Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Purification and <Emphasis Type="Italic">N</Emphasis>-Glycosylation Analysis of Melanoma Antigen Dopachrome Tautomerase

Dopachrome tautomerase (DCT) plays a critical role in lowering the oxidative stress resulting from melanogenesis. Levels of DCT are elevated in melanoma cell lines that are especially resistant to chemotherapy and radiation. DCT is processed as a melanoma antigen and is a potential target for immunotherapy. In order to establish a more complete understanding of the role that DCT may play in the etiology and treatment of melanoma skin cancer, isolation of highly pure and properly processed protein is necessary. Purification of native DCT has been problematic due to a hydrophobic transmembrane anchor and interactions with melanin. In this study, DCT was expressed, without its carboxy-terminal transmembrane region using an Sf9 insect cell protein expression system and its recombinant protein was purified by various chromatographic techniques. Analysis of DCT tryptic peptides by MALDI-TOF/TOF determined N-glycosylation as a primary post-translational modification. Our success in the expression of soluble mammalian DCT and the characterization of N-glycosylation sites is a useful reference toward the comprehensive understanding of the structure/function relationship of mammalian DCT.     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.     

Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.     

Novel Immunodominant Peptide Presentation Strategy: a Featured HLA-A*2402-Restricted Cytotoxic T-Lymphocyte Epitope Stabilized by Intrachain Hydrogen Bonds from Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β₂-microglobulin (β₂m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.In 2003, severe and acute respiratory syndrome (SARS), emerging from China, caused a global outbreak, affecting 29 countries, with over 8,000 human cases and greater than 800 deaths (5, 9, 24, 33, 37). Thanks to the unprecedented global collaboration coordinated by the WHO, SARS coronavirus (SARS-CoV), a novel member of Coronaviridae family, was rapidly confirmed to be the etiological agent for the SARS epidemic (36). Soon after the identification of the causative agent, SARS was controlled and then quickly announced to be conquered through international cooperation on epidemiological processes (9). However, the role that human immunity played in the clearance of SARS-CoV and whether the memory immunity will persist for the potential reemergence of SARS are not yet well understood.In viral infections, CD8⁺ cytotoxic T lymphocytes (CTLs) are essential to the control of infectious disease. Virus-specific CD8⁺ T cells recognize peptides which have 8 to 11 amino acids, in most cases presented by major histocompatibility complex (MHC) class I molecules. However, identification of virus-specific CD8⁺ T-cell epitopes remains a complicated and time-consuming process. Various strategies have been developed to define CTL epitopes so far. One of the most common practices to determine immunodominant CTL epitopes on a large scale is based on screening and functional analysis of overlapping 15- to 20-mer peptides covering an entire viral proteome or a given set of immunogenic proteins (19, 23, 32). However, peptides identified through this method are too long to be naturally processed CTL epitopes, and the definition of MHC class I restriction of these peptides still requires further analysis. Rapid and efficient strategies should be developed for the determination of naturally presented CTL epitopes in the context of any given HLA allele of interest. Furthermore, no other HLA alleles except HLA-A2-restricted CTL epitopes have been reported for SARS-CoV-derived proteins (16, 22, 31, 43, 46, 47, 49). This is primarily because of the limitation of the experimental methods for the other HLA alleles. HLA-A24 is one of the most common HLA-A alleles throughout the world, especially in East Asia, where SARS-CoV emerged, second only to HLA-A2 (30). The development of a fast and valid method to screen and identify HLA-A24-restricted epitopes would greatly contribute to the understanding of the specific CTL epitope-stimulated response and widen the application of the epitope-based vaccine among a more universal population (17). A genomewide scanning of HLA binding peptides from SARS-CoV has been performed by Sylvester-Hvid and colleagues, through which dozens of peptides with major HLA supertypes, including HLA-A24 binding capability, have been identified (41).There are strong indications that different peptide ligands, such as peptides with distinct immunodominance, can elicit a diverse specific T-cell repertoire, and even subtle changes in the same peptide can have a profound effect on the response (25, 44). Furthermore, a broader T-cell receptor (TCR) repertoire to a virus-specific peptide-MHC complex can keep the host resistant to the virus and limit the emergence of virus immune-escape mutants (29, 34, 38). Recent studies have demonstrated that the diversity of the selected TCR repertoire (designated as T-cell receptor bias) is clearly influenced by the conformational characteristics of the bound peptide in the MHC groove. Peptides with a flat, featureless surface when presented by MHC generate only limited TCR diversity in a mature repertoire, while featured peptides with exposed residues (without extreme bulges) protruding outside the pMHC landscapes are rather associated with the more diverse T-cell repertoire (15, 28, 39, 44, 45). Therefore, being able to determine the binding features of a peptide to MHC and describe the peptide-MHC topology will help us understand the immunodominance of a given peptide and demonstrate the peptide presentation strategy of the host.Structural proteins of SARS-CoV, such as spike, membrane, and nucleocapsid (N), have been demonstrated as factors of the antigenicity of the virus, as compared with the nonstructural proteins (12, 20). Coronavirus nucleocapsid (N) protein is a highly phosphorylated protein which not only is responsible for construction of the ribonucleoprotein complex by interacting with the viral genome and regulating the synthesis of viral RNA and protein, but also serves as a potent immunogen that induces humoral and cellular immunity (13, 14, 26, 48). The CD8⁺ T-cell epitopes derived from SARS-CoV N protein defined so far mainly cluster in two major immunogenic regions (4, 21, 23, 31, 32, 43). One of them, residues 219 to 235, comprises most of the N protein-derived minimal CTL epitopes identified so far—N220-228, N223-231, N227-235, etc.—all of which are HLA-A*0201 restricted (4, 43). The other region, residues 331 to 365, also includes high-immunogenicity peptides that can induce memory T-lymphocyte responses against SARS-CoV (21, 23, 32). However, until now, no minimal CTL epitope with a given HLA allele restriction has been investigated in this region.Here, based on previously defined immunogenic regions derived from SARS-CoV N protein (21), we identified an HLA-A*2402-restricted epitope, N1 (residues 346 to 354), in the region through a distinct strategy using structural and functional approaches. The binding affinity with HLA-A*2402 molecules and the cellular immunogenicity of the peptide were demonstrated in a series of assays. The X-ray crystal structure of HLA-A*2402 complexed with peptide N1 has shown a novel host strategy to present an immunodominant CTL epitope by intrachain hydrogen bond as a featured epitope.     

