A novel biosensor for quantitative monitoring of on-target activity of paclitaxel.

This study describes a system for quantifying paclitaxel activity using the C-terminus of α-tubulin as a biomarker. Following stabilization of microtubules with paclitaxel, a specific detyrosination reaction occurs at the C-terminus of α-tubulin which could be used to assess efficacy. A fluorescence resonance energy transfer (FRET) based biosensor was synthesized comprising a short peptide that corresponded to the C-terminus of α-tubulin, a fluorophore (Abz), and a quencher (Dnp). The fluorophore added to the end of the peptide can be released upon enzymatic detyrosination. In addition, a single fluorophore-tagged peptide was also conjugated to mesoporous silica nanoparticles to examine the feasibility of combining the drug with the peptide biomarker. As a proof of concept, we found that the degree of peptide cleavage, and therefore enzymatic activity, was directly correlated with exogenous bovine carboxypeptidase (CPA) an enzyme that mimics endogenous detyrosination. In addition, we show that cell lysates obtained from paclitaxel-treated cancer cells competed with exogenous CPA for biosensor cleavage in a paclitaxel dose-dependent manner. Our work provides strong evidence for the feasibility of combining paclitaxel with a novel biosensor in a multi-load nanoparticle.

Delayed ulnar nerve palsy secondary to ulnar artery pseudoaneurysm distal to Guyon's canal following penetrating trauma to the hand.

Compression of the ulnar nerve in Guyon's canal is an uncommon phenomenon. Reports of ulnar nerve palsy secondary to ulnar artery pseudoaneurysm at this anatomical location are very rare and equivalent pathology just distal to this site is unheard of. Here we present such a case, which featured a delayed onset of symptoms. This followed penetrating trauma to the hand. Our methods for diagnosis, operative planning and surgical treatment are included.

Electrospray deposited fibronectin retains the ability to promote cell adhesion.

Scaffolds for tissue engineering require the correct biochemical cues if the seeded cells are to migrate into the scaffold and proliferate. For complex tissues this would require precise patterning of the scaffold structure with the particular biochemical cue required at each location on the scaffold. Electrospray enables the deposition of a wide number of biomolecules onto surfaces and can be used for precise patterning. We assessed the functionality of a key cell-adhesion molecule, fibronectin, after depositing it onto a surface using the electrospray technique. The addition of polypropylene glycol allowed a stable spray to be obtained from solutions with a range of fibronectin concentrations. Immunoassay tests showed that the amount of fibronectin retained on the surface was proportional to that sprayed from the solution. Increasing the surface density of fibronectin deposited onto silicon surfaces enhanced fibroblast attachment. The fibronectin thus appears to have retained its cell attachment functionality after undergoing the electrospray process. Since recent advances allow electrospray to pattern material from solution with micrometre accuracy this may allow materials to be biologically functionalized on a similar scale.

Preparation and characterization of a magnetic and optical dual-modality molecular probe.

Multi-modality imaging probes combine the advantages of individual imaging techniques to yield highly detailed anatomic and molecular information in living organisms. Herein, we report the synthesis and characterization of a dual-modality nanoprobe that couples the magnetic properties of ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) with the near infrared fluorescence of Cy5.5. The fluorophore is encapsulated in a biocompatible shell of silica surrounding the iron oxide core for a final diameter of approximately 17 nm. This silica-coated iron oxide nanoparticle (SCION) has been analyzed by transmission electron microscopy, dynamic light scattering, and superconducting quantum interference device (SQUID). The particle demonstrates a strong negative surface charge and maintains colloidal stability in the physiological pH range. Magnetic hysteresis analysis confirms superparamagnetic properties that could be manipulated for thermotherapy. The viability of primary human monocytes, T cells, and B cells incubated with the particle has been examined in vitro. In vivo analysis of agent leakage into subcutaneous A431 tumors in mice was also conducted. This particle has been designed for diagnostic application with magnetic resonance and fluorescence imaging, and has future potential to serve as a heat-sensitive targeted drug delivery platform.

Growth and morphology control of carbon nanotubes at the apexes of pyramidal silicon tips.

