Filter Assay Research Articles

The cytosolic tryparedoxin peroxidase from Trypanosoma cruzi induces a pro-inflammatory Th1 immune response in a peroxidatic cysteine-dependent manner.

Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin (Prx) with an important role in detoxifying host cell oxidative molecules during parasite infection. c-TXNPx is a virulence factor, as its overexpression enhances parasite infectivity and resistance to exogenous oxidation. As Prxs from other organisms possess immunomodulatory properties, we studied the effects of c-TXNPx in the immune response and analysed whether the presence of the peroxidatic cysteine is necessary to mediate these properties. To this end, we used a recombinant c-TXNPx and a mutant version (c-TXNPxC52S) lacking the peroxidatic cysteine. We first analysed the oligomerization profile, oxidation state and peroxidase activity of both proteins by gel filtration, Western blot and enzymatic assay, respectively. To investigate their immunological properties, we analysed the phenotype and functional activity of macrophage and dendritic cells and the T-cell response by flow cytometry after injection into mice. Our results show that c-TXNPx, but not c-TXNPxC52S, induces the recruitment of IL-12/23p40-producing innate antigen-presenting cells and promotes a strong specific Th1 immune response. Finally, we studied the cellular and humoral immune response developed in the context of parasite natural infection and found that only wild-type c-TXNPx induces proliferation and high levels of IFN-γ secretion in PBMC from chronic patients without demonstrable cardiac manifestations. In conclusion, we demonstrate that c-TXNPx possesses pro-inflammatory properties that depend on the presence of peroxidatic cysteine that is essential for peroxidase activity and quaternary structure of the protein and could contribute to rational design of immune-based strategies against Chagas disease.

Decontamination and re-use of surgical masks and respirators during the COVID-19 pandemic

ObjectivesThe coronavirus disease 2019 pandemic increased global demand for personal protective equipment (PPE) and resulted in shortages. The study evaluated the re-use of surgical masks and respirators by analysing their performance and safety before and after reprocessing using the following methods: oven, thermal drying, autoclave, and hydrogen peroxide plasma vapour. MethodsIn total, 45 surgical masks and 69 respirators were decontaminated. Visual integrity, air permeability, burst resistance, pressure differential and particulate filtration efficiency of new and decontaminated surgical masks and respirators were evaluated. In addition, 14 used respirators were analysed after work shifts before and after decontamination using reverse transcription polymerase chain reaction (RT-PCR) and viral culturing. Finally, reprocessed respirators were evaluated by users in terms of functionality and comfort. ResultsOven decontamination (75 °C for 45 min) was found to be the simplest decontamination method. Physical and filtration assays indicated that all reprocessing methods were safe after one cycle. Oven decontamination maintained the characteristics of surgical masks and respirators for at least five reprocessing cycles. Viral RNA was detected by RT-PCR in two of the 14 used respirators. Four respirators submitted to viral culture were PCR-negative and culture-negative. Reprocessed respirators used in work shifts were evaluated positively by users, even after three decontamination cycles. ConclusionOven decontamination is a safe method for reprocessing surgical masks and respirators for at least five cycles, and is feasible in the hospital setting.

Adriamycin does not damage podocytes of zebrafish larvae

Podocytes are highly specialized epithelial cells that are essential for an intact glomerular filtration barrier in the kidney. Several glomerular diseases like focal segmental glomerulosclerosis (FSGS) are initially due to podocyte injury and loss. Since causative treatments for FSGS are not available until today, drug screening is of great relevance. In order to test a high number of drugs, FSGS needs to be reliably induced in a suitable animal model. The zebrafish larva is an ideal model for kidney research due to the vast amount of offsprings, the rapid development of a simple kidney and a remarkable homology to the mammalian glomerulus. Zebrafish larvae possess a size-selective glomerular filtration barrier at 4 days post fertilization including podocytes with interdigitating foot processes that are connected by a slit membrane. Adriamycin is an anthracycline which is often used in mice and rats to induce a FSGS-like phenotype. In this study, we aimed to induce a similar phenotype to zebrafish larvae by adding adriamycin to the tank water in different concentrations. Surprisingly, zebrafish larvae did not develop glomerular injury and displayed an intact filtration barrier after treatment with adriamycin. This was shown by (immuno-) histology, our filtration assay, in vivo imaging by 2-photon microcopy, RT-(q)PCR as well as transmission electron microscopy. To summarize, adriamycin is unable to induce a podocyte-related damage in zebrafish larvae and therefore major effort must be made to establish FSGS in zebrafish larvae to identify effective drugs by screenings.

