The assessment of scapular radiographs: analysis of anteroposterior radiographs of the shoulder and the effect of rotational offset on the glenopolar angle.

The glenopolar angle assesses the rotational alignment of the glenoid and may provide prognostic information and aid the management of scapula fractures. We have analysed the effect of the anteroposterior (AP) shoulder radiograph rotational offset on the glenopolar angle in a laboratory setting and used this to assess the accuracy of shoulder imaging employed in routine clinical practice. Fluoroscopic imaging was performed on 25 non-paired scapulae tagged with 2 mm steel spheres to determine the orientation of true AP views. The glenopolar angle was measured on all the bony specimens rotated at 10° increments. The mean glenopolar angle measured on the bone specimens in rotations between 0° and 20° and thereafter was found to be significantly different (p < 0.001). We also obtained the AP radiographs of the uninjured shoulder of 30 patients treated for fractures at our centre and found that none fitted the criteria of a true AP shoulder radiograph. The mean angular offset from the true AP view was 38° (10° to 65°) for this cohort. Radiological AP shoulder views may not fully project the normal anatomy of the scapular body and the measured glenopolar angle. The absence of a true AP view may compromise the clinical management of a scapular fracture.

Delayed operative management of fractures of the scapula.

Between 1998 and 2007, 22 patients with fractures of the scapula had operative treatment more than three weeks after injury. The indications for operation included displaced intra-articular fractures, medialisation of the glenohumeral joint, angular deformity, or displaced double lesions of the superior shoulder suspensory complex. Radiological and functional outcomes were obtained for 16 of 22 patients. Disabilities of the Arm, Shoulder, Hand (DASH) and Short form-36 scores were collected for 14 patients who were operated on after March 2002. The mean delay from injury to surgery was 30 days (21 to 57). The mean follow-up was for 27 months (12 to 72). At the last review the mean DASH score was 14 (0 to 41). Of the 16 patients with follow-up, 13 returned to their previous employment and recreational activities without restrictions. No wound complications, infection or nonunion occurred. Malunion of the scapula can be prevented by surgical treatment of fractures in patients with delayed presentation. Surgery is safe, effective, and gives acceptable functional results.

Effect of LNA modifications on small molecule binding to nucleic acids.

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2'-O, 4'-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.

Hybridization of PNA to structured DNA targets: quadruplex invasion and the overhang effect.

Peptide nucleic acid (PNA) probes have been synthesized and targeted to quadruplex DNA. UV-vis and CD spectroscopy reveal that the quadruplex structure of the thrombin binding aptamer (TBA) is disrupted at 37 degrees C by a short PNA probe. The corresponding DNA probe fails to bind to the stable secondary structure at this temperature. Thermal denaturation experiments indicate surprisingly high thermal and thermodynamic stabilities for the PNA-TBA hybrid. Our results point to the nonbonded nucleobase overhangs on the DNA as being responsible for this stability. This "overhang effect" is found for two different PNA-DNA sequences and a variety of different overhang lengths and sequences. The stabilization offered by the overhangs assists the PNA in overcoming the stable secondary structure of the DNA target, an effect which may be significant in the targeting of biological nucleic acids, which will always be much longer than the PNA probe. The ability of PNA to invade a structured DNA target expands its potential utility as an antigene agent or hybridization probe.

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove.

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.

Investigation of DNA binding modes for a symmetrical cyanine dye trication: effect of DNA sequence and structure.

The DNA binding behavior of a tricationic cyanine dye (DiSC3+(5)) was studied using the [Poly(dA-dT)]2, [Poly(dI-dC)]2 and Poly(dA) x Poly(dT) duplex sequences and the Poly(dA) x 2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA) x Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]2 duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]2 sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987-2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.