We describe the development of catalysed chemical vapour deposition (cCVD) growth schemes suitable for the production of carbon nanotube atomic force microscopy (CNT-AFM) probes. Growth and sample processing conditions are utilized that both incorporate safety in the process, e.g. the use of ethanol (EtOH) vapour as a carbon feedstock and hydrogen at only 4% (flow proportion), and simplicity, e.g. no catalyst patterning is required. Cobalt is employed as the growth catalyst and thin films of aluminium on silicon as the substrate material. Purpose-fabricated silicon substrates containing large numbers of tip structures are used as models of AFM probes. This enables growth to be carried out on many tips at once, facilitating a thorough investigation of the effect of different growth schemes on yields. cCVD growth schemes are chosen which produce stabilizing high density networks of carbon nanotubes on the sidewalls of the pyramidal tips to aid in anchoring the apex protruding carbon nanotube(s) in place. This results in long-lasting AFM imaging tips. We demonstrate that through rational tailoring of cCVD conditions it is possible to tune the growth conditions such that CNTs which protrude straight from tip apexes can be obtained at yields of greater than or equal to 78%. Application of suitable growth schemes to CNT growth on commercially available AFM probes resulted in CNT-AFM probes which were found to be extremely useful for extended lifetime metrological profiling of complex structures.

Immuno-analysis of phospho-Akt in primary human breast cancers.

The aim of this study was to investigate the expression of the activated (phosphorylated) form of Akt (Ser473) in primary breast cancer and to correlate the results with clinicopathological and prognostic variables for clinically relevant associations. Phospho-Akt expression was studied using immunoblot analysis in 49 invasive breast carcinomas (median follow-up time 55 months, range 7-74 months). We assessed the level of phospho-Akt in different types of primary breast cancers and compared the use of autoradiograph X-ray film with a PVDF-DAB-staining system. Twelve percent of the tumours had no phospho-Akt protein, 25% had low phospho-Akt expression, 51% had intermediate expression and 12% had high phospho-Akt expression. No relationship was observed between phospho-Akt and tumour grade, tumour size or nodal status. A significant relationship was demonstrated between phospho-Akt score and oestrogen receptor status (P=0.014). Univariate analysis demonstrated that intermediate levels of phospho-Akt in breast tumour tissue are associated with a lower probability of developing recurrences (P=0.035), while in multivariate analyses, none of the phospho-Akt levels appeared to be independent predictors of disease recurrence or death. In addition, it has been clearly established that a suitable composition of reagents and components such as PVDF membranes treated with DAB substrate will enable the performing of sensitive immuno-analyses.

Mass spectrometry tools and metabolite-specific databases for molecular identification in metabolomics.

The chemical identification of mass spectrometric signals in metabolomic applications is important to provide conversion of analytical data to biological knowledge about metabolic pathways. The complexity of electrospray mass spectrometric data acquired from a range of samples (serum, urine, yeast intracellular extracts, yeast metabolic footprints, placental tissue metabolic footprints) has been investigated and has defined the frequency of different ion types routinely detected. Although some ion types were expected (protonated and deprotonated peaks, isotope peaks, multiply charged peaks) others were not expected (sodium formate adduct ions). In parallel, the Manchester Metabolomics Database (MMD) has been constructed with data from genome scale metabolic reconstructions, HMDB, KEGG, Lipid Maps, BioCyc and DrugBank to provide knowledge on 42,687 endogenous and exogenous metabolite species. The combination of accurate mass data for a large collection of metabolites, theoretical isotope abundance data and knowledge of the different ion types detected provided a greater number of electrospray mass spectrometric signals which were putatively identified and with greater confidence in the samples studied. To provide definitive identification metabolite-specific mass spectral libraries for UPLC-MS and GC-MS have been constructed for 1,065 commercially available authentic standards. The MMD data are available at http://dbkgroup.org/MMD/.

Glomus tumor and knee pain: a report of four cases.

We present four cases of glomus tumors presenting as knee pain. All cases were treated by surgical excision of the tumor. All patients made an immediate recovery with return to full normal function. The presentation of this is unique in that the patient has exquisite pain and tenderness when the area affected is palpated. Occasionally, local infiltration or an ischaemia test can assist with diagnosis. To our knowledge, this is the largest case series in the literature.

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide.

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.

Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica.

