Western Blot Procedure Research Articles

UV radiation in HCT 116 cells influences intracellular H2O2 and glutathione levels, antioxidant expression, and protein glutathionylation.

UV radiation influences cellular levels of hydrogen peroxide (H2O2) and glutathione (GSH) and alters the expression of antioxidant genes in the human colorectal cancer cell line HCT116. In this study, cells were irradiated with UV light of different wavelength (A, B, or C). A surge in H2O2 concentration and total glutathione (level occurred 6 hours later. Consequently, protein glutathionylation increased above control levels. Expression of the antioxidant enzymes: glutathione peroxidase (GPX) and glutathione reductase (GSR), assessed by real-time quantitative PCR, increased by 1.5-2 times after 24 hours post-irradiation, in comparison to the untreated controls. Glutathionylation of proteins was enhanced after UV radiation and the set of biotinylated glutathione ethyl ester (BioGEE) tagged proteins was detected by Western Blot procedure. This specific glutathione analogue is conjugated with antioxidant proteins during glutathionylation especially under oxidized conditions in cells. A pool of glutathionylated proteins in the treated cells showed peculiar characteristics. These proteins exhibited varying molecular weights. For UVA-irradiated cells, 24 hours after the treatment we observed two additional ~60 and ~72 kDa bands of glutathionylated proteins from NADPH oxidases (NOX family). Total glutathione level in the UV-irradiated HCT116 cells was higher than in the control. This correlates with the detection of glutathionylation in UV-irradiated cells in the first and twelfth hour of post-irradiation, and can be defined as a specific antioxidant element activation for cellular protection.

Abundances of autophagy-related protein LC3B in granulosa cells, cumulus cells, and oocytes during atresia of pig antral follicles

In mammals, apoptosis has been accepted as the type of programmed cell death (PCD) that occurs in ovarian follicles undergoing atresia. Results of recent studies, however, indicate autophagy may be an alternative mechanism involved in follicle depletion through independent or tandem actions with apoptosis. Western blotting and immunofluorescence procedures were used in the present study to investigate the abundances of LC3B protein in freshly collected granulosa cells (GCs), cumulus cells (CCs), and oocytes to evaluate whether autophagy is an important process of antral follicle atresia in sexually mature sows. Furthermore, apoptosis was analyzed using annexin V and TUNEL assays in the same cellular cohorts to evaluate the correlation between the two processes. Immunostaining results indicate autophagy was induced in the majority of GCs, CCs, and oocytes from early and advanced stage atretic follicles. The quantitative results of western blot analysis indicate there is a progressive increase (P < 0.05) in abundance of autophagy-related protein (LC3B-II) in these cells compared with cells in non-atretic follicles. Furthermore, there is confirmation that apoptosis occurs in the GCs of atretic follicles, thus indicating that in pigs apoptosis and autophagy are processes in GCs that regulate PCD and as a consequence antral follicle depletion. There was a greater abundance of LC3B-II in CCs and oocytes of atretic follicles, while apoptosis was not detected. It, therefore, is suggested that in these cells the two processes function independently, with autophagy having a cytoprotective rather than PCD mechanism of action.

Co-culture of males with late premolt to early postmolt female giant freshwater prawns, Macrobrachium rosenbergii resulted in greater abundances of insulin-like androgenic gland hormone and gonad maturation in male prawns as a result of olfactory receptors

Insulin-like androgenic gland hormone (IAG) controls development of primary and secondary male sex-characteristics in decapod crustaceans. In male giant freshwater prawns, Macrobrachium rosenbergii, the IAG concentration correlates with male reproductive status and aggressiveness. When female prawns are co-cultured with males this can result in male size variations while this variation does not occur when males are cultured in monosex conditions. It was hypothesized that pheromone-like factors from female prawns may affect the abundance of IAG mRNA and protein in co-cultured males which would affect the pattern of sexual maturation of these males. In the present study, late premolt to postmolt females co-cultured with males for 7 days had a greater abundance of MrIAG mRNA transcript in all male phenotypes as well as for the gonad-somatic indexes (GSI). The abundance of MrIAG mRNA gradually increased from days 1 to 7 and using Western blot procedures MrIAG protein also increased in a similar pattern. Furthermore, with use of BrdU labeling, there was an increased cell proliferation in the spermatogenic zone of testicular tubules and in the spermatic duct epithelium during the 1 to 7 day co-culture period when there were increases in MrIAG mRNA and protein. In contrast, these effects were negated if short lateral antennules of males were ablated. Thus, results of the present study provide evidence that there might be female-molting factors which function as important regulators of androgenic gland function and gonadal maturation that were perceived by males via their short lateral antennules which are the olfactory organs.

