Agarose Method Research Articles

Quality of cell blocks prepared from residual pleural effusion and bronchial washing samples for immunocytochemistry

Cell blocks (CBs) enable the long-term preservation of cytological samples. The aim of this study was to analyse the quality of CBs prepared from leftover fluid from lung adenocarcinoma pleural effusion samples and residual bronchial washing sediment for immunocytochemistry. The residual part of 455 lung adenocarcinoma pleural effusion samples and sediment from 384 bronchial washing samples were used to prepare CBs following the agarose method. The quality of CBs was evaluated based on the quantity of malignant cells in haematoxylin and eosin-stained slides and interpreted as optimal or insufficient for immunocytochemistry. Immunocytochemistry on CBs was performed using the Dako EnVision™ FLEX detection visualisation system. The CB results for TTF-1, ALK, and PD-L1 immunocytochemistry were compared with the corresponding cytological smears. Among all CBs, 202 (44.4%) from leftover pleural effusion fluid and 85 (22.1%) from residual bronchial washing sediment had an optimal number of lung adenocarcinoma cells. Eight pleural effusion CBs were stained for TTF-1. Four pleural effusion and two bronchial washing CBs were stained for ALK and PD-L1. All tested pleural effusion CBs were confirmed positive for TTF-1 and negative for ALK. The PD-L1 tumour proportion score (TPS) was ≥ 50% in two pleural effusions. ALK was confirmed negative in bronchial washing CBs. One bronchial washing CB was interpreted as PD-L1-negative while the corresponding smear was positive (TPS ≥1%; 2%). The CB results of TTF-1, ALK, and PD-L1 corresponded to the findings for the smears. The inclusion of CBs prepared from leftover fluid from pleural effusion samples and residual bronchial washing sediment in routine cytological practice could provide a source of high-quality material for immunocytochemistry in addition to smears and cytospins.

Western Agarose Native GeELution (WANGEL) with beta-amyloid and tau: Novel method to elute proteins or peptides using native agarose gels followed by Lumipulse assay

Alzheimer´s disease is characterized by hyperphosphorylated tau neurofibrillary tangles and beta-amyloid plaques. Both molecules can be easily measured in human fluids or tissue extracts by immunoassays. However, the different molecular weight species can only be differentiated on Western Blot gels. Analysis of native proteins from polyacrylamide gels is also not well characterized. Hence, we developed a modified method to elute proteins or peptides from native agarose gels. Initially, full-length tau (60 kDa) and beta-amyloid(42) (4 kDa) were separated on a Western Blot gel and eluted from native agarose gels (WANGEL) using an elution system inside a polypropylene tube. The eluates were analyzed with the Lumipulse immunoassay. Both molecules were successfully eluted into 1% agarose gels to the cathode and were detected in the eluate. Additionally, tau was eluted from mouse cortical extracts, but was below the detection limit when eluted from human cerebrospinal fluid. Beta-amyloid(40) was eluted from CSF extracts and detected by Lumipulse. In cortical extracts taken from transgenic mice (APP_SweDI) beta-amyloid(42) was detectable as a native peptide and small oligomeric aggregates. Taken together, our novel WANGEL method enables fast, easy and cheap elution of protein/peptides from polyacrylamide/agarose gels with a subsequent analysis by Lumipulse immunoassay.Three bullet points:•Beta-amyloid and tau are major hallmarks in Alzheimer´s disease and are established cerebrospinal fluid biomarkers.•Lumipulse is a method to measure beta-amyloid and tau in cerebrospinal fluid in the pg/mL range.•Western Blot and our novel combined native agarose method (WANGEL) allows an easy and fast determination of the molecular size in combination with Lumipulse.

Hepatotoxicity of copper sulfide nanoparticles towards hepatocyte spheroids using a novel multi-concave agarose chip method.