Synthetase of the methyl donor S-adenosylmethionine from nitrogen-fixing α-rhizobia can bind functionally diverse RNA species

ABSTRACT Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti. The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans-sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme.

Sandwich dot-immunogold filtration assay (DIGFA) for specific immunodiagnosis of active neuroangiostrongyliasis.

Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10-15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present 'AcDIGFAAg' enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.

Detection of endogenous lipids in chicken feathers distinct from preen gland constituents

Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong reaction with osmium tetroxide (OsO4), even after treatment with detergents. This leads us to postulate the existence of endogenous feather lipids distinct from preen gland lipids. In order to substantiate our hypothesis, we investigated down feathers from a 1-day-old chicken as their uropgygial gland is not functionally active. The results confirmed the osmiophilic reaction, which was concentrated in the center of barbs and strongly reduced after lipid extraction. In these lipid extracts, we identified using thin layer chromatography, cholesterol, various ceramides, glycolipids, phospholipids, and fatty acids, which closely resembled the lipid composition of the water barrier in the chicken-cornified epidermal envelope. This composition is clearly distinct from chicken uropygeal gland secretion (UGS) known to consist of fatty alcohols as part of aliphatic monoester waxes and of free, predominantly saturated, fatty acids. A filter assay showed a strong reactivity between OsO4 and the fatty acids C18:1 and C18:2 and with feather lipid extracts, but not with UGS. These observations were confirmed by gas chromatography detecting unsaturated fatty acids including C18:1 and C18:2 as well as cholesterol exclusively in chicken feathers. Our results indicate that (1) endogenous lipids are detectable in chicken feathers and distinct from UGS and (2) in analogy to the morphogenesis of the cornified envelope of chicken feather lipids that may have derived from cellular feather-precursors, apparently enduring the specific cell death during developmental feather cornification.

Agglutination of Human Polyomaviruses by Using a Tetravalent Glycocluster as a Cross-Linker.

Two kinds of tetravalent double-headed sialo-glycosides with short/long spacers between the Neu5Acα2,6Galβ1,4GlcNAc unit and ethylene glycol tetraacetic acid (EGTA) scaffold were found to be capable of binding to virus-like particles of Merkel cell polyomavirus (MCPyV-LP). The binding process and time course of interaction between the tetravalent ligand and MCPyV-LP were assessed by dynamic light scattering (DLS). On the addition of increasing concentrations of ligand to MCPyV-LP, larger cross-linked aggregates formed until a maximum size was reached. The binding was stronger for the tetravalent ligand with a short spacer than for that with a long spacer. The binding of the former ligand to the virus was observed to proceed in two stages during agglutination. The first step was the spontaneous formation of small aggregates comprising the cross-linked ligand–virus complex. In the second step, the aggregates grew successively larger by cooperative binding among the initially produced small aggregates. In transmission electron microscopy, the resulting complex was observed to form aggregates in which the ligands were closely packed with the virus particles. The cross-linked interaction was further confirmed by a simple membrane filtration assay in which the virus-like particles were retained on the membrane when complexed with a ligand. The assay also showed the effective capture of particles of pathogenic, infectious human polyomavirus JCPyV when complexed with a ligand, suggesting its possible application as a method for trapping viruses by filtration under conditions of virus aggregation. Collectively, these results show that the tetravalent glycocluster serves as a ligand not only for agglutinating MCPyV-LP but also for trapping the pathogenic virus.

Cholera Toxin Subunit B for Sensitive and Rapid Determination of Exosomes by Gel Filtration.

We developed a sensitive fluorescence-based assay for determination of exosome concentration. In our assay, Cholera toxin subunit B (CTB) conjugated to a fluorescence probe and a gel filtration technique (size-exclusion chromatography) are used. Exosomal membranes are particularly enriched in raft-forming lipids (cholesterol, sphingolipids, and saturated phospholipids) and in GM1 ganglioside. CTB binds specifically and with high affinity to exosomal GM1 ganglioside residing in rafts only, and it has long been the probe of choice for membrane rafts. The CTB-gel filtration assay allows for detection of as little as 3 × 108 isolated exosomes/mL in a standard fluorometer, which has a sensitivity comparable to other methods using advanced instrumentation. The linear quantitation range for CTB-gel filtration assay extends over one order of magnitude in exosome concentration. Using 80 nM fluorescence-labeled CTB, we quantitated 3 × 108 to 6 × 109 exosomes/mL. The assay ranges exhibited linear fluorescence increases versus exosome concentration (r2 = 0.987). The assay was verified for exosomal liposomes. The assay is easy to use, rapid, and does not require any expensive or sophisticated instrumentation.

