Nonidet P-40 Research Articles

Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and β-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

Optimization and evaluation of tear protein elution from Schirmer's strips in dry eye disease.

Extraction of tear protein from Schirmer's strip is a prerequisite for the proper identification and screening of biomarkers in dry eye disease. The study compares different methods of extraction of tear proteins from the Schirmer's strip. Reflex tear was collected from healthy controls (HC; n = 12), Stevens-Johnson syndrome (SJS; n = 3) and dry eye disease (DED; n = 3) patients using capillary tube. This tear was used to measure the volume absorbed by Schirmer's strip per microliter. Different buffers (6) were used to compare the protein yield from the Schirmer's strip in four different conditions. The tear proteins extracted using the highest protein yield buffer were analyzed by mass spectrometry. A linear relationship between the tear volume and wetting length was observed (r = 0.0.997, n = 6). The highest yield was observed after incubation of the Schirmer's strip in 100 mM ammonium bicarbonate (ABC) with 0.25% Nonidet P-40(NP-40) at 4°C for an hour (P < 0.00005). The in-solution digestion of tear eluted in the above condition 100 Mm ABC + 0.25% NP-40 with one-hour incubation yielded a total of 2119 proteins in HC, SJS, and DED. The unique protein observed in SJS and DED was 0.6% and 17.9%, respectively. The significantly expressed proteins are associated with innate immune response, proteolysis, wound healing, and defense response. A method for extraction of protein from Schirmer's strip was optimized for increase in protein yield from the tear sample. SJS and DED tear samples have unique protein signature. The study will aid in better design of tear protein-based experimental study.

Removal of nonspecific binding proteins is required in co-immunoprecipitation with nuclear proteins.

Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the decrease of ion concentrations but reduced by Nonidet P40. Western blot indicated that p65 and β-actin were present as nonspecifically bound protein to the beads. p53 and β-actin were present in the CO-IP precipitates of nuclear proteins but pretreatment cleared the nonspecifically pulled down p53 and β-actin. These data suggest that magnetic beads are better for CO-IP, but preclearing is necessary to minimize false positive regardless of which bead is used, particularly for nuclear proteins.

A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates

The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-β-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.

Dynamics of Apolipoprotein B-100 in Interaction with Detergent Probed by Incoherent Neutron Scattering.

Apolipoprotein B-100 (apo B-100) is the protein moiety of both low- and very-low-density lipoproteins, whose role is crucial to cholesterol and triglyceride transport. Aiming at the molecular dynamics' details of apo B-100, scarcely studied, we performed elastic and quasi-elastic incoherent neutron scattering (EINS, QENS) experiments combining different instruments and time scales. Similar to classical membrane proteins, the solubilization results in remaining detergent, here Nonidet P-40 (NP40). Therefore, we propose a framework for QENS studies of protein-detergent complexes, with the introduction of a combined model, including the experimental apo B-100/NP40 ratio. Relying on the simultaneous analysis of all QENS amplitudes, this approach is sensitive enough to separate both contributions. Its application identified two points: (i) apo B-100 slow dynamics and (ii) the acceleration of NP40 dynamics in the presence of apo B-100. Direct translation of the exposed methodology now makes the investigation of more membrane proteins by neutron spectroscopy achievable.

Ionic Liquid-Based Extraction System for In-Depth Analysis of Membrane Protein Complexes.

Limited by the rare efficient extraction system in extracting hydrophobic membrane protein complexes (MPCs) without compromising the stability of protein-protein interactions (PPIs), the in-depth functional study of MPCs has lagged far behind. In this study, the first systematic screening of ionic liquids (ILs) was performed and showed that triethylammonium acetate (TEAA) IL exhibited excellent performance in stabilizing PPIs, which was further confirmed by molecular docking simulations. By combining TEAA with the conventional detergent Nonidet P-40 (NP-40), a novel IL-based extraction system, i-TAN (TEAA IL with 1% NP-40), was proposed, which demonstrated superior performance in extracting and stabilizing MPCs, attributed to its larger size, more uniform distribution, and closer-to-neutral microenvironment of micelles. Extraction of MPCs with i-TAN allowed the confident identification of more hydrophobic EGFR-interacting proteins that are easily dissociated during the extraction process. Quantitative analysis of the difference in EGFR complexes between trastuzumab-sensitive and trastuzumab-resistant breast cancer cells provided comprehensive insights to understand the drug resistance mechanism, suggesting that i-TAN has great potential in interactomics and functional analysis of MPCs. This study provides a novel strategy for MPC extraction and downstream processing.

