Percutaneous transluminal angioplasty of subclavian arteries

The clinical records and procedural details of 30 subclavian angioplasty procedures attempted in 27 patients were reviewed. Long-term follow-up was obtained through referring physician records and direct telephone contact with the patients. Eight patients presented with neurologic symptoms only, six had arm claudication only, nine had both neurologic and arm symptoms, three underwent dilations to provide graft inflow, and one was asymptomatic. Procedural complications included a stroke in the contralateral carotid distribution, occurring during follow-up arch aortography, and an embolus in the fifth digit of the left hand, which was of no clinical significance. Long-term success did not correlate well with degree of stenosis, lesion length, or postangioplasty appearance. The patients with arm and neurologic symptoms who were followed up for 3 years experienced immediate relief and remained symptom free or improved, except for the patient who suffered the stroke and one patient with labyrinthitis.     

Effective corneal retrieval in a general hospital The Royal Melbourne Hospital Eye Bank

An active program of corneal transplantation depends upon a constant supply of high quality donor tissue. We describe an effective and inexpensive system of corneal donor retrieval in a major teaching hospital, and present an analysis of its donor population. During the five-month period studied in detail (May to October 1991), 365 deaths occurred in the hospital. The relatives of the deceased were contacted and consent for donation was requested after 323 (88.5%) of these deaths. Consent was given in 212 cases (65.5% of requests), with a mean age of the donor population of 69 years. After screening donor tissue for quality, 110 pairs of corneal buttons and 10 globes of sclera were despatched for surgery during the study period, giving an overall procurement rate of over 30%. Further, the efficient notification and 'on-call' retrieval system led to very rapid corneal retrieval, resulting in an average death to storage medium time of less than two hours.     

The medical assessment of bruising in suspected child maltreatment cases: A clinical perspective

Bruises commonly occur in children and are most often the result of a minor accidental injury. However, bruises can also signal an underlying medical illness or an inflicted injury (maltreatment). Although bruising is the most common manifestation of child physical maltreatment, knowing when to be concerned about maltreatment and how to assess bruises in this context can be challenging for clinicians. Based on current literature and published recommendations, this practice point will help clinicians to distinguish between accidental and inflicted bruises, to evaluate and manage bruising in the context of suspected child maltreatment, and to evaluate for an underlying medical predisposition to bruising.     

The use of surfactant-filled mesoporous silica as an immobilising medium for a fluorescence lifetime pH indicator,providing long-term calibration stability

Surfactant filled mesoporous silica is applied as a matrix for immobilizing the fluorescence lifetime pH-indicator acridine. We demonstrate that this type of encapsulation provides a stable and uniform chemical environment for the indicator and has good proton transport properties leading to rapid pH response times. Furthermore, the immobilising medium effectively prevents leaching of the indicator, facilitates high long-term stability and does not influence the pH sensing-range of the indicator.

A material support is proposed which grants long-term stability for fluorescence lifetime pH measurements due to a homogeneous chemical microenvironment.