Ultra-small superparamagnetic iron oxide nanoparticles (SPIOs) were synthesized by co-precipitation of iron chloride salts with ammonia and then encapsulated with thin (~2nm) layers of silica. The particles have been characterized for size, diffraction pattern, surface charge, and magnetic properties. This rapid and economical synthesis has a number of industrial applications; however, the silica-coated particles have been optimized for use in medical applications as MR contrast agents, biosensors, DNA capturing, bioseparation and enzyme immobilization.

Intraoperative red blood-cell salvage in revision hip surgery. A case-matched study.

BACKGROUND: Revision hip arthroplasty is commonly associated with substantial blood loss and the subsequent need for transfusion. This leads to an increased risk of blood-borne infection and hemolytic reactions. The purpose of this study was to demonstrate whether the use of intraoperative red blood-cell salvage in revision hip arthroplasty reduces the overall rate of allogeneic transfusion. METHODS: Forty-seven patients who had undergone revision hip arthroplasty with the use of intraoperative cell salvage were identified. A computer database was used to individually match these patients, for age, sex, and eleven operative variables, to control patients who had undergone revision hip arthroplasty in the same unit without intraoperative cell salvage. Data gathered included the total allogeneic transfusion requirement for each patient, preoperative and postoperative hemoglobin levels, and operative time. RESULTS: The total allogeneic transfusion requirement was significantly lower in the group that had intraoperative cell salvage than in the control group (median, 2 compared with 6 U of packed red blood cells, p = 0.0006), with a median reduction in allogeneic transfusion of 4 U. There was no significant difference in preoperative or postoperative hemoglobin levels between the groups. CONCLUSIONS: The use of intraoperative cell salvage significantly lowered the allogeneic transfusion requirement, which can lead to substantial cost savings. To our knowledge, this is the first study in which the use of intraoperative red blood-cell salvage in revision hip arthroplasty was evaluated by matching patients on the basis of age, sex, and operative variables.

National comparative audit of the use of platelet transfusions in the UK.

The objective of this national audit was to examine the use of platelet transfusions against audit standards developed from national guidelines. Hospitals were asked to provide data on 40 consecutive patients receiving platelet transfusions (15 haematology patients, 10 cardiac, 10 critical care and five in other clinical specialties). One hundred and eighty-seven UK hospitals participated, including 168/263 (64%) hospitals in England. A total of 4421 patients receiving platelet transfusions were audited. The reason for transfusion was documented in the medical records for 93% of transfusions and 57% were used for prophylaxis (in the absence of bleeding). Overall 3726/4421 (84%) of the transfusions were evaluable and 43% (1601/3726) were found to be non-compliant with the audit standards. A major non-compliance was failure to measure the platelet count before transfusion (29% of transfusions). Other non-compliances included the use of platelet transfusion in the absence of bleeding in 11% of cardiac surgery patients receiving platelet transfusions, the use of a threshold platelet count more than 10 x 10(9)/L for 60% of prophylactic platelet transfusions in haematology patients without risk factors indicating the need for a higher threshold, and a threshold platelet count more than 30 x 10(9)/L for 59% of prophylactic platelet transfusions in critical care. The reasons for the high rate of non-compliance were not explored in this audit, but this is a topic worthy of further study. The main recommendations were that hospitals should ensure there are written local guidelines for platelet transfusions, clinicians must be provided with training about their appropriate use, and hospitals should carry out regular audits of practice. More research should be carried out to develop the evidence base for the use of platelet transfusions, more detailed guidelines should be developed for platelet transfusions in critical care and cardiac surgery, and the audit should be repeated in about three years.

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria.

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.

Anthocyanins from red wine--their stability under simulated gastrointestinal digestion.

