Electron microscopy and electron microscopical measurements of collagen mineralization in hard tissues

Summary The best focussed members of a through focus series of electron micrographs of ultra-thin sections of freeze-dried forming rat incisor dentine and glutaraldehyde-fixed 10 months old human femoral cortex were selected for measurements. The original micrographs were prepared at magnifications of x20000 or x40000 and were photographically enlarged to a magnification from x200000 to x600000. The number of dot-like calcium phosphate nuclei per hole zone and per overlapping zone of the 64 nm collagen macro-period was determined; we found a range of 6–8 nuclei per hole zone and 3–5 nuclei per overlapping zone. Thus a total of 9–13 nuclei per 64 nm macro-period was found; this is in agreement with the total number of cross bandings per macro-period reported by previous authors. The distances between neighbouring dot-like calcium phosphate nuclei were measured and found to lie in the range of 4.0 to 8.5 nm, which is the range in which most of the distances between the collagen cross-bandings appear. The distances between close, parallel calcium phosphate rows within the collagen fibres were found to lie in the range 4.6–8.0 nm which seems to be a good deal shorter than the distances between the typical microholes of the Hodge-Petruska collagen model. Our results and ideas concerning the active sites of nucleation also are in agreement with previous amino acid sequence analyses in respect of the occurrence of neighbouring polar amino acids which would be free to bind ions.We thank Professor S. Heuck for valuable bone specimens. We thank Frau G. Neinhardt, Frau R. Höhling and Mr. P. S. Reynolds for their valuable technical assistance and Mr. H. Pittorf for building the metal model of Fig. 10. We thank the Deutsche Forschungsgemeinschaft and the Medical Research Council for their financial support.     

Quantitative electron microscopic investigations of mineral nucleation in collagen

Summary It has been previously shown that the distances between the nuclei within the collagen bundles of mineralizing tissues were in good agreement with the repeat distances of the cross-banding pattern of collagen, which supports the assumption that the distances between the mineral deposits reflect to a good approximation the distances between nucleation centres on the collagen macromolecule. However, the lateral separation of the nuclei were significantly higher than the distances between close-packed triple helices.Recently a new model of collagen aggregation has been proposed in which the smallest morphological units are subfibrils (Ø approx. 39 Å) packed in tetragonal array. This led us to measure once again the lateral separation between a) close-packed calcium phosphate needles lying in bundles at (1) the mineralizing front of mantle dentine and (2) at the mineralizing front of rat tail bone, and b) between the uranyl-lead nuclei produced in the staining of rat tail tendon.The mean lateral distances separating these nuclei fell within the range of 39–47 Å, which is a little higher than the distances of 39 Å which separate the microholes between the subfibrils in the tetragonal packing model, which are regarded as the likely sites of nucleation. If, however, it is assumed that the forces generated during mineralization can cause the collagen fibres to swell, then the lateral separation of the nuclei and the distances between the microholes would correspond very closely.We thank the Deutsche Forschungsgemeinschaft for financial support. We thank Prof. Dr. K. Heckmann and Dr. U. Mays, Dept. of Zoology, Münster, for allowing us to use their Siemens-Elmiskop 101 sponsored by Stiftung Volkswagenwerk, and Frau Dr. Weichan, Applikationslabor Siemens, Berlin, for performing the tilting experiments at their Siemens-Elmiskop 102. We thank Fräulein Ute Sporman for valuable technical help.     

Analysis of the crystal arrangement in collagen fibrils of mineralizing turkey tibia tendon