The most common pH sensor is the electrochemical sensor. Although electrochemical pH sensors are used for a range of analytical purposes, they have limitations that make them unsuitable for some applications. One obvious application is physiological pH measurements, where e.g. pH is a potential tool for in situ diagnostics.¹ Moreover, electrochemical pH sensors require frequent calibration and are therefore not ideal for environmental applications, such as for water quality monitoring in rivers, the ocean, fish farms or pipelines, where long-term continuous operation is desired and frequent field maintenance is a drawback. Standalone optical sensors comprising membrane-immobilized indicators are attracting an increasing amount of attention for environmental applications due to their higher stability, small size, low power consumption and moderate cost.^2,3Such optical sensors often incorporate fluorophores as pH indicators, where both the fluorescence intensity and lifetime can depend on pH over a limited pH range of about 2–3 pH units about the pK_a of the indicator.⁴ Although such a limited pH sensing range excludes a number of applications, it is sufficient for most environmental and physiological purposes. A drawback with fluorescence intensity-based pH monitoring is that the intensity of the emitted light will also be sensitive to other variables that are difficult to bypass completely, such as leaching of the indicator, photobleaching and variations in the probe light intensity. Fluorescence lifetime (FL), on the other hand, is an intrinsic property of the indicator and will be independent of these durability concerns, providing a sufficient signal-to-noise ratio is maintained.The possibility of a more reliable pH sensor based on FL has been proposed frequently in literature, both as standalone sensors^5,6 and as physiological pH probes when combined with imaging techniques.^1,7 However, a source of uncertainty for such measurements is the dependence of FL on the chemical environment of the indicator, potentially causing instability and uncertainty. For example, in the complicated environment of a living cell, interactions between fluorophores and cell constituents lead to variable FL and unreliable pH measurements.⁷ The FL of an indicator will depend on solvation dynamics, molecular rotation, polarity of the environment, Förster resonance energy transfer and charge transfer, among other things.⁸ Hence, the FL depends not only on pH, but also on the environment where the indicator is located.In the case of standalone sensors, the properties of the film that immobilizes the indicator is therefore of crucial importance for the sensor performance.^5,9 The non-uniform nature of most polymer-based films or coatings leads to additional instability, as slow migration of the indicator between different environments may occur over time. Changes in pH can potentially initiate such migrations.^10,11Sol–gel silica is another common support material used to immobilize indicators.^12–15 However, potential indicators are often non-polar, which restricts the loading capacity in porous silica. Moreover, in absence of covalent bonds, the indicator may interact weakly with the pore surface, which makes leaching an issue for long-term use.The intact surfactant template of mesoporous silica has been proposed previously as host for chemo-sensing molecules in general.^16,17 Here, we present a similar way to encapsulate a FL indicator in order to keep it in a more constant chemical environment. This removes many reliability concerns and allows more accurate external pH calibration. Moreover, non-polar indicators will dissolve in the surfactant non-polar phase, which drastically increases the loading capacity of the material. When used as part of a standalone sensor, the encapsulation could protect the indicator from the influence of other compounds within the membrane that may otherwise influence the pH response characteristics.Acridine is among the few organic fluorophores with a large lifetime variation with pH.^5,18 Hence, this fluorophore was chosen for the study. The protonated and deprotonated forms of this amine have fluorescence lifetimes of 10 and 31 ns, respectively.In the synthesis of the silica, acridine is dissolved in the surfactant (tetradecyltrimethylammonium bromide, TTAB) phase (1 : 10 mole ratio) prior to addition of the silicon alkoxide. The TTAB phase is kept intact in the final powder material. Transmission electron microscopy (TEM) and ²⁹Si magic angle spinning (MAS) NMR was used to characterize the material. The NMR data gives a Q3/Q4 ratio of 0.52, which is similar to e.g. MCM-41 (ESI^†).¹⁹ For comparison, the Q3/Q4 ratios of e.g. MCM-48 (cubic) and MCM-50 (lamellar) are expected around 1.1 and 1.4, respectively.¹⁹ However, the transmission electron micrographs reveal a more amorphous pore structure (ESI^†). A more spherical pore structure is expected as TTAB does not form a liquid crystalline phase similar to that of cetyltrimethylammonium bromide (CTAB) used as a template for MCM41, although the inclusion of acridine may favor a more elongated pore structure. Fig. 1 compares the pH dependency of the fluorescence lifetime of free acridine and acridine immobilized in the surfactant phase of the mesoporous silica material. The data shows that the pH sensing range of the indicator is effectively unchanged by incorporation in the material. A very high acridine loading was achieved, with 3.7 wt% acridine in the dry powder.Open in a separate windowFig. 1Fluorescence lifetime versus pH for acridine (Δ) and acridine in the surfactant phase of the silica (●). Measurements are done in 100 mM phosphate buffers (NaH₂PO₄/Na₂HPO₄ from pH 5.8–8, and Na₃PO₄/BH₃O₃ below pH 5.8).Consequently, the high fluorescence intensity enables a low uncertainty on the lifetime measurements. This particular system will be most accurate in the pH range between pH 5.3 to 7.3, where FL and pH are linearly dependent and a change in pH of 0.1 gives a change in FL of 0.5 ns. The total change in FL between pH 3 and 8 of about 20 ns for acridine meaning that it greatly outperforms other known FL indicators in this pH range, that typically have a maximum change in FL of 1–2 ns.