Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

Si1-xGex anode synthesis on plastic films for flexible rechargeable batteries.

SiGe is a promising anode material for replacing graphite in next generation thin-film batteries owing to its high theoretical charge/discharge capacity. Metal-induced layer exchange (LE) is a unique technique used for the low-temperature synthesis of SiGe layers on arbitrary substrates. Here, we demonstrate the synthesis of Si1-xGex (x = 0-1) layers on plastic films using Al-induced LE. The resulting SiGe layers exhibited high electrical conductivity (up to 1200 S cm-1), reflecting the self-organized doping effect of LE. Moreover, the Si1-xGex layer synthesized by the same process was adopted as the anode for the lithium-ion battery. All Si1-xGex anodes showed clear charge/discharge operation and high coulombic efficiency (≥ 97%) after 100 cycles. While the discharge capacities almost reflected the theoretical values at each x at 0.1 C, the capacity degradation with increasing current rate strongly depended on x. Si-rich samples exhibited high initial capacity and low capacity retention, while Ge-rich samples showed contrasting characteristics. In particular, the Si1-xGex layers with x ≥ 0.8 showed excellent current rate performance owing to their high electrical conductivity and low volume expansion, maintaining a high capacity (> 500 mAh g-1) even at a high current rate (10 C). Thus, we revealed the relationship between SiGe composition and anode characteristics for the SiGe layers formed by LE at low temperatures. These results will pave the way for the next generation of flexible batteries based on SiGe anodes.

Community pharmacists' measurement of health-related quality of life in outpatients taking high-risk drugs.

Patients experiencing severe side effects when taking high-risk drugs may have a significantly reduced health-related quality of life (QOL); therefore, it is important to identify changes in the health-related QOL in these patients. This study aimed to determine the health-related QOL in community pharmacy outpatients taking high-risk drugs. This prospective observational study was conducted in 29 community pharmacies with 71 pharmacists in 12 regions and cities in Japan from October to December 2020 and 760 patients were enrolled. Using descriptive questionnaires of EuroQOL-5-dimensions-5-levels (EQ-5D-5L), community pharmacists obtained health-related QOL data from outpatients taking high-risk drugs. The mean health-related QOL of all outpatients was 0.869. The health-related QOL decreased with increasing age. The outpatient health-related QOL was 0.700, 0.763, 0.785, and 0.817 when taking antiepileptic, antidepressant, digitalis, and antiarrhythmic drugs, respectively, which was lower than the average health-related QOL of all outpatients. Mobility and pain/ discomfort accounted for a large proportion of the decline in the health-related QOL with increasing age. There were no significant differences in personal care with increasing age; however, the number of outpatients with mobility, normal activity, and pain challenges decreased with age. In contrast, outpatients aged <65 years with anxiety/depression showed a lower than overall average health-related QOL. To the best of our knowledge, this is the first study in Japan to report an investigation by community pharmacists regarding health-related QOL assessment in outpatients taking high-risk drugs.

Panel of novel urine biomarkers for incident microalbuminuria in people with type 2 diabetes mellitus.

AIM: The need to identify novel biomarkers for early diagnosis and treatment of diabetic nephropathy is widely recognized. However, only a few studies have investigated the association between biomarkers and the onset of albuminuria. In this study, we aimed to investigate a panel of biomarkers suitable for predicting microalbuminuria. METHODS: Some 346 Japanese people with type 2 diabetes exhibiting normoalbuminuria were studied. The endpoint was defined as the onset of microalbuminuria. Thirty biomarkers were selected from among urinary biomarkers described in previous studies. A panel of biomarkers was selected using least absolute shrinkage and selection operator (LASSO). The prognostic performance of the developed panel was evaluated. RESULTS: During a mean follow-up of 6.2 years, 45 people progressed to microalbuminuria. A composite panel of six biomarkers (monocyte chemoattractant protein-1, osteopontin, soluble human tumour necrosis factor receptor-1, tenascin C, vascular endothelial growth factor-A and kidney injury molecule-1) was developed using LASSO. Compared with the basal model comprising estimated GFR and urinary albumin-to-creatinine ratio, addition of these six biomarkers significantly increased the overall C index from 0.773 to 0.824 (P = 0.019). Net reclassification improvement and integrated discrimination improvement were estimated to be 0.412 (P = 0.049) and 0.040 (P = 0.040), respectively. Decision curve analysis also showed that the model with the six novel biomarkers had a better prognostic value for predicting the onset of microalbuminuria. The optimism was moderate or negligible according to measures. CONCLUSIONS: The panel consisting of six novel urinary biomarkers effectively predicted incident microalbuminuria in people with type 2 diabetes.