Summary Mineralized pieces of tendons from the tibio-tarsus of turkeys were (i) shock-frozen, freeze-dried, embedded and cut without staining, or (ii) fixed, embedded and stained after sectioning. Micrographs were taken with an electron microscope on longitudinally cut sections. The center-to-center distances of neighboring apatitic needles within collagen fibrils were measured. For shock-frozen and freeze-dried specimens, the average of these distances is 4.7 nm and the most frequent value 4.2 nm; for fixed and stained specimens, 3.8 nm and 3.6 nm, respectively. Laser diffraction of the electron micrographs showed a dumbbell-like intensity pattern (two diffuse maxima of intensity on the equator, one on each side of the central spot), giving an average distance of about 6 nm. This value represents the upper range of the direct measurements. The measurements demonstrate that the arrangement of the collagen microfibrils is mainly preserved during mineralization. However, using laser diffraction, distances of 9–11 nm were also observed. Such large distances can also be demonstrated by X-ray diffraction on collagen fibrils stained under special conditions. This may indicate that special conditions of apatitic mineralization or staining may alter the arrangement of the microfibrils.The authors thank the Deutsche Forschungsgemeinschaft for financial support     

Transmission microscopy of freeze dried,unstained epiphyseal cartilage of the guinea pig

Summary The question of the initial mineralization in the epiphyseal plate has been investigated to date in specimens prepared by conventional electron microscopical techniques. As conventional techniques can produce artifacts, either a loss of mineral substance or a secondary nucleation, the mineralization process was investigated using freeze dried, vacuum embedded growth cartilage which was neither contrasted nor stained and which had a very short contact with water.The prevailing theory that the first mineralization begins within extracellular matrix vesicles and that the mineralization outside these vesicles is a secondary process was confirmed. Mineralized matrix vesicles were found in the fully mineralized long septa down to the opening zone. In several cases a mineralization could be observed in those transverse septa in which organic substance was present between the cells. The typical radial arrangement of the apatitic needles and platelets in the matrix vesicles could be explained by the formation of a mineralization in an ionotropic gel, the orientation of the matrix macromolecules to be produced by a vectorial influx of calcium ions and phosphate groups coming from different directions. Thin strands of mineral substance with low contrast, which follow the direction of the longitudinal septum, were assumed to be the mineralized collagen fibrils. In several needles dot-like formations were seen and the distance between the middle of neighbouring dots was found to lie mainly in the range 30–56 Å, while the lateral separation distance between the middle of closely packed parallel chains and needles was found to lie mainly in the range 30-42 Å. Parallel periodic structures which could be visualized in apatitic chains and needles 20–40 Å in diameter were assumed to be the 8.2 Å-(100)-lattice planes of apatite, being an indication that these formations already possess criteria of the apatite lattice.We express our thanks to the Deutsche Forschungsgemeinschaft for financial support and to Dr. A. Boyde, London, for valuable discussions.     

Elektronenmikroskopie und Laserbeugungs-Untersuchungen zur Charakterisierung der organischen Matrix im Speichelstein und Hartgewebe

Zusammenfassung Elektronenmikroskopische Ultradünnschnittbilder vom Speichelstein zeigten — wie solche vom Dentin, Knochen usw. — nadelbzw. kettenförmige Ca-Phosphatbildungen mit globulären Keimen. Wir bestimmten in der Grundsubstanz a) die Länge der Nadeln als Werte für die Länge der kettenartigen Matrix, b) die Abstände zwischen den punktförmigen Keimen innerhalb der Nadeln als Wiedergabe der Abstände zwischen den aktiven Baugruppeneinheiten, die auf der Matrix die Mineralkeimbildung einleiten, und c) die Seitenabstände zwischen dicht zusammenliegenden, parallelen Ketten als Wiedergabe der Seitenabstände zwischen den Hauptsträngen der Matrix (s. Abb. 2). Die Bestimmungen zu b) und c) wurden sowohl über morphologische Vermessungen wie Laserbeugung an den elektronenmikroskopischen Platten durchgeführt. Die unter a) bezeichneten langen Nadeln (damit die Kettenlänge der Matrixleitstruktur) erreichten Werte über 1000 Å. Die unter b) bezeichneten morphologischen Vermessungen zwischen den Keimen ergaben Werte zwischen 40 und 85 Å und die unter c) bezeichneten Seitenabstände Werte zwischen 37 und 75 Å; die Laserbeugung führte zu Werten zwischen 37 und 54 Å, die also bevorzugt im unteren Bereich der morphologischen Vermessungen zu b) und c) lagen. Anhand dieser Meßdaten wurden erste Überlegungen darüber angestellt, ob die Leitstruktur in der organischen Matrix die Eiweiß- oder Polysaccharidkette im Proteinpolysaccharid bzw. Glykoprotein ist. Entsprechende Untersuchungen wurden auch innerhalb der Bakterien und der Bakterienmembran durchgeführt.

