC laboratories. Furthermore, our results also help define more precisely the possibility for assay interchangeability and to what degree the assays may be harmonized. Citation Format: Emina E. Torlakovic, Seshi Sompuram, Kodela Vani, Steve Bogen. Analysis of PD-L1 IHC tests using NIST SRM 1934-traceable reference materials: A new paradigm for development of predictive IHC biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB034.

Antibiofilm activity against Staphylococcus aureus and content analysis of Taraxacum Officinale phenolic extract.

Taraxacum Officinale, commonly called dandelion, is herbaceous perennial belonging to the family of Asteraceae, having good antibacterial effects which are related to its phenolic substances. In this study, the effect of phenolic contents as well as the antibiofilm activity against Staphylo- coccus aureus of phenolic extract from T. Officinale were evaluated in vitro. With 70% metha- nol-water (v/v) as a solvent, the dandelion was extracted by ultrasonic assisted extraction method. Subsequent identification and quantification of phenol in extract was carried out using High Performance Liquid Chromatography (HPLC). The minimum inhibitory concentration and anti- bacterial kinetic curve of dandelion phenolic extract were analyzed by spectrophotometry. Changes in extracellular alkaline phosphatase (AKP) contents, electrical conductivity, intracellular protein contents, and DNA of S. aureus after the action of dandelion phenolic extract were determined to study its effect on the permeability of S. aureus cell wall and cell membrane. The results showed that chlorogenic acid (1.34 mg/g) was present in higher concentration, followed by lute- olin (1.08 mg/g), ferulic acid (0.22 mg/g), caffeic acid (0.21 mg/g), and rutin (0.19 mg/g) in the dandelion phenolic extract. The minimum inhibitory concentration (MIC) of dandelion phenolic extract against S. aureus was 12.5 mg/mL. The antibacterial kinetic curve analysis showed that the inhibitory effect of dandelion phenolic extract on S. aureus was mainly in the exponential growth phase. After applying the dandelion phenolic extract, the growth of S. aureus was signifi- cantly inhibited entering into the decay phase early. Furthermore, after the action of dandelion, the extracellular AKP contents of S. aureus, the electrical conductivity and the extracellular protein contents were all increased. The phenolic extract also affected the normal reproduction of S. aureus. These results suggest that dandelion has an inhibitory effect on S. aureus, and the mechanism of its action was to destroy the integrity of the cell walls and cell membranes.

Fermentation of Saccharomyces cerevisiae in a 7.5 L ultrasound-enhanced fermenter: Effect of sonication conditions on ethanol production, intracellular Ca2+ concentration and key regulating enzyme activity in glycolysis

In this study, the effect of sonication on the fermentation process of a single-celled fungus was examined. During the experiment, Saccharomyces cerevisiae (S. cerevisiae) was used as the starting strain for ethanol fermentation (batch fermentation) in a 7.5 L automated fermentation tank. The fermentation tank connected with a six-frequency ultrasonic equipment. Non-sonication treatment was set up as the control. Sonication treatment with power density of 280 W/L and 48 h of treatment time were set up as trial groups for investigating the influence of different ultrasound frequency including 20, 23, 25, 28, 33 and 40 kHz on the changes in dry cell-weight, glucose consumption rate, and ethanol yield. The results showed that the dry cell-weight, glucose consumption rate, and ethanol content reached the best results under the ultrasonic condition of 28 kHz ultrasound frequency in comparison with other ultrasound frequency. The dry cell-weight and ethanol content of the 28 kHz ultrasonic treatment group increased by 17.30% and 30.79%, respectively in comparison with the control group The residual sugar content dropped to a lower level within 24 h, which was consistent with the change in ethanol production. Besides, the results found that the glucose consumption rate increased compared to the control. It indicated that ultrasound accelerated glucose consumption contributed to increase the rate of ethanol output. In order to explore the mechanism of sonication enhanced the content of ethanol output by S. cerevisiae, the morphology, permeability of S. cerevisiae and key enzyme activities of ethanol synthesis were investigated before and after sonication treatment. The results showed that after sonication treatment, the extracellular nucleic acid protein content and intracellular Ca2+ concentration increased significantly. The morphology of S. cerevisiae was observed by SEM and found that the surface of the strain had wrinkles and depressions after ultrasonic treatment. furthermore after sonication treatment, the activities of three key enzymes which catalyze three irreversible reactions in glycolysis metabolism, namely, hexokinase, phosphofructokinase and pyruvate kinase increased by 59.02%, 109.05% and 87.27%, respectively. In a word, low-intensity ultrasound enhance the rate of ethanol output by S. cerevisiae might due to enhancing the growth and cell permeability of strains, and increasing the activities of three key enzymes of ethanol biosynthesis.

