Endotoxin Removal Research Articles

Development of boronic acid-functionalized mesoporous silica-coated core/shell magnetic microspheres with large pores for endotoxin removal

Endotoxins are found almost everywhere and possess high toxicity in vivo and in vitro. Here we design a novel boronate affinity material, called boronic acid-functionalized mesoporous silica-coated core/shell magnetic microspheres (Fe3O4@nSiO2@mSiO2-BA) with large pores (pore size > 20 nm) based on the chemical structure and physical properties of endotoxins, for facile and highly efficient removal of endotoxins. Dual modes for endotoxin removal were proposed and confirmed in this work: the endotoxin aggregates with size < 20 nm were bound with boronic acid ligands chemically modified on the inner and outer surface of the large pores of Fe3O4@nSiO2@mSiO2-BA microspheres; while the larger endotoxin micelles (size >20 nm) were absorbed on the outer surface of the prepared material based on boronate affinity. Transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen adsorption/desorption isotherms and Fourier transform infrared (FT-IR) spectroscopy confirm that Fe3O4@nSiO2@mSiO2-BA microspheres possess core/shell structure, uniform diameter (520 nm), high surface area (205.57 m2/g), large mesopores (21.8 nm) and boronic acid ligands. The purification procedures of Fe3O4@nSiO2@mSiO2-BA microspheres for endotoxin were optimized, and 50 mM NH4HCO3 (pH 8.0) and 0.05 M fructose were selected as loading/washing, elution buffers, respectively. The binding capacity of Fe3O4@nSiO2@mSiO2-BA microspheres for endotoxin was calculated to be 60.84 EU/g under the optimized conditions. Finally, the established analytical method was applied to remove endotoxins from plasmid DNA. After endotoxin removal, the endotoxin content in plasmid DNA was reduced from 0.0026 to 0.0006 EU/mL for two-fold concentration, and from 0.0088 to 0.0022 EU/mL for five-fold concentration after binding, respectively. Additional advantages of the prepared boronate affinity material include excellent stability, reusability/repeatability, and low cost. Boronate affinity materials with large pores could thus prove to be powerful adsorbents for endotoxin removal and the potential applications in the aspects of biological research, pharmaceutical industry, and life health.

Reduction of Endotoxin from Human Root Canals by Calcium Hydroxide Nanoparticles

This study was aimed to evaluate the reduction of endotoxin using the passive ultrasonic irrigation (PUI) of calcium hydroxide nanoparticles (CHNPs) compared with the PUI of calcium hydroxide (CH) and the CH medication. Forty-seven single-root human premolars were used. The root canals were enlarged and sterilized. Escherichia coli endotoxin was inoculated into the root canals. Thirty-six root canals were assigned into three experimental groups: CHNPs irrigation, CH irrigation, and CH medication. For the irrigation groups, the PUI was activated for three min. For the CH medication, the CH was filled into each canal and incubated at 37°C for seven days. The reduction of endotoxin was evaluated by quantitative chromogenic limulus amoebocyte lysate assay. Data was analysed by Kruskal-Wallis and pairwise comparison tests, with a level of significance set at P < 0.05. The CHNPs irrigation was more effective in against endotoxin than the CH irrigation (P < 0.05). No significant difference was found between the CHNPs irrigation and the CH medication (P > 0.05). The irrigation of CHNPs could eliminate most of the endotoxin from the root canals and approximately from 300 μm of root dentin. CHNPs might be potential as an adjunctive irrigation for endotoxin removal.

Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament From Uropathogenic Escherichia coli for Immune Assay.

