Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods.

PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. Materials and Mehtods: In this study, we use two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. RESULTS: Raf kinase inhibitor protein (RKIP), an inhibitor of Raf-mediated activation of mitogen-activated protein kinase/extracellular signal-regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time-polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. CONCLUSION: These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down-regulated in liver cancer cell.

Solid freeform-fabricated scaffolds designed to carry multicellular mesenchymal stem cell spheroids for cartilage regeneration.

Three-dimensional (3D) cellular spheroids have recently emerged as a new trend to replace suspended single cells in modern cell-based therapies because of their greater regeneration capacities in vitro. They may lose the 3D structure during a change of microenvironment, which poses challenges to their translation in vivo. Besides, the conventional microporous scaffolds may have difficulty in accommodating these relatively large spheroids. Here we revealed a novel design of microenvironment for delivering and sustaining the 3D spheroids. Biodegradable scaffolds with macroporosity to accommodate mesenchymal stem cell (MSC) spheroids were made by solid freeform fabrication (SFF) from the solution of poly(D,L-lactide-co-glycolide). Their internal surface was modified with chitosan following air plasma treatment in order to preserve the morphology of the spheroids. It was demonstrated that human MSC spheroids loaded in SFF scaffolds produced a significantly larger amount of cartilage-associated extracellular matrix in vitro and in NOD/SCID mice compared to single cells in the same scaffolds. Implantation of MSC spheroid-loaded scaffolds into the chondral defects of rabbit knees showed superior cartilage regeneration. This study establishes new perspectives in designing the spheroid-sustaining microenvironment within a tissue engineering scaffold for in vivo applications.

A compact computational model for cell construct development in perfusion culture.

A problem nowadays tissue engineers encounter in developing sizable tissue implants is the nonuniform spread of cells and/or extracellular matrices. Research shows such a nutrients transport restriction may be improved by employing hydrodynamic culture systems. We propose a compact model for the simulation of cell growth in a porous construct under direct perfusion. Unlike the previous model proposed in the literature, which composes a cellular scaffold sandwiched between two culture media layers, the current model includes only the scaffold layer to simplify the mathematical and computational complex. Results show the present single-layer model can predict cell spreads and the nutrient and metabolic waste distribution as accurately as does the three-layer model. Only if the hydrodynamic aspects such as the pressure and viscous stress are prominent to know, should the more sophisticated analyses with the three-layer model be employed. The compact model provides comparable investigations for the tissue-engineering construct developments.

Combined UGT1A1 and UGT1A7 variant alleles are associated with increased risk of Gilbert's syndrome in Taiwanese adults.

Gilbert's syndrome (GS) is caused by a reduction in the activity of hepatic bilirubin UDP-glucuronosyltransferase (UGT). This reduction is associated with UGT1A1*28 and UGT1A1*6 polymorphisms. Recent research also showed that carriage of UGT1A1*6 allele were significantly related with UGT1A7*3. Polymerase chain reaction-restriction fragment length polymorphism were utilized to determine UGT1A7 and UGT1A1 genes for 207 patients with GS and 207 gender/age-matched healthy controls. For the 207 healthy controls, linkage disequilibrium was observed between -57UGT1A7 and 622UGT1A7 loci (D' = 1.00 and r(2) = 1.00), -57UGT1A7 and 211UGT1A1 loci (D' = 0.72 and r(2) = 0.36), respectively. A dose-response effect for number of at-risk allele of UGT1A1 and risk for GS was noted (odds ratio (OR) = 8.19 for heterozygous UGT1A1*28 genotype; OR = 124.96 for homozygous UGT1A1*28 genotype; and p for trend <0.05). Patients with combined genotypes carrying UGT1A7 variant alleles and UGT1A1 variant alleles (including UGT1A1*28 and UGT1A1*6) are associated with increased risk of GS (OR = 13.96 for patients with combined genotype carrying at least one variant allele of UGT1A1 and UGT1A7). In conclusion, the -57UGT1A7 (T>G) is highly associated with UGT1A7*3 and moderately associated with 211UGT1A1 (G>A). Certain UGT1A1/UGT1A7 combined genotypes are risk factors of GS.

Enhancement of cell growth in tissue-engineering constructs under direct perfusion: Modeling and simulation.