The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes

Bile acids are synthesized from cholesterol in the liver and are excreted into bile via the hepatocyte canalicular bile salt export pump. After their passage into the intestine, bile acids are reabsorbed in the ileum by sodium-dependent uptake across the apical membrane of enterocytes. At the basolateral domain of ileal enterocytes, bile acids are extruded into portal blood by the heterodimeric organic solute transporter OSTalpha/OSTbeta. Although the transport function of OSTalpha/OSTbeta has been characterized, little is known about the regulation of its expression. We show here that human OSTalpha/OSTbeta expression is induced by bile acids through ligand-dependent transactivation of both OST genes by the nuclear bile acid receptor/farnesoid X receptor (FXR). FXR agonists induced endogenous mRNA levels of OSTalpha and OSTbeta in cultured cells, an effect that was not discernible upon inhibition of FXR expression by small interfering RNAs. Furthermore, OST mRNAs were induced in human ileal biopsies exposed to the bile acid chenodeoxycholic acid. Reporter constructs containing OSTalpha or OSTbeta promoters were transactivated by FXR in the presence of its ligand. Two functional FXR binding motifs were identified in the OSTalpha gene and one in the OSTbeta gene. Targeted mutation of these elements led to reduced inducibility of both OST promoters by FXR. In conclusion, the genes encoding the human OSTalpha/OSTbeta complex are induced by bile acids and FXR. By coordinated control of OSTalpha/OSTbeta expression, bile acids may adjust the rate of their own efflux from enterocytes in response to changes in intracellular bile acid levels.