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Concept: Western blot

166

Optic nerve atrophy caused by abnormal intraocular pressure (IOP) remains the most common cause of irreversible loss of vision worldwide. The aim of this study was to determine whether topically applied IOP-lowering eye drugs affect retinal ganglion cells (RGCs) and retinal metabolism in a rat model of optic neuropathy. IOP was elevated through cauterization of episcleral veins, and then lowered either by the daily topical application of timolol, timolol/travoprost, timolol/dorzolamide, or timolol/brimonidine, or surgically with sectorial iridectomy. RGCs were retrogradely labeled 4 days prior to enucleation, and counted. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization mass spectrometry, Western blotting, and immunohistochemistry allowed the identification of IOP-dependent proteomic changes. Genomic changes were scrutinized using microarrays and qRT-PCR. The significant increase in IOP induced by episcleral vein cauterization that persisted until 8 weeks of follow-up in control animals (<0.05) was effectively lowered by the eye drops (<0.05). As anticipated, the number of RGCs decreased significantly following 8 weeks of elevated IOP (<0.05), while treatment with combination compounds markedly improved RGC survival (<0.05). 2D-PAGE and Western blot analyses revealed an IOP-dependent expression of crystallin cry-βb2. Microarray and qRT-PCR analyses verified the results at the mRNA level. IHC demonstrated that crystallins were expressed mainly in the ganglion cell layer. The data suggest that IOP and either topically applied antiglaucomatous drugs influence crystallin expression within the retina. Neuronal crystallins are thus suitable biomarkers for monitoring the progression of neuropathy and evaluating any neuroprotective effects.

Concepts: Molecular biology, Western blot, Gel electrophoresis, Retina, Eye, SDS-PAGE, Retinal ganglion cell, Southern blot

148

The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-27312A. This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy.

Concepts: HIV, AIDS, Immune system, Antibody, Immunology, Western blot, Primate, Gp120

81

The mechanisms that contribute to selective vulnerability of the magnocellular basal forebrain cholinergic neurons in neurodegenerative diseases, such as Alzheimer’s disease, are not fully understood. Because age is the primary risk factor for Alzheimer’s disease, mechanisms of interest must include age-related alterations in protein expression, cell type-specific markers and pathology. The present study explored the extent and characteristics of intraneuronal amyloid-β accumulation, particularly of the fibrillogenic 42-amino acid isoform, within basal forebrain cholinergic neurons in normal young, normal aged and Alzheimer’s disease brains as a potential contributor to the selective vulnerability of these neurons using immunohistochemistry and western blot analysis. Amyloid-β1-42 immunoreactivity was observed in the entire cholinergic neuronal population regardless of age or Alzheimer’s disease diagnosis. The magnitude of this accumulation as revealed by optical density measures was significantly greater than that in cortical pyramidal neurons, and magnocellular neurons in the globus pallidus did not demonstrate a similar extent of amyloid immunoreactivity. Immunoblot analysis with a panel of amyloid-β antibodies confirmed accumulation of high concentration of amyloid-β in basal forebrain early in adult life. There was no age- or Alzheimer-related alteration in total amyloid-β content within this region. In contrast, an increase in the large molecular weight soluble oligomer species was observed with a highly oligomer-specific antibody in aged and Alzheimer brains when compared with the young. Similarly, intermediate molecular weight oligomeric species displayed an increase in aged and Alzheimer brains when compared with the young using two amyloid-β42 antibodies. Compared to cortical homogenates, small molecular weight oligomeric species were lower and intermediate species were enriched in basal forebrain in ageing and Alzheimer’s disease. Regional and age-related differences in accumulation were not the result of alterations in expression of the amyloid precursor protein, as confirmed by both immunostaining and western blot. Our results demonstrate that intraneuronal amyloid-β accumulation is a relatively selective trait of basal forebrain cholinergic neurons early in adult life, and increases in the prevalence of intermediate and large oligomeric assembly states are associated with both ageing and Alzheimer’s disease. Selective intraneuronal amyloid-β accumulation in adult life and oligomerization during the ageing process are potential contributors to the degeneration of basal forebrain cholinergic neurons in Alzheimer’s disease.

Concepts: Neuron, Molecular biology, Death, Senescence, Western blot, Acetylcholine, Pyramidal cell, Basal forebrain

60

Polyclonal anti-immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class.

Concepts: Immune system, Antibody, Protein, Molecular biology, Western blot, Escherichia coli, Immunohistochemistry, Immunostaining

44

Transgender persons are at high risk for human immunodeficiency virus (HIV) infection; in a recent analysis of the results of over nine million CDC funded HIV tests, transgender women* had the highest percentage of confirmed positive results (2.7%) of any gender category (1). Transgender men,(†) particularly those who have sex with cisgender(§) men, are also at high risk for infection (2). HIV testing is critical for detecting and treating persons who are infected and delivering preventive services to those who are uninfected. CDC recommends that persons at high risk for HIV infection be screened for HIV at least annually, although transgender persons are not specified in the current recommendations. CDC analyzed data from the Behavioral Risk Factor Surveillance System (BRFSS) to describe HIV testing among transgender women and men and two cisgender comparison groups in 27 states and Guam. After adjusting for demographic characteristics, transgender women and men had a lower prevalence of ever testing and past year testing for HIV (35.6% and 31.6% ever, and 10.0% and 10.2% past year, respectively) compared with cisgender gay and bisexual men (61.8% ever and 21.6% past year) and instead reported testing at levels comparable to cisgender heterosexual men and women (35.2% ever, and 8.6% past year). This finding suggests that transgender women and men might not be sufficiently reached by current HIV testing measures. Tailoring HIV testing activities to overcome the unique barriers faced by transgender women and men might increase rates of testing among these populations.

