High reliability in digital planning of medial opening wedge high tibial osteotomy, using Miniaci's method.

PURPOSE: Pre-operative planning is essential in high tibial osteotomy (HTO). Miniaci's method employs Mikulicz's weight-bearing line and is advantageous because the point of mechanical loading is related to the known degenerative condition of the knee. Miniaci's geometrical method has been modified for an opening wedge and described for use with a digital picture archiving and communications system viewer. Reliability for this method was hypothesised to be equivalent to published reliability for landmark-based commercial software and independent of observer experience. METHODS: Twenty-four patients awaiting HTO had standardised long-leg radiographs. Mikulicz's weight-bearing line was projected through the lateral compartment of the knee at Fujisawa's point. The correction angle was generated at the hinge point subtending the current and proposed ankle centres. The opening wedge was plotted to measure an opening distance. Observations were recorded twice by three observers. Agreement was reported as intraclass correlation coefficients with 95 % confidence intervals. RESULTS: Intra-rater agreement was excellent for the correction angle (0.965-0.985) and opening distance (0.928-0.980). If no set hinge point was used, then the inter-rater reliability was 0.986 for the correction angle and 0.984 for the opening distance. There was no discernible pattern demonstrating improved reliability from the experienced observer. CONCLUSIONS: Reliability is comparable to commercially based landmark software and independent of observer experience. This makes such geometrical pre-operative planning accessible to surgeons who perform HTO with insufficient frequency to justify the investment in commercial software. LEVEL OF EVIDENCE: Diagnostic study, Level II.

A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers.

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [(18)F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [(18)F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [(18)F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.

Hurling-related hand injuries.

Hurling is a contact sport, associated with significant morbidity. We have identified specific hand injuries sustained by participants and quantified the functional and financial implications of these injuries. Over a 3-month period, all hand injuries seen in the fracture clinic of our regional trauma unit were studied prospectively. Of the 123 consecutive injuries, 41 (33%) were sustained during hurling matches. Metacarpal (47%) and proximal phalangeal (37%) fractures were the most frequent. Eight hurlers (20%) required surgical intervention. Only four (10%) of the injured players were wearing hand protection. The mean cost of injury to the player was pound 615. We suggest the introduction of the mandatory use of hand protection for hurling.

Magnetic resonance microscopy of morphological alterations in mouse trabecular bone structure under conditions of simulated microgravity.

This work describes the use of magnetic resonance (MR) microscopy to examine changes in tibial trabecular bone structure in mice following 28 days of hindlimb suspension, a model simulating the effects of microgravity in rodents. In this first MR study involving mice, analysis of 3D images showed that apparent bone volume fraction, trabecular number, and trabecular thickness were decreased, and apparent trabecular spacing increased, significantly (P < 0.05) in hindlimb-suspended mice compared to controls. These changes agreed well with light microscopy measurements from an independent study and also with actual spaceflight experiments with rats.

The production of novel sordarin analogues by biotransformation.

The biotransformation of the fungal protein synthesis inhibitor sordarin is reported. Nine taxonomically diverse organisms supported the isolation and identification of twelve modified products. The structural diversity of the biotransformation products observed and their value in supporting further chemistry is discussed.

Cofactor recycling with immobilized heterologous cytochrome P450 105D1 (CYP105D1).

Immobilisation of cells and enzymes can be a convenient and rapid way for testing and transforming substances. Cytochromes P450 may be useful in numerous biotransformations of varied lipophilic substrates, performing both regio- and stereo-specific monooxygenation reactions. However, one limitation of their use in vitro is the requirement of cofactor for the supply of electrons in the catalytic cycle. Here we report CYP105D1 from Streptomyces griseus expressed in Escherichia coli can be immobilised from cell-free extracts using DE52, that the immobilised protein is active in bioconversions and that a requirement for cofactor can be sustained by a recycling system for NADH regeneration.

Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

Accuracy of 1H and 31P MRS analyses of lactate in skeletal muscle.

