Heterogeneity in Thymic Emigrants: Implications for Thymectomy and Immunosenescence

The development of mature, antigen-inexperienced (naive) T cells begins in the thymus and continues after export into the periphery. Post-thymic maturation of naive T cells, in humans, coincides with the progressive loss of markers such as protein tyrosine kinase 7 (PTK7) and platelet endothelial cell adhesion molecule-1 (CD31). As a consequence, subpopulations of naive T cells can be recognised raising questions about the processes that give rise to the loss of these markers and their exact relationship to recent thymic emigrants (RTE). Here, we combine a mathematical survival analysis approach and data from healthy and thymectomised humans to understand the apparent persistence of populations of ‘veteran’ PTK7⁺T cells in thymectomised individuals. We show that a model of heterogeneity in rates of maturation, possibly linked to natural variation in TCR signalling thresholds or affinity for self-antigens, can explain the data. This model of maturation predicts that the average post-thymic age of PTK7⁺T cells will increase linearly with the age of the host suggesting that, despite the immature phenotype, PTK7⁺cells do not necessarily represent a population of RTE. Further, the model predicts an accelerated increase in the average post-thymic age of residual PTK7⁺T cells following thymectomy and may also explain in part the prematurely aged phenotype of the naive T cell pool in individuals thymectomised early in life.     

Estimating Orientation Factors in the FRET Theory of Fluorescent Proteins: The TagRFP-KFP Pair and Beyond

The orientation factor κ², one of the key parameters defining Förster resonance energy transfer efficiency, is determined by the transition dipole moment orientations of the donor and acceptor species. Using the results of quantum chemical and quantum mechanical/molecular mechanical calculations for the chromophore-containing pockets in selected colored proteins of the green fluorescent protein family, we derived transition dipole moments corresponding to the S_0,min → S₁ excitation for green fluorescent protein, red fluorescent protein (TagRFP), and kindling fluorescent protein, and the S_1,min → S₀ emission for TagRFP. These data allowed us to estimate κ² values for the TagRFP-linker-kindling fluorescent protein tetrameric complex required for constructing novel sensors.     

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.     

The Impact of Sediment Characteristics on PCB-dechlorinating Cultures: Implications for Bioaugmentation

Polychlorinated Biphenyl (PCB)-dechlorinating cultures with complimentary activities, previously derived from estuarine Baltimore Harbor (B), marine Palos Verdes (P), and riverine Hudson River (H) sediments, were mixed and then inoculated into sterile sediments from the same sources. In the treatments containing sterile B sediment, the different inocula had limited impact on the bacterial community development and on dechlorination patterns, all of which were similar. In treatments containing sterile P or H sediment, however, different inocula resulted in significantly different PCB dechlorination patterns and bacterial communities. The B sediment appeared to support not only the most extensive and rapid dechlorination of the three sediments, but also supported a more diverse bacterial community. This was thought to be a result of nutritional richness, as it was high in organic carbon and micronutrients such as zinc and cobalt. Although mixing three PCB-dechlorinating cultures was able to produce a culture capable of enhanced PCB-dechlorinating activity as compared to single cultures, some activities were lost upon culture transfer. This indicates that care must be taken to establish robust PCB-dechlorinating cultures capable of extensive dechlorination prior to pursuing bioaugmentation. In addition, our results indicate that the concentration and availability of macro-and micronutrients could have a significant impact on the microbial community structure, and thus a thorough characterization of the sediment at contaminated sites is essential for implementing bioaugmentation for PCB bioremediation.     

Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4–6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.     

Impact of Emission Anisotropy on Fluorescence Spectroscopy and FRET Distance Measurements

The objective of this report is to provide a practical and improved method for estimating Förster resonance energy transfer distance measurement error due to unknown angles in the dipole orientation factor based on emission anisotropy measurements. We improve on the method of Dale et al. (1979), which has minor mistakes and is frequently interpreted in overly optimistic ways in the literature. To facilitate proper fluorescence intensity measurements, we also evaluated instrument parameters that could impact the measurement. The apparent fluorescence intensity of isotropic samples depends on the sample emission anisotropy, fluorometer geometry, and optical apertures. We separate parameters of the sample, and those of the cylindrically symmetric illumination source and detector in the equations describing results of unpolarized and polarized fluorescence intensity measurements. This approach greatly simplifies calculations compared with the more universal method of Axelrod (1989). We provide a full computational method for calculating the Förster resonance energy transfer distance error and present a graph describing distance error in the simplest case.     

