Precocious sexual signalling and mating in Anastrepha fraterculus (Diptera: Tephritidae) sterile males achieved through juvenile hormone treatment and protein supplements.

Sexual maturation of Anastrepha fraterculus is a long process. Methoprene (a mimic of juvenile hormone) considerably reduces the time for sexual maturation in males. However, in other Anastrepha species, this effect depends on protein intake at the adult stage. Here, we evaluated the mating competitiveness of sterile laboratory males and females that were treated with methoprene (either the pupal or adult stage) and were kept under different regimes of adult food, which varied in the protein source and the sugar:protein ratio. Experiments were carried out under semi-natural conditions, where laboratory flies competed over copulations with sexually mature wild flies. Sterile, methoprene-treated males that reached sexual maturity earlier (six days old), displayed the same lekking behaviour, attractiveness to females and mating competitiveness as mature wild males. This effect depended on protein intake. Diets containing sugar and hydrolyzed yeast allowed sterile males to compete with wild males (even at a low concentration of protein), while brewer´s yeast failed to do so even at a higher concentration. Sugar only fed males were unable to achieve significant numbers of copulations. Methoprene did not increase the readiness to mate of six-day-old sterile females. Long pre-copulatory periods create an additional cost to the management of fruit fly pests through the sterile insect technique (SIT). Our findings suggest that methoprene treatment will increase SIT effectiveness against A. fraterculus when coupled with a diet fortified with protein. Additionally, methoprene acts as a physiological sexing method, allowing the release of mature males and immature females and hence increasing SIT efficiency.

The structure of the cytoplasm of lens fibers as determined by conical tomography.

Studies using conventional electron microscopy describe the cytoplasm of lens fiber cells as having essentially an amorphous structure. We hypothesized that significant structural detail might have been lost as a result of projecting the entire thickness of the section (50-100 nm) onto a single plane (the "projection artifact"). To test this hypothesis, we studied the 3D-structure of rat lens cortical fibers before and after extracting the "soluble" crystallins with low ionic strength buffers to make "ghosts." Tomographic series in conical geometry were collected at 55 degrees tilts and by 5 degrees rotations until completing a 360 degrees turn by low dose methods. They were aligned using fiduciary points, reconstructed with the weighted back projection algorithm and refined by projection matching. Analysis of the 3D-maps included semiautomatic density segmentation using a computer program based on the watershed algorithm. We found that the cytoplasm of cortical fibers, though appearing amorphous in regions of the highest density, was in fact comprised of an ordered structure resembling a "clustered matrix." The matrix was comprised of thin ( approximately 6 nm diameter) filaments bent sharply at 110-120 degrees angles and studded with cube-shaped particles (the "beaded" filaments). In cortical fibers, the particles measured a=14+/-2, b=13+/-2 and c=10+/-2.4 nm (n=30, mean+/-SD) and were spaced at distances measuring 27.5+/-2.4 nm apart (n=8, mean+/-SD), center-to-center. The matrix was formed as "beaded" filaments, bound to clusters of "soluble" proteins, crossed each other at nearly perpendicular angles. The matrix also made contact with the plasma membrane at a large number of distinct regions. We thus concluded that the cytoplasm of cortical lens fibers is comprised of a cytoskeletal matrix of "beaded" filaments that organize the "soluble" crystallins in separate regions. The association of this matrix with the plasma membrane allows the lens to maintain its structural integrity, while its association with crystallins yields its long-term transparency. Loss of either function likely would play a significant role in cataract formation.

Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused.