⁸The response in FL to changes in pH occurs quicker than the time it takes to set up and run a measurement, i.e. less than a minute. This observation indicates rapid transport of protons through the material.Due to the high sensitivity of FL to changes in the local chemical environment surrounding the indicator, the most important parameter for a FL support material is long-term stability. Moreover, this stability may change depending on the dispersing solution. The material used to obtain the data in Fig. 2 was stored in either distilled water (Milli-Q), 3.5% NaCl, 100 mM pH 5.8 and pH 8 buffers, and extracted and re-dispersed in a new solution prior to every measured data point. Fig. 2 shows that the measured fluorescence lifetime of the material was stable over one month in 100 mM pH 5.8 and pH 8 buffers, as well as in 3.5% NaCl. However, in Milli-Q water, the FL declines steadily. When dissolved in Milli-Q water without the immobilizing material, the FL of acridine is unchanged with time: a fresh concentrated aqueous solution (0.25 mM) of acridine was compared to one that had been stored for about two years. The FLs were identical within uncertainty (old = 15.22 ns; new = 15.37 ns). Hence, the observed decline in FL is related to the material. Since the material is analyzed in dispersion, electrolytes may facilitate greater material stability due to increased aggregation behavior. However, the reason for the observed instability in Milli-Q water is not fully understood.Open in a separate windowFig. 2Fluorescence lifetime long-term stability of the of the acridine-mesoporous silica material in four different solutions; Milli-Q water, 3.5% NaCl and 100 mM phosphate buffers at pH 5.8 and 8.0. The data points are averages of five consecutive measurements, and the error bars are 2× the standard deviation of these.Note that the lower FLs measured in NaCl (Fig. 2) are due to the presence of chloride; a well-known issue with acridine whereby the fluorescence is quenched by chloride.²⁰ The transport of chloride anions was investigated by adding NaCl solution to the dispersion to give a final NaCl concentration of 100 mM. As with protons, the effect of the chloride ions was observed instantly, indicating that cations and anions transport are equally effective in the material. The chloride caused the FL to drop instantly to around 10 ns when NaCl was added to the dispersion, with marginal dependence on pH (between pH 5.8 and 8.0). This is similar to free acridine in solution,⁵ and reflects that the dynamic quenching occurs for the protonated acridine only. Fluorescence is therefore only observed from the 10 ns lifetime neutral acridine species. Without use of chloride protective membranes, this system will only function as a FL pH indicator in environments with [Cl⁻] below 20 mM.⁵ This excludes use in extracellular environments ([Cl⁻] = 100 mM), but allows intracellular ([Cl⁻] = 4 mM) and blood ([Cl⁻] = 10 mM) applications. For sensor applications, the material can be further protected to prevent Cl⁻ quenching, e.g. by using a film of semipermeable Nafion.⁵ Note that acridine is not quenched by other halogen ions and is therefore not affected by the bromide present in the TTAB phase.²⁰The amount of acridine in the final samples was about 37 mg g⁻¹ material, which gives a TTAB : acridine mole ratio of 12 : 1. This is slightly less than the 10 : 1 ratio of the initial TTAB : acridine mixture. Fig. 3 shows that tumbling a small amount of powder in various solutions causes between 0.3–0.8% of the original acridine present to leach within 15 days. In 3.5% NaCl and pH 8 buffer, no further leaching is detected following the washing procedure in Milli-Q water. In Milli-Q water, the leaching seems to proceed at a rate of about 0.2% per month. When placed in a slightly acidic buffer of pH 5.8, about 0.2% additional acridine leaches, after which no further leaching is detected the following eight days of continued tumbling in the buffer. In fact, the leaching was found to be inversely proportional to pH, where the percentage of remaining acridine after 5 days (%_acr) can be estimated from: %_acr = 0.13 × pH + 98.4 (ESI^†). Still, at any relevant pH value, the leaching will be insignificant regarding fluorescence sensor applications.Open in a separate windowFig. 3Leaching of acridine from the material. The material was tumbled in Milli-Q water for 5 days as part of the washing procedure. Following this, the material was divided in four solutions; Milli-Q water, 3.5% NaCl, 100 mM phosphate buffers of pH 5.8 and 8.0, and tumbled for another ten days.The location of acridine inside the material was evaluated by MAS ¹H NMR. Fig. 4 indicates the changes in chemical shift of the resonances corresponding to protons a–d when acridine is incorporated into the TTAB phase of the mesoporous silica. The most notable changes are a large upfield shift (−0.26 ppm) for proton ‘a’ and a similar downfield shift (+0.26 ppm) for proton ‘b’. This can be rationalized by an interaction between the nitrogen and hydroxyls at the surface. This will alter the bond polarization about the nitrogen which will mostly affect the nuclear shielding of the proton in the same ring (proton a) as well as those in close proximity to the nitrogen (proton b). Acridine is therefore located in the palisade region of the TTAB phase, with the nitrogen pointing towards the pore walls (Fig. 4). This makes the nitrogen accessible to protons diffusing between the pore walls and the TTAB head groups.Open in a separate windowFig. 4 ¹H NMR spectra of acridine (500 MHz) in solution and incorporated in the MCM-41/CTAB material: (A) MAS spectrum (10 kHz MAS) of powder wetted with D₂O. (B) Solution ¹H NMR spectrum. Both spectra are recorded with water suppression and using DSS as chemical shift reference. The changes in chemical shift upon incorporation in the silica material is indicated in the molecular structure of acridine for protons a–d. The location of acridine inside a pore is schematically illustrated.For real applications, the material may need further refinement, such as incorporation into a film or substrate in the case of standalone sensors, or addition of target-specific substituents in the case of physiological pH measurements. An advantage with the system presented here is that the indicator is protected in the interior of the material, and changes to the material exterior will not affect its chemical environment. This particular material can function in several physiological environments, as well as in seawater applications following additional protection from chloride by e.g. Nafion. The material may further be an interesting candidate as host material for other types of molecules, both regarding indicators for pH and other sensor applications, and as a carrier for drugs. One potential application in this respect is for example photodynamic therapy for cancer treatment, where the hydrophobicity of the photosensitizers limits the amount and effect of the light therapy in cancer cells.²¹     