Subjective Evaluation of Denture Adhesives: A Multicenter Randomized Controlled Trial.

INTRODUCTION: Many reports show that denture adhesives improve the retention and stability of dentures. However, few randomized controlled trials have examined the effects of denture adhesives. OBJECTIVE: This 10-center randomized controlled trial with parallel groups involving 200 edentulous patients wearing complete dentures aimed to evaluate the effects of short-term use of cream and powder denture adhesives. METHODS: Patients were allocated into 2 cream- and powder-type adhesive groups and 1 control group. Intervention groups were treated with the 2 adhesives (1 each), and the control group received saline solution. Adhesive or control was applied to the denture-mucosal surface for 4 d, and data at baseline and after day 4 of intervention (i.e., 8 meals) were obtained. Patient satisfaction was evaluated with a 100-mm visual analog scale. Oral health-related quality of life was measured with the Japanese version of the Oral Health Impact Profile for Edentulous Patients. Perceived chewing ability was evaluated by a questionnaire regarding ease of chewing and swallowing food. Between-group comparisons were performed with Kruskal-Wallis tests with the Mann-Whitney U test adjusted by Bonferroni correction. Within-group comparisons of pre- and postintervention measurements were performed with the Wilcoxon signed-rank test. Intention-to-treat analysis was also performed. RESULTS: Between-group comparisons showed no significant differences for general satisfaction or Oral Health Impact Profile for Edentulous Patients. However, significant differences in satisfaction with various denture functions with cream- and powder-type adhesives were seen in pre- and postintervention comparisons (P < 0.05). Significant differences were also observed for perceived chewing ability of hard foods (P < 0.05). CONCLUSION: These results suggest that although denture adhesives do not invariably improve denture function, they do affect subjective evaluations and possibly chewing of hard foods. Therefore, the effects of denture adhesive use are insufficient to resolve any fundamental dissatisfaction with dentures ( ClinicalTrials.gov NCT01712802 ). KNOWLEDGE TRANSFER STATEMENT: The results of this study suggest that denture adhesives should be applied under certain conditions; however, an appropriate diagnosis is important before application. These practice-based data provide information to establish evidence-based guidelines for applying denture adhesives.

Development of a microwave ion source for ion implantations.

A microwave ion source is expected to have a long lifetime, as it has fewer consumables. Thus, we are in the process of developing a microwave ion source for ion implantation applications. In this paper, we report on a newly developed plasma chamber and the extracted P(+) beam currents. The volume of the plasma chamber is optimized by varying the length of a boron nitride block installed within the chamber. The extracted P(+) beam current is more than 30 mA, at a 25 kV acceleration voltage, using PH3 gas.

YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes.

OBJECTIVE: YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. SUBJECTS AND METHODS: The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. RESULTS: YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. CONCLUSIONS: GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis.

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

BACKGROUND AND OBJECTIVE: Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. MATERIAL AND METHODS: To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. RESULTS: Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. CONCLUSION: We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.

Development of microwave ion source for industrial applications.