We have used thin sectioning and conical electron tomography to determine the three-dimensional structure of synaptic vesicles that were associated (docked) at release sites of the presynaptic membrane, called active-zones. Vesicles docked at the active zone occupied a strategic location: they formed regions of contact with the plasma membrane on one side and with that of one or more vesicles located deeper within the presynaptic terminal on the other side. The region of contact with the active zone measured approximately 15 nm in diameter ( approximately 2% of the vesicle's surface) and contained a smaller approximately 6 nm region where the proximal leaflets merged (hemi-fused). Hemi-fusion was only observed on the side of vesicles in contact with the active zone; at the side of contact between neighboring vesicles, the membranes were not hemi-fused. Approximately three-fourths of the docked vesicles contained hemi-fused regions. Vesicles fully fused to the active zone (exhibiting pores that appeared as interruptions of a single membrane) were less frequently observed ( approximately 1 of 10 hemi-fused vesicles). In conclusion, our observations in cortical synapses strengthen the hypothesis that hemi-fusion is a stable intermediary that precedes full fusion and release.

Insights into structural proteins of 936-type virulent lactococcal bacteriophages.

bIL41 and bIL170, virulent phages of Lactococcus lactis belonging to the 936 group, possess a late gene named l12, coding a putative fiber sharing partial similarity to diverse gene products of dairy phages, including host-range determinants, but whose function is unknown in this group. We observed that the full-size gpl12 gene product is a minor protein constitutive of both phage particles. A derivative of bIL41 deleted for part of this gene was constructed by homologous recombination. The recombinant bIL41DeltaL12 showed normal propagation on strain IL1403 and no altered head and tail structures, demonstrating its non-essential role under our laboratory conditions. bIL170 was investigated for major structural components. Tails were characterized by electron microscopy and image analysis, which indicated that the major repeat unit of the tail occupied a maximum volume of 18.5 nm3, corresponding to a size of 20 kDa for a globular protein. Total protein profiles and head-enriched fractions of bIL170 exhibited a major 38 kDa protein, identified by N-terminal sequence as the product of l13. This result questions some of the functional predictions deduced from synteny relationships assumed for the lambda-supergroup of the family Siphoviridae to which the 936-type phages were proposed to belong.

Conical tomography II: A method for the study of cellular organelles in thin sections.

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.

Conical tomography of freeze-fracture replicas: a method for the study of integral membrane proteins inserted in phospholipid bilayers.

We have used conical tomography to study the structure of integral proteins in their phospholipid bilayer environments. Complete conical series were collected from replicas of the water channel aquaporin-0 (AQP0), a 6.6 nm side tetramer with a molecular weight of approximately 120 kDa that was purified and reconstituted in liposomes. The replicas were tilted at 38 degrees , 50 degrees or 55 degrees and rotated by 2.5 degrees , 4 degrees , or 5 degrees increments until completing 360 degrees turns. The elliptical paths of between 6 and 12 freeze-fracture particles aligned the images to a common coordinate system. Using the weighted back projection algorithm, small volumes of the replicas were independently reconstructed to reconstitute the field. Using the Fourier Shell Correlation computed from reconstructions of even and odd projections of the series, we estimated a resolution of 2-3 nm, a value that was close to the thickness of the replica (approximately 1.5 nm). The 3D reconstructions exhibited isotropic resolution along the x-y plane, which simplified the analysis of particles oriented randomly in the membrane plane. In contrast to reconstructions from single particles imaged using random conical tilt [J. Mol. Biol. 325 (2003) 210], the reconstructions using conical tomography allowed the size and shape of individual particles representing the AQP0 channel to be identified without averaging or imposing symmetry. In conclusion, the reconstruction of freeze-fracture replicas with electron tomography has provided a novel experimental approach for the study of integral proteins inserted in phospholipid bilayers.

Correspondence analysis of sinogram lines. Sinogram trajectories in factor space replace raw images in the orientation of projections of macromolecular assemblies.

The lines of a large group of sinograms of projections with random orientation can be submitted to correspondence analysis. Since the number of samples per line is small, a small matrix is accumulated which is quick to orthogonalise. In the eigenvector space, the lines of a sinogram are represented by points describing a closed trajectory. Two trajectories of different sinograms intersect in the position of their common line. Determining where two trajectories cross each other is a problem of minimum chi2 distance in the space of 5-7 eigenvectors. An algorithm to determine common lines has been implemented and tested with phantom projections oriented at random, and corrupted with noise. The images were simulating a set collected with the two exposures technique, already proposed by the authors for three dimensional reconstruction from random projections. The preliminary models obtained with the new algorithm have been refined by a projection matching based on trajectories. This step requires determining which trajectory, in a set representing computed projections, matches at best with that of an experimental projection. This is a problem of minimum distance in a space with low dimensionality. The present algorithms, based on chi2 distances, run much faster than those based on correlation analysis and the quality of the reconstructed phantoms looks satisfactory.