Concepts: HIV, AIDS, Immune system, Viral load, Infectious disease, Western blot, Gender, Transgender

29

Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub-clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi-centred study. A coded panel of 241 well-characterized sera from infected and non-infected bovines was provided by all participants (SALUVET-Madrid, FLI-Wusterhausen, ENV-Toulouse, IPB-Berne). The tests evaluated were as follows: an in-house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non-reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests (‘Majority of tests’) and (ii) the majority of test results plus pre-test information based on clinical signs (‘Majority of tests plus pre-test info’). Relative to the gold standard ‘Majority of tests’, almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET-Madrid and FLI-Wusterhausen tachyzoite- and bradyzoite-based Western blot tests under non-reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in-house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI-Wusterhausen performed better than IFAT SALUVET-Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard ‘Majority of test plus pre-test info’, Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.

Concepts: Antibody, Epidemiology, Infection, Type I and type II errors, Apicomplexa, Western blot, Transmission and infection of H5N1, Serology

28

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, β-nerve growth factor (β-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-β-NGF primary antibody.

Concepts: Protein, Molecular biology, Western blot, Gel electrophoresis, Electrophoresis, Laboratory techniques, Two-dimensional gel electrophoresis, Isoelectric focusing

28

Seven mAbs with specific reaction patterns against each of the four genotypes and eight subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24 reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57 reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of mAb VHS-1.24 to be aa 32-34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57 and VHS-3.75 were found to be aa 43 and 45-48, aa 117 and 121, and aa 103, 118 and 121 of the N-protein, respectively.

Concepts: Immune system, Antibody, Gene, Monoclonal antibodies, Western blot, Antigen, Viral hemorrhagic septicemia

28

Enterovirus 71 (EV71) is a member of the Picornaviridae family and one of the main causative agents of hand, foot, and mouth disease (HFMD). Currently, EV71 infection is prevalent in the Asia-Pacific regions where it affects millions of children under the age of five, causing significant morbidity and mortality. No specific vaccine or antiviral drugs are available for EV71. The development of murine monoclonal antibodies (mAbs) with potent neutralization effects on EV71 is described. Mab-secreting hybridomas were generated from mice immunized with EV71 recombinant virus-like particles. Three IgG1 mAbs, D5, H7, and C4, capable of binding to and neutralizing EV71, were identified. In ELISA and Western blot assays, these mAbs reacted with recombinant VP1 protein, but not with VP0. They also detected cells infected with EV71 by immunofluorescent staining. In addition, these three mAbs had potent EV71 neutralization capacity, with 95% inhibitory concentrations of 0.3125, 0.3125, and 1.25μg/ml for D5, H7, and C4, respectively. The presented data demonstrate that the anti-EV71 mAbs are not only promising candidates for development into humanized mAb for treatment but also useful reagents for development of diagnostic tests.

Concepts: Immune system, Antibody, Enterovirus, Monoclonal antibodies, Immunology, Western blot, Monoclonal antibody therapy, Immunohistochemistry

28

Tubulointerstitial macrophage infiltration is a hallmark of chronic kidney disease (CKD) involved in the progression of renal fibrosis. Pirfenidone is a newly identified anti-fibrotic drug, the potential mechanism of which remains unclear. The aim of this study was to investigate the effects of pirfenidone on M1/M2 macrophage infiltration in nephrectomized rats. Nephrectomized rats were treated with pirfenidone by gavage for 12 weeks. 24-h urinary protein, NAG activity, systolic blood pressure (SBP) and CRP were determined. Paraffin-embedded sections were stained for CD68 (-), CCR7 (-) and CD163 (-) macrophages. MCP-1 and MIP-1α, as well as M1 and M2 macrophages secretory markers were evaluated by real-time RT-PCR and western blotting analysis. Pirfenidone significantly improved the elevated proteinuria and NAG activity from week 2 onward after surgery. Pirfenidone attenuated interstitial fibrosis and decreased expression of fibrotic markers including TGF-β1, CTGF, α-SMA, fibronectin and FSP-1. Pirfenidone significantly decreased the infiltrating macrophages. The number of M1 and M2 macrophages was significantly lower after pirfenidone treatment. MCP-1 and MIP-1α were increased in nephrectomized rats at mRNA and protein levels. Pirfenidone treatment significantly inhibited their expression. The TNF-α, IL-6 and iNOS expressed by M1 macrophages were increased in nephrectomized rats, and pirfenidone significantly attenuated their expression. Pirfenidone treatment also significantly decreased arginase-1, dectin-1, CD206 and CD86 expressed by M2 macrophages. Thus, Pirfenidone inhibits M1 and M2 macrophage infiltration in 5/6 nephrectomized rats, which suggests its efficacy in the early and late periods of renal fibrosis.

Concepts: Chronic kidney disease, Kidney, Molecular biology, Atherosclerosis, Fibrosis, Macrophage, Blood pressure, Western blot