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Electron microscopic and laser diffraction measurement and study of the organic matrix of salivary concretions and hard body tissues

Summary Electron micrographs of ultrathin sections of salivary concretions showed needle-like Ca-phosphate formations with globular nuclei. Similar formations were observed in dentine, bone, and other hard tissues. We determined in the ground substance (a) the lengths of the needles as values for the length of the chain-like matrix backbone, (b) the distances between the dotlike nuclei within the needles as distances between the active sites of the chain-like matrix which induce the phosphate nucleation, and (c) the side distances between the close-packed, parallel needles as values for the distances between the parallel matrix backbones. The investigation of (b) and (c) was carried out by morphological measurement as well as by laser diffraction method on electron micrographs. The long needles of (a) (the length of the matrix backbone) reached values of more than 1,000 Å. Measurement of the distances between the small globular nuclei within the needles (b) gave values of 40 to 85 Å, and of those on the side distances of 37–75 Å. The values for (b) and (c) derived by laser diffraction appeared in the range of 37–54 Å, which was the lower range of the morphological values of (b) and (c). The results were analysed to discuss whether the main chain of the matrix is the proteinor the polysaccharide chain in the protein-polysaccharide or the glycoprotein. Corresponding measurements were carried out in areas within the bacteria and in the bacterial membrane.

Herrn Prof. Dr. L. Reimer und Herrn cand. phys. H. G. Heine, Physikalisches Institut der Universität Münster, danken wir für die Durchführung der Laserbeugungs-Untersuchungen und für wertvolle Diskussionen, Fräulein Gisela Rehsöft für sorgfältige Assistenz. Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Evidence against a Looped Structure of the Inactive Human X-Chromosome Territory

Multicolor fluorescencein situhybridization with a whole chromosome composite probe for the X-chromosome and microdissection probes for the Xp and Xq arms, as well as for the Xp terminal, Xq terminal, and X centromer specific subregional probes, was applied to three-dimensional (3D) preserved human female amniotic fluid cell nuclei. Confocal laser scanning microscopy and three-dimensional image analysis demonstrated distinctly separated Xp arm and Xq arm domains. 3D distance measurements revealed a high variability of intrachromosomal distances between Xpter, Xcen, and Xqter specific probes within both X territories. A 3D distance measurement error of ±70 nm was found in control experiments using quartz glass microspheres labeled with different fluorochromes. Our data argue against the hypothesis of Walkeret al.(1991,Proc. Natl. Acad. Sci. USA88, 6191–6195) that a looped structure of the inactive X territory is formed by tight telomere–telomere associations.     

ON THE ROLE OF MICROTUBULES IN MOVEMENT AND ALIGNMENT OF NUCLEI IN VIRUS-INDUCED SYNCYTIA

Infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes rapid and extensive cell fusion. Time-lapse cinematography shows that when cells fuse, their nuclei migrate straight to the center of the syncytium at rates of 1–2 µ/min. Nuclei are often arranged in long, tightly packed, parallel rows in syncytia derived from the fibroblastic BHK21-F cells. Polarization microscopy shows birefringent material between and parallel to these rows of nuclei, and electron microscopy shows bundles of cytoplasmic microtubules, ~250 A in diameter, and filaments, ~80 A in diameter, parallel to and between the rows of nuclei. Colchicine treatment causes disappearance of microtubules from BHK21-F cells and an apparent increase in the number of 80-A filaments. Although colchicine-treated, SV5-infected cells fuse, their nuclei do not migrate or form rows but remain randomly scattered through the syncytial cytoplasm. Incubation at 4°C does not disrupt microtubules in BHK21-F cells. Rows of nuclei have been isolated from SV5-induced syncytia, and the nuclei in them have been found to be intimately associated with microtubules but not with other cytoplasmic structures. These results suggest that microtubules demarcate cytoplasmic channels through which nuclei migrate and that they may also be involved in the mechanism of nuclear movement.     