Malaria Drug Resistance: The Impact of Human Movement and Spatial Heterogeneity

Human habitat connectivity, movement rates, and spatial heterogeneity have tremendous impact on malaria transmission. In this paper, a deterministic system of differential equations for malaria transmission incorporating human movements and the development of drug resistance malaria in an \(n\) patch system is presented. The disease-free equilibrium of the model is globally asymptotically stable when the associated reproduction number is less than unity. For a two patch case, the boundary equilibria (drug sensitive-only and drug resistance-only boundary equilibria) when there is no movement between the patches are shown to be locally asymptotically stable when they exist; the co-existence equilibrium is locally asymptotically stable whenever the reproduction number for the drug sensitive malaria is greater than the reproduction number for the resistance malaria. Furthermore, numerical simulations of the connected two patch model (when there is movement between the patches) suggest that co-existence or competitive exclusion of the two strains can occur when the respective reproduction numbers of the two strains exceed unity. With slow movement (or low migration) between the patches, the drug sensitive strain dominates the drug resistance strain. However, with fast movement (or high migration) between the patches, the drug resistance strain dominates the drug sensitive strain.     

The Impact of Using Alternative Forages on the Nutrient Value within Slurry and Its Implications for Forage Productivity in Agricultural Systems

Alternative forages can be used to provide valuable home-grown feed for ruminant livestock. Utilising these different forages could affect the manure value and the implications of incorporating these forages into farming systems, needs to be better understood. An experiment tested the hypothesis that applying slurries from ruminants, fed ensiled red clover (Trifolium pratense), lucerne (Medicago sativa) or kale (Brassica oleracea) would improve the yield of hybrid ryegrass (Lolium hybridicum), compared with applying slurries from ruminants fed ensiled hybrid ryegrass, or applying inorganic N alone. Slurries from sheep offered one of four silages were applied to ryegrass plots (at 35 t ha⁻¹) with 100 kg N ha⁻¹ inorganic fertiliser; dry matter (DM) yield was compared to plots only receiving ammonium nitrate at rates of 0, 100 and 250 kg N ha⁻¹ year⁻¹. The DM yield of plots treated with 250 kg N, lucerne or red clover slurry was significantly higher than other treatments (P<0.001). The estimated relative fertiliser N equivalence (FNE) (fertiliser-N needed to produce same yield as slurry N), was greatest for lucerne (114 kg) >red clover (81 kg) >kale (44 kg) >ryegrass (26 kg ha⁻¹ yr⁻¹). These FNE values represent relative efficiencies of 22% (ryegrass), 52% (kale), 47% (red clover) and 60% for lucerne slurry, with the ryegrass slurry efficiency being lowest (P = 0.005). Soil magnesium levels in plots treated with legume slurry were higher than other treatments (P<0.001). Overall, slurries from ruminants fed alternative ensiled forages increased soil nutrient status, forage productivity and better N efficiency than slurries from ruminants fed ryegrass silage. The efficiency of fertiliser use is one of the major factors influencing the sustainability of farming systems, these findings highlight the cascade in benefits from feeding ruminants alternative forages, and the need to ensure their value is effectively captured to reduce environmental risks.     

Investigation of Endoglin Wild-Type and Missense Mutant Protein Heterodimerisation Using Fluorescence Microscopy Based IF,BiFC and FRET Analyses

The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing of other membrane receptors, such as TβRII, ALK5 or ALK1.     

