DNA-hybridization electron microscopy. Localization of five regions of 16 S rRNA on the surface of 30 S ribosomal subunits

DNA-hybridization electron microscopy has been used to locate five regions of 16 S rRNA on the surface of 30 S ribosomal subunits. Biotinylated DNA probes that are complementary to selected regions of 16 S rRNA were hybridized to activated 30 S ribosomal subunits. These hybridized probes were reacted with avidin and localized by electron microscopy. The specificity of DNA binding was monitored with RNase H, which recognizes RNA-DNA hybrids and cleaves the RNA. Three of the five sequences examined were mapped on the platform. These sequences are 686-703, 714-733 and 787-803. Region 1492-1505 is mapped in the cleft and region 518-533 is at the neck on the side opposite the platform, respectively.     

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols. Download video file.^{(68M, mov)}     

Automatic physiological waveform processing for FMRI noise correction and analysis

Functional MRI resting state and connectivity studies of brain focus on neural fluctuations at low frequencies which share power with physiological fluctuations originating from lung and heart. Due to the lack of automated software to process physiological signals collected at high magnetic fields, a gap exists in the processing pathway between the acquisition of physiological data and its use in fMRI software for both physiological noise correction and functional analyses of brain activation and connectivity. To fill this gap, we developed an open source, physiological signal processing program, called PhysioNoise, in the python language. We tested its automated processing algorithms and dynamic signal visualization on resting monkey cardiac and respiratory waveforms. PhysioNoise consistently identifies physiological fluctuations for fMRI noise correction and also generates covariates for subsequent analyses of brain activation and connectivity.     

Interleukin-7-aggravated joint inflammation and tissue destruction in collagen-induced arthritis is associated with T-cell and B-cell activation

Introduction

We sought to investigate the capacity of interleukin (IL)-7 to enhance collagen-induced arthritis and to study by what mechanisms this is achieved.

Methods

Mice received multiple injections with IL-7 or phosphate-buffered saline (PBS) as a control. Arthritis severity and incidence were determined by visual examination of the paws. Joint destruction was determined by assessing radiographs and immunohistochemistry of the ankle joints. Total cellularity and numbers of T-cell and B-cell subsets were assessed, as well as ex vivo production of interferon-γ (IFN-γ), IL-17, and IL-4. Proinflammatory mediators were measured in serum with multianalyte profiling.

Results

IL-7 increased arthritis severity and radiology-assessed joint destruction. This was consistent with IL-7-increased intensity of cell infiltrates, bone erosions, and cartilage damage. Splenic CD19⁺ B cells and CD19⁺/GL7⁺ germinal center B cells, as well as CD4 and CD8 numbers, were increased by IL-7. IL-7 expanded memory T cells, associated with increased percentages of IFN-γ-, IL-4-, and IL-17-producing CD4⁺ T cells. On antigen restimulation of draining lymph node cells in vitro IL-7 treatment was found to increase IFN-γ and IL-17 production, whereas IL-4 was reduced. IL-7 also increased concentrations of proinflammatory mediators, indicative of T-cell activation (sCD40L), vascular activation (VCAM-1, VEGF), tissue destruction (fibroblast growth factor-basic (FGF-b), LIF), and chemotaxis (MIP-1γ, MIP-3β, lymphotactin, MDC, and MCP-5).

Conclusions

In arthritic mice, IL-7 causes expansion of T and B cells, associated with increased levels of proinflammatory mediators. IL-7 intensifies arthritis severity and joint destruction, accompanied by increased Th1 and Th17 activity. These data indicate that IL-7 could be an important mediator in arthritic conditions and that targeting IL-7 or its receptor represent novel therapeutic strategies.     

Causes of sulfur isotope variability in the seagrass, Zostera capricorni

Sulfur has been proposed as a useful element to employ in addition to carbon and nitrogen in stable isotope studies of marine food webs, but variability in δ³⁴S of primary producers may prevent food web resolution. δ³⁴S values in green leaves of the seagrass, Zostera capricorni, showed considerable variability (12.7-17.6‰) in a survey in Moreton Bay, Australia. We demonstrated that δ³⁴S values were correlated with sediment organic matter (OM) content and height of seagrass on the tidal gradient, but these relationships were opposite to those expected from work elsewhere. In our survey, δ³⁴S values were relatively depleted at sites higher on the shore and with lower OM content. We did find the expected relationship of depleted δ³⁴S values where sediment porewater sulfide concentrations were higher. Any influence of OM content on δ³⁴S values would have been confounded in the survey by the relationship between height on shore and OM content itself. We separated the effects of height and OM content by creating the following treatments at one height on the shore: (1) OM added, (2) procedural control, and (3) untouched control. δ³⁴S values of seagrass in OM added plots were significantly depleted (5.6‰) relative to procedural (10.1‰) and untouched (11.0‰) controls 8 weeks after the manipulation. This demonstrated that OM content on its own does have the expected effect on δ³⁴S values of seagrass, so in the initial survey another factor, probably related to height on shore, must have overridden the influence of OM content. Seagrass roots are able to exude excess oxygen produced during photosynthesis, reoxidising sulfides in surrounding porewater. We demonstrated that the above and below-ground biomass of seagrass was higher low on the shore, and contend that higher seagrass productivity low on the shore results in greater reoxidation of sulfides and leads to more enriched δ³⁴S values of seagrass.     

Tuning the strength of a bacterial N-end rule degradation signal

The N-end rule is a degradation pathway conserved from bacteria to mammals that links a protein's stability in vivo to the identity of its N-terminal residue. In Escherichia coli, the components of this pathway directly responsible for protein degradation are the ClpAP protease and its adaptor ClpS. We recently demonstrated that ClpAP is able to recognize N-end motifs in the absence of ClpS although with significantly reduced substrate affinity. In this study, a systematic sequence analysis reveals new features of N-end rule degradation signals. To achieve specificity, recognition of an N-end motif by the protease-adaptor complex uses both the identity of the N-terminal residue and a free alpha-amino group. Acidic residues near the first residue decrease substrate affinity, demonstrating that the identity of adjacent residues can affect recognition although significant flexibility is tolerated. However, shortening the distance between the N-end residue and the stably folded portion of a protein prevents degradation entirely, indicating that an N-end signal alone is not always sufficient for degradation. Together, these data define in vitro the sequence and structural requirements for the function of bacterial N-end signals.