The Geriatric Nutritional Risk Index is Independently Associated with Prognosis in Patients with Critical Limb Ischemia Following Endovascular Therapy.

OBJECTIVES: Patients with critical limb ischemia (CLI) have poor overall and limb prognosis. Although nutritional status influences overall prognosis, and the Geriatric Nutritional Risk Index (GNRI) is a widely used, simple and well established nutritional status screening method, the association between the GNRI and the overall and limb prognosis of patients with CLI following endovascular therapy (EVT) has not been explored. METHODS: Clinical outcomes were retrospectively evaluated in 473 consecutive patients (74 ± 10 years; 59% male) with CLI who underwent EVT. The GNRI on admission was calculated as follows: [14.89 × albumin (g/dL)] + [41.7 × (body weight/ideal body weight)]. Cox proportional hazard analysis was performed to explore the independent association between the GNRI and mortality and major amputation. RESULTS: Patients (53% ambulatory, 38% wheelchair bound, and 9% bedridden) were divided into two groups based on the median GNRI: the higher group (GNRI ≥ 91.2, n = 237) and the lower group (GNRI < 91.2, n = 236). Median follow up duration after EVT was 11.3 months. Three years after EVT, the survival rate (74% in the higher GNRI, and 48% in the lower GNRI, respectively), and limb salvage rate (92% in the higher GNRI, and 84% in the lower GNRI) were significantly lower in the lower GNRI group. GNRI (hazard ratio [HR], 1.03; 95% confidence interval [CI], 1.01-1.05), along with being wheelchair bound (HR, 1.87; 95% CI 1.17-2.97; vs. ambulatory status), being bedridden (HR, 3.10; 95% CI, 1.63-2.97; vs. ambulatory status), being on hemodialysis (HR, 2.33; 95% CI, 1.49-3.64), and having chronic heart failure (HR, 2.22; 95% CI, 1.44-3.43) were the independent predictors of mortality. The GNRI (HR, 1.04; 95% CI, 1.01-1.07), being bedridden (HR, 4.15; 95% CI, 1.67-10.3; vs. ambulatory status), isolated below knee disease (HR, 2.49; 95% CI, 1.30-4.77), and hemodialysis (HR, 2.44; 95% CI, 1.23-4.85) were independently associated with major amputation. CONCLUSIONS: The GNRI on admission was independently associated with mortality and major amputation after EVT in patients with CLI.

Predictors of delayed wound healing after endovascular therapy of isolated infrapopliteal lesions underlying critical limb ischemia in patients with high prevalence of diabetes mellitus and hemodialysis.

OBJECTIVES: Acceptable limb salvage rates underlie the widespread use of endovascular therapy (EVT) for patients with critical limb ischemia (CLI) secondary to isolated infrapopliteal lesions; however, post-EVT delayed wound healing remains a challenge. Predictors of delayed wound healing and their use in risk stratification of EVT in patients with CLI due to isolated infrapopliteal lesions are explored. METHODS: This was a retrospective multicenter study. 871 consecutive critically ischemic limbs were studied. There was tissue loss in 734 patients (age: 71 ± 10 years old; 71% male) who had undergone EVT between April 2004 and December 2012. The wound healing rate after EVT was estimated by the Kaplan-Meier method. The association between baseline characteristics and delayed wound healing was assessed by the Cox proportional hazard model. RESULTS: Diabetes mellitus and regular dialysis were present in 75% (553/734) and 64% (476/734) of patients, respectively; 67% of limbs (585/871) had Rutherford class 5 CLI; 8% (67/871) of wounds were located in the heel only; 25% (219/871) of limbs had Rutherford 6 (involving not only the heel); and 42% (354/871) of wounds were complicated by infection. The rate of freedom from major amputation at 1 year reached 88%, whereas the wound healing rate was 67%. Median time to wound healing was 146 days. By multivariate analysis, non-ambulatory status (hazard ratio [HR], 1.58; 95% confidence interval [CI] 1.31-1.91) serum albumin <3 g/dL (HR 1.42; 95% CI 1.08-1.86), Rutherford 6 (not only heel) (HR 1.68; 95% CI 1.33-2.14), wound infection (HR 1.24; 95% CI 1.03-1.50), EVT not based on angiosome concept (HR 1.28; 95% CI 1.06-1.55), and below the ankle (BTA) 0 vessel runoff after EVT (HR 1.45; 95% CI 1.14-1.86) were independent predictors of delayed wound healing. CONCLUSIONS: Non-ambulatory status, low albumin level, Rutherford 6 (not only heel), wound infection, indirect intervention, and poor BTA runoff were independent predictors for delayed wound healing after EVT in patients with CLI secondary to infrapopliteal lesions, and their use in risk stratification allows estimation of the wound healing rate.