Flagella are expressed on the surface of a wide range of bacteria, conferring motility and contributing to virulence and innate immune stimulation. Host-pathogen interaction studies of the roles of flagella in infection, including due to uropathogenic Escherichia coli (UPEC), have used various methods to purify and examine the biology of the major flagella subunit protein, FliC. These studies have offered insight into the ways in which flagella proteins interact with host cells. However, previous methods used to extract and purify FliC, such as mechanical shearing, ultracentrifugation, heterologous expression in laboratory E. coli strains, and precipitation-inducing chemical treatments have various limitations; as a result, there are few observations based on highly purified, non-denatured FliC in the literature. This is especially relevant to host-pathogen interaction studies such as immune assays that are designed to parallel, as closely as possible, naturally-occurring interactions between host cells and flagella. In this study, we sought to establish a new, carefully optimized method to extract and purify non-denatured, native FliC from the reference UPEC strain CFT073 to be suitable for immune assays. To achieve purification of FliC to homogeneity, we used a mutant CFT073 strain containing deletions in four major chaperone-usher fimbriae operons (type 1, F1C and two P fimbrial gene clusters; CFT073Δ4). A sequential flagella extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal was applied to CFT073Δ4. Protein purity and integrity was assessed using SDS-PAGE, Western blots with anti-flagellin antisera, and native-PAGE. We also generated a fliC-deficient strain, CFT073Δ4ΔfliC, to enable the concurrent preparation of a suitable carrier control to be applied in downstream assays. Innate immune stimulation was examined by exposing J774A.1 macrophages to 0.05-1 μg of purified FliC for 5 h; the supernatants were analyzed for cytokines known to be induced by flagella, including TNF-α, IL-6, and IL-12; the results were assessed in the context of prior literature. Macrophage responses to purified FliC encompassed significant levels of several cytokines consistent with prior literature reports. The purification method described here establishes a new approach to examine highly purified FliC in the context of host-pathogen interaction model systems.

Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones

The most common approaches to improve soluble expression of heterologous proteins are applications of molecular chaperones such as DnaK, DnaJ, GrpE, GroEL and GroES. The aim of present study was to enhance soluble expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Escherichia coli by different approaches including modification of cultivation and induction conditions, and thermally, genetically and chemically enhancement of expression of cellular chaperones. To genetically enhance amount of molecular chaperones, co-expression of pET28-GM-CSF and pKJE7 plasmids was performed. The soluble expressed protein was affinity purified and subjected to endotoxin removal. Co-expression with molecular chaperones significantly increased soluble expression of GM-CSF. Addition of chemical chaperones and osmolytes like NaCl (0.5 M), sucrose (0.5 M), sorbitol (0.5 M) and MgCl2 (1 mM) to growing media could improve solubility of GM-CSF. Biological activity of purified GM-CSF was confirmed based on its proliferative effect on HL-60 cell lines. The approach developed in the present study can be applied to improve soluble expression of other recombinant protein proteins.

In vitro assessment of mixed matrix hemodialysis membrane for achieving endotoxin-free dialysate combined with high removal of uremic toxins from human plasma

For a single hemodialysis session nearly 500 L of water are consumed for obtaining pyrogen-free dialysis fluid. However, many efforts are required to avoid biofilm formation in the system and risk of contamination can persist. Water scarcity and inadequate water purification facilities worsen contamination risk in developing countries. Here, we investigated the application of an activated carbon (AC)/polyethersulfone/polyvinylpyrrolidone mixed matrix membrane (MMM) for achieving for the first time endotoxin-free dialysate and high removal of uremic toxins from human plasma with a single membrane. The MMM, thanks to sorbent AC, can remove approximately 10 times more endotoxins from dialysis fluid compared to commercial fibers. Pyrogens transport through the MMM was investigated analyzing inflammation in THP-1 monocytes incubated with samples from the dialysis circuit, revealing safety-barrier properties of the MMM. Importantly, endotoxins from dialysate and protein-bound toxins from human plasma can be removed simultaneously without compromising AC adsorption capacity. We estimated that only 0.15 m2 of MMM is needed to totally remove the daily production of the protein-bound toxins indoxyl sulfate and hippuric acid and to completely remove endotoxins in a wearable artificial kidney (WAK) device. Our results could open up new possibilities for dialysis therapy with low water consumption including WAK and where purity and scarcity of water are limiting factors for hemodialysis treatment. Statement of SignificanceHemodialysis is a life-sustaining extracorporeal treatment for renal disease, however the production of pyrogen-free dialysate is very costly and water demanding. Biofilm formation in the system worsens bacteria contamination risk. Pyrogens could be transferred into the patients’ blood and trigger inflammation. Here, we show for the first time that a mixed matrix membrane composed of polyethersulfone/polyvinylpyrrolidone and activated carbon can achieve simultaneous complete removal of endotoxins from dialysate and high removal of uremic toxins from human plasma without compromising activated carbon adsorption capacity. The mixed matrix membrane could find future applications for simultaneous blood purification and dialysate depyrogenation thus lowering water consumption as for wearable artificial kidney devices and where purity and scarcity of water hamper hemodialysis treatment.