A microwave ion source is one of the long-life ion sources. In this paper, we report on the characteristics of the extracted Ar ion beam produced by a microwave ion source under various conditions, in terms of magnetic flux distribution and mass flow, and the stability of the ion beam. The measured spectra show that, under the experimental condition, almost all of produced ions were Ar(+) ions. For more than 6 h, the ion beam was stable.

(62)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) PET in human gliomas: comparative study with [(18)F]fluorodeoxyglucose and L-methyl-[(11)C]methionine PET.

BACKGROUND AND PURPOSE: (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) was developed as a hypoxic radiotracer in PET. We compared imaging features among MR imaging and (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-PET, FDG-PET, and L-methyl-[(11)C]methionine)-PET in gliomas. MATERIALS AND METHODS: We enrolled 23 patients who underwent (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-PET and FDG-PET and 19 (82.6%) who underwent L-methyl-[(11)C]methionine)-PET, with all 23 patients undergoing surgery and their diagnosis being then confirmed by histologic examination as a glioma. Semiquantitative and volumetric analysis were used for the comparison. RESULTS: There were 10 newly diagnosed glioblastoma multiforme and 13 nonglioblastoma multiforme (grades II and III), including 4 recurrences without any adjuvant treatment. The maximum standardized uptake value and tumor/background ratios of (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone), as well as L-methyl-[(11)C]methionine, were significantly higher in glioblastoma multiforme than in nonglioblastoma multiforme (P = .03 and P = .03, respectively); no significant differences were observed on FDG. At a tumor/background ratio cutoff threshold of 1.9, (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) was most predictive of glioblastoma multiforme, with 90.0% sensitivity and 76.9% specificity. The positive and negative predictive values, respectively, for glioblastoma multiforme were 75.0% and 85.7% on (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone), 83.3% and 60.0% on L-methyl-[(11)C]methionine, and 72.7% and 75.0% on MR imaging. In glioblastoma multiforme, volumetric analysis demonstrated that (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) uptake had significant correlations with FDG (r = 0.68, P = .03) and L-methyl-[(11)C]methionine (r = 0.87, P = .03). However, the (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-active region was heterogeneously distributed in 50.0% (5/10) of FDG-active and 0% (0/6) of L-methyl-[(11)C]methionine)-active regions. CONCLUSIONS: (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) may be a practical radiotracer in the prediction of glioblastoma multiforme. In addition to FDG-PET, L-methyl-[(11)C]methionine)-PET, and MR imaging, (62)Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)-PET may provide intratumoral hypoxic information useful in establishing targeted therapeutic strategies for patients with glioblastoma multiforme.

Use of ultrasound-guided intercostal nerve block as a sole anaesthetic technique in a high-risk patient with Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a progressive neuromuscular disease. Mortality is typically related to combined respiratory failure and dilated cardiomyopathy. Surgery under general anesthesia or deep sedation presents increased risks for pulmonary complications or ventilator dependency postoperatively. We describe the utility of ultrasound guided intercostal nerve blocks for surgery on the chest wall in a patient with Duchenne muscular dystrophy and severe respiratory compromise.

Oncological outcome of patients with deeply located soft tissue sarcoma of the pelvis: a follow up study at minimum 5 years after diagnosis.

AIMS: The treatment of pelvic soft tissue sarcomas (STS) presents one of the most challenging problems in musculoskeletal oncology because of the complex anatomy of the pelvis, late diagnosis and large tumor size. Our study was designed to determine the outcome and prognostic factors for survival and local recurrence in patients with pelvic STS located deep to the fascia and deemed suitable for curative surgical treatment. PATIENTS AND METHODS: Ninety consecutive pelvic STS patients with at least 5-year possible follow-up from diagnosis were studied. Mean age at diagnosis was 54 years. Mean follow-up and tumor size were 69 months and 13 cm, respectively. Histological grades were grade 3 in 51, grade 2 in 22 and grade 1 in 17 patients. Tumor locations were extra-pelvic or outside pelvic brim (n=67), intra-pelvic or within pelvic brim (n=10), and combined or involving both outside and within pelvic brim (n=13). RESULT: Surgical treatment was excision in 84 patients and hindquarter amputation in 6 patients. In 84 patients who underwent excision, surgical margin was wide in 21 patients, marginal in 33, and intralesional in 30. Radiotherapy was used for all high grade tumors. Disease-specific survival was 53.3% at 5 years. Local recurrence occurred in 23%. Development of local recurrence was related to surgical margin (p=0.03). Local recurrence, tumor histological grade and metastasis at diagnosis independently influenced disease-specific survival (p=0.0008, p<0.0001, p=0.02, respectively). CONCLUSION: The patients with high grade tumors and positive surgical margins represent a particular group with high risk of local recurrence even with radiotherapy.

