Multimodal-3D imaging based on µMRI and µCT techniques bridges the gap with histology in visualization of the bone regeneration process.

Bone repair/regeneration is usually investigated through X-ray computed microtomography (µCT) supported by histology of extracted samples, to analyse biomaterial structure and new bone formation processes. Magnetic resonance imaging (µMRI) shows a richer tissue contrast than µCT, despite at lower resolution, and could be combined with µCT in the perspective of conducting non-destructive 3D investigations of bone. A pipeline designed to combine µMRI and µCT images of bone samples is here described and applied on samples of extracted human jawbone core following bone graft. We optimized the coregistration procedure between µCT and µMRI images to avoid bias due to the different resolutions and contrasts. Furthermore, we used an Adaptive Multivariate Clustering, grouping homologous voxels in the coregistered images, to visualize different tissue types within a fused 3D metastructure. The tissue grouping matched the 2D histology applied only on 1 slice, thus extending the histology labelling in 3D. Specifically, in all samples, we could separate and map 2 types of regenerated bone, calcified tissue, soft tissues, and/or fat and marrow space. Remarkably, µMRI and µCT alone were not able to separate the 2 types of regenerated bone. Finally, we computed volumes of each tissue in the 3D metastructures, which might be exploited by quantitative simulation. The 3D metastructure obtained through our pipeline represents a first step to bridge the gap between the quality of information obtained from 2D optical microscopy and the 3D mapping of the bone tissue heterogeneity and could allow researchers and clinicians to non-destructively characterize and follow-up bone regeneration.

The role of water coordination in binary mixtures. A study of two model amphiphilic molecules in aqueous solutions by molecular dynamics and NMR.

Two binary aqueous mixtures which contain the small amphiphilic molecules TMAO (trimethylamine-N-oxide) and TBA (tert-butyl alcohol) have been investigated by molecular dynamics simulations and NMR chemical shift and self-diffusion measurements. TMAO is an osmolyte, while TBA is a monohydrate alcohol. Both possess bulky hydrophobic groups and polar heads, namely, NO in TMAO and OH in TBA. The hydrophilic/hydrophobic content of these isosteric molecules strongly modulates the structure and dynamics of the hydration shell, which is thought to be responsible for the effects observed on proteins and phospholipids. Simulation results, especially on hydrogen-bond networking, spatial correlations, and self-diffusivity, are consistent with NMR data and agree well with previous numerical studies on similar solutions. The methods employed allow the elucidation of the microscopic features of the solutions. For TBA solutions, the hydration shell is found to have a low density and a large spatial spread, and thus, above the molar fraction of 0.03, reduction of hydrophobic hydration drives self-aggregation of the solute. This effect does not take place in TMAO solutions, where the hydration shell is more compact and stable, maintaining its structure over a wider range of solute concentrations.

Characterization of two maize HSP90 heat shock protein genes: expression during heat shock, embryogenesis, and pollen development.

We have isolated two genes from Zea mays encoding proteins of 82 and 81 kD that are highly homologous to the Drosophila 83-kD heat shock protein gene and have analyzed the structure and pattern of expression of these two genes during heat shock and development. Southern blot analysis and hybrid select translations indicate that the highly homologous hsp82 and hsp81 genes are members of a small multigene family composed of at least two and perhaps three or more gene family members. The deduced amino acid sequence of these proteins based on the nucleotide sequence of the coding regions shows 64-88% amino acid homology to other hsp90 family genes from human, yeast, Drosophila, and Arabidopsis. The promoter regions of both the hsp82 and hsp81 genes contain several heat shock elements (HSEs), which are putative binding sites for heat shock transcription factor (HSF) commonly found in the promoters of other heat shock genes. Gene-specific oligonucleotide probes were synthesized and used to examine the mRNA expression patterns of the hsp81 and hsp82 genes during heat shock, embryogenesis, and pollen development. The hsp81 gene is only mildly heat inducible in leaf tissue, but is strongly expressed in the absence of heat shock during the pre-meiotic and meiotic prophase stages of pollen development and in embryos, as well as in heat-shocked embryos and tassels. The hsp82 gene shows strong heat inducibility at heat-shock temperatures (37-42 degrees C) and in heat shocked embryos and tassels but is only weakly expressed in the absence of heat shock. Promoter-GUS reporter gene fusions made and analyzed by transient expression assays in Black Mexican Sweet (BMS) Maize protoplasts also indicate that the hsp82 and hsp81 are regulated differentially. The hsp82 promoter confers strong heat-inducible expression of the GUS reporter gene in heat-treated cells (60- to 80-fold over control levels), whereas the hsp81 promoter is only weakly heat inducible (5- to 10-fold over control levels).

Isolation and characterization of a small heat shock protein gene from maize.

A maize (Zea mays L.) genomic clone (Zmempr 9') was isolated on the basis of its homology to a meiotically expressed Lilium sequence. Radiolabeled probe made from the maize genomic clone detected complementary RNA at high fidelity. Furthermore, it hybridized to RNA isolated from staged (an interval that is coincident with meiotic prophase) maize tassel spikelets. Complimentary RNA was strongly (at least 50-fold) induced during heat shock of maize somatic tissue and appeared as a single size class in Northern blot hybridizations. Sequencing of the complete coding region of Zmempr 9' confirmed the homology of the inferred amino acid sequence to other small heat shock proteins. Consensus sequences found in the flanking regions corresponded to the usual signals for initiation of RNA transcription, polyadenylate addition, and the induction of heat shock genes. The latter sequences conferred heat shock-specific transient expression in electroporated protoplasts when cloned into promoterless reporter gene plasmid constructs. Hybrid-selected translations revealed specific translation products ranging from 15 to 18 kilodaltons, providing evidence that this gene is a member of a related multigene family. We therefore conclude that this maize genomic DNA clone, recovered through its homology to clones for meiotic transcripts in lily, represents a genuine maize small heat shock protein gene.