LDPE:PLA and LDPE:PLA:OMMT polymer composites: Preparation, characterization, and its biodegradation using Bacillus species isolated from dumping yard

AbstractThe focus of the present research is on the biodegradation of low density polyethylene (LDPE):polylactic acid (PLA) and LDPE:PLA:organo modified montmorillonite (OMMT) polymer nanocomposites. PLA was synthesized using the polycondensation method. The surface modification of nanomaterials is affected by various factors such as modifying agent, dispersion medium, and the dispersion of nanoparticles. Ultrasound cavitation technique improves the better dispersion of MMT in reaction mixture and enhances the interaction rate of a modifying agent with clay layered structure. A mixture of aminopropyltriethoxysilane and octadecyl amine was used for the surface modification of MMT. Polymer nanocomposite was prepared using the melt mixing method and used further for the biodegradation study. The mesophilic bacterium isolated from the dumping yard, identified by the 16rRNA technique, was capable of biodegradation of LDPE:PLA and LDPE:PLA:OMMT polymer nanocomposites. Isolated bacterial AP1 is 96.05% identical to the current MCCC1A02146 strain of Bacillus albus. After 50 days of incubation, the polymer nanocomposite sheet was characterized using Fourier transform infrared, X‐ray powder diffractometer, UV–Visible spectroscopy, Field Emission Scanning Electron Microscope (FESEM), etc. Further, LDPE:PLA30% (17.5%) and LDPE:PLA30%:OMMT4.5phr (19.20%), shows maximum percentage degradation. It has been observed that the addition of OMMT contributes to an enhancement in the polymer nanocomposite degradation because it has a large d‐spacing gap that is (21 Å) that enables the water to penetrate in the polymer matrix and increases the growth of bacteria within the polymer matrix. The highest change in biomass and change in extracellular protein content was found to be (39.11 mg/L and OD 0.38) LDPE:PLA30%:OMMT4.5phr composites. In addition, the mechanical properties decrease effectively after the incubation period, for LDPE:PLA 30% and LDPE:PLA30%:OMMT4.5phr.

Lumbar and ventricular CSF concentrations of extracellular matrix proteins before and after shunt surgery in idiopathic normal pressure hydrocephalus

BackgroundIdiopathic normal pressure hydrocephalus (iNPH) is a reversible CNS disease characterized by disturbed cerebrospinal fluid (CSF) dynamics. Changes in the extracellular matrix (ECM) composition might be involved in the pathophysiology of iNPH. The aim of this study was to explore possible differences between lumbar and ventricular CSF concentrations of the ECM markers brevican and neurocan, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and their relation to clinical symptoms in iNPH patients before and after shunt surgery.MethodsPaired lumbar and ventricular CSF was collected from 31 iNPH patients, before and four months after shunt surgery. CSF was analysed for concentrations of tryptic peptides originating from brevican and neurocan using a mass spectrometry-based panel, and for MMP-1, -2, -9, -10 and TIMP-1 using fluorescent or electrochemiluminescent immunoassays.ResultsBrevican and neurocan peptide levels were not influenced by CSF origin, but MMP-1, -2, -10 and TIMP-1 were increased (p ≤ 0.0005), and MMP-9 decreased (p ≤ 0.0003) in lumbar CSF compared with ventricular CSF. There was a general trend of ECM proteins to increase following shunt surgery. Ventricular TIMP-1 was inversely correlated with overall symptoms (rho = − 0.62, p < 0.0001). CSF concentrations of the majority of brevican and neurocan peptides were increased in iNPH patients with a history of cardiovascular disease (p ≤ 0.001, AUC = 0.84–0.94) compared with those without.ConclusionLevels of the CNS-specific proteins brevican and neurocan did not differ between the lumbar and ventricular CSF, whereas the increase of several CNS-unspecific MMPs and TIMP-1 in lumbar CSF suggests contribution from peripheral tissues. The increase of ECM proteins in CSF following shunt surgery could indicate disturbed ECM dynamics in iNPH that are restored by restitution of CSF dynamics.

Quantifying the Influence of rpoF Overexpression on Extracellular Biomolecules in PHL628 E. coli Biofilms by Confocal Microscopy

Biofilms form when planktonic, or freely swimming, bacteria adhere to a surface. Once adhered, connections form with other bacteria through extracellular polymeric substances (EPS). Biofilms can form on medical devices, causing persistent infections in patients, and on industrial equipment, which is known as biofouling. Biofilms present a unique challenge, as they can exhibit a higher tolerance for antibiotics than their planktonic counterparts. Curli are extracellular quaternary protein structures associated with robust biofilm formation in E. coli. They have been shown to aid in initial adhesion of the bacterial cells to a surface.Here, we induced the overexpression of rpoF, also known as fliA and σ28, which codes for a flagellar transcription factor important in late-stage flagellar assembly, in PHL628 E. coli, which overexpress curli at growth temperatures less than 30 °C. Because rpoF increases cell motility, we originally predicted that rpoF overexpression would decrease the amount of biofilm formation. However, we observed that rpoFoverexpression increased the amount of biofilm formed. We now suspect that the nature of rpoF overexpression's effects on biofilm formation may be through changing the composition of the EPS. To test this hypothesis, we grew biofilm under different conditions, and then analyzed the biomolecule composition of the EPS, as well as the overall biofilm volume using confocal laser scanning microscopy (CLSM). We used the dyes Thioflavin T and Calcofluor White to quantify extracellular amyloid proteins and carbohydrates, respectively. We found that rpoF overexpression increases the amount of extracellular carbohydrates. We also observed more curli formation when rpoF was overexpressed at lower temperatures, but not at higher growth temperatures. This suggests that curli does not account for increased biofilm formation at higher temperatures. In future experiments, we will continue similar imaging and analysis methods with Sypro Ruby (extracellular protein concentration), propidium iodide (eDNA), and DiD’ oil (lipid concentration) to obtain a more complete picture of how rpoF changes the composition of the biofilm EPS.

Lignocellulolytic activity of Pleurotus ostreatus under solid state fermentation using silage, stover, and cobs of maize

Lignocellulolytic white-rot fungi allow the bioconversion of agricultural wastes into value-added products that are used in a myriad of applications. The aim of this work was to use corn residues (Zea mays L.) to produce valuable products under solid-state fermentation (SSF) with Pleurotus ostreatus. White-rot fungus P. ostreatus was isolated from maize silage (MS) and thereafter it was inoculated on MS as substrate and compared with maize stover (MSt) and maize cobs (MC) to determine the best lignocellulosic substrate for the production of lignocellulolytic enzymes and extracellular protein. The MS gave the highest productivity of CMCase (368.2 U/mL), FPase (170.5 U/mL), laccase (11.4 U/mL), and MnPase (6.6 U/mL). This is compared to productivity on MSt of 222 U/mL, 50.2 U/mL, 4.55 U/mL, and 2.57 U/mL, respectively; and productivity on MC at the same incubation period as 150.5 U/mL, 48.2 U/mL, 3.58 U/mL, and 2.5 U/mL, respectively. The levels of enzyme production declined with increasing incubation period after 15 and 20 days using MS and MC, respectively, as substrates. Maximum liberated extracellular protein content (754 to 878 µg/mL) was recorded using MS, while a low amount (343 to 408 µg/mL) was liberated with using MSt and MC.

HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy

BackgroundThe human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is...

Characterization of extracellular polymeric substances from Synechocystis sp. PCC6803 under Cd (II), Pb (II) and Cr (VI) stress

Research regarding interactions between heavy metal ions and cyanobacteria is significant for the industrial application of cyanobacteria in wastewater treatment. The effects of Cd (II), Pb (II) and Cr (VI) on Synechocystis sp. PCC6803 extracellular polymeric substances (EPS) was investigated. In the absence of the EPS on cell surface, the adsorption amount of cyanobacterial cells to heavy metal ions reduced. Lots of metal ions precipitation on cyanobacterial cells surface were observed by SEM-EDX. The increase of exopolysaccharide contents was more obvious than extracellular protein contents under the heavy metal ions following metal ion exposure. The amide I was most sensitive to metal ion stimulation in functional group of extracellular proteins, but the interaction of metal ions with polysaccharides occurred prior to that with proteins. These results comprehensive proved that the EPS have significant impacts on cyanobacteria, thereby affecting the combination with heavy metals. Such insights will benefit our knowledge of the interactions between heavy metal ions and cyanobacterial EPS and provided theoretical guidance on the application of heavy metal sewage treatment by cyanobacteria.

569 A novel technique using raman spectroscopy for assessing cervical change during labor

We sought to use Raman spectroscopy, a light-based technology, for in vivo assessment of changes in the biochemical composition of the cervix as a function of labor progression based on spectral data. Pregnant women without history of cervical insufficiency, preterm labor, or rupture of membranes were recruited upon admission to labor and delivery. Demographic data including age, BMI, parity, and gestational age at delivery were collected. Raman spectroscopy measurements, using a fiber optic probe placed on the cervical face, were serially acquired during routine cervical exams until rupture of membranes. Generalized linear models using a restricted cubic spline with 65 degrees of freedom were fit to the data and spectral features were analyzed using generalized estimating equations. 30 women were included in the study, with average age of 27 years, BMI of 32, and gestational age of 39 weeks at delivery (Table 1). Raman spectral signatures associated with collagen, located at 1265, 1441, and 1650 cm-1, decreased approaching delivery. Similar decreases in peaks associated with actin and other proteins are noted, indicating a decline in extracellular matrix protein (ECM) concentration (Figure 1A). The ratio of 1657 to 1668 cm-1 increased closer to delivery, corresponding to collagen fiber reorientation and decreasing compactness. A greater extent of spectral change across all regions was observed in nulliparous compared to multiparous patients, suggesting a greater extent of cervical remodeling in the nulliparous population (Figure 1B). Patients with a BMI >30 at term displayed less change in spectral features associated with collagen compared to BMI<30 (Figure 1C). Raman spectroscopy objectively probes changes in chemical composition, identifying decreases in collagen, actin, and other ECM proteins as the cervix prepares for delivery. This technique shows differences in the cervical remodeling process based on parity and BMI, demonstrating the potential to quantitatively evaluate labor progression and utility for assessing cervical insufficiency in the future.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Discovering and efficiently promoting the extracellular secretory expression of Thermobacillus sp. ZCTH02-B1 sucrose phosphorylase in Escherichia coli

Sucrose phosphorylase (SPase, EC2.4.1.7) is a promising transglycosylation biocatalyst used for producing glycosylated compounds that are widely used in the food, cosmetics, and pharmaceutical industries. In this study, a recombinant SPase from the Thermobacillus sp. ZCTH02-B1 (rTSPase), which was previously reported to have high thermostability and the catalytic ability to synthesize ascorbic acid 2-glucoside, was attempted to be extracellularly expressed in Escherichia coli BL21(DE3) by fusion of endogenous osmotically-inducible protein Y. Unexpectedly, the rTSPase itself was produced outside the cells with an underestimated performance, although no typical signal peptide was predicted. Further N- and C-terminal truncation experiments revealed that both termini of rTSPase have an important role in protein folding and enzymatic activity, while its secretion was N-terminus associated. Extracellular protein concentration and rTSPase activity achieved 1.8 mg/mL and 6.2 U/mL after induction of 36 h in a 5-L fermenter. High-level extracellular rTSPase production could also be obtained from E. coli within 24 h by inducing overexpression of D, D-carboxypeptidase for cell lysis.

Bioactivity effects of extracellular matrix proteins on apical papilla cells.

Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs).Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05).Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all.Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Long-term biofouling formation mediated by extracellular proteins in Nannochloropsis gaditana microalga cultures at different medium N/P ratios.