Effect of two different root canal instruments on removal of endotoxins from necrotic root canals (A Randomized clinical trial)

Aim of the study: The aim of the present randomized clinical study was to evaluate the effect of OneShape and Protaper Next rotary system on the removal of endotoxins from teeth with asymptomatic necrotic pulp.Materials and Methods: Thirty patients were randomly allocated into two groups (n=15) according to the type of rotary system used in mechanical preparation; either Group OS (OneShape) or Group PN (Protaper Next). Samples were taken before and after mechanical preparation and quantification of endotoxins was done using Human Elisa kit. Statistical analyses were performed using Mann-Whitney U test to compare between the two groups then Wilcoxon signed- rank test was used to study the changes after treatment within each group. The significance level was set at P ≤ 0.05.Results: Group OS, showed a statistically significant reduction in endotoxins levels after treatment (P-value < 0.001, Effect size = 0.880). In Group PN, there was a statistically significant reduction in endotoxins levels after treatment (P-value <0.001, Effect size = 0.931) and Group OS showed statistically significant lower median % reduction in endotoxins level than Group PN.Conclusion: Protaper Next was more effective than the Oneshape in reducing the endotoxins from teeth with asymptomatic necrotic pulp.

Extracorporeal Blood Purification Therapies for Sepsis

Extracorporeal blood purification is proposed as an adjuvant therapy for sepsis, aiming at controlling the associated dysregulation of the immune system, which is known to induce organ dysfunctions. Different therapies have been developed to address certain steps of the immune dysregulation. Most of the available blood purification devices focus on a single target, such as the endotoxin that triggers the immune cascade, or the cytokine storm that causes organ damages. However, the highly adsorptive membrane named oXiris® is a unique 4-in-1 device that combines cytokine and endotoxin removal properties, renal replacement function, and antithrombogenic properties. More recently, promising treatments that focus on the pathogen itself or the immune cells have been developed and are currently under investigation. In this review, we aim to summarize, according to their target, the different extracorporeal blood purification techniques that are already available for use. We will also briefly introduce the most recent techniques that are still under development. Because of its unique ability to remove both endotoxins and cytokines, we will particularly discuss the highly adsorptive preheparinized oXiris® membrane. We will present its properties, advantages, pitfalls, as well as therapeutic perspectives based on experimental and clinical data. Video Journal Club “Cappuccino with Claudio Ronco” at https://www.karger.com/Journal/ArticleNews/223997?sponsor=52

Partitioning and recovery of an elongation factor (1-γ) of Leishmania infantum chagasi expressed in E. coli M15 with simultaneous endotoxin removal using aqueous two-phase system

ABSTRACTThe 503-antigen, the elongation factor (1-γ) found in the amastigote phase of Leishmania infantum chagasi, could integrate either kits of specific diagnostic assays or vaccines for visceral leishmaniasis. However, protein purification expressed in Escherichia coli is driven by several steps, namely, clarification and multisteps chromatography. Additionally, the removal of lipopolysaccharide (LPS/endotoxin) which triggers an immune response in the human body is needed since LPS interacts with the proteins impairing the chromatography steps. Aqueous two-phase systems (ATPSs) based on polyethylene glycol (PEG) 1500 and salt (K2HPO4 or (NH4), ethanol/salt, and acetonitrile/D-glucose were used for partitioning of 503-antigen expressed in recombinant E. coli M15 with simultaneous LPSs removal. System based on 30% PEG 1500 and 10% potassium phosphate with 20% (w/w) unclarified cell homogenate achieved protein recovery of 116.96 ± 3.45% and purification factor of 1.55 ± 0.01. Endotoxin was removed with values as high as 99.90% for all systems with partial purification of 503-antigen, thus eliminating a clarification step in the partial purification protocol of this antigen.

Efficacy of Coupled Plasma Filtration Adsorption in Treating Patients with Severe Intra-Abdominal Infection: A Retrospective Study.

Background: Coupled plasma filtration adsorption (CPFA) is an extracorporeal treatment based on plasma filtration associated with an adsorbent cartridge and hemofiltration. CPFA is able to remove inflammatory mediators and it has been used to treat severe sepsis and multiple organ dysfunction. Limited experience exists on the use of CPFA in treating intra-abdominal infection (IAI). Methods: In this study, the efficacy of CPFA in treating patients with severe IAI and liver failure was evaluated in a retrospective analysis of 76 cases. Results: The survival rate of patients treated with CPFA was 82.6%, with effective removal of endotoxin and inflammatory mediators. Conclusion: Our data suggest that CPFA can be safely and effectively used to lower morbidity and mortality rates of patients with severe IAI and liver failure.