As the end product of anaerobic metabolism and a source of H(+), lactic acid is important in metabolism and pH regulation. Several methods have been introduced to calculate changes in the lactate anion (Lac(-)) concentration in exercising skeletal muscle from information derived from the (31)P spectrum. Alternatively, Lac-may be observed directly with (1)H MRS. Both (1)H and (31)P spectroscopy have potential problems, which could prevent accurate determination of [Lac(-)]. It is demonstrated that quantitatively accurate (1)H MRS measurements of changes in [Lac(-)] due to exercise are possible in isolated muscle. In general, calculation by (31)P MRS overestimates Lac-production. An analysis is presented of possible sources of errors in the (1)H and (31)P MRS methods.

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase.

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.

Alkaline biocatalysis for the direct synthesis of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).

Integration between the alkaline epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-Acetyl-D-mannosamine (ManNAc) and the N-acetyl-D-neuraminic acid (Neu5Ac) aldolase-catalyzed biotransformation has been assessed experimentally. GlcNAc epimerization took place above pH 9.0, and the initial rate of ManNAc formation increased exponentially to 10.37 mmol/L per hour at pH 12. However, above this pH, severe degradation of pyruvate occurred. A value of 31.3% molar conversion on Pyr was achieved in an integrated biotransformation. The "pseudo"-steady state at the end of the reaction was comparable to the equilibrium achieved with a combination of an epimerase and aldolase enzymes. The integrated reaction proved feasible, but at the expense of pyruvate and Neu5Ac aldolase degradation.

Lactate quantitation in a gerbil brain stroke model by GSLIM of multiple-quantum-filtered signals. Generalized spectral localization by imaging.

Quantitative magnetic resonance imaging of lactate using a zero-quantum/double-quantum filter and generalized spectral localization by imaging (GSLIM) was applied to a model of unilateral stroke in gerbil brain. GSLIM lactate images at 4T clearly reveal elevated concentrations of lactate in the ischemic compared with the normal hemisphere 100-175 minutes after unilateral carotid ligation. These results indicate that the technique is capable of studies of brain infarcts, and that application to human ischemic pathology in brain and other tissues may be possible.

The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations.

IFI 16 is an interferon-inducible nucleoprotein expressed by human monocytes. IFI 16 and a related mouse protein, p202, control cellular proliferation by binding and modulating the functions of cell cycle regulatory factors including p53 and the retinoblastoma gene product, pRb. In this study, we examined IFI 16 expression in myeloid precursor cells cultured in vitro in colony-forming assays using granulocyte (G-) and granulocyte-macrophage (GM-) colony-stimulating factor (CSF). IFI 16 was expressed in 100% of CD34+ cells isolated from human bone marrow. When the CD34+ cells were induced to differentiate, two sub-populations of cells were identified by two-color cytofluorography: the CD14+ (monocytoid) cells all expressed IFI 16, whereas the CD14- (polymorphonuclear precursor) cells did not. The strongest expression of IFI 16 was in the cells staining brightest for CD14, whereas depletion of CD14+ monocytoid cells from mixed monocytic/granulocytic cultures largely abolished IFI 16-stained cells. Furthermore, in eight independent colony-forming assays, the number of IFI 16+ cells correlated closely with the numbers of monocyte precursors identified morphologically (R2 = 0.99), but was unrelated to the numbers of myelocytes, promyelocytes, and metamyelocytes; nor was IFI 16 expressed by erythroid or eosinophil precursors. We conclude that IFI 16 is expressed in CD34+ and monocytoid daughter cells, but is rapidly and markedly down-regulated at the corresponding stages of polymorphonuclear and erythroid development. This differential expression of IFI 16 in myeloid precursor subpopulations and its perceived molecular properties are consistent with a possible role in regulating myelopoiesis.

High-performance liquid chromatographic assay for the novel antitumor drug, bryostatin-1, incorporating a serum extraction technique.

An HPLC assay incorporating a solid-phase extraction technique has been devised for bryostatin-1. Quantitation of bryostatin was found to be linear over the concentration range 0.012-25 microg/ml (0.2-25 ng on column) and was found to have a limit of detection of 0.2 ng on column, with a correlation coefficient of 0.9999. Following extraction of bryostatin over a range of concentrations from horse serum (0.012-25 microg/ml) and human serum (0.01-0.32 microg/ml) using a 100-mg C18 solid-phase extraction cartridge, extraction efficiencies consistently greater than 90% were obtained for extraction from horse serum and varied between 57 and 85% from human serum. However, on extending this work to blood samples from patients undergoing therapy with bryostatin-1, the drug was not detectable even at the maximum dose given, demonstrating the rapid loss of this agent from peripheral circulation.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

The temperature dependence of intracellular pH in isolated frog skeletal muscle: lessons concerning the Na(+)-H+ exchanger.