SU-E-J-203: Determination of PTV Margin for Lung Tumor Using In-Treatment 4D CBCT.

PURPOSE: To determine a planning target volume (PTV) margin for lung cancer patients using a four-dimensional cone-beam CT (4D CBCT) acquired during volumetric modulated arc therapy (VMAT) treatment. METHODS: A VMAT plan for lung cancer patients was created by Pinnacle v9.0 (Philips) treatment planning system (TPS), where the gross target volume (GTVs) in each breathing phase was delineated by using 4D-planning CT scan (TOSHIBA and ANZAI). The VMAT treatment was performed with a stereotactic body frame after the registration using Elekta X-ray volume imaging (XVI) unit. Simultaneous cone-beam projection images were acquired for 3 or 4 fractions of 10 patients. The in-treatment 4D CBCT was reconstructed by dividing into four breathing phase bins. A total of 38 in-treatment 4D-CBCT sets were exported to Pinnacle TPS. The isocenter of in-treatment 4D CBCT was matched with that of 4D-planning CT. The tumor motion during treatment was manually tracked on in-treatment 4D CBCT, and the center-of-mass (COM) location of the tumor was estimated. Analyzing the tumor regions observed by in-treatment 4D CBCT, a PTV margin in our system was derived. RESULTS: The average difference in COM location of the tumor was less than 1mm for all directions, while the standard deviations (SD's) were about 1.3mm, 1.6mm, and 2.1mm for the lateral, the vertical, and the longitudinal directions, respectively. The large discrepancy more than 3mm was observed for one patient. The required PTV margin was about 3-4mm for the lateral and the vertical directions, whereas it was about 5mm for the longitudinal direction. CONCLUSIONS: The uncertainties of the tumor motion caused by respiration were observed by in-treatment 4D CBCT images. It was feasible to determine the PTV margin from 4D volume images. K. Nakagawa receives research funding from Elekta.

Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses.

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7(giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.

Bone and soft tissue tumours of the foot: review of 83 cases.

PURPOSE: To review cases of bone and soft tissue tumours of the feet managed at the Hyogo College of Medicine, Nishinomiya and Takarazuka Municipal Hospital, Takarazuka, Japan. METHODS: Retrospective analysis of 83 patient records treated for bone and soft tissue tumours of the feet between 1974 and 2000. RESULTS: There were 33 benign bone tumours, one primary malignant bone tumour, and 2 metastatic bone cancer. Marginal resection was performed in cases of osteochondroma and curettage in cases of other benign bone tumour. Despite below-knee amputation in the case of chondrosarcoma, the patient died because of pulmonary metastasis. Two patients with metastatic cancer also died, and 2 cases of osteochondroma and one of benign chondroblastoma recurred. There were 47 cases of soft tissue tumour. Treatment for benign soft tissue tumours was marginal resection; no cases recurred. In contrast, all patients with soft tissue sarcoma died after surgery. The majority of bone tumours were located in the toe and hindfoot areas, in the first and second decades of life, whereas soft tissue tumours occurred mainly in the midfoot area and in patients aged between 20 and 50 years. The sex distribution was almost even for bone tumours (male: female ratio, 19:17), whereas about half as many males as females had soft tissue tumours (14:33). CONCLUSION: Bone and soft tissue tumours of the feet are uncommon. Most bone tumours are chondrogenic, but differential diagnosis of malignant from benign disease is difficult and prognosis is poor. Management of benign tumours by marginal resection has good prognosis, whereas prognosis of soft tissue sarcomas is very poor.