Effects of chlorination and combined UV/Cl2 treatment on endotoxin activity and inhalation toxicity of lipopolysaccharide, gram-negative bacteria and reclaimed water

Disinfection processes were applied in reclaimed water plant to eliminate pathogens and control the related health risk during water reuse. However, extra problems might emerge such as the released free endotoxins from the ruptured cell wall of gram-negative bacteria. Endotoxins can induce lung inflammatory responses after inhalation, which has been neglected in the water quality regulation, and the removal of endotoxin was not under consideration in the process of reclamation. In the present study, two well-known disinfection processes, chlorination and combined UV/chlorine (UV/Cl2), were performed to test the removal efficiency of endotoxin activity, as well as the inflammation inducing ability. In the pure LPS solution, UV/Cl2 treatment significantly reduced both endotoxin activity and lung inflammation responses with better oxidizability of the generated hydroxyl radical. However, its performance on bacteria liquid and real secondary effluent was more complicated. The cell wall-bound LPS have lower endotoxin activities and inflammation inducing ability. Immediately after the cell wall was destroyed, the bound LPS were released to the solution to be free LPS, which dramatically increased both the endotoxin activity and inflammation inducing ability of the water. When these free endotoxins were continuously oxidized, the endotoxin activity and inflammatory response decreased again but not to the background level. Therefore, the inflammation inducing ability of reclaimed water could not be removed efficiently. These results suggest that in spite of its high oxidability, UV/Cl2 treatment is not capable of removing the endotoxin-based toxicity, and other technologies are necessary to control endotoxin levels in reclaimed water.

A new approach to purification of bacterial cellulose membranes: What happens to bacteria in supercritical media?

In this paper, we report our attempts to elaborate the method of purification and simultaneous sterilization of bacterial cellulose films by means of processing them under high pressure, including supercritical conditions for carbon dioxide, for future application in medicine. Bacterial cellulose films were produced by Gluconacetobacter hansenii. The processed samples were controlled by means of AFM, SEM, EDS, IR spectrometry, elemental and microbiological analysis.It was shown that conventional methods do not always provide complete removal of cells and endotoxins and the samples contain traces of compounds used for washing. It was revealed that the outer lipopolysaccharide membrane of the producent cells was destroyed during processing, but bacterial cells still preserved their shape and dimensions. The processed samples were sterile; the level of endotoxins was strongly reduced, though, unfortunately the degree of purification required for medical use was not achieved.

The effect of different treatments of lymph after intestinal ischemia-reperfusion in rats on macrophages in vitro.

BackgroundTo observe the effects of different treatments of lymph after intestinal I/R in rats on macrophages in vitro.MethodsForty-eight healthy SPF SD rats weighing 300 ± 20 g, were randomly divided into two groups: group A, and group B. The rats in group A were drained of lymph fluid for 180 min; the rats in group B were subjected to 60 min ischemia by clamping the SMA, followed by 120 min reperfusion and 180 min of lymph drainage. The lymph fluid collected was divided into 4 sub-groups: 1. no treatment (A1, Ly, and B1, I/R Ly); 2. protein degradation (A2, Ly PD, and B2 I/R PD); 3. endotoxin removal (A3, Ly ER, and B3, I/R ER); 4. protein degradation plus endotoxin removal (A4, Ly PD+ER, and B4, I/R PD+ER), then used to stimulate a monocyte-macrophage cell line.ResultsCompared with group A1, the levels of the inflammatory cytokines, chemokines, HMGB1 concentration, protein and mRNA expression of TLR4, HMGB1 and NF-κBp65 were significantly increased in group B1. There was a significant reduction in proinflammatory cytokines and of the expression of TLR4, NF-κBp65, and chemokines in groups A2, B2, A4, and B4. However, there were no significant decrease of these factors in groups A3 and B3.ConclusionsThe lymph fluid drained after intestinal I/R can cause inflammation in vivo and in vitro. Deproteinization of lymph fluid with proteinase K significantly reduced the concentration of proinflammatory cytokines, chemokines, TLR4 and NF-κBp65 in cell culture supernatant, exerting a protective effect on inflammatory reaction caused by the intestinal I/R. Passage of lymph fluid through an endotoxin removal column did not reduce the levels of active proinflammatory factors produced by macrophages in vitro.

Endotoxin removal from aqueous solutions with dimethylamine-functionalized graphene oxide: Modeling study and optimization of adsorption parameters.