Synergetic Impact of Combined 5-Fluorouracil and Rutin on Apoptosis in PC3 Cancer Cells through the Modulation of P53 Gene Expression.

Purpose: Prostate cancer is as far the most prevalent male cancer. Rutin (a glycoside from quercetin flavonoid) displays antioxidant activity leading to cell apoptosis. Combined effects of rutin with the widely used anti-cancer drug, 5-fluorouracil (5-FU), on prostate cancer cell line (PC3) was investigated herein. Methods: Different concentrations of combined 5-FU and rutin were applied to PC3 cells compared to separate treatment for 48 hours. Cell viability, as well p53 gene expression respectively were assessed by MTT assay and real-time quantitative polymerase chain reaction (qPCR). Changes of Bcl-2 signal protein and apoptosis were determined using western blot and flow cytometry procedures, respectively. Clonogenic assay was used to colony counts assessment. Results: 50% inhibitory concentration (IC50) of separate cell treatment with either rutin and 5-FU respectively were 900 μM and 3Mm, while combination index (CI) of combined 5-FU /rutin application reached a level of synergistic effects (0.33). Combination of 5-FU/rutin enhanced apoptosis and p53 gene expression in PC3 cells. PC3 cell colony counts and Bcl-2 signaling protein were decreased by 5-FU/rutin combination. Conclusion: Synergistic effects of 5-FU/rutin combination on PC3 cells line enhanced apoptosis, p53 gene expression, and down-regulation of Bcl-2 protein, compared to control separate application. 5-FU/rutin combination does seem an interesting therapeutic pathway to be further investigated.

Intracellular FMRpolyG-Hsp70 complex in fibroblast cells from a patient affected by fragile X tremor ataxia syndrome

BackgroundFragile X-associated tremor/ataxia syndrome is a late-onset neurodegenerative disorder that affects about 40% of carriers of CGG-repeat expansions in the premutation range within the fragile X gene (FMR1). Main clinical features include intention tremor, cerebellar ataxia, and parkinsonism. Recently, great emphasis on the deposition of soluble aggregates produced by a RAN translation process, as main pathogenic mechanism, has been given. These aggregates contain a small protein with a polyglycine stretch on the aminoterminal end named FMRpolyG and, so far, have been isolated and characterized in drosophila and mouse models, in post mortem brain of fragile X-associated tremor/ataxia syndrome patients, in fibroblasts of fragile primary ovarian insufficiency patients, but never in fibroblasts from a fragile X-associated tremor/ataxia living patients. In adult carriers the syndrome is frequently misdiagnosed due to the lack of specific markers. MethodsWe standardized immunocytochemistry, immunoprecipitation and western blot procedures to study and biochemically characterize the FMRpolyG protein in fibroblasts from human skin biopsy. ResultsWe demonstrate for the first time, in fibroblasts from a patient affected by Fragile X-associated tremor/ataxia syndrome, the presence ex vivo of inclusions consisting of FMRpolyG- Hsp70 soluble aggregates. ConclusionThese observations can pave the way to develop a cellular model for studying ex vivo and in vitro the mechanisms involved in the production of FMRpolyG aggregates, their toxicity, and the role of the FMRpolyG-Hsp70 interaction in the pathogenesis of fragile X-associated tremor/ataxia syndrome.