Perfusion bioreactors improve mass transfer in cell-scaffold constructs. We developed a mathematical model to simulate nutrient flow through cellular constructs. Interactions among cell proliferation, nutrient consumption, and culture medium circulation were investigated. The model incorporated modified Contois cell-growth kinetics that includes effects of nutrient saturation and limited cell growth. Nutrient uptake was depicted through the Michaelis-Menton kinetics. To describe the culture medium convection, the fluid flow outside the cell-scaffold construct was described by the Navier-Stokes equations, while the fluid dynamics within the construct was modeled by Brinkman's equation for porous media flow. Effects of the media perfusion were examined by including time-dependant porosity and permeability changes due to cell growth. The overall cell volume was considered to consist of cells and extracellular matrices (ECM) as a whole without treating ECM separately. Numerical simulations show when cells were cultured subjected to direct perfusion, they penetrated to a greater extent into the scaffold and resulted in a more uniform spatial distribution. The cell amount was increased by perfusion and ultimately approached an asymptotic value as the perfusion rates increased in terms of the dimensionless Peclet number that accounts for the ratio of nutrient perfusion to diffusion. In addition to enhancing the nutrient delivery, perfusion simultaneously imposes flow-mediated shear stress to the engineered cells. Shear stresses were found to increase with cell growth as the scaffold void space was occupied by the cell and ECM volumes. The macro average stresses increased from 0.2 mPa to 1 mPa at a perfusion rate of 20 microm/s with the overall cell volume fraction growing from 0.4 to 0.7, which made the overall permeability value decrease from 1.35 x 10(-2)cm(2) to 5.51 x 10(-4)cm(2). Relating the simulation results with perfusion experiments in literature, the average shear stresses were below the critical value that would induce the chondrocyte necrosis.

Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.

Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural studies of DDPPs, we have expressed yeast DDPPs using Escherichia coli as the host cell. Thioredoxin and His tag were utilized to increase the solubility of the recombinant protein and facilitate its purification using Ni-nitrilotriacetic acid (NTA) column. The protein was overexpressed in E. coli but mostly existed in pellet in the absence of detergent. The low quantity of soluble DDPPs was purified using Ni-NTA, Mono Q anion-exchange, and size-column chromatographies. The protein in the pellet was solubilized with 7 M urea and purified using Ni-NTA under denaturing condition. The protein refolding was achieved via the stepwise dialysis to remove the denaturant in the presence of 6 mM beta-mercaptoethanol. Detergent n-octyl-beta-d-glucopyranoside and Triton X-100 increased the solubility of the DDPPs so that refolding can be performed at higher protein concentration. Alternatively, on-column refolding was carried out in a single step to obtain the active protein in large quantities. beta-Mercaptoethanol and Triton were both required in this quick refolding process. The kinetic studies indicated that the soluble and refolded DDPPs have comparable activities (k(cat) = 2 x 10(-4) s(-1)). Unlike its bacterial homologue, undecaprenyl diphosphate synthase, yeast DDPPs activity was not enhanced by Triton.

A methodology for evaluation of boundary detection algorithms on breast ultrasound images.

Image segmentation is the partition of an image into a set of non-overlapping regions that comprise the entire image. The image is decomposed into meaningful parts, which are uniform with respect to certain characteristics, such as grey level or texture. This study presents a novel methodology to evaluate ultrasound image segmentation algorithms. The sonographic features can differentiate between various sized malignant and benign breast tumours. The clinical experiment can determine whether a tumour is benign or not, based on contour, shape, echogenicity and echo texture. Further study of the standardized sonographic features, especially the tumour contour and shape, will improve the positive predictive value and accuracy rate in breast tumour detection. The effectiveness of using this methodology is illustrated by evaluating image segmentation on breast ultrasound images. Via definite segmentation, the appreciated tumour shape and contour can be ascertained. Furthermore, this method can enhance the ability of ultrasound to distinguish between benign and malignant breast lesions.

Application of three-dimensional orthogonal neural network to craniomaxillary reconstruction.

The purpose of this investigation is to establish a practical method to predict and create surface a profile of bone defects by a well-trained 3-D orthogonal neural network. First, the coordinates of the skeletal positions around the boundary of bone defects are input into the 3-D orthogonal neural network to train it to learn the scattering characteristic. The 3-D orthogonal neural network avoids local minima and converges rapidly. After the neural network has been well trained, the mathematic model of the bone defect surface is generated, and the pixel positions are derived. Herein, to verify its performance the proposed method is applied on a patient with a craniofacial defect.