Biofouling represents an important limitation in photobioreactor cultures. The biofouling propensity of different materials (polystyrene, borosilicate glass, polymethyl methacrylate, and polyethylene terephthalate glycol-modified) and coatings (two spray-applied and nanoparticle-based superhydrophobic coatings and a hydrogel-based fouling release coating) was evaluated by means of a short-term protein test, using bovine serum albumin (BSA) as a model protein, and by the long-term culture of the marine microalga Nannochloropsis gaditana under practical conditions. The results from both methods were similar, confirming that the BSA test predicts microalgal biofouling on surfaces exposed to microalgae cultures whose cells secrete macromolecules, such as proteins, with a high capacity for forming a conditioning film before cell adhesion. The hydrogel-based coating showed significantly reduced BSA and N. gaditana adhesion, whereas the other surfaces failed to control biofouling. Microalgal biofouling was associated with an increased concentration of sticky extracellular proteins at low N/P ratios (below 15).

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Size-Dependent Bioactivity of Silver Nanoparticles: Antibacterial Properties, Influence on Copper Status in Mice, and Whole-Body Turnover.

PurposeThe ability of silver nanoparticles (AgNPs) of different sizes to influence copper metabolism in mice is assessed.Materials and MethodsAgNPs with diameters of 10, 20, and 75 nm were fabricated through a chemical reduction of silver nitrate and characterized by UV/Vis spectrometry, transmission and scanning electronic microscopy, and laser diffractometry. To test their bioactivity, Escherichia coli cells, cultured A549 cells, and C57Bl/6 mice were used. The antibacterial activity of AgNPs was determined by inhibition of colony-forming ability, and cytotoxicity was tested using the MTT test (viability, %). Ceruloplasmin (Cp, the major mammalian extracellular copper-containing protein) concentration and enzymatic activity were measured using gel-assay analyses and WB, respectively. In vitro binding of AgNPs with serum proteins was monitored with UV/Vis spectroscopy. Metal concentrations were measured using atomic absorption spectrometry.ResultsThe smallest AgNPs displayed the largest dose- and time-dependent antibacterial activity. All nanoparticles inhibited the metabolic activity of A549 cells in accordance with dose and time, but no correlation between cytotoxicity and nanoparticle size was found. Nanosilver was not uniformly distributed through the body of mice intraperitoneally treated with low AgNP concentrations. It was predominantly accumulated in liver. There, nanosilver was included in ceruloplasmin, and Ag-ceruloplasmin with low oxidase activity level was formed. Larger nanoparticles more effectively interfered with the copper metabolism of mice. Large AgNPs quickly induced a drop of blood serum oxidase activity to practically zero, but after cancellation of AgNP treatment, the activity was rapidly restored. A major fraction of the nanosilver was excreted in the bile with Cp. Nanosilver was bound by alpha-2-macroglobulin in vitro and in vivo, but silver did not substitute for the copper atoms of Cp in vitro.ConclusionThe data showed that even at low concentrations, AgNPs influence murine copper metabolism in size-dependent manner. This property negatively correlated with the antibacterial activity of AgNPs.

Bacillus amyloliquefaciens application to prevent biofilms in reclaimed water microirrigation systems*

AbstractReclaimed water (RW) applied in drip irrigation systems could be a solution to cope with the challenges of limited freshwater resources. Biofilm growth in irrigation systems is an unavoidable issue when using RW. To date, strong chemical bactericides mostly dominate the commercial market in controlling biofilms. However, recently scientists have been concerned about their real efficacy and environmental risks. This study assesses a low‐concentration microbial antagonist (i.e., Bacillus amyloliquefaciens inoculums, BAI) intermittently injected into the systems to mitigate biofilm formation in three types of drip emitters using two kinds of treated RW. The results indicated that the application of BAI significantly (p &lt; .05) mitigated the microbial biomass within biofilms when compared with the control groups (no‐BAI). In addition, BAI reduced the contents of extracellular polysaccharides and proteins of biofilms, which decreased the total biomass in BAI treatments by 44.9%–73.8%. Consequently, BAI effectively improved the emitter performances and increased the discharge variation rate by 31.9%–44.3%. These findings might provide a new perspective to control biofouling when applying RW in irrigation systems, with potential implications for sustainable water management in agricultural production.