绿色荧光蛋白的特性及其在信号转导中的应用

细胞和分子生物学的最终目标是 ,明确细胞内的各种事件是如何发生的 ,明了细胞内复杂的动态变化的生化机制。绿色荧光蛋白 (ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ ,ＧＦＰ)自从克隆、表达之后 ,以其良好的物理特性及荧光特性而成为良好的报告基因和荧光标记分子 ,并在探索生命现象过程中得到了非常广泛的应用。ＧＦＰ作为报告基因 ,可用在活细胞中直接观察蛋白质向细胞器 ,如细胞核、内质网中运动 ;作为荧光标记分子 ,ＧＦＰ既具有敏感的标记检测率 ,又没有放射性的危害 ;最近又发现ＧＦＰ是一个良好的细胞间信号传递的动态标记…     

FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells

Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, E_app, across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the E_app distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method—akin to that of mass spectrometry—for sorting protein complexes according to their size and shape.     

Homepeptide Repeats:Implications for Protein Structure,Function and Evolution

Analysis of protein sequences from Mycobacterium tuberculosis H37Rv(Mtb H37Rv) was performed to identify homopeptide repeatcontaining proteins(HRCPs).Functional annotation of the HRCPs showed that they are preferentially involved in cellular metabolism.Furthermore,these homopeptide repeats might play some specific roles in protein-protein interaction.Repeat length differences among Bacteria,Archaea and Eukaryotes were calculated in order to identify the conservation of the repeats in these divergent kingdoms.From the results,it was evident that these repeats have a higher degree of conservation in Bacteria and Archaea than in Eukaryotes.In addition,there seems to be a direct correlation between the repeat length difference and the degree of divergence between the species.Our study supports the hypothesis that the presence of homopeptide repeats influences the rate of evolution of the protein sequences in which they are embedded.Thus,homopeptide repeat may have structural,functional and evolutionary implications on proteins.     

In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.     

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual’s sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.     

Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

Background

The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity.

Results

The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome''s chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability.

Conclusion

Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.     

The Role of Protein Conformational Switches in Pharmacology: Its Implications in Metabolic Reprogramming and Protein Evolution

Besides pharmacogenomics and drug dynamics, pharmacological properties of a drug could also arise from protein conformational switches. These switches would arise from the following mechanisms: (a) slight shifts away from a protein’s native conformation induced by mutation, (b) changes in the protein’s environment allowing for structural rearrangements to form hitherto unknown conformations, (c) parsing the protein into foldable polypeptide fragment(s) by either proteolysis of the native structure or (d) perturbation of the native conformation to generate polypeptide fragment(s). These switches are modulated by changes in the protein’s matrix properties such as pH, temperature, ligands—their nature, concentration and complexes; micronutrients, oxidant/antioxidant status and metabolic products within the functional environment of the protein. The pharmacological implications of these are discussed in light of polypharmacology arising from protein isomerism, cross-pharmacology, possible decreases in both the expressible and expressed protein population and metabolic reprogramming—and ultimately, how these factors relate to diseases. Further implications include variational drug toxicity and drug response idiosyncrasies. Another important consequence is that the “whole life” history of the individual would play an active role in that individual’s response to disease severity and drug response up to that very moment and is prone to variations with changes in pre-disposing factors.     

The Unique Impact of Menstruation on the Female Voice: Implications for the Evolution of Menstrual Cycle Cues

Research shows that hormonal changes in women across the menstrual cycle affect vocal production. Most work has documented shifts at high fertility times (i.e., ovulation) or during premenstruation. However, hormonal changes at menstruation also affect female physiology and behavior and could affect vocal production. The present studies investigated perceptual differences in voices recorded during menstruation compared with recordings taken at other times of the menstrual cycle. Results show that male raters could reliably identify voices recorded during menstruation with or without the presence of a voice recorded closest to ovulation. In addition, voices recorded at menstruation were identified as being the most unattractive. These findings indicate that voice recordings taken at times of lowest fertility may uniquely impact women’s vocal production and that perceptions of voice based on cycle phase are not specific to the time of heightened fertility. Implications for the evolution of human menstrual cycle cues are discussed.