Novel graphene oxide (GO)-based adsorbent embedded with epichlorohydrin (ECH) as a coupling agent and dimethylamine (DMA) as a ligand (GO-ECH-DMA) were prepared and employed for endotoxin removal from aqueous solutions. The physicochemical properties of nanocomposite were fully characterized. The model attributed to batch adsorption process was optimized employing response surface methodology (RSM) via various parameters such as pH, GO-ECH-DMA dosage, and contact time and endotoxin concentration. The p-value with low probability (<0.00001), determination coefficient (R2=0.99) and the non-significant lack of fit (p > 0.05) showed a quadratic model with a good fit with experimental terms. The synergistic effects of the linear term of contact time and GO-ECH-DMA dosage on endotoxin removal were significant. The optimum condition for endotoxin removal was obtained at pH of 5.52, GO-ECH-DMA dosage of 21 mgL-1, contact time of 56 min and endotoxin concentration of 51.3 endotoxin units per milliliter (EUmL-1). The equilibrium was the better explained by Langmuir isotherm with the maximum monolayer adsorption capacity of 121.47 EUmg-1, while the kinetics of the endotoxin adsorption process was followed by the pseudo-second-order model. The adsorbent could be recycled with NaOH. The possible mechanisms of endotoxin adsorption were proposed by hydrogen-bonding, π-π stacking, and electrostatic interaction.

Chitosan based in situ and ex situ magnetic iron oxide nanoparticles for rapid endotoxin removal from protein solutions

Chitosan based iron oxide nanoparticles have been employed for reversible capture and removal of bacterial endotoxin from aqueous protein solutions.

Microwave-induced release and degradation of airborne endotoxins from Escherichia coli bioaerosol.

Endotoxins are widely distributed toxins in the outer cell-wall membranes of Gram-negative bacteria and other microorganisms. Chronic exposure to endotoxins can induce and exacerbate airway symptoms and diseases. However, the release and degradation of airborne endotoxins from bioaerosol by microwave (MW) irradiation have not yet been reported. This study investigated the distribution and fate of airborne endotoxins during MW irradiation process, as well as the kinetics and thermodynamics of the degradation of airborne endotoxins. Results showed that MW irradiation induced cell lysis, thus considerably increasing the proportion of cells with ruptured membranes. Furthermore, MW irradiation changed the distribution of airborne endotoxins, sharply decreased the concentration of bound endotoxins from 230 EU/m3 to 68 EU/m3, and increased the concentration of free endotoxins from 21 EU/m3 to 122 EU/m3. These results indicated that MW irradiation released endotoxins from cells into the atmosphere. MW irradiation likely degraded endotoxins by exerting thermal effects, which achieved a total endotoxin removal efficiency of as high as 35%. Endotoxin degradation was a first-order reaction and required the activation energy of 26.3 kJ/mol.

Inactivation of airborne bacteria using different UV sources: Performance modeling, energy utilization, and endotoxin degradation

Airborne bacteria-containing bioaerosols have attracted increased research attention on account of their adverse effects on human health. Ultraviolet germicidal irradiation (UVGI) is an effective method to inactivate airborne microorganisms. The present study models and compares the inactivation performance of three UV sources in the UVGI for aerosolized Escherichia coli. Inactivation efficiency of 0.5, 2.2 and 3.1 logarithmic order was obtained at an exposure UV dose of 370 J/m3 under UVA (365 nm), UVC (254 nm) and UVD (185 nm) sources, respectively. A Beer–Lambert law-based model was developed and validated to compare the inactivation performances of the UV sources, and modeling enabled prediction of inactivation efficiency and analysis of the sensitivity of several parameters. Low influent E. coli concentrations and high UV doses resulted in high energy consumption (EC). The change in airborne endotoxin concentration during UV inactivation was analyzed, and UVC and UVA irradiation showed no marked effect on endotoxin degradation. By contrast, both free and bound endotoxins could be removed by UVD treatment, which is attributed to the ozone generated by the UVD source. The results of this study can provide a better understanding of the air disinfection and airborne endotoxin removal processes.