Prismatic cristae and paracrystalline inclusions in mitochondria of myocardial cells of the oyster Crassostrea virginica Gmelin

Summary Several types of unusual mitochondrial configurations were found in myocardial cells of the oyster Crassostrea virginica Gmelin. These mitochondria include, in order of frequency, prismatic cristae, filamentous paracrystals in honeycomb and herringbone configurations, and paracrystals composed of rows of electron dense particles. The long, parallel, evenly spaced prismatic cristae are square or rhomboidal in cross section. In the space between the prismatic cristae are rodlike structures (4–6 nm in diameter) that are regularly spaced about 12nm apart and appear to pass between adjacent cristae. Filamentous paracrystals are observed in slender, elongated mitochondria. The filament spacing and form of these paracrystals suggest that they are composed of the intercristal rods. Alternatively, filamentous paracrystals might be tangential sections of prismatic cristae and intercristal rods. Particulate paracrystals which consist of dense lines or rows of particles are the least frequent type of unusual configuration. The particles are triangular, possibly pyramidal, in shape; their bases are 10–12 nm thick and repeat in rows every 17–18 nm. There is a close association between particulate paracrystals and prismatic cristae plus intercristal rods. Although similar mitochondrial configurations have been associated with disease or altered metabolism in a number of species, we have found no such association in the oyster as yet.Supported in part by the Mississippi-Alabama Sea Grant Consortium, through NOAA, Dept. of Commerce under grant no. NA 79AA-D-0049We wish to thank Ms. Barbara M. Hyde, Ms. Patricia A. Vermiere and Mr. Robert Allen for their technical assistance     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : II. Structural Details and Further Evidence for Intercellular Channels by Freeze-Fracture and Negative Staining

Low resistance junctions between axons of crayfish ganglia are studied by freeze-fracture and negative staining. In freeze-fracture, fracture planes that go through a junctional membrane expose two faces, both internal, called face A and face B. Face A belongs to the internal membrane leaflet and faces the gap. Face B belongs to the external membrane leaflet and faces the axoplasm. Face A displays pits, 60–100 Å in diameter, arranged in a hexagonal array with a unit cell of ~200 Å. An ~25 Å bump is frequently seen at the center of each pit. Some pits are occupied by a globule ~125 Å in diameter, which displays a central depression ~25 Å in size. Face B contains globules also arranged in a fairly regular hexagonal pattern. The center-to-center distance between adjacent globules is most frequently ~200 Å; however, occasionally certain globules are seen separated by a distance as short as ~125 Å. The top surface of the globules occasionally displays a starlike profile and seems to contain a central depression ~25 Å in diameter. In negatively stained preparations of membranes from the nerve cord, two types of membranes are seen containing a fairly regular pattern. In one, globules ~95 Å in diameter form a hexagonal close packing with a unit cell of ~95 Å. In the other, globules of the same size are organized in a larger hexagonal array with a unit cell of ~155 Å (swollen arrangement). Some of the globules forming the swollen arrangement are seen containing six subunits. The six subunits form a hexagon which is skewed with respect to the main rows of hexagons in such a way that the subunits lie on rows which make an angle of ~37° with the main rows.     

Untersuchungen zur Mineralisierungsdichte im Hartgewebe mit Protein-Polysaccharid bzw. mit Kollagen als Hauptbestandteil der Matrix