The stability of anthocyanins from red wine was assessed using an in vitro digestion system that simulated the physiochemical changes that occur in the upper gastrointestinal tract. Anthocyanins in red wine were stable to gastric conditions whereas there was a small loss in total phenol content. After pancreatic digestion, the total anthocyanins were very poorly recovered compared to the bulk phenols in the IN sample, which was previously described as the "serum-available" fraction, and the majority of the anthocyanins and phenols were recovered in the OUT fraction, previously described as the "colon-available" fraction. Removing alcohol from the wine samples prior to the procedure did not markedly affect this pattern. The composition of anthocyanins in the post gastric, IN and OUT samples was analysed using liquid chromatography mass-spectrometry. The red wine used contained over 20 identifiable anthocyanins of which the main components were 3-O-glucosides of malvidin, peonidin, petundin, delphidin and cyanidin. Coumaroylated-glucoside derivatives of malvidin, petundin, peonidin, and delphinidin were observed and acetylated glucosides of peonidin, petundin and malvidin were also identified. Anthocyanins with modified aglycones similar to vitisin A derivatives of delphinidin, peonidin, petunidin and malvidin were also identified. After the in vitro digestion procedure, only five anthocyanins could be detected in the IN (serum-available) and the OUT (colon-available) fractions, which were confirmed as malvidin-3-O-glucoside and the vitisin A adducts of malvidin-3-O-glucoside, malvidin-3-O-acetylglucoside, malvidin-3-O-coumaroylglucoside and peonidin-3-O-glucoside. Malvidin-3-O-glucoside was recovered at 0.2% in the IN fraction and 0.9% in the OUT fraction. However, the vitisin derivatives were much more stable to pancreatic digestion. Assuming that the vitisin A derivatives display similar biological properties to their parent anthocyanins, their enhanced gastrointestinal stability could lead to enhanced bioavailability and bio-effectiveness in vivo.

An isolated osteochondroma with underlying non-Hodgkin's lymphoma of bone.

Osteochondroma is the most common benign bone tumour. The risk of sarcomatous change in an isolated lesion is approximately 1%. We report a case of an isolated osteochondroma which appeared benign on clinical and plain radiographic examination but routine histological analysis revealed non-Hodgkin's lymphoma in the underlying bone. This association has not previously been reported and the case emphasises the importance of routine histological analysis, even if a lesion appears benign.

Arthrofibrosis following total knee replacement; does therapeutic warfarin make a difference?

Arthrofibrosis following total knee replacement (TKR) is a relatively common complication which results in a reduction in knee range of movement and patient dissatisfaction. A retrospective study examined the relationship between anticoagulation with therapeutic warfarin and rates of arthrofibrosis following TKR. Arthrofibrosis was defined as less than 80 degrees of knee flexion 6-8 weeks post-TKR. Patients were warfarinised if they had a history of thrombophilic tendencies or medical conditions necessitating anti-coagulation, rather than as routine thromboprophylaxis. All other patients received thromboprophylaxis using low molecular weight heparin. A total of 728 patients underwent 874 primary TKR between 1993 and 2002 in one centre, performed by four surgeons. Mean age was 68 years (range 48-89 years) and there were 483 female and 391 male knees. Eighty cases were warfarinised post-operatively (53 female, 27 male). Overall, 83 of 874 TKRs (9%) had arthrofibrosis (57 female, 26 male) requiring manipulation under anaesthetic (MUA). In the warfarinised group, 21 knees (26%) had an MUA (15 female, 6 male). This compared to 62 cases (8%) requiring MUA in the non-warfarinised group (42 female, 20 male). There was a statistically significant difference on Fisher's exact testing (P<0.0001) between groups. Following MUA, knee flexion improved in 95% cases to a minimum 95 degrees but 8 cases had a fixed flexion deformity of 5-10 degrees . In conclusion, therapeutic warfarinisation post-TKR leads to a statistically greater chance of the patient developing arthrofibrosis compared to prophylactic low molecular weight heparin and that patients should be counseled appropriately.

Patient-perceived outcome measures following unicompartmental knee arthroplasty with mini-incision.

We reviewed 150 patients (183 knees) who underwent mini-incision unicompartmental knee arthroplasty (Oxford). Mean age was 71.5 (36-92) years. Review was conducted at least 12 months following surgery. To assess results, we used the Oxford knee questionnaire, modified Grimby score, return to sport and work, knee "normality" and patient general health. The mean Oxford knee score was 22.17 (range 12-54). Kneeling scored worse than other activities. No significant age or gender difference was found. Mean modified Grimby score was 3.89, equating to moderate exercise less than 2 h a week. Patients with "artificial-feeling" knees had significantly worse scores than patients with normal/near-normal-feeling knees. Patients who returned to/increased sporting activity had better Oxford scores than those who did not. Ninety-four percent of patients working pre-operatively returned to work. Sixty-seven percent continued at the same level of or increased sporting activity. Oxford knee scores and return to sport compared well to published data. Results regarding modified Grimby score, return to work and pain relief were encouraging. The best results were achieved in active patients who felt their health was good and their knee felt normal or near normal following surgery.