Application of orthogonal neural network to craniomaxillary reconstruction.

Traditionally, the orthopaedist, according to their past experience, reconstructs damaged area while the operation is in progress. This may prolong the operation and cause the wound to become infected. Most importantly it is difficult to precisely match the skeletal defect. A well-disciplined network of prediction re-fabricates the damaged area through automation. This research is based on the CT image file, which is the product of X-ray computed tomography (CT), and computes the skeletal positions around the damaged area through image processing and boundary detection. The skeletal positions are inputted into the orthogonal neural network and discipline the network so that it possesses the scattering characteristic of bone. The network then calculates skeletal positions in the damaged area and revises the former CT image file to rebuild a 3D model. Accordingly, in comparison with a manual sketch, the orthogonal neural network forecast is more geometrically precise. Moreover, the forecast satisfies the second order derivative, which is a continuous function, and the edge of the fabricated bone is therefore kept smoother.

Development of a robotic navigation system for neurosurgery.

This paper presents a robotic navigation system for image-guided neurosurgery, which can be applied to the treatment of Parkinson's disease and biopsy of brain tumor. The system integrates a computer for real-time display of brain anatomy, a magnetic tracking device for measuring the positions and orientations of surgical instruments, and a robot manipulator for guiding surgical instruments to the preplanned positions and orientations. The computer display of brain anatomy offers a convenient tool for surgeons to diagnose brain disease and to plan safe surgical paths; while the tracking device assists the robot manipulator to automatically guide surgical instruments to the preplanned direction. The registrations among the tracking device, the image system, and the robot are completed on the base of coordination mappings of external markers. An experiment of using a skull model for simulating a robotic biopsy of brain tumor has been done to verify the performance of the navigation system. The result shows that the system can accomplish a positioning accuracy around 2 mm.

A PC-based surgical simulator for laparoscopic surgery.

Surgical simulators for minimally invasive surgery have been developing in the 1990s. Most of them use high-end UNIX workstations for real-time simulation of complex human organ models. Only few of them have input devices with force feedback. Recently, personal computer technologies have made real-time display of relatively complex models feasible. We are developing an Intel-based laparoscopic surgical simulator that provides near real-time intuitive interaction between the trainee and simulated models of human organs. The surgical simulator has a prototypical scenario of cholecystectomic surgery. It can interactively simulate the deformation and cutting of cystic duct and vein. In addition, a set of input devices with force feedback has been designed and tested to imitate the manipulation of surgical instruments. The input device has five degrees of freedom and three of them are driven by DC motors to produce force feedback.

Succinylcholine-induced cardiac arrest in unsuspected becker muscular dystrophy--a case report.

A five year-old boy undergoing elective tonsillectomy sustained cardiac arrest following the administration of a single dose of succinylcholine during induction of anesthesia. With a 10-minute cardiopulmonary resuscitation (CPR) during which intravenous calcium gluconate, epinephrine, and sodium bicarbonate were given and DC counter shock applied, we were successful to restore cardiac activity without neurological sequelae. The cause of cardiac arrest we speculated was hyperkalemia, possibly secondary to succinylcholine-induced rhabdomyolysis. It is suggested that succinylcholine should not be used in patients with known or suspected muscular dystrophy.

Unilateral pulmonary edema during general anesthesia--report of two cases.

Unilateral presentation of pulmonary edema, though well known to occur, is an uncommon entity. Previous reviews of this subject have discussed the different etiologies, which include rapid reexpansion of collapsed lung, down lung syndrome (gravitational edema), systemic-to-pulmonary arterial shunts, heart failure, compression or occlusion of pulmonary vasculatures, obstruction of a bronchus and an acute manifestation of neuropulmonary reaction (neurogenic pulmonary edema). Occurrence of this complication during surgery, however, is even rarer. We report 2 cases of unilateral pulmonary edema occurring during general anesthesia for elective surgery.

An orthogonal neural network for function approximation.

This paper presents a new single-layer neural network which is based on orthogonal functions. This neural network is developed to avoid the problems of traditional feedforward neural networks such as the determination of initial weights and the numbers of layers and processing elements. The desired output accuracy determines the required number of processing elements. Because weights are unique, the training of the neural network converges rapidly. An experiment in approximating typical continuous and discrete functions is given. The results show that the neural network has excellent performance in convergence time and approximation error.