Zusammenfassung Ultradünnschnitte von schmelznahem Dentin sowie von Osteopetroseknochen-Partien zeigten im Elektronenmikroskop, daß die proteinpolysaccharid-reichen Zonen des peritubulären Dentins sowie der sog. dichten Zonen des Osteopetrose-Knochens punktartige Calzium-Phosphat-Keime bzw. Ketten von Punktkeimen in dichter Zusammenlagerung besitzen, während in den kollagenreichen Zonen neben diesen Ketten und Nadeln größere Blättchen-Kristallite auftreten. Durch Entmineralisierung-Kontrastierung der Ultradünnschnitte mit Phosphorwolframsäure wurde gezeigt, daß im peritubulären Dentin und den dichten Partien des Osteopetroseknochens elektronenmikroskopisch keine Kollagenfasern mit Querstreifen vorkommen; diese sind aber im intertubulären Dentin und den normalen Partien des Osteopetroseknochens verbreitet. Durch Registrierung der Calzium- und Phosphor-Röntgenimpulse mit der elektronenmikroskopischen Mikrosonde (Cameca) konnte — bei Annahme angenähert gleicher Schnitt-Dicke in benachbarten Zonen — gefunden werden, daß der Calzium-Gehalt pro Volumeneinheit im Mittel von 100 Einzelmessungen im peritubulären Dentin um etwa 40 % höher liegt als im kollagenreichen intertubulären Dentin und in den proteinpolysaccharid-reichen Zonen der Osteopetrose-Proben um 65 % höher als in den normalen kollagenreichen Bezirken. Da entsprechende Werte für Phosphor gefunden wurden, bedeuten diese Prozentangaben zugleich dieselben Unterschiede in der Mineralisierungsdichte.

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Investigations of mineralization in hard tissues with protein-polysaccharides or collagen as main constituents of the matrix

Summary Ultrathin sections of mature dentine in the vicinity of enamel and sections of osteopetrotic bone were examined. In the protein-polysaccharide-rich regions of the peritubular dentine, as well as in the so-called dense zones of the osteopetrotic bone, dot-like nuclei, or short chains of them, in close packing were found, whereas in the intertubular collagen-rich dentine zones, as well as the normal regions of osteopetrotic bone, many platelike crystals were found among the chains of dots. By demineralization-staining experiments with phosphotungstic acid, it was found that in the peritubular dentine as well as the so-called dense zones of the osteopetrotic bone there were no collagen fibres visible, while they dominated in the intertubular dentine as well as the normal regions of the osteopetrotic bone. The calcium and phosphorous concentrations in the sections were examined in the Cameca electronmicroscopical probe. Assuming that neighbouring regions were of nearly equal thickness, the mean calcium content per unit volume from 100 single point measurements for each region was found to be about 40 % higher in the peritubular region than in the collagen-rich intertubular region, and about 65 % higher in the so-called dense regions of the osteopetrose bone than in the normal collagen-rich zones. The same was found for phosphorous.

Wir danken Frl. Ch. Lippert, Frl. I. Schütte und Herrn J. Schreiber für wertvolle Hilfe bei der technischen Durchführung der Untersuchungen. Der Deutschen Forschungsgemeinschaft danken wir für die Unterstützung unserer Arbeit.     

Freeze fracture studies on the annelid septate junction

Summary Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80–120 Å, the distance between two particles (centre to centre) is 150–250 Å. Additionally striking rows of large particles (long diameter 150–200 Å). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100–150 Å, centre to centre distance approx. 250 Å) whereas the particles of the extracellular face (EF, mean diameter 200–250 Å) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.     

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (T_m) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer T_m between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the T_m for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.     

Genetic distance as an alternative to physical distance for definition of gene units in association studies

Background

Some association studies, as the implemented in VEGAS, ALIGATOR, i-GSEA4GWAS, GSA-SNP and other software tools, use genes as the unit of analysis. These genes include the coding sequence plus flanking sequences. Polymorphisms in the flanking sequences are of interest because they involve cis-regulatory elements or they inform on untyped genetic variants trough linkage disequilibrium. Gene extensions have customarily been defined as ± 50 Kb. This approach is not fully satisfactory because genetic relationships between neighbouring sequences are a function of genetic distances, which are only poorly replaced by physical distances.

Results

Standardized recombination rates (SRR) from the deCODE recombination map were used as units of genetic distances. We searched for a SRR producing flanking sequences near the ± 50 Kb offset that has been common in previous studies. A SRR ≥ 2 was selected because it led to gene extensions with median length = 45.3 Kb and the simplicity of an integer value. As expected, boundaries of the genes defined with the ± 50 Kb and with the SRR ≥2 rules were rarely concordant. The impact of these differences was illustrated with the interpretation of top association signals from two large studies including many hits and their detailed analysis based in different criteria. The definition based in genetic distance was more concordant with the results of these studies than the based in physical distance. In the analysis of 18 top disease associated loci form the first study, the SRR ≥2 genes led to a fully concordant interpretation in 17 loci; the ± 50 Kb genes only in 6. Interpretation of the 43 putative functional genes of the second study based in the SRR ≥2 definition only missed 4 of the genes, whereas the based in the ± 50 Kb definition missed 10 genes.

Conclusions

A gene definition based on genetic distance led to results more concordant with expert detailed analyses than the commonly used based in physical distance. The genome coordinates for each gene are provided to maintain a simple use of the new definitions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-408) contains supplementary material, which is available to authorized users.     

Genetic variation of F1 hybrids from controlled crosses between Pinus montana var. rostrata and Pinus sylvestris in morphological needle traits

One hundred and seventeen hybrids, representing ten families from controlled crosses between Pinus montana var. rostrata and Pinus sylvestris, were examined in respect to six morphological traits of two-year-old needles. The biometric data obtained from the measurements provided a basis for multivariate statistical analyses, including discriminant analysis and Mahalanobis distances as principal methods. The analysed families formed two complexes, which were significantly different. The main traits responsible for the distinction were numbers of stomatal rows on both sides of the needles.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C^α and C^β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ_i, ψ_i and χ_i¹, were computed. Secondly, the frequency with which short distances occur between amide, C^α and C^β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein ¹H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.     

Computational Assessment of Transport Distances in Living Skeletal Muscle Fibers Studied In Situ

Transport distances in skeletal muscle fibers are mitigated by these cells having multiple nuclei. We have studied mouse living slow (soleus) and fast (extensor digitorum longus) muscle fibers in situ and determined cellular dimensions and the positions of all the nuclei within fiber segments. We modeled the effect of placing nuclei optimally and randomly using the nuclei as the origin of a transportation network. It appeared that an equidistant positioning of nuclei minimizes transport distances along the surface for both muscles. In the soleus muscle, however, which were richer in nuclei, positioning of nuclei to reduce transport distances to the cytoplasm were of less importance, and these fibers exhibit a pattern not statistically different from a random positioning of nuclei. We also simulated transport times for myoglobin and found that they were remarkably similar between the two muscles despite differences in nuclear patterning and distances. Together, these results highlight the importance of spatially distributed nuclei to minimize transport distances to the surface when nuclear density is low, whereas it appears that the distribution are of less importance at higher nuclear densities.     

Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome

Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10–40 mJ for a 1 cm² area, corresponding to fluences of 4.5 to 18 × 10⁹ W m^–2, to determine strand breakage yields and the frequency and pattern of RNA–RNA cross- linking in the 16S rRNA. Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 × 10^–3 over the incident intensity range studied. The yield for RNA–RNA cross-linking was 3 × 10^–4 cross-links/photon at the intensity of 4.5 × 10⁹ W m^–2, an ~4-fold higher yield per photon than obtained with a transilluminator. The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal. The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes. Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation.     

Survival of Hepatitis C Virus in Syringes Is Dependent on the Design of the Syringe-Needle and Dead Space Volume

Background

Many people who inject drugs (PWID) use syringes with detachable needles, which have high dead space (HDS). Contaminated HDS blood may substantially contribute to the transmission of HIV, hepatitis C (HCV), and other blood-borne viruses within this population. Newly designed low dead space (LDS) syringe-needle combinations seek to reduce blood-borne virus transmission among PWID. We evaluated the infectivity of HCV-contaminated residual volumes recovered from two LDS syringe-needle combinations.

Methods

We tested two different design approaches to reducing the dead space. One added a piston to the plunger; the other reduced the dead space within the needle. The two approaches cannot be combined. Recovery of genotype-2a reporter HCV from LDS syringe-needle combinations was compared to recovery from insulin syringes with fixed needles and standard HDS syringe-needle combinations. Recovery of HCV from syringes was determined immediately following their contamination with HCV-spiked plasma, after storage at 22°C for up to 1 week, or after rinsing with water.