Structural basis of the interaction between cyclodipeptide synthases and aminoacylated tRNA substrates.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic β2 and β7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.

DiSCount: computer vision for automated quantification of Striga seed germination

BackgroundPlant parasitic weeds belonging to the genus Striga are a major threat for food production in Sub-Saharan Africa and Southeast Asia. The parasite’s life cycle starts with the induction of seed germination by host plant-derived signals, followed by parasite attachment, infection, outgrowth, flowering, reproduction, seed set and dispersal. Given the small seed size of the parasite (< 200 μm), quantification of the impact of new control measures that interfere with seed germination relies on manual, labour-intensive counting of seed batches under the microscope. Hence, there is a need for high-throughput assays that allow for large-scale screening of compounds or microorganisms that adversely affect Striga seed germination.ResultsHere, we introduce DiSCount (Digital Striga Counter): a computer vision tool for automated quantification of total and germinated Striga seed numbers in standard glass fibre filter assays. We developed the software using a machine learning approach trained with a dataset of 98 manually annotated images. Then, we validated and tested the model against a total dataset of 188 manually counted images. The results showed that DiSCount has an average error of 3.38 percentage points per image compared to the manually counted dataset. Most importantly, DiSCount achieves a 100 to 3000-fold speed increase in image analysis when compared to manual analysis, with an inference time of approximately 3 s per image on a single CPU and 0.1 s on a GPU.ConclusionsDiSCount is accurate and efficient in quantifying total and germinated Striga seeds in a standardized germination assay. This automated computer vision tool enables for high-throughput, large-scale screening of chemical compound libraries and biological control agents of this devastating parasitic weed. The complete software and manual are hosted at https://gitlab.com/lodewijk-track32/discount_paper and the archived version is available at Zenodo with the DOI https://doi.org/10.5281/zenodo.3627138. The dataset used for testing is available at Zenodo with the DOI https://doi.org/10.5281/zenodo.3403956.

Co-existence of hepatocellular carcinoma and cystic echinococcosis

PurposeCo-existence of hepatocellular carcinoma (HCC) and cystic echinococcus (CE) is extremely rare. Echinococcus granulosus may exhibit a protective effect against cancer. Herein, this study aimed to evaluate the possible effects of echinococcal infection on HCC patients.MethodsThree thousand three hundred hepatic CE patients and 815 HCC patients were retrospectively reviewed between January 2010 and December 2018 in Xinjiang, China, and these patients were 1:5 matched according to their sex, age and tumor TMN stage, and only 13 patients coexisted both CE and HCC. Preoperative ultrasonography (US), computed tomography (CT), liver magnetic resonance imaging (MRI) and dot immune-gold filtration assay (DIGFA) were used for preoperative identification and intraoperative specimens from liver resections were pathologically examined for further confirmation. Survival time was analyzed through Cox proportional hazard model analysis.ResultsThe co-existing incidence rate of two diseases was 0.39%. For these concurrent cases, HCC was all at the advanced stage and CE lesions were inactive. Median survival time for HCC patients was 6 month (1–17). However, it was 8 month (3–90) for the co-existing cases and was much longer than the median survival time of HCC patients (P<0.05), which was closely associated with tumor size, location, TMN stage and hydatid size, location, classification. Four of the patients underwent surgical intervention and their median survival time was 17 month (3–68).ConclusionsEchinococcus granulosus may elicit a protective effect against the development and progression of HCC, while more basic and clinical researches are needed.

Noncovalent Spin-Labeling of DNA and RNA Triplexes.

Studying nucleic acids often requires labeling. Many labeling approaches require covalent bonds between the nucleic acid and the label, which complicates experimental procedures. Noncovalent labeling avoids the need for highly specific reagents and reaction conditions, and the effort of purifying bioconjugates. Among the least invasive techniques for studying biomacromolecules are NMR and EPR. Here, we report noncovalent labeling of DNA and RNA triplexes with spin labels that are nucleobase derivatives. Spectroscopic signals indicating strong binding were detected in EPR experiments in the cold, and filtration assays showed micromolar dissociation constants for complexes between a guanine-derived label and triplex motifs containing a single-nucleotide gap in the oligopurine strand. The advantages and challenges of noncovalent labeling via this approach that complements techniques relying on covalent links are discussed.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, γ-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