IL‐15 Activates Akt Independent of AMPK Signaling in Cardiomyocytes

Obesity is a major contributor to the onset and progression of many diseases, such as cardiovascular disease (CVD). Diet and exercise are known to be successful treatments for obesity and ultimately CVD. However, implementation of lifestyle interventions is difficult to maintain. Thus, focus has turned to examine the role of molecular mediators of physical activity to prevent obesity and its related disorders. Following exercise, many factors are secreted into circulation and act to regulate various metabolic processes. Interleukin‐IL‐15 (IL‐15) is a myokine, secreted from skeletal muscle (SKM), and has been shown to increase in circulation as a result of exercise. Further, IL‐15 acts to reduce fat mass while increasing lean mass. On a cellular level, IL‐15 induces mitochondrial activity and glucose metabolism in various cell types. However, it is not known if IL‐15 recapitulates its effects in cardiomyocytes (CM). The objective of the current study was to determine if IL‐15 regulates metabolic processes in CM. Specifically, we aimed to examine the activation of enzymes associated with glucose metabolism following treatment with IL‐15 in CM. AMPK and Akt have been verified to be involved in regulating glucose metabolism and are suggested to be downstream of IL‐15 signaling. H9c2 CM were cultured and differentiated into mature myocytes. Following differentiation, CM were treated for 24 hrs with 100 ng/ml of IL‐15. Total protein lysates were harvested from the CM and a Bradford assay was used to quantify the protein concentrations. Western blotting procedures were carried out to asses both activation and total content of the proteins. Thirty‐five micrograms of total protein was loaded onto 4–20% polyacrylamide gels for electrophoresis. Proteins were then transferred from the gel onto a PVDF membrane. Membranes were blocked with 5% BSA for 1 hour and primary antibodies (phospho‐AMPK, total AMPK, phospho‐Akt and phospho‐Akt substrates) were applied (1:1000). Subsequently, membranes were probed with secondary antibodies (1:2000). The membranes were then imaged using enhanced chemiluminescence. Image J was used to quantify the density of the visualized protein bands. An unpaired t‐test (P&lt;0.05) was used to determine if IL‐15 had significant effects on protein levels. IL‐15 did not alter either phospho‐AMPK or total AMPK levels (P&gt;0.05). Phosphorylation of Akt and its substrates were induced (nearly 10‐fold) with IL‐15 treatment. Our results indicate that IL‐15 acts to increase phosphorylation of Akt and its downstream substrates without activating AMPK. Since Akt is on the insulin signaling pathway, it is feasible that IL‐15 acts to induce glucose metabolism in CM. However, it is not clear if IL‐15 directly mediates glucose uptake in CM. Nevertheless, it appears that IL‐15 may play a role in regulating CM glucose metabolism in a similar manner as in SKM cells. Further studies are needed to determine if our results translate into a whole‐body in vivo situation. If IL‐15 is involved in substrate metabolism in CM, it is necessary to determine if its effects alter heart function, especially in models of obesity.Support or Funding InformationAmerican Heart Association 16SDG30680003This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cells.

Myosin II is the main molecular motor in the actomyosin-dependent motility in cells. Phosphorylation of the myosin regulatory light chain (RLC) at Ser19 is a prerequisite for smooth muscle/non-muscle myosin II activation and serves as a biochemical equivalent of myosin II activity. Simultaneous phosphorylation at Thr18 further promotes the myosin II ATPase activity. A number of methods have been developed to measure myosin RLC phosphorylation at Ser19 or di-phosphorylation at Thr18/Ser19. While these methods are straightforward and robust in myosin-rich muscle tissues, they demonstrate limited applicability in non-muscle cells that have low myosin II content and are usually available in lesser amounts than muscle tissue. Because of this, dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells is difficult and requires large number of cells. The use of phospho-specific antibodies increases detection sensitivity but allows estimation of only relative levels of RLC phosphorylation at specific residues, which makes it difficult to estimate the physiologic relevancy of the observed changes in RLC phosphorylation. To measure RLC phosphorylation in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in vitro phosphorylated RLC in standard SDS-PAGE and Western blot procedures with phospho-specific RLC antibodies. Here, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation in endothelial cells in response to natural agonists (thrombin or insulin) and intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for measuring myosin RLC phosphorylation in non-muscle cells.

Extraction and purification of the H9N2 virus nucleoprotein: A simple and practical method.