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

Alignment of 3D structures of macromolecular assemblies.

MOTIVATION: A number of macromolecular assemblies are being reconstructed in 3D from electron micrographs. The analysis yields a 3D matrix representing the protein density map. In reconstruction processes and in comparing the results of different experiments, it is often necessary to obtain all models oriented the same way in three dimensions. The problem is not trivial since there exist no 3D counterpart of correlation analysis used for 2D images. It is usually solved by time consuming trial and error algorithms. RESULTS: 3D density distributions can be brought to a 'canonical' orientation. The tensor of inertia of the distribution is determined and its eigenvectors are oriented along the coordinate axes. The method is fast and essentially free of reference. It is suitable for structures whose inertial axes do not completely degenerate as they do in icosahedral viruses or if symmetry is cubic. Applications are presented for asymmetric objects and for molecules possessing symmetry axes higher than twofold. IMPLEMENTATION: The implementation simply requires the accumulation of the inertial tensor and its diagonalisation. Volume data rotation has been already illustrated in this journal by the authors.

Fast and accurate three-dimensional reconstruction from projections with random orientations via radon transforms

A new algorithm for three-dimensional reconstruction from randomly oriented projections has been developed. The algorithm recovers the 3D Radon transform from the 2D Radon transforms (sinograms) of the projections. The structure in direct space is obtained by an inversion of the 3D Radon transform. The mathematical properties of the Radon transform are exploited to design a special filter that can be used to correct inconsistencies in a data set and to fill the gaps in the Radon transform that originate from missing projections. Several versions of the algorithm have been implemented, with and without a filter and with different interpolation methods for merging the sinograms into the 3D Radon transform. The algorithms have been tested on analytical phantoms and experimental data and have been compared with a weighted back projection algorithm (WBP). A quantitative analysis of phantoms reconstructed from noise-free and noise-corrupted projections shows that the new algorithms are more accurate than WBP when the number of projections is small. Experimental structures obtained by the new methods are strictly comparable to those obtained by WBP. Moreover, the algorithm is more than 10 times faster than WPB when applied to a data set of 1000-5000 projections. Copyright 1999 Academic Press.

A two-exposure technique for ice-embedded samples successfully reconstructs the chlorocruorin pigment of Sabella spallanzanii at 2. 1 Nm resolution.

A technique for reconstructing ice-embedded macromolecules from electron micrographs taken at two specimen tilts (+/-23 degrees ) has been used to determine the structure of chlorocruorin isolated from the Polychaete annelid Sabella spallanzanii. Images of individual molecules were extracted in couples from two micrographs of the same field of view so each couple consists of two projections of the same molecule. One couple was used as a fixed reference for alignment. Different references yielded reconstructions with different orientations. These were merged to give a model against which the orientation of 1624 first-exposure images was refined to give a final reconstruction at 2.1 nm resolution. The structure of this hematic pigment, essentially the same as that for Lumbricus terrestris, is a bilayer structure with overall symmetry D6, containing six hollow groups per layer. A hollow group is formed by six globular masses and has approximate threefold symmetry. Other structural elements connect the two layers and the hollow groups in a layer. This non-globin material occupies about 15% of the total molecular volume. The results show that the double-exposure strategy, previously described by some of the authors and tested in computer simulations, performs well in real experiments and could be used to obtain preliminary reconstructions in a semiautomatic way.

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.

Fast computation of 3D radon transform via a direct Fourier method.