Anaerobic digestion of dairy manure in a fixed bed CSTR: Methane production performance and microbial diversity

To improve the fixed bed and continuous stirred-tank reactor (CSTR) technology relative to the rate of treatment of dairy manure (DM), a continuous stirred tank-treated DM with immobilized carrier biofilm was investigated for 20 days (hydraulic retention time (HRT) was 10 d). Methane productivity, biofilm characteristics, and microbial diversities of the biofilm and digestate were measured. The highest content of extracellular polymeric substances (EPS), proteins, and polysaccharides occurred at 15 days of digestion. An agglomeration phenomenon was observed using a scanning electron microscope on the biofilm. This indicated that the biofilm consisting of EPS was stable during the second HRT. Microbial diversities in digestate and carrier biofilm were characterized using a 16S rRNA sequencing technique. Results showed that the dominant bacterial communities were Pseudomonas (17% to 26%), Clostridium (13% to 21%), and Bacteroidetes (7% to 16%), while the archaea communities were Methanocorpusculum (25% to 37%), Methanosarcina (15% to 33%), Methanoculleus (11% to 15%), and Methanosaeta (13% to 18%). The methane production rate was significantly correlated with bacterial communities (Pseudomonas, Clostridium, Altererythrobacter atlanticus), archaeal communities (Methanosarcina, Methanoculleus, Methanosaeta, and Methanoplanus), and biofilm characteristics (chemical oxygen demand (COD) and EPS). These findings showed that a carrier biofilm could efficiently increase methane production.

Basic information for the development of a toxicity assay in inactivated bacterial vaccines

This study dealt with the toxicity of inactivated bacteria intended for veterinary autogenous vaccines toward a suitable control assay. Two in vitro methods were used. The [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] (MTT) test, based on the metabolic reaction of a tetrazolium salt in vital cells, was adopted on the basis of previous positive results. The Interleukin (IL)-1 beta release assay on monocyte-derived pig macrophages was carried out for comparative purposes, to evaluate the possible role of the inflammatory response. MTT and IL-1 beta responses showed a significant correlation (P < 0.05) at defined test dilutions of bacterial antigens, whereas no correlation was demonstrated using MTT responses normalized on bacterial cell concentration. Furthermore, the toxic effects shown in the MTT test were positively correlated to the extracellular protein content. On the whole, the above results could be a useful basis for the development of a toxicity assay on inactivated bacterial vaccines. Also, our data point at bacterial autolysis as a major component underlying toxicity.

Effects of endurance racing on horse plasma extracellular particle miRNA.

Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. To evaluate the effects of 160km endurance racing on horse plasma extracellular particles and their miRNA population. Cohort study. We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2hours after the race, which returned to baseline 15hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.

BolA gene involved in curli amyloids and fimbriae production in E. coli: exploring pathways to inhibit biofilm and amyloid formation

BackgroundBiofilm formation is a complex phenomenon of bacterial cells, involved in several human infections. Its formation is regulated and controlled by several protein factors. The BolA-like proteins (bolA gene) are conserved in both prokaryotes and eukaryotes. The BolA protein is a transcription factor involved in bacterial cell motility and biofilm formation. This study was initiated to elucidate the role of the bolA gene in the curli biogenesis and amyloid production as well as to observe changes in the expression of fimH, a fimbriae gene.MethodsKnockdown mutants of Escherichia coli MG1655 bolA gene (bolA-KD) were generated using CRISPR interference. The results obtained, were validated through gene expression using RT-PCR, microscopic analysis and different biofilm and amyloid assays.ResultsThe bolA knockdown mutants showed a decrement in curli amyloid fibers, in fimbriae production and biofilm formation. We have also observed a reduction in EPS formation, eDNA production and extracellular protein content. Gene expression data showed that bolA downregulation caused the suppression of csgA and csgD of curli that led to the reduction in curli fiber and the amyloid formation and also the suppression of fimH, leading to the loss of fimbriae.ConclusionsCurli fibers and fimbriae are found to be involved in biofilm formation leading to the pathogenicity of the bacterial cell. BolA is a conserved protein and is found to play a significant role in curli and fimbriae formation in E. coli. This study further proved that CRISPRi mediated suppression of the bolA gene leads to inhibition of biofilm formation through curli and fimbriae inhibition. Hence, it may be proposed as a possible target for intervention of biofilm mediated infections.