Evaluation of Six Protocols for Protein Extraction from Rice Young Panicles byTwo-Dimensional Electrophoresis

ABSTRACT For most reported proteomics approaches, protein extraction and sample preparation are of crucial importance for optimal results. However, extraction of protein from crop plant tissues remains a great challenge due to low protein content and abundant secondary metabolites that prominently interfere with isoelectric focusing and subsequent proteomic analysis. Up until now, no attempts are focused on comparison of protein extraction procedures from rice young panicles. To establish a high-efficiency protein extraction protocol suitable for two-dimensional electrophoresis (2-DE) in rice young panicles, six protocols for protein preparation were evaluated: (1) Phenol extraction; (2) Mg/ Nonidet P-40 (NP-40) extraction; (3) Tris-Base/acetone precipitation; (4) SDS extraction; (5) trichloroacetic acid (TCA)/acetone/phenol extraction; (6) TCA/acetone precipitation. The study explicitly demonstrates that TCA/ acetone/phenol method provides a high-enhanced protein extraction efficacy from rice young panicles than other protocols in terms of the higher quality of 1-DE gel, the maximum number (450) of well-resolved protein spots, greater resolution and spot abundance. In addition, these methods also generated remarkably different twodimensional gel electrophoresis protein spot patterns. Twenty-nine of 30 visible differential extracted proteins were identified by MALDI-TOF-MS/MS analysis and were divided into eight categories according to molecular function. Accumulated data suggested that different extraction methods respectively necessitate certain special plant tissues due to different physicochemical properties of each protocol. Overall, this study, which is presented in this paper, will facilitate to providing a cornerstone of comparative proteomic analysis from rice young panicles, including other complicated plant tissues.

Deficient Incorporation of Rabies Virus Glycoprotein into Virions Enhances Virus-Induced Immune Evasion and Viral Pathogenicity.

Previous studies have shown that wild-type (wt) rabies virus (RABV) evades the host immune response by restricting expression of glycoprotein (G), which blocks activation of dendritic cells (DCs) and induces production of virus-neutralizing antibodies (VNAs). In the present study, wt RABVs not only restricted G expression but also reduced incorporation of G into mature virions compared with laboratory-adapted viruses. A recombinant RABV expressing triple G was used to further determine whether G expression relates to incorporation. The recombinant virus showed higher expression and incorporation of G and activated more DCs than the virus that expressed a single copy of G. Removal of G from viruses using subtilisin or Dithiothreitol (DTT)/ Nonidet P-40 (NP40) almost completely abolishes DC activation and VNA production. Consequently, these G-depleted viruses cause lethal infection in mice. Thus, wt RABVs can subvert DC-induced antiviral immune response and maintain pathogenicity by decreasing G expression in infected cells and G incorporation into virions.

Evaluation of viral contamination in a baculovirus expression system.

Insect expression systems based on baculovirus are widely used for generating recombinant proteins. Here, the infectivity of baculoviruses under the physiological stresses of 'freeze-thaw' and sonication and the baculoviral contamination of recombinant proteins after protein purification were evaluated. Our findings suggest that Nonidet P-40 (NP-40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses. We therefore suggest that baculovirus is completely inactivated by NP-40 treatment and that recombinant proteins are unlikely to be contaminated with infectious baculoviruses after their affinity purification.