MOTIVATION: Arrays of three-dimensional (3D) data are ubiquitous in structural biology, biomedicine and clinical imaging. The Radon transform can be implied in their manipulation mainly for the solution of the inverse tomographic problem, since experimental data are often collected as projections or as samples of the Radon space. In electron tomography, new applications of the transform may become convenient if a fast and accurate transformation algorithm is adopted. RESULTS: A direct Fourier method (DFM) is proposed to compute the 3D Radon transform from a sampled function with compact support. This paper describes an already known two-step algorithm and illustrates its DFM implementation by coordinate transformations in 2D Fourier space. The algorithm is easily inverted to obtain a density distribution from the Radon transform. The main applications are in the field of electron tomography, especially in processes of angular refinement, since whatever projection of a structure can be retrieved from its Radon transform in a fast and accurate way. The times required to compute a number of projections with use of the Radon transform are compared with those required by other algorithms. Further uses of the Radon transform can be foreseen in applications based on 'projection onto convex sets' (POCS). AVAILABILITY: Software is available free of charge upon request to the authors. CONTACT: salvator@csmtbo.mi.cnr.it

Three-dimensional reconstruction of metal replicas of the Helicobacter pylori vacuolating cytotoxin.

The Helicobacter pylori vacuolating cytotoxin (VacA) forms high molecular weight homooligomers which contain either six or seven copies of a 95-kDa polypeptide. Electron microscope visualization of carbon platinum replicas of quick-freeze, deepetched, preparations of VacA has revealed that the oligomers are arranged in flower-like structures with six- or sevenfold radial symmetry, depending on the number of 95-kDa oligomers that they contain. Each monomer is structured in two subunits of 37 and 58 kDa connected by an exposed loop which is a site for proteolytic cleavage. In preparations of VacA which had undergone extensive cleavage at the exposed loop, oligomers of both six- and seven-fold symmetry which appeared flatter were observed; these latter were interpreted as molecules which had lost a complete set of one of the subunits. We exploited a 3D reconstruction of metal replicas of quick-freeze, deep-etched, oligomers, representing the four types of molecules described. All the molecules appear to adhere with the same face toward the mica. Images of rotary shadowed oligomers were processed by multivariate statistical analysis to evidence clusters of equivalent and homogeneous oligomers. 3D reconstructions of the replicas so classified were performed by random conical tilt tomography. In the case of intact molecules (not cleaved) the reconstructions represent both the outer and the inner surfaces of the mold; the latter gives a reasonably accurate sense of the upper surface of the VacA oligomers. These data support the hypothesis that VacA is an AB type toxin and suggest a model in which the smaller of the two subunits is arranged in a uniform ring on the surface of the molecule in such a way as to contribute to the overall stability of the molecule.

Fast direct Fourier methods, based on one- and two-pass coordinate transformations, yield accurate reconstructions of x-ray CT clinical images.

The conversion from polar to Cartesian coordinates can be carried out with two-pass algorithms. The paper describes two different methods based on concentric square frames and octagonal frames and their results, obtained with accurate interpolations based on the "moving window Shannon reconstruction' (MWSR). The embedding of these algorithms in direct Fourier methods (DFMs) of tomographic reconstruction is discussed. With respect to one-pass methods and to the use of octagonal frames, the square frame method makes it possible to carry out the first pass, a radial resampling, in the direct space, before computing 1D Fourier transforms (FTs) of projections. Reconstructions of clinical images from the raw data of a third-generation x-ray tomograph are presented and compared with those obtained with one-pass DFMs and with the convolution back-projection method (CBPM) performed by the instrument. The simple algorithm using square frames yields results in complete agreement with other DFM protocols and the CBPM. On a general-purpose computer, the execution of DFM protocols based on one-pass and two-pass coordinate transformations is 35 to 55 times faster than the CBPM and make the algorithms attractive for modern instrumentation.

Fast sinogram computation and the sinogram-based alignment of images.

A direct Fourier method (DFM) to compute sinograms is described. Since the DFM is much faster than the customary rotation and projection process, novel uses of sinograms can be devised. As an example and an exercise on the properties of sinograms, a fast sinogram-based method to align large sets of images is described. The method is based on shift-invariant functions to detect rotations, and on tomographic reconstructions of cross-correlation functions to detect relative shifts. It has been implemented in a novel library, SIGNAL, used to align images of macromolecular assemblies observed in the electron microscope. A comparative analysis of the complexity of sinogram- and imagebased methods, as well as quite a few tests with EM images, show that SIGNAL runs faster, the accuracy being the same.

Image and volume data rotation with 1- and 3-pass algorithms.

Three different implementations of the 3-pass algorithm of image and volume data rotation are illustrated and discussed. The three protocols use interpolation in real domain, with a peculiar implementation of the Shannon reconstruction, or phase shifts in Fourier domain. Accuracy and speed of the three methods are compared with corresponding values obtained with a 1-pass method. The results indicate that for low or moderate accuracy, 1-pass is more convenient than 3-pass rotation for both accuracy and speed. Very accurate rotations can be obtained in reasonable time if all steps of 3-pass rotation are performed in the Fourier domain.

Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin.

Disease-associated strains of Helicobacter pylori produce a potent toxin that is believed to play a key role in peptic ulcer disease in man. In vitro the toxin causes severe vacuolar degeneration in target cells and has thus been termed VacA (for vacuolating cytotoxin A). Cytotoxic activity is associated with a > 600-kD protein consisting of several copies of a 95-kD polypeptide that undergoes specific proteolytic cleavage after release from the bacteria to produce 37- and 58-kD fragments. Quick freeze, deep etch electron microscopy has revealed that the native cytotoxin is formed as regular oligomers with either six- or seven-fold radial symmetry. Within each monomer, two domains can clearly be distinguished, suggesting that the 37- and 58-kD fragments derive from proteolytic cleavage between discrete subunits of the monomer. Analysis of preparations of the toxin that had undergone extensive cleavage into the 37- and 58-kD subunits supports this interpretation and reveals that after cleavage the subunits remain associated in the oligomeric structure. The data suggest a structural similarity with AB-type toxins.

Three-dimensional reconstruction of helical structures with fast inversion of very large Fourier transforms.

A single projection of a helical distribution of matter allows one to obtain the complete three-dimensional reconstruction of the structure. This task is usually performed by a Fourier-Bessel algorithm, which is more efficient than a customary fast Fourier transform inversion. This article describes how to achieve such a result by a direct Fourier method in a reasonable time. Once the two-dimensional transform of the projection is obtained from the source image, it is possible to build up the three-dimensional transform array, in Cartesian coordinates, that yields the reconstruction by a straightforward Fourier inversion. Images of projected helices should be studied with high sampling rates to enhance the resolution, and the segments of helix should be long enough to give a satisfactory signal-to-noise ratio. These conditions result in three-dimensional transform arrays that would require one or more gigabytes of storage. The strategy proposed here requires much less storage and is fast enough to allow the reconstruction to be performed with different parameters and filters in a very short time without any sacrifice in resolution.

Flagellar structure in normal human spermatozoa and in spermatozoa that lack dynein arms.

Human sperm flagella were analyzed by electron microscopy and computer averaging in order to characterize normal flagella and to detect differences between normal and mutated spermatozoa. The A-tubules of normal spermatozoa were seen to have 13 protofilaments and a lumen containing a 'pentagon' and a 'sickle'. The incomplete B-tubule was seen to have 10 protofilaments with an angular separation such that a complete circle would have 16 protofilaments. A thin '11th filament' is located at the inner border between A- and B-tubules and, in the centriole, also between B- and C-tubules. The tail end piece has 18 microtubules of a conventional appearance. We conclude that the 9 axonemal doublets split distally into 2 microtubules and that normal microtubules with 13 protofilaments can grow from the incomplete B-tubules. The cell membrane in the end piece has a glycocalyx with regular periodicity. Spermatozoa from a man suffering from the immotile-cilia syndrome was also analyzed. His sperm flagella were seen to be abnormal in that the dynein arms are lacking, and, that the sickle is incomplete. In other respects his immotile spermatozoa were normal; spokes and central projections have the same appearance as in normal spermatozoa.