Aim: To explore the hepatotoxicity of copper sulfide nanoparticles (CuSNPs) toward hepatocyte spheroids. Materials & methods: Other than thetraditional agarose method to generate hepatocyte spheroids, we developed a multi-concave agarose chip (MCAC) method to investigate changes in hepatocyte viability, morphology, mitochondrial membrane potential, reactive oxygen speciesand hepatobiliary transporter by CuSNPs. Results: The MCAC method allowed a large number of spheroids to be obtained per sample. CuSNPs showed hepatotoxicity in vitro through a decrease in spheroid viability, albumin/urea production and glycogen deposition. CuSNPs also introduced hepatocyte spheroid injury through alteration of mitochondrial membrane potential and reactive oxygen species, that could be reversed by N-acetyl-l-cysteine. CuSNPs significantly decreased the activity of BSEP transporter by downregulating its mRNA and protein levels. Activity of theMRP2 transporter remained unchanged. Conclusion: We observed the hepatotoxicity of CuSNPs in vitro with associated mechanisms in an advanced 3D culture system.

Application of quantitative transcriptomics in evaluating the ex vivo effects of per- and polyfluoroalkyl substances on Atlantic cod (Gadus morhua) ovarian physiology

Because of their global consumption and persistence, per- and polyfluoroalkyl substances (PFASs), are ubiquitously distributed in the environment, as well as in wildlife and humans. In the present study, we have employed an ex vivo organ culture technique, based on the floating agarose method, of Atlantic cod ovarian tissue to investigate the effects of three different concentrations of PFOS, PFOA (1, 5 and 25 μM) and PFNA (0.5, 5 and 50 μM), used singly and in also in combination (1×, 20× and 100×). In the 1× exposure mixture, concentrations were decided based on their proportional levels (in molar equivalents) relative to PFOS, which is the most abundant PFAS in cod liver from a 2013 screening project. To investigate the detailed underlying mechanisms and biological processes, transcriptome sequencing was performed on exposed ovarian tissue. The number of differentially expressed genes (DEGs) having at least 0.75 log2-fold change was elevated in high, compared to low and medium concentration exposures. The highest PFNA, PFOA and PFOS concentrations, and the highest (100×) mixture exposure, showed 40, 68, 1295, and 802 DEGs, respectively. The latter two exposure groups shared a maximum of 438 DEGs. In addition, they both shared the majority of functionally enriched pathways belonging to biological processes such as cellular signaling, cell adhesion, lipid metabolism, immunological responses, cancer, reproduction and metabolism. Shortlisted DEGs that were specifically annotated to reproduction associated gene ontology (GO) terms were observed only in the highest PFOS and mixture exposure groups. These transcripts contributed to ovarian key events such as steroidogenesis (star, cyp19a1a), oocyte growth (amh), maturation (igfbp5b, tgfβ2, tgfβ3), and ovulation (pgr, mmp2). Contrary to other PFAS congeners, the highest PFOS concentration showed almost similar transcript expression patterns compared to the highest mixture exposure group. This indicates that PFOS is the active component of the mixture that significantly altered the normal functioning of female gonads, and possibly leading to serious reproductive consequences in teleosts.

Young Investigator Challenge: A novel, simple method for cell block preparation, implementation, and use over 2 years.

The cell block is an essential adjunct to conventional cytopreparatory techniques. The need for molecular analysis and immunostains will increase the need for successful cell block preparation. Even with this need, to the authors' knowledge very little has changed regarding the way in which cell blocks are produced. The authors developed A Formalin-Fixed, paraffin Embedded Cytology cell block Technique (AFFECT) that uses a cytospin centrifuge and funnel to deposit a cell pellet into a well on a piece of open-cell, absorbent foam. The foam and the pellet are then sent through normal processing. Herein, the authors present the implementation of this method and some of their experience with its performance over the course of 2 years. Although a comparison of the methods indicated good correlation for the production of a cell block between AFFECT and the agarose method, the AFFECT blocks demonstrated markedly improved cellular morphology. Over the first 6 months of use, AFFECT produced a successful cell block in 74% of cases overall, and in 65% of cases with a cell pellet measuring ≤0.1 mL. The year preceding the implementation of AFFECT and its first year of use were compared for endoscopic and bronchoscopic ultrasound-guided fine-needle aspiration specimens, and demonstrated an improved success rate. The authors developed a novel method of cell block preparation that demonstrates improved histology and has increased the success rate of cell block production compared with the agarose method. Cancer Cytopathol 2016;124:885-892. © 2016 American Cancer Society.