空气细颗粒物PM2.5水溶成分对人黑素细胞系PIG1增殖、迁移和黑素合成的影响

Objective To evaluate the effect of water-soluble components of atmospheric fine particulate matter PM2.5 on proliferation, migration, tyrosinase activity and melanin content of a human melanocyte line PIG1. Methods PM2.5 was collected during haze weather in heating seasons, and processed into suspensions. PIG1 melanocytes were cultured and divided into 5 experimental groups and 1 control group. PIG1 melanocytes in the 5 experimental groups were treated with 10, 20, 50, 100 and 200 mg/L PM2.5 suspensions respectively for 48 hours, while cells in the control group were not treated with PM2.5 suspensions. In cell migration assay, there was only 1 experimental group treated with 10 mg/L PM2.5 suspensions. After treatment, methyl thiazol tetrazolium (MTT) assay, micropore filtration assay, DOPA oxidase assay and NaOH lysis method were performed to determine the cell proliferation rate, migration rate, tyrosinase activity and melanin content respectively. Statistical analysis was carried out by using t test for comparison of means of two samples, one-way analysis of variance for means of multiple samples, Student-Newman-Keuls (SNK) -q test for multiple comparisons, and linear correlation analysis for analysis of correlations. Results Compared with the control group ([100 ± 1.41]%) , the proliferation rate of PIG1 cells significantly decreased in the 20-, 50-, 100- and 200-mg/L PM2.5 groups ([93.41 ± 2.13]%, [88.31 ± 1.3557]%, [79.75 ± 1.89]%, [69.83 ± 2.50]% respectively, all P < 0.05) . Linear correlation analysis showed that the proliferation rate and tyrosinase activity of PIG1 cells decreased with the increase in PM2.5 concentrations (r = -0.98, -0.93, respectively, both P < 0.01) . After the treatment with 10 mg/L PM2.5, the migration rate of PIG1 cells significantly decreased (66.23% ± 1.11%) compared with the control group ([76.86 ± 1.81]%, t = 7.55, P < 0.01) . With the increase in PM2.5 concentrations (50-200 mg/L) , the melanin content of PIG1 cells gradually decreased (r = -0.97, P < 0.01) . Conclusion Atmospheric fine particulate matter PM2.5 can affect the normal functions of melanocytes by inhibiting their proliferation and migration, and reducing their tyrosinase activity and melanin content. Key words: Melanocytes; Particulate matter; Cell proliferation; Melanins; Cell migration assays; PM2.5

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

Downregulation of Cytoskeletal Protein Kazrin in Lung Endothelial Cells of Preclinical Sickle Cell Disease Mouse Model - Essential Role of Endothelial Barrier Function

Background: Lung capillary barrier dysfunction is common to all causes of acute chest syndrome (ACS), a leading cause of morbidity and mortality in individuals with sickle cell disease (SCD). Our published data (Haematologica 2017, 102, e26-e29) demonstrated, for the first time, an impaired barrier function in freshly isolated endothelial cells (EC) from the lungs of Townes transgenic sickle cell anemia (SS) mice, compared to heterozygote (AS) or control (AA) mice. In addition, we showed a sub-stimulatory dose (0.05 mg/kg) of bacterial toxin lipopolysaccharide, which did not cause prominent acute lung injury in control AS/AA mice, was fatal to SS mice suggesting an enhanced hypersensitivity of sickle EC to infection. Hemolysis is a fundamental feature of SCD that contributes to its pathophysiology. Hemolysis results in the leakage of vast amounts of hemoglobin, and subsequently, heme into the plasma. Although, previous studies have demonstrated the detrimental effect of heme in SCD, the mechanisms of heme-mediated lung EC barrier dysfunction have not been studied. Recently, Kazrin was identified as a widely expressed protein influencing intercellular adhesion and co-localizes with the cortical actin ring to stabilize cell-cell junctions. In this study, we investigated the mechanisms of heme-induced EC barrier dysfunction related to Kazrin function, using primary EC isolated from the lungs of SCD mice and controls. Methods: Primary EC from the lungs of SS, AS, and AA mice (6-8 weeks old) were isolated according to methods described in our recent publication. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) and Evans blue dye transendothelial permeability was done using a transwell filter assay system. Western blot was performed to measure total and phosphorylated expression levels of protein involved in the regulation of EC barrier function. Immunocytochemistry was performed using specific antibodies and actin cytoskeleton was identified using a known F-actin binding fluorescent marker Phalloidin. Results: Our data demonstrated a significantly reduced basal expression of the Kazrin mRNA and protein in freshly isolated SS-EC compared to AS-EC or AA-EC. To determine the effects of Kazrin downregulation on the EC barrier function, we depleted the Kazrin protein in AS-EC using Kazrin specific siRNA and scrambled control siRNA. Kazrin depleted cells demonstrate an attenuated EC barrier function as evidenced by reduced trans-endothelial electrical resistance by ECIS and increased Evans blue dye leak by transwell filter assay (p&lt;0.05). By contrast, control siRNA treated AS-EC displayed normal barrier function. Therefore, our data, for the first time, suggest that Kazrin is essential for the EC barrier function. Given the crucial role of hemolysis in SCD, we detected that clinically relevant dose of heme (5 µM; 60 min) significantly decreases the AS-EC barrier function (p&lt;0.05) and the heme-mediated decrease of barrier function is dose-dependent. Moreover prolonged treatment of AS-EC with heme (5 µM; 24-48 hrs) downregulated the Kazrin protein levels. These results are intriguing since the SS-EC inherently show downregulated Kazrin when compared with AS-EC. Therefore, it is possible that impaired barrier function in SS-EC that we recently published could be an effect of Kazrin downregulation since hemolysis is a constant process in SCD. We also found that incubation of AS-EC with heme upregulated the efficacy of barrier disruption agent RhoA. Previous studies have indicated that activated RhoA promotes the actin stress fibers by stimulating actin-myosin contractility and increased endothelial permeability. Our immunocytochemistry data reveal that heme incubated AS-EC develop severe actin stress fibers. Studies in progress to determine the mechanistic link between Kazrin downregulation, RhoA activation and impaired EC barrier function in SS-EC. Conclusions and Future Direction: Our findings support the notion that Kazrin participates in the EC barrier function and its downregulation cause an impaired EC barrier function. Our future studies will focus on to identify protective mechanisms of lung EC barrier function in SCD. Disclosures Kutlar: Bluebird Bio: Other: DSMB Member; Novo Nordisk: Research Funding; Novartis: Consultancy; Micelle Biopharma: Other: DSMB Chair; Global Blood Therapeutics, Inc. (GBT): Research Funding.