Post-remission intensive consolidation with high-dose cytarabine-based chemotherapy and granulocyte colony-stimulatory factor in adults with acute myelogenous leukemia: a preliminary report.

BACKGROUND: Modern induction chemotherapy produce 60% to 80% complete remission in adults with newly diagnosed acute myelogenous leukemia (AML). A major challenge is to eradicate subclinical disease in remission and prevent leukemic relapse. Intensive post-remission chemotherapy was proved of comparable disease-free survival as BMT. METHODS: From February 1992 to to March 1995, twelve patients with AML, aged 15 to 57 y/o, received intensive consolidation chemotherapy immediately after the first complete remission. The chemotherapy included either 4 courses of high dose Arac (HiDAC), 3 gm/m2 q12h x3 days, or 2 courses of HiDAC (4 days) plus mitoxantrone for 3 days and etoposide for 7 days (HiDAC-3-7). Granulocyte colony-stimulating factor (G-CSF) used used 24 hours after chemotherapy until absolute neutrophile count greater than 500/mm3. RESULTS: Totally 24 courses of high dose chemotherapy were given. The median duration of severe neutropenia (absolute neutrophile count < or = 500/mm3) was 12 days, thrombocytopenia (< or = 50,000/mm3) 18 days, fever > or = 38 degrees C 6 days, and from severe neutropenia (absolute neutrophile count < or = 500/mm3) was 12 days, thrombocytopenia (< or = 50,000/mm3) 18 days, fever > or = 38 degrees C 6 days, and from severe neutropenia (< or = 500/mm3) to infection 4 days. Infection was the most frequent complication during HiDAC treatment. No toxic death was noted. After a median follow-up of 16 months, early relapse was noted in 3 patients (2, 4, and 5 months, respectively), and late relapse in two patients (11 and 20 months, respectively). Seven patients remained in complete remission status after a median follow-up of 14+ months (7+ to 37+ months). CONCLUSIONS: Intensive consolidation chemotherapy is well tolerable and may prolong remission duration when used in the early post-remission phase of AML.

Swollen head syndrome in Taiwan-isolation of an avian pneumovirus and serological survey.

Outbreaks of swollen head syndrome (SHS) were observed in two broiler and two broiler-breeder farms in Taiwan. The disease was characterized by oedematous swelling of the head, especially surrounding the eyelids, the neck and wattles. Avian pneumovirus and Escherichia coli were isolated from birds in all four farms. In addition, Staphylococcus aureus and infectious bursal disease virus were each isolated from one farm. A serological survey of 398 birds from 11 broiler breeder farms showed 86.4% (344) of them had ELISA antibodies against turkey rhinotracheitis virus.

Effects of opioid agonists and opioid antagonists in endotoxic shock in rats.

Endotoxic shock is presented with a complex pathophysiology and is associated with high mortality. Recently, it has been reported that endogenous opioids play an important role in endotoxic shock. Pressor effect of naloxone in shock may be mediated through antagonism of endogenous opioid inhibition of the sympatho-adrenal catecholaminergic system. In endotoxemic animal, circulating catecholamine levels were not elevated by naloxone. It is possible that naloxone acts upon opiate receptors to enhance catecholamine actions at the receptor level or post-receptor level. We investigated endotoxic shock using a rat model. The animals anesthetized with phenobarbital were infused with E. coli LPS for 30 minutes. They were divided into 5 groups. After an endotoxin i.v. infusion of 15 mg/kg (LD 60), a significant fall in mean arterial pressure (MAP), heart rate and pH occurred in all groups. Treatment with naloxone or buprenorphine or naloxone + epinephrine resulted in significant improvement in MAP, pH and base excess. Treatment with morphine resulted in a decrease in MAP and an increase in heart rate. The pressor response to epinephrine 10, 30, 60 microgram/kg i.v. caused an increase of 62%, 48% and 17% of control values respectively in endotoxic treated rats. The duration of the pressor response to epinephrine was significantly increased by naloxone, although no significant effects on survival were seen at 4 hours after the start of treatment. These findings suggest that the buprenorphine may prove to be an alternative to naloxone, the co-administration of naloxone and epinephrine may be of benefit in the management of septic shock.