Factors predicting compensatory vascular remodelling of the carotid artery affected by atherosclerosis.

OBJECTIVE: To investigate factors predicting the development of outward remodelling of the carotid artery in patients with atherosclerosis. DESIGN: 130 patients with carotid artery stenosis (15-85% of the vessel diameter) were divided into two groups, based on the presence or absence of outward remodelling of the sclerotic carotid segment on high resolution ultrasonography. Logistic regression analysis was used to evaluate the contribution of haemodynamic, laboratory, and clinical measurements on the development of remodelling, including age, sex, type of stenosis, extent of plaque, per cent diameter stenosis, underlying disease, selected drug treatment, and plasma concentrations of total cholesterol, high density lipoprotein cholesterol, triglyceride, and uric acid. RESULTS: 64 patients (49%) had outward remodelling. Multivariate regression analysis showed that hypertension, the type of plaque, the thickness of the plaque, and the extent of stenosis were independent factors predicting remodelling. The odds ratios of hypertension, unstable shape of plaque, thickness of plaque, and the extent of the stenosis were 6.70, 3.02, 2.04, and 1.05, respectively. Other measurements did not contribute significantly to the estimation of remodelling. CONCLUSIONS: Compensatory enlargement of the vessel occurs in about 50% of carotid artery segments with a diameter stenosis of 15-85%. Hypertension and the shape of the plaque are major determinants of the development of outward remodelling.

Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing.

We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

Relation of QT-interval variability to ventricular arrhythmias during percutaneous transluminal coronary angioplasty.

The present study investigated the role of the dispersion of QT interval in percutaneous transluminal coronary angioplasty (PTCA)-induced ventricular tachyarrhythmias. Patients with effort angina without a previous myocardial infarction (n = 22), who had single-vessel disease of the anterior descending coronary artery (LAD), underwent PTCA if the coronary lesion was 75% or more stenosed in segment 6 or 7 of the LAD. The standard 12-lead ECG was continuously recorded during the procedure. Averaged QTc and QTac intervals, where QTac was the interval from the beginning of QRS complex to the nadir of T wave corrected by Bazett's formula, did not change significantly during PTCA. Of the 22 patients, 7 showed ventricular arrhythmias during PTCA. The maximum difference (deltaQTc) and the standard deviation (QTcSD) of the corrected QT interval in the standard 12-lead ECG increased significantly during PTCA in the 7 patients with ventricular arrhythmias, whereas they decrreased in the 15 patients without ventricular arrhythmias. deltaQTac and QTacSD were not affected by PTCA regardless of ventricular arrhythmias, which shows that the increases in the variation of the ventricular repolarization process play a role in PTCA-induced ventricular arrhythmias.

Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.

A p53 polymorphism associated with increased risk of hepatitis C virus infection.

Incidence of polymorphisms on exon 4 (CGC vs. CCC, p53Arg vs. p53Pro, A2 allele vs. A1 allele at codon 72, respectively) of the p53 gene was compared in 75 cases with hepatitis C virus (HCV) infection and 232 noninfected control subjects in a defined geographical area in Japan. Polymorphism was analyzed by the polymerase chain reaction-single strand conformation polymorphism method using DNA from peripheral blood leukocytes. When all cases and controls were compared, there was no significant correlation between hepatitis C virus (HCV) infection and the p53 polymorphism in question. However, when male cases infected with HCV type 1b, the most common viral genotype, were compared with controls matched by sex and age, significantly higher homozygotes for p53Pro were found in cases compared with controls (P=0.039). Significantly higher allelic frequency of this polymorphism was also observed with cases (P=0.010). We found no significant statistical difference between the p53 polymorphism and other genotypes of HCV (2a, 2b and others). On the basis of our study we believe there exists a significant correlation between male homozygotes for p53Pro with HCV type 1b infection.

Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.

Coexpression of microsomal-type prostaglandin E synthase with cyclooxygenase-2 in brain endothelial cells of rats during endotoxin-induced fever.

Fever is triggered by an elevation of prostaglandin E(2) (PGE(2)) in the brain. However, the mechanism of its elevation remains unanswered. We herein cloned the rat glutathione-dependent microsomal prostaglandin E synthase (mPGES), the terminal enzyme for PGE(2) biosynthesis, and examined its induction in the rat brain after intraperitoneal injection of pyrogen lipopolysaccharide (LPS). In Northern blot analysis, mPGES mRNA was weakly expressed in the brain under the normal conditions but was markedly induced between 2 and 4 hr after the LPS injection. In situ hybridization study revealed that LPS-induced mPGES mRNA signals were mainly associated with brain blood vessels, especially vein or venular-type ones, in the whole brain area. Immunohistochemical study demonstrated that mPGES-like immunoreactivity was expressed in the perinuclear region of brain endothelial cells, which were identified as von Willebrand factor-positive cells. Furthermore, in the perinuclear region of the endothelial cells, mPGES was colocalized with cyclooxygenase-2 (COX-2), which is the enzyme essential for the production of the mPGES substrate PGH(2). Inhibition of cyclooxygenase-2 activity resulted in suppression of both PGE(2) level in the CSF and fever (Cao et al., 1997), suggesting that the two enzymes were functionally linked and that this link is essential for fever. These results demonstrate that brain endothelial cells play an essential role in the PGE(2) production during fever by expressing COX-2 and mPGES.

LiV2O4 spinel as a heavy-mass fermi liquid: anomalous transport and role of geometrical frustration

Transport and specific heat measurements on hydrothermally grown single crystals reveal the formation of a heavy-mass Fermi liquid in the LiV2O4 spinel, below a coherence temperature of T* = 20-30 K. A few observations which illustrate the uniqueness of this spinel are discussed in connection with the origin of the heavy mass, such as the anomalous absence of resistivity saturation above T* and the close proximity to a spin glass phase where the influence of the magnetic frustration is evident.

l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons.

We investigated the effects of l-menthol on cultured dorsal root ganglion (DRG) cells, instead of free nerve endings of sensory fibers. Using Fura-2 microfluorimetry, we identified a few DRG neurons that showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) in response to l-menthol. They made up only 10% of the neurons activated by a high K+ solution. l-Menthol induced the [Ca2+]i increase in a dose-dependent manner, with an EC50 of 37.9 microM and a Hill coefficient of 0.97. A related compound, cyclohexanol, had no effect. When extracellular Ca2+ was removed, l-menthol did not induce the [Ca2+]i increase. Whole-cell current-clamp recordings revealed that l-menthol induced depolarization (13.2 mV, receptor potential) leading to impulses. We conclude that l-menthol induced the impulses through activation of menthol receptors in a small subset of the cultured sensory neurons.

Cyclooxygenase in the vagal afferents: is it involved in the brain prostaglandin response evoked by lipopolysaccharide?

The vagal afferents are proposed to transmit abdominal immune signals to the brain. In this immune-brain communication, prostaglandins might play a mediator role. In fact, prostaglandin receptors are abundant in the vagal afferents. We examined here the presence of cyclooxygenase, an enzyme necessary for prostaglandin biosynthesis, in the vagal afferents of rats. We also tested whether the vagal afferents contribute to the elevation of prostaglandin E2 in the brain after intraperitoneal injection of lipopolysaccharide. Under normal conditions, cyclooxygenase-1-like immunoreactivity was constitutively expressed in the vagal afferents at their central terminals and in their cell bodies. Cyclooxygenase-2-like immunoreactivity was absent in the vagal afferents under normal as well as lipopolysaccharide-challenged conditions. Instead, cyclooxygenase-2-like immunoreactivity was induced in brain endothelial cells by the lipopolysaccharide challenge. The elevation of prostaglandin E2 in the cerebrospinal fluid after lipopolysaccharide challenge was not inhibited, but was rather enhanced, by the bilateral vagotomy. These results suggest that the vagal afferents potentially generate prostaglandins, which may locally modulate the vagal signal transmission, but that the vagal afferents are not essential to the elevation of prostaglandin E2 in the brain after intraperitoneal challenge with LPS.