Leukoencephalopathy and raised brain lactate from heroin vapor inhalation ("chasing the dragon")

BACKGROUND: Inhalation of heated heroin vapor ("chasing the dragon"), which is gaining popularity among drug users seeking to avoid the risks of parenteral drug administration, can produce progressive spongiform leukoencephalopathy. METHODS: We studied the clinical phenotype and course, MRI, MRS, and brain pathology in the first American patients described with this syndrome. RESULTS: Two of the three heroin users studied inhaled heroin pyrolysate together daily over the course of 2 weeks. They developed ataxia, dysmetria, and dysarthria. Patient 1 progressed to an akinetic mute state with decorticate posture and subsequent spastic quadriparesis. Patient 2 developed a mild spastic quadriparesis and gait freezing. Patient 3 was asymptomatic following less heroin exposure. Brain MRI showed diffuse, symmetrical white matter hyperintensities in the cerebellum, posterior cerebrum, posterior limbs of the internal capsule, splenium of the corpus callosum, medial lemniscus, and lateral brainstem. MRS showed elevated lactate. Brain biopsy (Patient 1) showed white matter spongiform degeneration with relative sparing of U-fibers; electron microscopy revealed intramyelinic vacuolation with splitting of intraperiod lines. Progressive deterioration occurred in Patients 1 and 2 over 4 weeks. Both were treated with antioxidants including oral coenzyme Q, and clinical improvement occurred. Patient 1 recovered nearly completely over 24 months. Patient 2 improved, but developed a delayed-onset cerebellar hand tremor. Both still have white matter abnormalities on MRI and MRS. CONCLUSIONS: Elevated lactate in white matter and the possible response to antioxidants suggests mitochondrial dysfunction in progressive spongiform leukoencephalopathy following inhalation of heated heroin vapor.

Presynaptic induction and expression of homosynaptic depression at Aplysia sensorimotor neuron synapses.

The cellular mechanisms underlying the induction and expression of homosynaptic depression at the glutamatergic synapse between Aplysia sensory and motor neurons were studied in dissociated cell culture. Intracellular microelectrodes were used to stimulate action potentials in the presynaptic sensory neuron and record the depolarizing EPSP from the motor neuron. Homosynaptic depression (HSD) was induced by repeatedly stimulating the sensory neuron at rates as low as one action potential per minute. Activation of postsynaptic Glu receptors was neither sufficient nor necessary to induce HSD. Thus, repeated applications of exogenous Glu did not depress the synaptically evoked EPSP. Moreover, normal HSD was observed when the sensory neuron was stimulated during a period when the Glu receptors were blocked with the antagonist DNQX. The induction of HSD is thus likely to occur within the presynaptic terminal. We explored the role of presynaptic calcium in the induction of HSD by injecting the sensory neuron with EGTA, a relatively slow calcium chelator that does not alter rapid release but effectively buffers the slow residual calcium transient thought to be important for plasticity. EGTA had little effect on HSD, indicating that residual Cai is not involved. HSD does not appear to involve a decrease in presynaptic calcium influx, because there was no change in the presynaptic calcium transient, measured by calcium indicator dyes, during HSD. We conclude that HSD is induced and expressed in the presynaptic terminal, possibly by a mechanism directly coupled to the release process.

Hairpin-forming peptide nucleic acid oligomers.

A series of partially self-complementary peptide nucleic acid (PNA) oligomers was prepared. Examination of their melting behavior, circular dichroism spectra, and fluorescence properties reveals that these PNA oligomers exist as stem-loop ("hairpin") structures. Fluorescence is readily observed in hairpins containing a covalently linked, emissive acridine derivative which is, at least partially, intercalated in the duplex region of the PNA hairpin. The acridine fluorescence is quenched when an anthraquinone derivative is covalently attached to the PNA so that it is bound near the acridine in the hairpin structure. Acridine fluorescence is restored in hairpins containing both the anthraquinone and the acridine by increasing the temperature and melting the structure to its linear form or by opening the hairpin through formation of a hybrid duplex with complementary DNA. The latter process may form the basis for development of selective and sensitive DNA assays.

Selective photocleavage of DNA by anthraquinone derivatives: targeting the single-strand region of hairpin structures.