Results

Insulin syringes with fixed needles had the lowest proportion of HCV-positive syringes before and after storage. HCV recovery after immediate use ranged from 47%±4% HCV-positive 1 mL insulin syringes with 27-gauge ½ inch needles to 98%±1% HCV-positive HDS 2 mL syringes with 23-gauge 1¼ inch detachable needles. LDS combinations yielded recoveries ranging from 65%±5% to 93%±3%. Recovery was lower in combinations containing LDS needles than LDS syringes. After 3 days of storage, as much as 6-fold differences in virus recovery was observed, with HCV recovery being lower in combinations containing LDS needles. Most combinations with detachable needles required multiple rinses to reduce HCV infectivity to undetectable levels whereas a single rinse of insulin syringes was sufficient.

Conclusions

Our study, the first to assess the infectivity of HCV in residual volumes of LDS syringes and needles available to PWID, demonstrates that LDS syringe-needle combination still has the greater potential for HCV transmission than insulin syringes with fixed needles. Improved LDS designs may be able to further reduce HCV recovery, but based on the designed tested, LDS needles and syringes remain intermediate between fixed-needle syringes and HDS combinations in reducing exposure to HCV.     

Structural Proton Diffusion along Lipid Bilayers

For H⁺ transport between protein pumps, lateral diffusion along membrane surfaces represents the most efficient pathway. Along lipid bilayers, we measured a diffusion coefficient of 5.8 × 10⁻⁵ cm² s⁻¹. It is too large to be accounted for by vehicle diffusion, considering proton transport by acid carriers. Such a speed of migration is accomplished only by the Grotthuss mechanism involving the chemical exchange of hydrogen nuclei between hydrogen-bonded water molecules on the membrane surface, and the subsequent reorganization of the hydrogen-bonded network. Reconstitution of H⁺-binding sites on the membrane surface decreased the velocity of H⁺ diffusion. In the absence of immobile buffers, structural (Grotthuss) diffusion occurred over a distance of 100 μm as shown by microelectrode aided measurements of the spatial proton distribution in the immediate membrane vicinity and spatially resolved fluorescence measurements of interfacial pH. The efficiency of the anomalously fast lateral diffusion decreased gradually with an increase in mobile buffer concentration suggesting that structural diffusion is physiologically important for distances of ~10 nm.     

The extent of linkage disequilibrium in beef cattle breeds using high-density SNP genotypes

Background

The extent of linkage disequilibrium (LD) between molecular markers impacts genome-wide association studies and implementation of genomic selection. The availability of high-density single nucleotide polymorphism (SNP) genotyping platforms makes it possible to investigate LD at an unprecedented resolution. In this work, we characterised LD decay in breeds of beef cattle of taurine, indicine and composite origins and explored its variation across autosomes and the X chromosome.

Findings

In each breed, LD decayed rapidly and r² was less than 0.2 for marker pairs separated by 50 kb. The LD decay curves clustered into three groups of similar LD decay that distinguished the three main cattle types. At short distances between markers (< 10 kb), taurine breeds showed higher LD (r² = 0.45) than their indicine (r² = 0.25) and composite (r² = 0.32) counterparts. This higher LD in taurine breeds was attributed to a smaller effective population size and a stronger bottleneck during breed formation. Using all SNPs on only the X chromosome, the three cattle types could still be distinguished. However for taurine breeds, the LD decay on the X chromosome was much faster and the background level much lower than for indicine breeds and composite populations. When using only SNPs that were polymorphic in all breeds, the analysis of the X chromosome mimicked that of the autosomes.

Conclusions

The pattern of LD mirrored some aspects of the history of breed populations and showed a sharp decay with increasing physical distance between markers. We conclude that the availability of the HD chip can be used to detect association signals that remained hidden when using lower density genotyping platforms, since LD dropped below 0.2 at distances of 50 kb.