A case-control study of hepatocellular carcinoma in Hyogo Prefecture.

We conducted a case-control study to elucidate factors contributing to the development of hepatocellular carcinoma (HCC) in Hyogo Prefecture, Japan where its incidence is high. In this study, the incidence of hepatitis virus infection in HCC patients and lifestyle factors including dietary and drinking habits were investigated. One hundred and eight patients with HCC (89 males and 19 females) and 93 controls (63 males and 30 females) were enrolled. Hepatitis C virus (HCV) antibody was identified in 87% of cases of which HCV-RNA was further detected in more than 95%. Genotype distribution of HCV was similar to that reported for the Japanese infected with HCV. There was a positive correlation between HCC and history of blood transfusion (both male and female) and surgery (male). Alcohol consumption over a period of 30 years was higher in the HCC patients than in controls; but not significant. These data indicated that HCV infection is a primary contributing factor to the development of HCC in Hyogo Prefecture.

Morphologic correlation between atherosclerotic lesions of the carotid and coronary arteries in patients with angina pectoris.

The morphology of atherosclerosis between the carotid and coronary artery systems was studied in 63 patients with ischemic heart disease to determine if there was a correlation with coronary heart disease. The sclerotic lesions of the carotid and coronary artery systems were imaged with ultrasonography and coronary arteriography, respectively, and divided into 4 types. Hemodynamic variables, serum lipid levels, and serum uric acid concentration were not different among the groups, but the serum C-reactive protein (CRP) concentration in patients without significant atheroma in the carotid artery system was lower than the mean concentration of the other 3 groups with carotid atheroma. The morphological stability of carotid arterial plaques correlated well to coronary artery stenosis. Morphologically unstable plaques of the carotid artery predicted unstable forms of coronary obstruction with a sensitivity of 68%, specificity of 85%, predictive power of 72% and a likelihood ratio of 4.5. These results suggest that ultrasonic examination of the carotid artery is useful for predicting the presence or absence of unstable lesions in coronary arteries.

Yeast gene YRR1, which is required for resistance to 4-nitroquinoline N-oxide, mediates transcriptional activation of the multidrug resistance transporter gene SNQ2.

We have cloned and characterized a Saccharomyces cerevisiae gene YRR1 that is important for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO). The wild-type YRR1 gene encodes a protein that contains a Zn(II)2Cys6-type zinc-finger motif. Disruption of the YRR1 gene leads to hypersensitivity to 4-NQO. A dominant mutation (YRR1-1) that confers strong resistance to 4-NQO has been identified. Epistasis analysis demonstrated that 4-NQO resistances mediated by the YRR1 and YRR1-1 alleles require the presence of the SNQ2 gene that encodes a multidrug resistance ATP binding cassette superfamily protein responsible for 4-NQO export. Northern blot analysis of SNQ2 mRNA levels indicated that Yrr1p is involved in basal and drug-induced transcriptional activation of SNQ2, whereas Pdr1p/Pdr3p transcription factors are mainly involved in basal SNQ2 expression. In the YRR1-1 mutant, the level of SNQ2 mRNA is constitutively elevated. These results establish that Yrr1p is important for 4-NQO resistance by mediating transcriptional activation of the SNQ2 gene in response to the stress imposed by 4-NQO. The gain-of-function mutation of Yrr1-1p was attributable to the duplication of a 12-amino-acid sequence generated near the carboxy terminus.

Endothelin-induced apoptosis of A375 human melanoma cells.

Endothelin-1 (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nM ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G1/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ETB expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides.