Protein Array Patterning by Diffusive Gel Stamping

Proteins and small molecules are the effectors of physiological action in biological systems and comprehensive methods are needed to analyze their modifications, expression levels and interactions. Systems-scale characterization of the proteome requires thousands of components in high-complexity samples to be isolated and simultaneously probed. While protein microarrays offer a promising approach to probe systems-scale changes in a high-throughput format, they are limited by the need to individually synthesize tens of thousands of proteins. We present an alternative technique, which we call diffusive gel (DiG) stamping, for patterning a microarray using a cellular lysate enabling rapid visualization of dynamic changes in the proteome as well protein interactions. A major advantage of the method described is that it requires no specialized equipment or in-vitro protein synthesis, making it widely accessible to researchers. The method can be integrated with mass spectrometry, allowing for the discovery of novel protein interactions. Here, we describe and characterize the sensitivity and physical features of DiG-Stamping. We demonstrate the biologic utility of DiG-Stamping by (1) identifying the binding partners of a target protein within a cellular lysate and by (2) visualizing the dynamics of proteins with multiple post-translational modifications.     

Identification and organization of the components in the isolated microvillus cytoskeleton

We have examined the effects of ATP and deoxycholate (DOC) on the cytoskeletal organization of Triton-demembranated microvilli (MV) isolated from chicken intestine brush borders. Isolated MV are composed of a core of tightly bundled microfilaments from which arms project laterally to the plasma membrane with a 33-nm periodicity. These lateral arms spiral around the core microfilaments as a helix with a 25 degrees pitch. Demembranated MV consist of four polypeptides with mol wt of 110,000, 95,000, 68,000, and 42,000, present in molar ratios of 1.1:1.6:1.3:10.0. After addition of 50 microM ATP and 0.1 mM Mg++, the cytoskeletons are organized as a tight bundle of microfilaments from which lateral arms are missing. In these ATP-treated cytoskeletons, the 110-kdalton polypeptide is reduced in amount and the 95,000, 68,000, and 42,000 polypeptides are present in a 1.3:1.2:10.0 ratio. In contrast, after incubation with 0.5% DOC, the core microfilaments are no longer tightly bundled yet the lateral arms remain attached with a distinct 33-nm periodicity. These DOC-treated cytoskeletons are depleted of the 95,000 and 68,000 polypeptides and are composed of the 110,000 and 42,000 polypeptides in a 2:10 molar ratio. These results suggest that the microfilaments are associated into a core bundle by the 95- and 68-kdalton polypeptides and from this core bundle project the lateral arms composed of the 110-kdalton polypeptide.     

Scaling structure factor amplitudes in electron cryomicroscopy using X-Ray solution scattering

The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.     

Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney.

The complete cDNA sequence of human intestine-specific plastin (I-plastin) was determined from a clone derived by PCR. It consists of a 97-bp 5' untranslated region, a 1,887-bp coding region, and a 1,655-bp 3' untranslated region. The coding region predicts a 629-residue polypeptide whose sequence displays 86, 75, and 73% identities with chicken intestine fimbrin, human T-plastin, and human L-plastin, respectively. Recombinant I-plastin cross-linked actin filaments into bundles in the absence but not in the presence of calcium. The I-plastin gene was mapped by PCR to human chromosome 3; the L- and T-plastin genes were previously mapped to chromosomes 13 and X, respectively. I-plastin mRNA was detected in the small intestine, colon, and kidneys; relatively lower levels of expression were detected in the lungs and stomach. In contrast, L-plastin expression was restricted to the spleen and other lymph node-containing organs, while T-plastin was expressed in a variety of organs, including muscle, brain, uterus, and esophagus. In contrast to the situation for the intestine, high levels of L- and T-plastin mRNAs were detected in Caco-2, a human colon-derived cell line. Immunofluorescence microscopy detected I-plastin in the brush border of the small intestine and colon. These results identify I-plastin as the human homolog of chicken intestine fimbrin and as a third plastin isoform in humans.     

Thirty-cycle temperature optimization of a closed-cycle capillary PCR machine

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm i.d., oil-filled capillary using a multi-element scattered light detector and active feedback. The design permits time and number of cycles to be changed without hardware modification, unlike other flow-in-capillary PCR systems. Temperature optimization has been performed on the three PCR heat steps. The optimal denaturation temperature is 94 degrees C-96 degrees C, which is identical to commercial machines. The optimal extension temperature of 62 degrees C-66 degrees C is lower than reported for Taq DNA polymerase (70 degrees C-80 degrees C) because of the high enzyme concentration and/or the absence of detergent in the PCR mixture. The optimal annealing temperature seems to be the same as the optimal extension temperature. This is because extension occurs when the sample is inside of the annealing heat zone. Annealing takes place as the sample travels between heat zones. Device speed (23 minfor 30 cycles without time optimization) is competitive with other rapid PCR designs for efficiencies comparable to a commercial machine.     

Resistance to collagen-induced arthritis in SHPS-1 mutant mice

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Here we show that mice expressing a mutant form of SHPS-1 fail to develop type-II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans. Histological examinations of the arthritic paws from immunized wild-type mice revealed that cartilage was destroyed in association with marked mononuclear cell infiltration, while only mild cell infiltration was observed in immunized SHPS-1 mutant mice. Consistently, the serum levels of both IgG and IgG2a specific to CII and of IL-1β in immunized SHPS-1 mutant mice were markedly reduced compared with those apparent for wild-type mice. The CII-induced proliferation of, and production of cytokines by, T cells from immunized SHPS-1 mutant mice were reduced compared to wild-type cells. These results suggest that SHPS-1 is essential for development of CIA.     