Performance of electroremediation in real contaminated sediments using a big cell, periodic voltage and innovative surfactants

The present work focused on evaluating the electrokinetic (EK) treatment of real contaminated sediments with toxic metals and polycyclic aromatic hydrocarbons (PAHs), using a big laboratory EK cell, periodic voltage and recently tested non-ionic surfactants. The results indicated that the “day on-night off” application mode of voltage, in conjunction with the selected solubilising agents, favoured the overall EK process. Arsenic, nickel and chromium exhibited the highest removal percentages, obtaining 83%, 67% and 63%, respectively, while zinc and lead attained 54% and 41% at the maximum. Furthermore, in the experiments where the non-ionic surfactants were introduced in the electrolyte chambers, there was a major uniformly removal of PAHs from the entire sediment across the EK cell, indicating the high solubilisation capacity of the enhancing agents. Essentially, transport and in some cases removal of PAHs (particularly from sections adjacent to the electrolyte compartments) also occurred in the unenhanced EK run, mainly due their negative charge, their potential weak bonds to the soil matrix and to the periodic application of voltage. Maximum removal was obtained by the use of Nonidet P40 where app. 1/3 (ca. 6498μg out of 20145μg) of the total initial amount of PAHs were removed from the cell.

My Early Days with Ari Helenius: Detergents and Viruses.

My Early Days with Ari Helenius: Detergents and Viruses.

Mutations of rat surfactant protein A have distinct effects on its glycosylation, secretion, aggregation and degradation

AimsSurfactant protein A (SP-A) plays critical roles in the innate immune system and surfactant homeostasis of the lung. Mutations in SP-A2 of the carbohydrate recognition domain (CRD) impair its glycosylation and are associated with pulmonary fibrosis in humans. We aim to examine how mutations in SP-A that impair its glycosylation affect its biological properties and lead to disease. Main methodsWe generated rat SP-A constructs with two types of mutations that impair its glycosylation: N-glycosylation site mutations (N21T, N207S and N21T/N207S) and disease-associated CRD mutations (G231V, F198S). We transfected these constructs into Chinese hamster ovary (CHO)-K1 cells and assessed biochemical differences in cellular and secreted wild-type and mutant SP-As by western blot, immunofluorescence, and sensitivity to enzymatic digestion. Key findingsMutations of the CRD completely impaired SP-A secretion, whereas mutations of N-glycosylation sites had little effect. Both types of mutations formed nonidet p-40 (NP-40) insoluble aggregates, but the aggregates only from CRD mutations could be partially rescued by a chemical chaperone, 4-phenylbutyrate acid (4-PBA). The majority of CRD mutant SP-A was retained in the endoplasmic reticulum. Moreover, both types of mutations reduced SP-A stability, with CRD mutant SP-A being more sensitive to chymotrypsin digestion. Both types of soluble mutant SP-A could be degraded by the proteasome pathway, while insoluble aggregates could be additionally degraded by the lysosomal pathway. SignificanceOur data provide evidence that the differential glycosylation of SP-A may play distinct roles in SP-A secretion, aggregation and degradation which may contribute to familial pulmonary fibrosis caused by SP-A2 mutations.

Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting

In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting.Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14+ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes.This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14+ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14+ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs.Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.

Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification

Eukaryotic cilia/flagella are ideal organelles for the analysis of membrane trafficking, membrane assembly, and the functions of a variety of signal transduction molecules. Cilia are peninsular organelles and the membrane lipids, membrane proteins, and microtubular-associated components are selectively transported into cilia through the region formed by the basal body/transition region and tightly associated ciliary membrane. Cilia can be isolated from many organisms without disrupting cells and many will rapidly regenerate cilia (with the ciliary membrane lipids and proteins) to replace those that are released. Despite their ease of isolation, we have relatively little understanding of the mechanisms that regulate lipid and protein transport into ciliary membranes (Pazour and Bloodgood, 2008; Bloodgood, 2009; Bloodgood, 2012).Chlamydomonas flagella shed membrane vesicles, also called ectosomes (Wood et al., 2013) from flagellar tips and these vesicles can be purified from the culture medium without damaging or deflagellating cells (McLean et al., 1974; Bergman et al., 1975; Snell, 1976; Kalshoven et al., 1990). Based on a comparison of biotinylated proteins on the shed vesicles with biotinylated proteins isolated from purified flagella and cell bodies, the ectosomes contain most, but not all, flagellar surface proteins and none of the major cell body proteins (Dentler, 2013). Although ectosomes have only been purified from Chlamydomonas cells, preliminary evidence indicates that similar vesicles are released from Tetrahymena cilia (Dentler, unpublished). Flagellar (and ciliary) membranes or membrane proteins also can be released from purified flagella/cilia. Most membrane proteins can be solubilized by extracting purified cilia with nonionic detergent [Triton X-100 or X-114 or Nonidet P-40 (NP-40)] and pelleting the microtubules (axonemes). However, not all membranes are released by detergent (Dentler, 1980) and the supernatant also contains all of the flagellar proteins that are not attached to the microtubules. Intact membrane vesicles can be released from flagella by agitation of flagella, often with low concentrations of nonionic detergents or freeze-thawing (Witman et al., 1972; Snell, 1976; Dentler, 1980; Dentler, 1995; Bloodgood and May, 1982; Pasquale and Goodenough, 1987; Iomini et al., 2006; Huang et al., 2007). Once released, they can be purified from axonemes by differential centrifugation.Each of these methods may enrich for different populations of axonemal and membrane proteins and lipids. The different solubility of membranes may reveal local differences in lipid or protein composition (Bloodgood, 2009). The ectosomes contain most but not all surface proteins found on purified Chlamydomonas flagella (Dentler, 2013). The ectosomes vesicles may be enriched in different soluble flagellar proteins than those trapped as vesicles are released from purified flagella. The detergent-solubilized “membrane+matrix” will contain all soluble membrane proteins as well as all of the soluble proteins in the flagellar compartment. In this paper, a method to purify ectosomes vesicles released from the tips of living Chlamydomonas cells is presented as are two methods to release flagellar membrane vesicles and proteins from purified flagella., 真核纤毛/鞭毛是分析膜运输，膜组装和各种信号转导分子的功能的理想细胞器。纤毛是半岛细胞器，并且膜脂质，膜蛋白和微管相关组分通过由基底体/过渡区和紧密相关的睫状膜形成的区域选择性地转运到纤毛中。纤毛可以从许多生物体中分离而不破坏细胞，并且许多将快速再生纤毛（具有睫状膜脂质和蛋白质）以替代释放的纤毛。尽管它们容易分离，但我们对调节脂质和蛋白质转运到睫状膜的机制的理解相对较少（Pazour和Bloodgood，2008; Bloodgood，2009; Bloodgood，2012）。 鞭毛脱落的膜囊泡，也称为来自鞭毛尖端的卵母细胞（Wood等人，2013），并且可以从培养基中纯化这些囊泡而没有损伤或去鞭毛细胞（McLean等人， ，1974; Bergman et al。，1975; Snell，1976; Kalshoven et al。，1990）。基于脱落囊泡上的生物素化蛋白质与从纯化鞭毛和细胞体分离的生物素化蛋白质的比较，外周体含有大多数但不是全部的鞭毛表面蛋白质，并且没有主要的细胞体蛋白质（Dentler，2013）。虽然ectosomes只从衣原体细胞中纯化，但初步证据表明类似的囊泡从四膜虫纤毛释放（Dentler，未发表）。 鞭毛（和睫状体）膜或膜蛋白也可以从纯化的鞭毛/纤毛释放。大多数膜蛋白可以通过用非离子去污剂[Triton X-100或X-114或Nonidet P-40（NP-40）]提取纯化的纤毛并将微管（轴突）造粒来溶解。然而，不是所有的膜都被去污剂释放（Dentler，1980），并且上清液还包含所有未附着到微管的鞭毛蛋白。通过搅动鞭毛，通常使用低浓度的非离子型洗涤剂或冷冻融化，可以从鞭毛中释放完整的膜囊泡（Witman等人，1972; Snell，1976; Dentler，1980 ; Dentler，1995; Bloodgood和May，1982; Pasquale和Goodenough，1987; Iomini等人，2006; Huang等人，2007）。一旦释放，可以通过差速离心从轴突中纯化它们。这些方法中的每一种可以富集轴突和膜蛋白和脂质的不同群体。膜的不同溶解度可以揭示脂质或蛋白质组成的局部差异（Bloodgood，2009）。外来体含有在纯化的衣藻鞭毛上发现的大多数但不是全部表面蛋白（Dentler，2013）。外来体囊泡可以富集在不同的可溶性鞭毛蛋白中，而不是当从纯化的鞭毛中释放囊泡时捕获的那些。洗涤剂增溶的&quot;膜+基质&quot;将包含鞭毛区室中的所有可溶性膜蛋白以及所有可溶性蛋白。 在本文中，提出了从活的衣藻细胞的末端释放的外来体囊泡的方法，以提供从纯化的鞭毛中释放鞭毛膜囊泡和蛋白质的两种方法。

CAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress

Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.

Sequential application of chelating agents and innovative surfactants for the enhanced electroremediation of real sediments from toxic metals and PAHs

This study focused on the sequential application of a chelating agent (citric acid) followed by a surfactant in the simultaneous electroremediation of real contaminated sediments from toxic metals and Polycyclic Aromatic Hydrocarbons (PAHs). Furthermore, the efficiency evaluation of two innovative non-ionic surfactants, commercially known as Poloxamer 407 and Nonidet P40, was investigated. The results indicated a removal efficacy of approximately 43% and 48% for the summation of PAHs (SUM PAHs), respectively for the aforementioned surfactants, much better than the one obtained by the use of Tween 80 (nearly 21%). Individual PAHs (e.g. fluorene) were removed in percentages that reached almost 84% and 92% in the respective electrokinetic experiments when these new surfactants were introduced. In addition, the combined-enhanced sequential electrokinetic treatment with citric acid improved dramatically the removal of Zn and As, compared to the unenhanced run, but did not favor the other toxic metals examined. Since no improvement in metal removal percentages occurred when Tween 80 was used, significant contribution to this matter should also be attributed to the solubilization capacity of these innovative, in electrokinetic remediation, non-ionic surfactants.

Transmission of visceral leishmaniasis in a previously non-endemic region of Tunisia: Detection ofLeishmaniaDNA inPhlebotomus perniciosus

Visceral leishmaniasis (VL) has been endemic in northern Tunisia and has occurred sporadically in the center of Tunisia. Recently, there have been several cases from areas known to be free of VL. We report in this work all human and canine cases of VL recorded between 2003 and 2011 and an entomological study of phlebotomine fauna in a previously non-endemic region. Sixty-three cases of VL were diagnosed and identified as L. infantum using several different methods. Eight species of 179 sand flies were caught and identified by both morphological and molecular methods. Two genera were present, Phlebotomus and Sergentomya, with an abundance of the subgenus Phlebotomus (Larrousius) spp., a classic vector of VL in Tunisia. Moreover, Leishmania DNA was detected in seven unfed Phlebotomus pernicousus and L. infantum was identified in three of them. This result confirms the establishment of a transmission cycle of VL in the studied region by the coexistence of infected vectors with infected hosts.

Use and comparison of the non-ionic surfactants Poloxamer 407 and Nonidet P40 with HP-β-CD cyclodextrin, for the enhanced electroremediation of real contaminated sediments from PAHs