Application of 62Cu-diacetyl-bis (N4-methylthiosemicarbazone) PET imaging to predict highly malignant tumor grades and hypoxia-inducible factor-1α expression in patients with glioma.

BACKGROUND AND PURPOSE: Hypoxic tissue evaluation in glioma is important for predicting treatment response and establishing antihypoxia therapy. In this preliminary study, (62)Cu-ATSM PET was used to determine its validity as a biomarker for distinguishing tumor grade and tissue hypoxia. MATERIALS AND METHODS: (62)Cu-ATSM PET was performed in 22 patients with glioma, and the (62)Cu-ATSM SUV(max) and T/B ratio were semiquantitatively evaluated. (62)Cu-ATSM uptake distribution was qualitatively evaluated and compared with MR imaging findings. HIF-1α expression, a hypoxia marker, was compared with (62)Cu-ATSM uptake values. RESULTS: The (62)Cu-ATSM SUV(max) and T/B ratio were significantly higher in grade IV than in grade III gliomas (P = .014 and .018, respectively), whereas no significant differences were found between grade III and grade II gliomas. At a T/B ratio cutoff threshold of 1.8, (62)Cu-ATSM uptake was predictive of HIF-1α expression, with 92.3% sensitivity and 88.9% specificity. The mean T/B ratio was also significantly higher in HIF-1α-positive glioma tissue than in HIF-1α-negative tissue (P = .001). Using this optimal threshold of T/B ratio, (62)Cu-ATSM PET showed regional uptake in 61.9% (13/21) of tumors within the contrast-enhanced region on MR imaging, which was significantly correlated with presence of a necrotic component (P = .002). CONCLUSIONS: Our results demonstrated that (62)Cu-ATSM uptake is relatively high in grade IV gliomas and correlates with the MR imaging findings of necrosis. Moreover, the (62)Cu-ATSM T/B ratio showed significant correlation with HIF-1α expression. Thus, (62)Cu-ATSM appears to be a suitable biomarker for predicting highly malignant grades and tissue hypoxia in patients with glioma.

Measurement of dynamic bite force during mastication.

Efficient mastication of different types and size of food depends on fast integration of sensory information from mechanoreceptors and central control mechanisms of jaw movements and applied bite force. The neural basis underlying mastication has been studied for decades but little progress in understanding the dynamics of bite force has been made mainly due to technical limitations of bite force recorders. The aims of this study were to develop a new intraoral bite force recorder which would allow the study of natural mastication without an increase in the occlusal vertical dimension and subsequently to analyze the relation between electromyographic (EMG) activity of jaw-closing muscles, jaw movements and bite force during mastication of five different types of food. Customized force recorders based on strain gauge sensors were fitted to the upper and lower molar teeth on the preferred chewing side in fourteen healthy and dentate subjects (21-39 years), and recordings were carried out during voluntary mastication of five different kinds of food. Intraoral force recordings were successively obtained from all subjects. anova showed that impulse of bite force as well as integrated EMG was significantly influenced by food (P<0·05), while time-related parameters were significantly affected by chewing cycles (P<0·001). This study demonstrates that intraoral force recordings are feasible and can provide new information on the dynamics of human mastication with direct implications for oral rehabilitation. We also propose that the control of bite force during mastication is achieved by anticipatory adjustment and encoding of bolus characteristics.