Endotoxin removal from human-like collagen by allantoin

ABSTRACTHuman-like collagen(HLC) has an important role in biomedical field, while endotoxin contamination limits it use. In this study, the saturated allantoin as an adsorbent was applied in adsorbing endotoxins from human-like collagen in Tris-acetate-EDTA(TAE) buffer.Finally, the endotoxins carried by allantoin and soluble allantoin could be removed by centrifugation and ultrafiltration. Treated by 400mg/ml of allantoin, at pH6.5 and 25mM NaCl the endotoxins residual in HLC was decreased to 2.38+0.11EU/mg and protein recovery achieved 98.7% in TAE buffer. Endotoxins could be adsorbed on the surface of allantoin crystal by the hydrogen-bond. At pH8.0, the main force was hydrophobic and bivalent bridge between HLC and endotoxins. When the pH was 6.5, the negative-electron repulsion became the main force. And then, Transmission Electron Microscopy(TEM) was used to observe the ultrastruction of HLC,illustrating the protein ultrastruction had not been affected in TAE buffer. In addition, the method of endotoxins removed by allantoin adsorptoin in TAE buffer was safety and greatly reduced the costs,time consumption in contrast with affinity chromatography. As a whole, this paper provided a good idea to establish a large-scale industrial process to remove endotoxin in biomedical field for its simple operation and low cost.

Polymyxin B immobilized nanofiber sponge for endotoxin adsorption

Nanofiber sponge, which showed high porosity and excellent blood compatibility, was successfully prepared by electrospinning and freeze-drying technology. The APTT (activated partial thromboplastin time) and PT (prothrombin time) of human plasma with nanofiber sponge were a little longer than control groups, which revealed the nanofiber sponge has excellent blood compatibility. By activating the carboxyl groups on the fibers, PMB (polymyxin B) was successfully immobilized on the nanofiber sponge. The adsorption performance was researched in different solutions. The adsorption capacity in physiological saline solution was around 17,889 EU/mL and the adsorption equilibrium time was about 40 min. The adsorption behavior not only obeyed to second-order kinetic equation, but also was conformed to Langmuir model, which turned out to be chemical adsorption and monolayer adsorption. The endotoxin removal rate in BSA (bovine serum albumin) physiological saline solution was about 96.6%, and the BSA recovery rate was around 75.6%, which indicated the PMB immobilized nanofiber sponge adsorbed endotoxin selectively. The endotoxin removal rate of normal nanofiber sponge was about 17.3%, while it was around 99.6% when it comes to PMB immobilized-nanofiber sponge, which manifested PMB played an important role in endotoxin adsorption. The endotoxin removal rate in human plasma reached 90%, and the adsorption reached equilibrium within 60 min, which proved the PMB immobilized-nanofiber sponge would have great potential for applying in clinical blood purification.

Abstract 17171: Kruppel-Like Factor 4 Deletion Regulates Lymphatic Endothelial Cell Homeostasis

Introduction and Hypothesis: Lymphatic vasculature exquisitely controls the clearance of metabolic end products, removal of bacterial endotoxins, and trafficking of immune cells. Signaling pathways that converge into endothelial cell kruppel-like factor 4 (KLF4) provide an atheroprotective role; however, the role of lymphatic endothelial cell (LEC)-KLF4 in these processes has not been clarified. Thus, we examined whether LEC-specific deletion of Klf4 alleles LECs. Methods and Results: After several generations of breeding, we transferred ROSA mT/mG (dual-fluorescent reporter allele mT/mG) to Prox1 CreERT2 (tamoxifen, TAM) inducible mice in C57 background to generate ROSA mT/mG ::Prox1 CreERT2 mice. In the next breeding scheme, ROSA mT/mG ::Prox1 CreERT2 female mice were crossed with male Klf4 fl/wt (Klf4 floxed allele) mice to generate heterozygous ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt offspring (50%). Thereafter, we bred male ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt with female ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/wt to produce ROSA mT/mG ::Prox1 CreERT2 ::Klf4 fl/fl genotype (25%). These mice (12-14 weeks old) received TAM for five consecutive days, and on 21 st or 28 th day all mice were sacrificed. To confirm LEC Cre activation after TAM administration, we prepared cryosections of lung and the heart tissues. Mice receiving TAM showed lively and clear separation of green fluorescent protein (GFP) and tomato-Red colors, showing LEC specificity of Prox1 CreERT2 transgenic methodology. Cryosectioning combined with low- and high-resolution microscopy, and morphometric analyses of H&amp;E sections showed disruption of LEC function and integrity in these mice. Biochemical experiments underway will address the role of KLF4 in lymphatic vessels homeostasis. Conclusion: These results indicate a key role for Klf4 in the regulation of lymphatic vessels.

Endotoxin-Free E. coli-Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production.

Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.