Real contaminated sediments often come with the simultaneous existence of heavy metals and PAHs requiring, thereby, appropriate choice of flushing agents when subjected to electroremediation. The application and efficiency evaluation of the innovative non-ionic surfactants octylphenoxypolyethoxyethanol (Nonidet P40) and 2-methyloxirane (Poloxamer 407), in comparison with the already known Tween 80 and HPCD cyclodextrin, were examined during enhanced electrokinetic remediation of surficial sediments, mainly from PAHs. Heavy metal removal was also assessed. The results indicated a removal efficacy for SUM PAHs of approximately 48% and 43% with the use of Nonidet P40 and Poloxamer 407 respectively, which was far better than the ones taken from both the cyclodextrin and Tween 80. Furthermore, removal percentages for individual PAHs, e.g. fluorene and chrysene, reached almost 83% or 92%, respectively. As far as heavy metals are concerned, unenhanced treatment was the only one demonstrating a removal efficacy in all metals examined, however, giving “poor to medium” percentages (5% for Zn to 43% for Cr). On the other hand, the enhanced runs exhibited sufficient removal only in some of the metals examined, e.g. Zn and As, indicating that although surfactants favor the removal of PAHs are not suitable for all heavy metals.

Alteration of the Serum N-glycome of Mice Locally Exposed to High Doses of Ionizing Radiation

Exposure of the skin to ionizing radiation leads to characteristic reactions that will often turn into a pathophysiological process called the cutaneous radiation syndrome. The study of this disorder is crucial to finding diagnostic and prognostic bioindicators of local radiation exposure or radiation effects. It is known that irradiation alters the serum proteome content and potentially post-translationally modifies serum proteins. In this study, we investigated whether localized irradiation of the skin alters the serum glycome. Two-dimensional differential in-gel electrophoresis of serum proteins from a man and from mice exposed to ionizing radiation showed that potential post-translational modification changes occurred following irradiation. Using a large-scale quantitative mass-spectrometry-based glycomic approach, we performed a global analysis of glycan structures of serum proteins from non-irradiated and locally irradiated mice exposed to high doses of γ-rays (20, 40, and 80 Gy). Non-supervised descriptive statistical analyses (principal component analysis) using quantitative glycan structure data allowed us to discriminate between uninjured/slightly injured animals and animals that developed severe lesions. Decisional statistics showed that several glycan families were down-regulated whereas others increased, and that particular structures were statistically significantly changed in the serum of locally irradiated mice. The observed increases in multiantennary N-glycans and in outer branch fucosylation and sialylation were associated with the up-regulation of genes involved in glycosylation in the liver, which is the main producer of serum proteins, and with an increase in the key proinflammatory serum cytokines IL-1β, IL-6, and TNFα, which can regulate the expression of glycosylation genes. Our results suggest for the first time a role of serum protein glycosylation in response to irradiation. These protein-associated glycan structure changes might signal radiation exposure or effects.

Clinical and Immunological Analysis of Cutaneous Leishmaniasis before and after Different Treatments

Amastigotes from L. (L.)amazonensis (La), L. (L.)venezuelensis (Lv), L. (V.)brasiliensis (Lb), and L. (L.)chagasi (Lch) were cultured in a free cells liquid culture medium. Patients (n = 87) from a cutaneous leishmaniasis (CL) hyperendemic region receiving different treatments were followed up from January 1994 to August 2000. Time for remission of lesions were spontaneous remission (SR) 7 weeks; Glucantime (Glu) chemotherapy 9 weeks; immunotherapy with La, Lv, Lb, and Lch amastigotes Tosyl-Lysil Chloromethyl-ketone (TLCK) treated and Nonidet P-40(NP-40) extracted (VT) 7 weeks. Delayed type hypersensitivity (DTH) response with leishmanine intradermic reaction (IDR) was higher in CL patients than healthy controls (P < 0.05) and increased in active secondary versus primary infection (P < 0.001) with diagnostic value 1.74 for active infection and 1.81 postclinical remission. Antibodies to amastigotes characterized by Enzyme Linked Immunosorbent Assay (ELISA) decreased in sera postclinical remission versus active infections (P < 0.001), with a diagnostic value from 1.50 to 1.84. Immunoblottings antigenic bands frequency as well as Integral Optical Density (IOD) Area Densitometry decreased with sera from SR, after Glu or VT treatments in CL volunteers. Intracellular parasitism is due to normal antibodies recognizing parasite antigens after inoculation by vector. VT vaccine induced mainly cellular immunity, for remission of lesions and protection from CL infection.