Effects of grain size and porosity on strength of Li2TiO3 tritium breeding pebbles and its grain growth behavior

Tons of Li2TiO3 tritium breeding pebbles will be filled in the blanket for obtaining tritium fuel. In this work, isothermal sintering was carried out to study the grain growth behavior of the Li2TiO3 pebbles fabricated by agarose method. The grain growth exponent (n) and the activation energy (Q) calculated by the phenomenological kinetic equation were 2 and 435.65 kJ/mol, respectively. The grain growth was controlled by vapor transport (p = 2S/r). In addition, effects of porosity and grain-size on the strength of Li2TiO3 pebbles were investigated. The strength was affected by the grain size and the porosity of Li2TiO3 pebbles, and high strength (about 72 MPa) depended partly on achieving the optimum balance between the porosity (about 10%) and grain size (about 2 μm).

Microscopic study of Alternaria brassicae infection processes in Brassica juncea cultivars by drop plus agarose method

Alternaria blight caused by Alternaria brassicae (Berk) Sacc. is one of the most serious diseases of Indian mustard [Brassica juncea (L.) Czern and Coss.] grown as edible oilseed crop in India. Poorly characterized tolerance mechanisms against this pathogen further confirm the strategies that can be undertaken to design durable resistance or effective disease control measures. The host-pathogen interaction was studied and showed that the initial infection processes involving conidium germination and mode of penetration were similar in both cultivars, although considerably late in ‘PAB 9511’ evidently due to tolerance. However, apparent differences between the two cultivars were noticeable by 1 dpi. At the plant surface, impeded fungal growth, less proliferation of spores and active suppression of the dense hyphal moulds are the factors involved in the expression of tolerance against A. brassicae in cultivar PAB 9511. Our findings have notably advanced our understanding of the mechanism of tolerance in this patho-system. Key words: Artificial inoculation, Brassica juncea, Alternaria brassicae, pathogenicity,plant-pathogen interaction.

Comparison of artificial inoculation methods for studying pathogenesis of Alternaria brassicae (Berk.) Sacc on Brassica juncea (L.) Czern. (Indian mustard)

Establishment of disease by artificial inoculation is essential for studies of various aspects of plant pathology. Keeping this in mind, five inoculation methods were compared namely spraying, infiltration, wounding, spore suspension drop and spore suspension drop along with agarose method. The findings of the present study suggest that out of five inoculation methods used, spore suspension drop along with agarose inoculation method was most ideal as this fixed the inoculum on the target site. The mean value of number of initial disease lesions in drop plus agarose method was highest in all of the time intervals of observation namely 312.2, 484.2, 664.2, 734.2 and 799.2 at 24, 48, 72, 96 and 120 h after pathogen inoculation compared to other methods respectively. Statistical analysis software (SAS) was used to find out the significant comparison among the different methods. The spore suspension drop along with agarose method has the advantage of being accurate and precise, and it was also easy to handle the inoculated plants. Keywords : Artificial inoculation, Brassica , Alternaria , pathogenicity, screening African Journal of Biotechnology Vol. 12(18), pp. 2422-2426

Plant asparaginase-based asparagine biosensor for leukemia

The present work aims at the development of plant asparaginase-based asparagine biosensor for leukemia. It is a novel diagnostic for monitoring asparagine levels in patients suffering from acute lymphoblastic leukemia (ALL). Various immobilization strategies have been applied to improve the stability of the asparaginase. The latest and updated information including some new techniques of immobilization related to L-asparaginase such as gelatin, agarose, agar, and calcium alginate methods are described in detail along with response time studies and comparative data. Furthermore these immobilization techniques have been applied for the detection of asparagine in normal and leukemia serum samples.