Background: Avian Influenza disease annually entails a significant economic loss to the poultry industry around the world. Influenza virus is a polymorphic virus of the orthomyxoviridae family (single-stranded RNA genome), and nucleoprotein (NP) is the structural and internal protein of the virus. The aim of the work was to purify nucleoprotein for further investigations with a simple, low-cost, fast and practical method. Methods: In this study, H9N2 influenza virus was isolated in specific pathogen-free embryonated chicken eggs by allantoically inoculating 103 to 105 egg-infective doses (EID50) for 9 to 11 days, purified by 10% (W/V) polyethylene glycol (PEG) 6000 with a sucrose gradient of 60% to 30%. The influenza virus proteins were collected and prepared as fractions by preparative electrophoresis. Finally, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot procedures. Results: The protein analysis with SDS-PAGE and silver nitrate staining indicated that the desired samples contained purified nucleoprotein and lacked other viral proteins. The results of the investigation of lyophilized fractions containing nucleoprotein on the SDS-PAGE revealed the absence of viral RNA in nucleoprotein and its high purity. Conclusion: According to this study, purified nucleoprotein can be used to produce nucleoprotein vaccines, as well as to study structural, molecular and diagnostic and therapeutic materials.

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.

Western blot is a principal technique for the detection of specific proteins in various biology disciplines. The antibody incubation is an imperative step in western blot. Antibody incubation by mild shaking on a rocker is generally used to facilitate mixing of antibodies. However, mild shaking is an inefficient process to remove antibody depletion layer on blotting membrane and requires hours of incubation time to achieve antibody binding to target proteins. We propose an alternative method of cyclic draining and replenishing (CDR) incubation of antibody solution using a rotational incubation chamber. The study demonstrated that rotational CDR incubation could shorten antibody incubation time with enhanced sensitivity. Moreover, rotational CDR incubation could achieve a stronger antibody binding with lower antibody concentration when compared with batch incubation. In addition, rotational CDR incubation significantly improved the detection of low abundance proteins. This simple modification in western blot procedure makes it rapid, sensitive, and cost-efficient.

Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease.

Accurate laboratory diagnosis of Lyme disease (Lyme borreliosis), caused by the spirochete Borrelia burgdorferi (BB), is difficult and yet important to prevent serious disease. The US Centers for Disease Control and Prevention (CDC) presently recommends a screening test for serum antibodies followed by confirmation with a more specific Western blot (WB) test to detect IgG and IgM antibodies against antigens in whole cell lysates of BB. Borrelia species related to BB cause tick-borne relapsing fever (TBRF). TBRF is increasingly recognized as a health problem in the US and occurs in areas where Lyme disease is prevalent. The two groups of Borrelia share related antigens. We have developed a modified WB procedure termed the Lyme immunoblots (IBs) using recombinant antigens from common strains and species of the BB sensu lato complex for serological diagnosis of Lyme disease. A reference collection of 178 sera from 26 patients with and 152 patients without Lyme disease were assessed by WB and IB in a blinded manner using either criteria for positive antibody reactions recommended by the CDC or criteria developed in-house. The sensitivity, specificity, positive and negative predictive values obtained with the reference sera suggest that the Lyme IB is superior to the Lyme WB for detection of specific antibodies in Lyme disease. The Lyme IB showed no significant reaction with rabbit antisera produced against two Borrelia species causing TBRF in the US, suggesting that the Lyme IB may be also useful for excluding TBRF.

The role of Toll-receptor 4 in tumor-associated macrophages-induced migration and invasion of Hep3B cell

Objective The invasion and migration of Hepatocellular carcinoma (HCC) is remarkable characteristic. Tumor-associated macrophages has been found to be associated with progression and metastasis of HCC patients. Our data propose a new mechanism of tumor-associated macrophages (TAMs) promoting Hep3B invasion and migration and suggest TAMs as a new target to reverse this process. Methods (1) We successfully induced tumor-associated macrophages (TAMs) from THP-1 cells. (2) The Western blotting procedures were performed to test the Toll-receptor 4 (TLR4) protein expression of TAMs. The expression of TLR4 were down-regulated via plasmid transfection in TAMs. (3) Hep 3B was treated with TAM-conditioned medium (TAM-CM) and small hairpin RNA TLR4-conditioned medium (shRNA TLR4-CM), respectively. The wound healing assay reflected the ability of migration of Hep3B while transwell assay reflected invasion. Results Successful Induction of TAMs. Compared with control groups, Hep3B cells migrated at a much longer distance and more invasion cells in TAM-CM groups, P=0.000. After TLR4 expression of TAMs were down-regulated, the distance Hep3B migrated and the numbers Hep3B invaded were less than TAM-CM groups, P=0.001. Conclusion TAMs facilitated migration and invasion of Hep3B cell, the mechanism of which is related to the TLR4 of TAMs. Key words: Hepatocellular carcinoma; Tumor-associated macrophages; Invasion and migration; Toll-receptor 4