Transfection screening for primary defects in the pyruvate dehydrogenase E1alpha subunit gene

In a significant number of patients with biochemical evidence of a defect in the E1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex, it has not proved possible to identify a mutation in the gene coding regions. To assess the need for more extensive genetic analysis in these patients and to establish a test system in which to study the biochemical consequences of mutations in the E1alpha subunit gene (which is responsible for the great majority of defined cases of pyruvate dehydrogenase deficiency), we have developed a method to screen for E1alpha gene defects based on complementation of the enzyme deficiency in transformed fibroblast cell lines following transfection and expression of the normal cDNA. Using this system, cell lines from patients with a variety of different defined mutations in the E1alpha gene show restoration of enzyme activity. A number of patients have been identified in whom deficient enzyme activity is not restored by expression of the normal cDNA indicating that an alternative explanation for the enzyme defect must be sought.      

Regional changes in vertebra morphology during ontogeny reflect the life history of Atlantic cod (Gadus morhua L.)

This study examined vertebra formation, morphology, regional characters, and bending properties of the vertebral column of Atlantic cod throughout its life cycle (0–6 years). The first structure to form was the foremost neural arch, 21 days post hatching (dph), and the first vertebra centrum to form – as a chordacentrum – was the 3rd centrum at 28 dph. Thereafter, the notochord centra developed in a regular sequence towards the head and caudal fin. All vertebrae were formed within 50 dph. The vertebral column consisted of 52 (± 2) vertebrae (V) and could be divided into four distinct regions: (i) the cervical region (neck) (V1 and V2), characterized by short vertebra centra, prominent neural spines and absence of articulations with ribs; (ii) the abdominal region (trunk) (V3–V19), characterized by vertebrae with wing‐shaped transverse processes (parapophyses) that all articulate with a rib; (iii) the caudal region (tail) (V20–V40), where the vertebra centra have haemal arches with prominent haemal spines; (iv) the ural region (V41 to the last vertebra), characterized by broad neural and haemal spines, providing sites of origin for muscles inserting on the fin rays – lepidotrichs – of the tail fin. The number of vertebrae in the cervical, abdominal and caudal regions was found to be constant, whereas in the ural region, numbers varied from 12 to 15. Geometric modelling based on combination of vertebra lengths, diameters and intervertebral distances showed an even flexibility throughout the column, except in the ural region, where flexibility increased. Throughout ontogeny, the vertebra centra of the different regions followed distinct patterns of growth; the relative length of the vertebrae increased in the cervical and abdominal regions, and decreased in the caudal and ural regions with increasing age. This may reflect changes in swimming mode with age, and/or that the production of large volumes of gametes during sexual maturation requires a significant increase in abdominal cavity volume.     

Cellular infiltration and cytokine mRNA expression in perennial allergic rhinitis

BACKGROUND: Allergen challenge in allergic rhinitis patients leads to local eosinophilia and Th2-type cytokine expression. Natural exposure to grass pollen is additionally characterized by epithelial mast-cell infiltration. We hypothesized that perennial allergic rhinitis is also associated with T-cell and eosinophil infiltration of the nasal mucosa, local Th2-type cytokine expression, and increased numbers of nasal epithelial mast cells. METHODS: Nasal biopsies from perennial allergic rhinitis patients and controls were analysed by immunocytochemistry for different cell populations and in situ hybridization for cytokine mRNA-expressing cells. RESULTS: Perennial allergic rhinitis was associated with increased numbers of submucosal CD3+ T cells (P=0.05), EG2+ activated eosinophils (P=0.01), and CD68+ macrophages (P=0.01) compared to controls. Epithelial, but not submucosal, tryptase-positive mast cells were also elevated in rhinitics compared to controls (P=0.01). The numbers of cells expressing interleukin (IL)-5 were higher (P=0.01) and the numbers of cells expressing IL-2 were lower (P=0.04) in rhinitic patients than controls. There were no significant differences for either IL-4 or interferon-gamma between the groups. CONCLUSIONS: Perennial allergic rhinitis is characterized by mast-cell migration into the epithelium; submucosal infiltration by T cells, eosinophils, and macrophages; and an imbalance in local T-cell cytokine production in favour of enhanced IL-5 and reduced IL-2 expression.