A tetracationic anthraquinone derivative (27AQS2) binds to hairpin DNA and irradiation of the bound quinone leads to selective strand cleavage. NMR spectroscopy reveals that 27AQS2 binds at the loop and to the stem-loop junction of hairpin DNA. UV irradiation of the bound quinone causes cleavage of the DNA in the loop region and at guanines in the stem region. Inclusion of ethidium bromide in the reaction mixture leads to a greatly increased selectivity for loop cleavage. Spectroscopic and chemical evidence suggests a three component mechanism for reaction. The ability to target single-stranded regions of DNA structures is an important property of this photonuclease.

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.

Peptide nucleic acid-DNA duplexes: long range hole migration from an internally linked anthraquinone.

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA-DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA-DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.

Anthraquinone photonucleases: a surprising role for chloride in the sequence-neutral cleavage of DNA and the footprinting of minor groove-bound ligands.

Irradiation of water-soluble anthraquinone (AQ) reagents in the presence of chloride ions results in the spontaneous, sequence-neutral cleavage of DNA. Mechanistic studies indicate that cleavage is initiated by chlorine atoms, produced by charge transfer interaction between chloride anion and AQ triplet states. High-resolution gel electrophoresis suggests that cleavage arises from abstraction of a hydrogen atom from C-4' of deoxyribose units. The targeting of this hydrogen, which is located in the minor groove of duplex DNA, can be effectively blocked by netropsin and, to a lesser degree, berenil, leading to photofootprinting of these minor groove-binding drugs.

Variation in fertility between different types of local area.

Trends in fertility at the national level have been described in a recent article in the Population Review series. This article examines variations in the pattern of fertility experienced in different kinds of area within England and Wales. For this purpose use is made of the new ONS classification of local authorities into 11 groups and 29 clusters of areas with similar socio-economic characteristics. The classification was developed from statistics collected in the 1991 Census of Population. Although proportions of residents aged 0-4, 5-14, and 25-44 were among the 37 Census variables used to define groups and clusters of similar areas, the analysis did not make direct use of any measure of fertility related to ages of women at childbirth.

PIP: This study examines variations in fertility in local authority areas in England and Wales. Data were aggregated into 11 groups and 29 clusters of local authority areas (LAAs). English fertility historically shows that the manufacturing group of local authorities during 1974-94 had fertility above the natural average. The total period fertility rate in England and Wales has remained stable at about 1.8 children/woman during 1974-94. Age-specific fertility rates (ASFRs) have shown greater variation over time and in 1994 between the subgroups. 1994 ASFRs by subgroups compared to corresponding national averages reveal that the Services and Education group of LAAs, which includes Outer London boroughs and some University Towns, had low fertility but higher rates among women over 30 years old. Four LAAs located southwest of London and affluent areas in the southeast of England comprise the Most Prosperous group. Fertility was below average for women aged under 30 years and above average for women aged over 30 years. Variations occurred between each of the four London clusters. The Growth Areas of Corridors, Transients, Overspill, Market Towns, and Satellite Towns showed fertility around the national average. The Coast and Country group and the Mixed Economy group showed fertility close to the national average. Fertility in the Resort and Retirement group and the Ports and Industry group was slightly below average. Mixed Urban and Rural Areas showed fertility typical of prosperous areas and depressed areas. Coalfield's fertility was slightly above average. Manufacturing had the highest fertility at 1.93. The highest proportions of live births within marriage occurred in Manufacturing, Services and Education, and Inner London. Central London had the lowest proportions. The percentage of multiple births was strongly correlated with affluence or economic difficulties.

Selective stabilization of triplex DNA by anthraquinone sulfonamide derivatives.

A series of cationic anthraquinone derivatives was investigated for their ability to stabilize duplex and triplex DNA. Thermal denaturation experiments demonstrate that each of these compounds stabilizes the [poly(dT) x poly(dA) x poly(dT)] triplex without significantly affecting the [poly(dT) x poly(dA)] duplex. The amount of stabilization is determined by the number and placement of the cationic substituents on the anthraquinone skeleton. The stabilization arises primarily from higher affinity binding of the quinones to the triplex relative to the duplex structures. Phosphorescence quenching and viscometric titrations indicate that the quinones bind to the triplex by intercalation.

Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA.

A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.

Population review: (4). Trends in fertility.

Fertility in the United Kingdom has now been at a level below that needed for natural replacement of the population (TPFR of 2.1) for more than twenty years. Whilst fertility in many parts of the world (eg most of Europe, and much of the developing world) has declined sharply in recent years, the overall rate in the United Kingdom has been fairly stable (TPFR about 1.8) since 1980. Women are having their children at older ages: fertility rates have been falling for women aged under 30 and rising for women above that age. The mean age at motherhood rose by 1.4 years to 28.4 in England and Wales between 1984 and 1994. The proportion of women in England and Wales who were still childless at age 45 rose to 13 per cent in 1994, and will increase further in the future. One third of births are now to women who are not married, but the recent rapid increase in this proportion may not continue. The percentage of all births which are both outside marriage and registered solely by the mother has been fairly stable (around 7 per cent) for several years. Fertility rates are higher in Northern Ireland than in the rest of the United Kingdom, and lowest in Scotland and in the North of England. Northern Ireland has the lowest proportion of births outside marriage.

PIP: This article describes fertility trends in the United Kingdom during 1944-94. Fertility is examined in terms of the total period fertility rate (TPFR), cohort fertility, maternal age, family size, nonmarital fertility, and country of birth. The TPFRs for regions in 1994 were 1.95 in Northern Ireland, 1.58 in Scotland, 1.79 in Wales, and 1.74 in both England and the entire United Kingdom. Northern Ireland had the highest TPFR but the lowest proportion of nonmarital births (22.0%). The highest proportion of nonmarital births was in north and northwest England (over 38%). The national average was 32.0%. Although the United Kingdom had under replacement fertility in 1994, it had the highest fertility, excepting the Republic of Ireland (1.86), in the European Community. TPFR peaked in 1947 at 2.75 and 1964 at 2.95. The lowest TPFR was 1.69 in 1977, but TPFR rose to 1.89 in 1980. Fertility stabilized around 1.80 after 1980. Fertility decline during 1964-77 is attributed to greater proportions of women receiving a higher education, greater female participation in the work force, and greater control over fertility. Age specific fertility rates were variable. In 1994, the highest fertility was among women 25-29 years old. Fertility was lowest among women over 40 years old. During 1980-94, rates for younger women declined, and rates for women 35-39 years old increased slightly. The rates for women 30-34 are now close to rates for women 25-29. The mean age of the mother rose 1.4 years during 1984-94 to 28.4 years. The mean age of first birth in 1994 was 26.5 years. Cohorts born in 1944 were more fertile at younger ages and less fertile after the age of 25 years. Average completed family size increased for cohorts born during 1920-34 and decreased for cohorts born after 1944. The lowest level of childlessness was among the 1944 cohort. There were high rates of childlessness in 1964 and 1994. Fertility was higher among immigrant women.

Proteins functioning in synaptic transmission at the sensory to motor synapse of Aplysia.

Over expression of Aplysia synaptotagmin in acutely dissected cholinergic neurons from the buccal ganglia, or in primary co-cultures of glutaminergic sensory neurons and motor neurons, causes a reduction synaptic transmission. Anti-sense oligonucleotide treatment of similar cultures produced an enhancement of synaptic transmission. The interaction between Aplysia VAMP/synaptobrevin and syntaxin is reconstructed using the yeast two hybrid system, and used to identify amino acid residues of VAMP/synaptobrevin that are required for this interaction. Point mutations around residue 50, close to the site of cleavage by botulinum toxins specifically disrupt the interaction with syntaxin. An additional VAMP/synaptobrevin binding protein, VAP33, is identified using the yeast two hybrid system. Intracellular injection of VAP33 specific antisera inhibits synaptic transmission in sensory-motor neuron co-cultures.