Transient expression of a bovine leukemia virus envelope glycoprotein in plants by a recombinant TBSV vector

Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle. Here we evaluate the potential for using a viral vector based on the genome of Tomato bushy stunt virus (TBSV) for the efficient expression of BLV envelope glycoprotein rgp51 in Nicotiana benthamiana plants. The codon-optimized gene encoding rgp51 was synthesized by the de novo DNA synthesis to replace the GFP gene in the TBSV-derived viral vector that was then delivered into 4–5 week old N. benthamiana plants by agroinfiltration. Expression of recombinant his-tagged rgp51 was verified by protein extraction followed by western blot procedures, and by purification using Ni2+-affinity chromatography. The molecular weight of this plant-expressed rgp51 ranged from 43 to 55 kDa and it was shown to be glycosylated. Important for potential use in diagnostic tests, purified rgp51 specifically reacted with BLV infected bovine sera while no reaction was observed with the negative serum samples.

Effects of Thymoquinone on IL-6 Gene Expression and Some Cellular Signaling Pathways in Prostate Cancer PC3 Cells

Background: Thymoquinone (TQ), a bioactive compound, has many pharmacological actions especially anticancer effects. Prostate cancer (PC) is one of the most common malignancies among males in the world. Objectives: The current study aimed at evaluating the effects of TQ treatment on interleukin-6 (IL-6) expression, IL-6 secretion, cell viability, phosphorylated extracellular signal–regulated kinases (pERK1/2), phosphorylated AKT (pAKT), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) signaling pathways in human prostate cancer PC3 cells. Materials and Methods: PC3 cells were cultured in different concentrations of TQ for 48 hours. Cell viability, expression, and secretion of IL-6 were assessed by MTT assay, real-time quantitative polymerase chain reaction (RT-q PCR) analysis, and the enzyme-linked immunosorbent assay (ELISA) technique, respectively. The changes of pERK1/2, pAKT, and pSTAT3 were determined using Western blot procedure. Results: Treatment with TQ caused the reduction of viability (inhibitory concentration [IC50] = 45 µM) and survival in PC3 cells. RT-q PCR had a significant reduction (P < 0.05) in IL-6 gene expression compared with the control cells (50%, 59%, 68%, and 70% at 30, 40, 45, and 50 µM of TQ, respectively). TQ treatment resulted in a significant decline (P < 0.05) in the secretion of IL-6 compared with the control cells (56.89%, 71.12%, 84.32%, and 85.89% at 30, 40, 45, and 50 µM of TQ, respectively). Western blot showed a reduction in the phosphorylated STAT3, AKT, and ERK proteins. Conclusions: The obtained results suggested that TQ can effectively reduce IL-6 signaling pathways (pSTAT3, pAKT, and pERK1/2). Also, TQ can decrease IL-6 gene expression, which leads to the reduction of cell proliferation and viability. Therefore, TQ may be beneficial to treat prostate cancer.

An alkaloid extract obtained from Phlegmariurus Saururus induces neuroprotection after status epilepticus

BackgroundThe brain is exposed to many excitotoxic insults that can lead to neuronal damage. Among these, Epilepsy is a neurological disease that affects a large percentage of world population and is commonly associated with cognitive disorders and excitotoxic neuronal death. Most experimental strategies are focused on preventing Status Epilepticus (SE), but once it has already occurred, the key question is whether it is possible to save neurons. PurposeThe aim of this study was to determine if a purified alkaloid extract (AE) obtained from Phlegmariurus saururus, a genus of Lycophyte plants (sometimes known as firmossesor fir club mosses) could induce neuroprotection following SE. MethodsIn vitro and in vivo techniques were applied for this purpose. Protein levels were measured by western blotting procedures. Neuronal death analysis was performed by calcein-ethidium staining and the presence of the NeuN protein as a marker for presence or absence of cells (in vitro experiments) and by Fluoro Jade B staining for the in vivo experiments. ResultsThe effect of AE in the hippocampal neurons culture was the first determination, where we found an increase in neuronal survival and in the level of pErk and TrkB activation, 24 h after the addition of AE. In a well-established in vitro model of SE, we found that 24 h after being added to the hippocampal neuron-astrocyte co-culture, the AE induces a significant increase in neuronal survival. In addition to this, in the in vivo Li-pilocarpine model of SE, the AE induced a remarkable neuroprotection in areas such as the entorhinal cortex and hippocampal CA1 area. ConclusionThese results make the AE an excellent candidate for potential clinical use in neurological disorders where memory impairment and neuronal death occurs.