Phosphopeptide shotgun analysis using mass spectrometry and Phos-tag agarose beads

Enrichment of phosphopeptides is an important process for mass spectrometric analysis. Here, we used the enrichment method using the biphasic Phos-tag/C18 tip, which consists of Phos-tag agarose beads overlaid on the C18 disk filter in a micropipet tip. In the cell lysates of GIST882 cells, 3187 phosphopeptides (1688 unique phosphopeptides) were identified by nano LC-MS/MS analysis coupled with phosphopeptide enrichment using this method. This method was comparable to another phosphopeptide enrichment method using Titansphere Phos-TiO Kit in numbers of peptides detected. A total number of unique phosphopeptides detected by the two methods was 3202, and the overlap between phosphopeptides enriched by two methods was 36%. Therefore, it is concluded that Phos-tag agarose method is an alternative method for identification of phosphoproteins.

Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping

TILLING (Targeting Induced Local Lesions IN Genomes) exploits the fact that CEL I endonuclease cleaves heteroduplexes at positions of single nucleotide or small indel mismatches. To detect single nucleotide polymorphisms (SNPs) across a population, DNA pools are created and a target locus under query is PCR-amplified and subjected to heteroduplex formation, followed by CEL I cleavage. Currently, the common method used to detect cleaved products is by polyacrylamide gel electrophoresis using a high-throughput genotyping platform. Exact SNPs are then determined by sequencing. We sought to simplify the detection of CEL I-cleaved products on conventional agarose gels to make the technique more accessible to collaborating partners in developing countries where access to instrumentation could be limiting. Here, we used a panel of stress-related genes to evaluate SNP detection across 48 rice genotypes by contrasting them individually against IR64 and Nipponbare. SNP detection calls corresponded perfectly with those obtained from the Li-Cor genotypers. We were able to detect SNPs in pools of eight DNA templates, suggesting that the agarose gel system could be used to screen for SNPs with comparable throughput as that of the Li-Cor genotypers and showed that the throughput can be increased by analyzing larger amplicons (∼3 kb). The agarose method offers a significant advantage by alleviating the need for labeled primers. We further demonstrated that the agarose method can be effectively used in gene mapping, an application particularly useful for parental lines with low levels of polymorphism. The lower cost and simplicity of the technique make it possible for broader applications of SNP-based markers for germplasm characterization and mapping studies.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Immobilisation of HEP-1 by covalent attachment to aldehyde?agarose gels

A procedure of covalent multipoint attachment has been performed for immobilisation of HEP-1 using two different pH conditions. This study revealed that in more alkaline media as pH 10 the antibody immobilises quickly. In this condition, supports are able to bind 4 mg of HEP-1 per mL of gel. In order to test the activity of this new immobilised derivative, it was used in the affinity purification of the recombinant Hepatitis B surface antigen. We made a comparison between immunosorbents activated by the CNBr and aldehyde–agarose methods and subsequently studied the recovery during 23 cycles. The amounts of eluted antigen per millilitre decrease 1.5 times with the increment of the cycles either aldehyde–agarose or CNBr gels. We also measured the leakage in each cycle and it was below 15 ng IgG/mL.

Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins.

Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni(2+)-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha' proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

Effect of Lansoprazole on Human Leukocyte Function

Recent findings on the capacity of omeprazole to mfluence various leukocyte functions, in vitro, raises the question on the potential use of protonic pump inhibitors, commonly used in the treatment of acid-secretion- related disorders, as immunomodulators. The aim of this study was to evaluate the in vitro effect of lansoprazole on human natural killer (NK) cell cytotoxix activity, chemotaxis and superoxide anion (02*−) generation exerted by polymorphonucleated cells (PMNs). NK cytotoxicity activity was assessed by a 51Cr release assay, PMN chemotaxis was determined by an under agarose method and 02*− generation was analyzed on the basis of reduced cytochrome C. Incubation times with lansoprazole was 30 min for PMNs and 1–4.5 hours for NK cells, respectively. Lansoprazole induced significant dose dependent bitiion of NK cell activity and PMN functions at concentrations ranging fiom 100 to 1,000μM.Ths study demonstrate that lansoprazole, like omeprazole, inhibits several leukocyte functions, in vitro, then suggesting that protonic pump inhibitors are able to provoke these effects, at least at certain doses.

Effects of pulmonary surfactant on macrophage migration: suppression of chemokinesis by surfactant phospholipid and enhancement of chemotaxis by surfactant protein.

We fractionated the bronchoalveolar lavage fluid (BALF) from normal rabbit lungs into several fractions by high speed centrifugation and ethanol-ether extraction. Random migration, chemokinesis and chemotaxis of freshly harvested alveolar macrophages (AM), 24 h cultured AM, and peritoneal exudate cells (PEC) were assayed in vitro using a modified, under agarose method and a blind well chemotactic chamber method. Freshly harvested AM demonstrated little random migration compared with PEC. However, when freshly harvested AM were pre-incubated in surfactant free medium for 24 h, the cells showed the same rate of migration as PEC. The increased migration of the 24 h cultured AM was partially suppressed by the presence of all BALF fractions containing high proportions of phospholipid. The inhibition by Fr-L (a fraction enriched in phospholipids) was reversed by normal serum, but not by heat-inactivated serum, cholesterol, synthetic dipalmitoyl phosphatidyl choline, or indomethacin. Fr-L markedly suppressed macrophage chemokinesis but did not affect on macrophage chemotaxis. Alternatively, Fr-P, a delipidated preparation of surfactant consisting mainly of protein, had no effect on macrophage chemokinesis but increased the chemotaxis of PEC to zymosan-activated serum. We conclude that surfactant phospholipid suppresses AM migration, while surfactant protein increases macrophage chemotaxis.

Urine protein analysis by capillary electrophoresis.

Increased concentration of proteins in urine as well as abnormal patterns are seen in many disorders such as various renal disorders and light chain disease. At the wavelength of 214 nm used for detection of the peptide bond, numerous compounds interfere in the analysis of urinary proteins. We show that either adsorptive filtration with a wash step or cold ethanol precipitation are two methods which can eliminate many of the interferences. The wash step is rapid, greatly reduces the interfering substances, and decreases the effect of sample matrix. Both of these methods yield results comparable to the traditional agarose method. Capillary electrophoresis (CE) is faster and more cost-effective than agarose electrophoresis.

Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly.

The migration of cells of chondrocyte lineage is believed to play a role in cartilage growth and repair. The present study examined 1) whether chondrocytes are capable of migration in vitro; and 2) the effects of nitric oxide (NO) on chondrocyte migration, adhesion, and cytoskeletal assembly. Chondrocyte migration was evaluated by 2 assays: 1) "centrifugal" migration within a 3-dimensional collagen matrix (dot culture); and 2) directed migration under agarose in response to bone morphogenetic protein. To assess the effects of NO, chondrocytes were treated with either exogenous NO (S-nitrosoglutathione [SNO-GSH]) or a mixture of cytokines known to induce endogenous NO production. The effects of NO on chondrocyte adhesion to fibronectin-coated surfaces, as well as on actin polymerization (determined by indirect immunofluorescence), were also examined. The capacity of chondrocytes to migrate was demonstrated both by the dot culture and by agarose methods. Both SNO-GSH and endogenous NO induced by cytokines inhibited this migration. Exposure to NO also inhibited attachment of chondrocytes to fibronectin and disrupted assembly of actin filaments. These effects of SNO-GSH and cytokine-induced NO production were reversed in the presence of hemoglobin and the NO synthase inhibitor NG-monomethyl arginine, respectively. NO interferes with chondrocyte migration and attachment to fibronectin, an extracellular matrix protein, probably via effects on the actin cytoskeleton. These effects of NO may result in impairment of cartilage repair, by interfering with the extracellular matrix regulation of chondrocyte function.

Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane.

Polymorphonuclear neutrophil (PMN) migration across basement membrane is thought to be dependent on the degradation of membrane constituents. PMN gelatinase B, a metalloproteinase able to degrade type IV collagen, may be involved in this phenomenon. PMN gelatinase B is released in the extracellular medium as a latent proform and then activated, mainly by PMN elastase. We investigated the role of gelatinase B in PMN migration across a Matrigel basement membrane matrix coated onto a filter, in a Boyden chamber. The effects of gelatinase and elastase inhibitors on PMN migration in this system were tested. Chemokinesis of PMN was tested in the same Boyden chamber across a filter free of basement membrane. The agarose method was used to test the same inhibitors for effects on PMN chemotaxis. In both systems, FMLP 10(-7)M was used as a chemoattractant. Addition of 10(-8)M TIMP-1 (the preferential gelatinase B inhibitor) inhibited trans-basement membrane PMN migration by 52 +/- 6% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. Also, (Ala)(2) Pro Val chloromethyl ketone (AAPVCK) 100 micron, a specific elastase inhibitor, inhibited trans-basement membrane PMN migration by 51 +/- 8% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. The AAPVCK-TIMP combination led to a decrease in migration across Matrigel basement membrane (46 +/- 2%, P,0.05)similar to that seen with TIMP alone. AAPVCK was responsible for inhibition of gelatinase B activation, leading to a decrease in activated gelatinase from 14% to 2% of total gelatinase release (P<0.05). All these results strongly suggest that gelatinase B is a major factor of PMN migration across basement membrane and that elastase may contribute to this process by activating pro-gelatinase B.

Reperfused Gut Elaborates PAF That Chemoattracts and Primes Neutrophils

Our in vivo model of mesenteric ischemia/reperfusion (I/R) has shown that the gut serves as a priming bed for neutrophils (PMN). Activation of phospholipase A 2 (PLA 2) during ischemia temporally precedes PMN sequestration in the gut and the appearance of primed PMN in the portal circulation. Therefore, we hypothesized that reperfused gut secretes platelet activating factor (PAF) via PLA 2 activation that is responsible for increased PMN chemotaxis and priming for superoxide (O - 2) generation. Sprague-Dawley rats underwent gut ischemia/reperfusion (45 min SMA occlusion/2 hr reperfusion) or sham laparotomy. Distal ileum was harvested, rinsed with bacteriostatic saline/neomycin, and incubated for 1 hr at 37°C in RPMI 1640 and the cellfree supernatant was collected. Normal human PMNs, isolated by plasma-Percoll gradients, were pretreated with or without a PAF receptor antagonist (WEB 2170). Chemotaxis toward gut supernatant was then measured by the agarose method. Additionally, PMNs were preincubated with or without WEB 2170 and their O - 2 release in response to 1 μ M FMLP was measured by the V max of SOD-inhibitable cytochrome c reduction. Reperfused gut produced a chemotactic index of 2.1 ± 0.1 compared to 0.2 ± 0.9 following sham laparotomy ( P < 0.05); this was reduced to 0.4 ± 0.9 with PAF receptor blockade. Similarly, gut I/R supernatant primed PMNs for O - 2 ( P < 0.05) compared to laparotomy, and this effect was abrogated by a PAF antagonist. These data suggest that reperfused gut can elaborate PAF which chemoattracts and primes PMNs for O - 2 generation.