Divergence and introgression in small apes,the genus Hylobates,revealed by reduced representation sequencing

Gibbons of the genus Hylobates, which inhabit Southeast Asia, show great diversity and comprise seven to nine species. Natural hybridisation has been observed in several species contact zones, but the history and extent of hybridisation and introgression in possibly historical and the current contact zones remain unclear. To uncover Hylobates species phylogeny and the extent of introgression in their evolution, genotyping by random amplicon sequencing-direct (GRAS-Di) was applied to 47 gibbons, representing seven Hylobates species/subspecies and two outgroup gibbon species. Over 200,000 autosomal single-nucleotide variant sites were identified. The autosomal phylogeny supported that divergence from the mainland species began ~3.5 million years ago, and subsequently occurred among the Sundaic island species. Significant introgression signals were detected between H. lar and H. pileatus, H. lar and H. agilis and H. albibarbis and H. muelleri, which all are parapatric and form ongoing hybrid zones. Furthermore, the introgression signals were detected in every analysed individual of these species, indicating a relatively long history of hybridisation, which might have affected the entire gene pool. By contrast, signals of introgression were either not detected or doubtful in other species pairs living on different islands, indicating the rarity of hybridisation and introgression, even though the Sundaic islands were connected during the Pliocene and Pleistocene glacial events.Subject terms: Phylogenetics, Population genetics     

Potential Risk Factors of Persistent Low Back Pain Developing from Mild Low Back Pain in Urban Japanese Workers

Study Design

Two-year, prospective cohort data from the Japan epidemiological research of occupation-related back pain study in urban settings were used for this analysis.

Objective

To examine the association between aggravated low back pain and psychosocial factors among Japanese workers with mild low back pain.

Summary of Background Data

Although psychosocial factors are strongly indicated as yellow flags of low back pain (LBP) leading to disability, the association between aggravated LBP and psychosocial factors has not been well assessed in Japanese workers.

Methods

At baseline, 5,310 participants responded to a self-administered questionnaire including questions about individual characteristics, ergonomic work demands, and work-related psychosocial factors (response rate: 86.5%), with 3,811 respondents completing the 1-year follow-up questionnaire. The target outcome was aggravation of mild LBP into persistent LBP during the follow-up period. Incidence was calculated for the participants with mild LBP during the past year at baseline. Logistic regression was used to explore risk factors associated with persistent LBP.

Results

Of 1,675 participants who had mild LBP during the preceding year, 43 (2.6%) developed persistent LBP during the follow-up year. Multivariate analyses adjusted for individual factors and an ergonomic factor found statistically significant or almost significant associations of the following psychosocial factors with persistent LBP: interpersonal stress at work [adjusted odds ratio (OR): 1.96 and 95% confidence interval (95%CI): 1.00–3.82], job satisfaction (OR: 2.34, 95%CI: 1.21–4.54), depression (OR: 1.92, 95%CI: 1.00–3.69), somatic symptoms (OR: 2.78, 95%CI: 1.44–5.40), support from supervisors (OR: 2.01, 95%CI: 1.05–3.85), previous sick-leave due to LBP (OR: 1.94, 95%CI: 0.98–3.86) and family history of LBP with disability (OR: 1.98, 95%CI: 1.04–3.78).

Conclusions

Psychosocial factors are important risk factors for persistent LBP in urban Japanese workers. It may be necessary to take psychosocial factors into account, along with physical work demands, to reduce LBP related disability.     

Computational model for cell migration in three-dimensional matrices

Although computational models for cell migration on two-dimensional (2D) substrata have described how various molecular and cellular properties and physiochemical processes are integrated to accomplish cell locomotion, the same issues, along with certain new ones, might contribute differently to a model for migration within three-dimensional (3D) matrices. To address this more complicated situation, we have developed a computational model for cell migration in 3D matrices using a force-based dynamics approach. This model determines an overall locomotion velocity vector, comprising speed and direction, for individual cells based on internally generated forces transmitted into external traction forces and considering a timescale during which multiple attachment and detachment events are integrated. Key parameters characterize cell and matrix properties, including cell/matrix adhesion and mechanical and steric properties of the matrix; critical underlying molecular properties are incorporated explicitly or implicitly. Model predictions agree well with experimental results for the limiting case of migration on 2D substrata as well as with recent experiments in 3D natural tissues and synthetic gels. Certain predicted features such as biphasic behavior of speed with density of matrix ligands for 3D migration are qualitatively similar to their 2D counterparts, but new effects generally absent in 2D systems, such as effects due to matrix sterics and mechanics, are now predicted to arise in many 3D situations. As one particular sample manifestation of these effects, the optimal levels of cell receptor expression and matrix ligand density yielding maximal migration are dependent on matrix mechanical compliance.     

Individual Variation in Behavioural Reactions to Unfamiliar Conspecific Vocalisation and Hormonal Underpinnings in Male Chimpanzees

It has been established that various species exhibit personality, defined as intra‐individual consistency and inter‐individual variation in behavioural phenotypes. For example, certain individuals may demonstrate consistently greater behavioural reactions and elevated stress responses. We conducted playback experiments and hormonal analyses on male chimpanzees (Pan troglodytes) in captivity to investigate the patterns and proximate mediators of individual variations in behavioural reactions. We found intra‐individual consistency and inter‐individual variation in behavioural reactions (intensive vigilance towards the direction of speakers) to vocalisations by unfamiliar chimpanzees. This behavioural reaction was positively correlated with changes in salivary cortisol concentration, suggesting that stress is a proximate factor mediating the variation in behavioural reactions. The males who were highly responsive to the conspecific vocalisation also exhibited high behavioural reactions towards the neutral broadcast stimulus (the jungle crow’s Corvus macrorhynchos ‘ka’ vocalisation). This observation is consistent with the notion that male chimpanzees vary in intrinsic behavioural tendency to different stimuli.