Expression of camel (Camelus bactrianus) and bovine (Bos Taurus) chymosin in Pichia (Komagataella) pastoris

Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle. Here we evaluate the potential for using a viral vector based on the genome of Tomato bushy stunt virus (TBSV) for the efficient expression of BLV envelope glycoprotein rgp51 in Nicotiana benthamiana plants. The codon-optimized gene encoding rgp51 was synthesized by the de novo DNA synthesis to replace the GFP gene in the TBSV-derived viral vector that was then delivered into 4–5 week old N. benthamiana plants by agroinfiltration. Expression of recombinant his-tagged rgp51 was verified by protein extraction followed by western blot procedures, and by purification using Ni2+-affinity chromatography. The molecular weight of this plant-expressed rgp51 ranged from 43 to 55 kDa and it was shown to be glycosylated. Important for potential use in diagnostic tests, purified rgp51 specifically reacted with BLV infected bovine sera while no reaction was observed with the negative serum samples.

BioSpotlight / Citations

BioTechniquesVol. 62, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114530AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Stabler expression in stable cell linesA major issue with stable cell lines generated by standard methods is the heterogeneous levels of recombinant protein expression in different cell clones due to the integration of variable numbers of the transgene and/or position effects caused by integration of the transgene into random locations in the genome. In addition, the use of strong heterologous promoters in transgenic vectors can drive expression to very high, non-physiological levels that harm the cells. In this month's issue, researchers led by Brian Chen at McGill University describe an efficient method for stable cell line generation that achieves controlled, physiological expression of a transgene. Using CRISPR/Cas9 gene editing, the team inserted a single copy of a transgene at a specific gene locus, placing it under the control of an endogenous promoter with the desired level of gene expression. Their transgenic vector also includes a protein quantitation reporter gene to produce an equimolar amount of a fluorescent protein, allowing easy quantitation of the recombinant protein.See “Generating stable cell lines with quantifiable protein production using CRISPR/Cas9 mediated knock-in” on page 165.Two colors are better than one for dna methylation assayThe cytosine extension assay (CEA) assesses global DNA methylation levels by tracking the methylation of a CpG-containing restriction enzyme site, for example CCGG, which when methylated cannot be digested by HpaII but can be digested by its isoschizomer, MspI. The relative digestion of CCGG sites in a DNA sample by HpaII compared to MspI reflects the extent of DNA methylation and is assessed in CEA by the incorporation of a fluorescently labeled cytosine through end-filling by DNA polymerase of the 5′ guanosine overhang at the HpaII/MspI cut site. In this issue of BioTechniques, Craig Parfett and his colleagues at Health Canada present an improved version of CEA. In standard CEA, the same fluorescently colored cytosine is used in the extension reactions following HpaII and MspI digestion. In their version, Parfett's team used a different fluorescently colored cytosine for the extension reaction of each digest, after which the reactions were combined for downstream purification and signal detection. This two-color CEA showed greater precision compared to the one-color CEA and is well-suited to high-throughput screening.See “Two-color fluorescent cytosine extension assay for the determination of global DNA methylation” on page 157.Root problemsPlant biologists have long been interested in understanding the interactions that occur in nature between microorganisms and plant roots. Plants both obtain information about their environment and send out signals to microbes through their roots. However, deciphering the interplay between plant roots and the microbial communities surrounding them has proven difficult. Now, researchers report in PNAS a new microfluidic-based device designed specifically for monitoring root–bacteria interactions over time. The device, which the authors named the tracking root interaction system (TRIS), is composed of 9 independent microfluidic chambers where roots can be established and into which bacteria are introduced for monitoring. The chambers are coupled to a microscope assembly that makes it possible to obtain both spatial and temporal information on root–bacteria interactions. In proof-of-concept experiments, the authors fluorescently labeled bacteria and were able to observe root colonization in real time over extended periods using their TRIS device.Massalha, H. et al. 2017. Live imaging of root-bacteria interactions in a microfluidics setup. Proc Natl Acad Sci USA. doi:10.1073/pnas.1618584114.Western blotting goes smallWestern blotting is a commonly used technique for studying cellular proteins. While effective when using large numbers of cells as starting material, western blot analysis has proven more difficult when scaled down for studies of single cells with their much lower protein concentrations. Seeking to overcome this limitation and extend the range of protein analysis, researchers describe a new rare-cell, single-cell western blotting procedure in Nature Communications. Building on previous work that established the feasibility of single-cell resolution western blotting, the authors sought to measure panels of proteins from extremely rare circulating tumor cell (CTC) isolates. For protein analysis, each CTC was placed into an individual microwell, where the cell was first lysed and then immediately electrophoresed into a polyacrylamide matrix. Following a miniaturized version of polyacrylamide gel electrophoresis (PAGE), in-gel immunoprobing was done on the size-separated CTC proteins. Using this method, the authors were able to successfully perform multiplexed protein analyses and report protein expression on a per-cell basis.Sinkala, E. et al. 2017. Profiling protein expression in circulating tumor cells using microfluidic western blotting. Nat Commun. 8:14622.FiguresReferencesRelatedDetails Vol. 62, No. 4 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print April 2017 Information© 2017 Author(s)PDF download