A heat shock protein is encoded within mitochondria of higher plants.

A temperature shift from 25 to 41 degrees C initiates the synthesis of a specific set of proteins in maize, including a peptide of 60 kilodaltons. Using an in vitro mitochondrial protein synthesizing system, we provide evidence that this 60-kDa heat shock protein is encoded within the organelle. Further support for this heat-induced protein being encoded within mitochondria is that its synthesis is inhibited in whole seedlings by chloramphenicol. This 60-kDa heat shock protein is induced in all lines of maize we examined. Additionally, a heat-induced peptide of similar size (62 kDa) can be detected in isolated mitochondria of a dicot plant, Brassica campestris. The function of the heat shock protein encoded within the mitochondria remains unknown.

One- and two-dimensional polyacrylamide gel analysis of the heat shock proteins of the virilis group of Drosophila.

The heat shock proteins of the virilis group of Drosophila are analyzed by one- and two-dimensional polyacrylamide gel analysis. This group consists of the two closely related but distinct virilis and montana phylads. The analysis reveals that some of the heat shock proteins are highly conserved among the two phylads while others are not. The 83-, 72-, and 69-kdalton proteins comigrate in all species examined. There is, however, a noticeable trend toward greater molecular weight variability in the smaller heat shock proteins. In general, the heat shock protein patterns within each phylad follow the proposed phylogenetic relationships with some exceptions. D. ezoana and D. littoralis, both members of the montana phylad, exhibit heat shock protein patterns more similar to those of the virilis phylad. The data also demonstrate that the montana phylad has almost two times the heat shock allele members that the virilis phylad has. It is also shown that F1 and F2 hybrid flies of crosses between Drosophila species having different patterns of heat shock proteins show Mendelian segregation of alleles. After several generations of inbred growth, however, the pattern of heat shock protein synthesis in reciprocal hybrids each resembles that of the paternal parent. The implications of these findings are discussed.

Putative function of Drosophila melanogaster heat shock proteins in the nucleoskeleton.

The cellular distribution in Drosophila Kc cells of [35S]methionine-labeled heat shock proteins has been examined by 0.2% Nonidet P-40-mediated cell lysis and Na-deoxycholate-Tween 40 extraction of the nuclei. The 83,000-dalton heat shock protein was limited to the detergent extracts while the remaining heat shock proteins were found both in a soluble pool in the detergent extracts and in a bound pool in the nuclei. The bound pool included the 70,000-68,000-, 27,000-, 26,000-, 23,000-, and 21,000-dalton heat shock proteins; these proteins accumulated in the nuclei during the time course of heat shock as assayed by [35S]methionine labeling and dye binding on gel electropherograms. DNA and histone-depleted nuclei were prepared by extensive nuclease digestion, 2.0 M NaCl extraction, and sedimentation of the original detergent-washed nuclei. Of the 35S-labeled bound pool, 69% remained associated in a rapidly sedimenting complex that retained only approximately 5% of the DNA. Thus, the binding of the 70,000-68,000-, 27,000-, 26,000-, 23,000-, and 21,000-dalton heat shock proteins appeared primarily to be with the nuclear scaffold rather than the chromatin. We conclude from the mass of heat shock protein synthesis that these proteins probably are structural elements of the nucleus.

Localization and characterization of rDNA in Drosophila tumiditarsus.

Using in situ nucleic acid hybridizations, the genes that code for 28, 18 and 5S rRNA have been localized in the polytene chromosomes of Drosophila tumiditarsus. The 5S genes are found at a single site near the centromere of the second chromosome, whereas the 28 and 18S genes are found at the nucleolar organizer region of the dot chromosome. The dot chromosome has been previously described as alpha-heterochromatic. However, our cytochemical and autoradiographic results do not support such a conclusion. The autoradiographic results reveal that the dot chromosome is transcriptionally active and is not late-replicating, as is expected of alpha-heterochromatin. Further, the dot chromosomes possess none of the usual staining characteristics of heterochromatin except for its lack of polytene bands. Using rRNA-DNA filter hybridizations, we find that the rDNA of D. tumiditarsus salivary glands is under-replicated. This is the first species of Drosophila where the rDNA in not found on the sex chromosomes, and is the first report of an under-replicated autosomal locus which is not located in heterochromatic blocks.

Characterization of the toromeric structure in Drosophila lummei.

The toromere, a structure previously reported only in several strains of Drosophila melanogaster, is found in salivary gland nuclei of three populations of Drosophila lummei, a member of the virilis group. The toromere is characterized by being quinacrine-bright and Feulgen-positive. Further staining and enzyme digestion procedures reveal that the toromere is composed of double-stranded DNA with little or no protein complexed with it. The toromere appears as a small quinacrine-bright dot in diploid cells and apparently undergoes polytenization, as it is observed as a large quinacrine-bright doughnut-shaped structure in salivary gland preparations. We describe the effect of larval culture temperature on the morphology and number of toromeres per cell, and lend support to the proposal that the toromere could be a highly replicated episome.