We used 31P NMR to investigate the temperature-dependence of intracellular pH (pHi) in isolated frog skeletal muscles. We found that Ln[H+i] is a linear function of 1/Tabs paralleling those of neutral water (i.e., H+ = OH-) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na(+)-H+ exchange shifted the intercept in the alkaline direction but had not effect on the slope. Acid loading followed by washout resulted in an amiloride-sensitive return to the (temperature dependent) basal pHi. These results show that the temperature dependence of activation of Na(+)-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/ [OH-] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point of the Na(+)-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (delta H degree) under both control and insulin-stimulated conditions that is similar to the delta H degree of the intracellular buffers and approximately half of the delta H degree for the dissociation of water. Thus, the temperature-dependent component of the standard free-energy change (delta F degree) is unaffected by insulin stimulation, suggesting that changes in Arrhenius activation energy (Ea) may not be a part of the mechanism of hormone stimulation.

Novel inhibitors of fungal protein synthesis produced by a strain of Graphium putredinis. Isolation, characterisation and biological properties.

The isolation and structure determination of 6 analogues of the fungal protein synthesis inhibitor GR135402, from Graphium putredinis, is described. The relative potencies of the compounds as protein synthesis inhibitors and as in vitro antifungal agents provide interesting insights into the structure-activity relationships in this series.

Quantitative lactate-specific MR imaging and 1H spectroscopy of skeletal muscle at macroscopic and microscopic resolutions using a zero-quantum/double-quantum coherence filter and SLIM/GSLIM localization.

Quantitative lactate imaging and spectroscopy were performed on phantoms and on electrically stimulated, excised frog skeletal muscle at macroscopic and microscopic resolutions. Lactate selectivity was achieved by use of a zero-quantum/double-quantum coherence (ZQC/DQC) lactate filter, which suppressed all signals besides lactate, including water and lipid, to below noise level. Three-dimensional lactate data sets were acquired in 1-3 h; one of these spatial dimensions was frequency-encoded and the other two were phase-encoded. High-resolution images were reconstructed using the spectral localization by imaging (SLIM) and generalized SLIM (GSLIM) techniques. Lactate quantitation was achieved by employing an external lactate concentration standard and was verified by comparison to quantitative STEAM-localized and nonlocalized spectra that used total creatine as an internal concentration reference. Additionally, quantitatively accurate behavior of the SLIM and GSLIM techniques as applied to data sets of low signal-to-noise ratio and to macroscopically heterogeneous objects was verified using simulations and real muscle lactate data sets with known heterogeneity.

Biochemical heterogeneity in hysterectomized uterus measured by 31P NMR using SLIM localization.

Ultrasound and magnetic resonance imaging show contrast between the inner and outer myometrium, which is useful in the diagnosis of gynecological disorders. To determine whether the image contrast is associated with biochemical differences between these myometrial regions, phosphorus metabolite concentrations in the inner one third of the myometrium (the junctional zone; JZ) were compared with the outermost one third of the myometrium (OM) in hysterectomized uteri using 31P spectral localization by imaging (SLIM). The technique was validated by comparing the results of SLIM with the results of standard Fourier-encoded spectroscopic imaging (FSI) analysis using phantoms, and by nonlocalized spectroscopy on biopsies taken from the same hysterectomy specimens. As expected theoretically, SLIM yielded better localization than FSI, as judged by spectral intensity and leakage measurements on phantom compartments of known composition. SLIM localization revealed that the JZ has a higher intracellular phosphomonoester (PME) concentration than does the OM, which was confirmed by nonlocalized spectroscopy, and that there is very little NMR-visible phosphorus in the cervix.

An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase.

N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation. Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac. The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction. Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach. This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin. In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc. ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate. Neu5Ac can be isolated from such reaction mixtures by a simple crystallization. This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.