Indocyanine Green-001 (ICG-001) Attenuates Wnt/β-catenin-induces Myocardial Injury Following Sepsis.

Objective:To investigate the mechanistic pathway of both indocyanine green (ICG)-001 in attenuated endotoxemia-induced cardiac depression through downregulation cardiac Wnt/ β-catenin cell signaling.Materials and Methods:Adult (4–6 months) male Albino-Webster mice, their weights ranged from 25 to 30 g, were pretreated with ICG-001 i.p., following cecal ligation and puncture (CLP). Left ventricle (LV) function was assessed using a microcatheter system. Monocyte chemoattractant protein-1 (MCP-1) and cytokines mediators in plasma and myocardium were analyzed by enzyme-linked immunosorbent assay. Further, the cardiac Wnt protein measured by quantitative real-time polymerase chain reaction while β-catenin analysis through Western blotting procedure. The pathological changes and cells injury in the myocardium were examined using hematoxylin and eosin staining.Results:CLP mice displayed worse LV function. The exaggerated cardiac depression in CLP mice was associated with higher levels of MCP-1 and cytokines in plasma and myocardium together with greater cardiac levels of cardiac troponin-I and Wnt/β-catenin. Neutralization of sepsis by either ICG-001resulted in improved LV function and reductions in inflammatory mediators.Conclusion:Taken together, these data showed that ICG-001 improved LV function following sepsis through downregulation of Wnt/β-catenin and serve as a potential mechanistic pathway ICG-001 in therapeutic cardiac endotoxemia in animal model.

Antibody Validation by Western Blotting.

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

Public health significance of echinococcosis

Echinococcosis is a cosmopolitan zoonosis caused by infection with adult larval form of Echinococcus granulosus, a cause of unilocular echinococcosis (hydatidosis), or, less often, Echinococcus multilocularis, a cause of multilocular echinococcosis (alveolar echinococcosis). Humans act as intermediate hosts and are infected through direct contact with definitive hosts, namely parasite-infested dogs. Echinococcosis is a disease that is prevalent throughout the world, including Croatia. As such it is categorized as a disease of great significance for public health. A timely and valid laboratory diagnostic testing to determine its seroprevalence is therefore of clinical and epidemiological importance in order to successfully monitor, control and treat the disease. Although sometimes limited, serological testing is increasingly used and ought to be accepted and implemented should the disease be suspected. In view of the above, within this study we have analysed IgG subclass antibodies of 599 patient serum samples by using the ELISA assay during 2015 to determine the presence of Echinococcus granulosus. Obtained results indicated that 6 % of samples were positive, 11 % borderline and 83 % negative. All ELISA reactive samples were further confirmed using the Western blot (WB) assay. By applying these procedures we have established that 14/599 samples were positive and calculated the prevalence of 2.3 % in general population (4.25 % among male and 4.8 % among female patients). The analysis of results according to age showed that the majority of tested patients and the highest seroprevalence determined by WB procedure for general population pertained to the age group of 61 years and older (4.1 %).