Performance of black ceramic water filters and their implementation in rural Ecuador

Abstract In rural Ecuador, microbial water contamination is associated with child morbidity mainly due to gastroenteritis. Black ceramic water filters (BCWF) are a new household water treatment recently developed to improve microbial removal from the classical model implemented worldwide. This study has assessed BCWF microbial performance at laboratory level by continuous filtering of spiked water with microbial surrogates (Escherichia coli and MS2 bacteriophage) and highly contaminated surface water to evaluate physicochemical pollutants' removal. At field level, baseline studies in Nanegal and Gualea districts have been performed to evaluate water quality and hygiene practices among communities and a six-month BCWF field implementation study in the Santa Marianita community. Results revealed poor drinking water quality in communities studied. Water treatment practices at household level were reported in low percentages. Conversely, results in BCWF filter assays at laboratory level for 600 litres of usage have shown 5.36 logarithms of bacterial removal and 3.83 logarithms for viral removal and significant reductions of physicochemical pollutants considering international standards. BCWF implementation in the Santa Marianita community reveals promising results on microbial water quality in households using this new technology. However, it is important to reinforce correct BCWF maintenance for better performance at field level.

Validation of a rapid, conservative transuranic alpha activity estimation method in air samples

In air filter assay for radiological emergency response, radon (222Rn) and thoron (220Rn) progeny are known interferents to transuranic activity estimation. Previous work detailed a conservative, graded approach for TRU alpha activity estimation from air samples void of transuranic activity yet containing varying amounts of radon and thoron progeny. Validation of this method to produce rapid, conservative and defensible transuranic alpha activity estimates was accomplished through introduction of surrogate transuranic activity, 239Pu and 230Th check sources, along with the naturally occurring radioactive progeny from an environmental air filter. Following air collection, the filter was centre hole-punched with the transuranic check source placed underneath the filter during counting. With the surrogate transuranic activity introduced into the measurement, verification of the previously studied methodology for rapid transuranic activity estimation was determined with quantifiable conservative bias. 70 environmental filters with various levels of radon progeny and air sampling duration were collected; 35 examined with the 239Pu check source and 35 studied with the 230Th check source. To characterise the expected transuranic activity introduced to the counting experiment without the environmental interferents of radon and thoron progeny, 30 blank filters were counted using the described experimental setup with each of the respective surrogate sources. Following characterisation of the sources with blank filters, transuranic activity estimation comparison against the 70 environmental filters with natural background radioactive progeny interferents was accomplished. This work contributes to the comprehensive analysis of operational air samples by detailing validation results for a rapid and conservative transuranic alpha activity estimation methodology.

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (>98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

026 Study on detection of IgM and IgG of the anti-42000 protein from egg nucleus antibody in SLE patients’s sera with Dot immunogold filtration assay

026 Study on detection of IgM and IgG of the anti-42000 protein from egg nucleus antibody in SLE patients’s sera with Dot immunogold filtration assay