Expression pattern of REIC/Dkk-3 in mouse squamous epithelia.

The REIC/Dkk (reduced expression in immortalized cells/Dickkopf-3) gene was originally identified as a tumour-suppressor gene with reduced expression in immortalized cells, cancer-cell lines and tumour tissues. Of the four members of the Dkk family, the REIC/Dkk-3 protein is unique in terms of DNA sequence, expression profiles and biological functions. In this study, we investigated and compared the expression patterns of the REIC/Dkk-3 protein in mouse squamous epithelia. Expression of REIC/Dkk-3 in the back skin was localized to the upper layer of the interfollicular epidermis, and the inner root sheath of hair follicles. Expression of REIC/Dkk-3 was detected in the ear skin, oral mucosa, tongue, oesophagus, uterine cervix, footpad and tail skin, but not in the cornea. Interestingly, expression was localized to the upper layers of these epithelial tissues. The physiological function of REIC/Dkk-3 is still unclear, but our detailed observation highlight its unique expression pattern in squamous epithelia.

Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils.

BACKGROUND AND OBJECTIVE: Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P-LPS). MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus-related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA(1c)) value was obtained from patients with diabetes mellitus-related periodontitis by a medical interview. Human neutrophils were cultured with P-LPS (0-1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P-LPS. The medium and cellular fractions were used for determination of resistin by ELISA. RESULTS: The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus-related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA(1c) value. The P-LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. CONCLUSION: We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P-LPS-induced resistin release from neutrophils.

Fabrication of a submicron-channel organic field-effect transistor using a controllable electrospun single fibre as a shadow mask.

We demonstrate a simple and versatile method for the fabrication of a submicron channel for an organic field-effect transistor (OFET) using a single electrospun fibre as a shadow mask. A single electrospun fibre is produced by an alternative switching electrospinning method and is stretched 2.5-fold. The average diameter of the stretched fibres is 302 nm. The stretched fibre is placed on ultrathin dielectric layers of aluminium oxide and a self-assembled monolayer (SAM). During electrode deposition the fibre acts as a very small shadow mask. After removing the fibre, electrodes with very narrow gaps of around 350 nm and with high uniformity are easily obtained. We fabricate an OFET by depositing pentacene as an active layer onto the electrodes. The OFET is operable at low voltages, with a threshold voltage of - 1.1 V and a subthreshold swing of 0.27 V decade(-1), values which are one order of magnitude lower than those obtained with a channel length of 75 µm.

Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression.

BACKGROUND: Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423-7). Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas. METHODS: We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen. We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAF(V600E) mutation, as well as by cloning and sequencing of separated alleles. Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients. RESULTS: Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells. Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas. Selection of BRAF mutant alleles during progression was demonstrated in all the three patients. CONCLUSION: Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.

Photodynamic therapy with acridine orange in musculoskeletal sarcomas.

Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. We have been studying alternative treatments for musculoskeletal sarcoma since 1990, and have recently established a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells. These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

Total femur replacement: primary procedure for treatment of malignant tumours of the femur.

We present our experience of treating patients with tumours involving the whole femur with excision and total femur endoprostheses over the last 30 years (1975-2005). There were 26 consecutive patients (14 men and 12 women). Average age was 40 years (14-82 years) at the time of surgery and 21 of the patients had primary malignant bone tumours with five having the procedure for metastases. 11 patients were still alive of which nine were free of disease at the time of review at a mean follow-up of 57 months (3-348). The overall patient survival at 10 years was 37%. The survival of patients with a primary localised tumour was 50% at 10 years. Revision of the prostheses was necessary in two patients (at 110 and 274 months) because of recurrent dislocation and aseptic loosening. Amputation was necessary in two patients but long term limb survival was 92% at 10 years. Nine patients alive with no evidence of disease had a mean MSTS functional score of 72%.