Reliability of industrial packaging for microsystems

Packaging concepts for silicon-based micromachined sensors exposed to harsh environments are explored. By exposing the sensors directly to the media and applying protection at the wafer level the packaging and assembly will be simplified as compared to conventional methods of fabrication.Protective coatings of amorphous silicon carbide and tantalum oxide are suitable candidates with etch rates below 0.1 Å/h in aqueous solutions with pH 11 at temperatures up to 140°C. Si-Ta-N films exhibit etch rates around 1 Å/h. Parylene C coatings did not etch but peeled off after extended exposure times at elevated temperatures. The best diamond-like carbon films we tested did not etch, but delaminated due to local penetration of the etchants.Several glue types were investigated for chip mounting of the sensors. Hard epoxies, such as Epotek H77, on the one hand exhibit high bond strength and least degradation and leakage, but on the other hand introduce large sensor output drift with temperature changes. Softening of the Epo-tek H77 was observed at 70°C.An industrially attractive thin-film anodic silicon-to-silicon wafer bonding process was developed. Glass layers are deposited at 20 nm/s (1.2 μm/min) by electron-beam evaporation and bond strengths in excess of 25 N/mm² are obtained for bonding temperatures higher than 300°C.Through-hole electrical feedthroughs with a minimum line width of 20μm and a density of 250 wires per cm were obtained by applying electro-depositable photo-resist. Hermetically sealed feedthroughs were obtained using glass frits, which withstand pressures of 4000 bar.     

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.     

Neurologic disease among women with breast implants

US angiography, enhanced with intraarterial CO2 microbubbles imaging, documented 40 nodules of hepatocellular carcinoma (HCC) lesser than 20 mm in diameter in 34 patients, which were convinced histopathologically. As to the imaging acuity of arterial vascularity in nodules, US angiography was compared with DSA and US color angio. The detection of arterial vascularity was possible in 34 (85.0%) of 40 nodules by US angiography, 26 (65.0%) by DSA, and 28 (70.0 %) by US color angio. US angiography was available for detection of HCC, particularly with small HCC lesser than 20 mm in size.     

Cardiovascular evaluation of the athlete. Issues regarding performance, screening and sudden cardiac death

Recent studies have reported ECG anomalies and a high prevalence of exercise-related arrhythmias among well trained, apparently healthy endurance athletes with superior levels of cardiorespiratory fitness. The occurrence of sudden and premature cardiac deaths in amateur and professional athletes, who appear to embody all of the virtues of health and fitness, ahs raised our consciousness regarding the underlying atherosclerotic or nonatherosclerotic causes, and the need for, and extent of, preparticipation screening in competitive athletes. It appears that strenuous physical activity may trigger acute cardiovascular events in some athletes. Coronary artery disease is the most frequent autopsy finding in those over the age of 35 years who die suddenly. In contrast, structural cardiovascular abnormalities, including hypertrophic cardiomyopathy and malformations of the coronary arteries, are the major cause of sudden death in younger athletes. This article reviews these issues, with specific reference to the assessment of cardiorespiratory fitness, legal and prohibited performance-altering medications, the pathophysiological basis of exertion-related untoward events, the athlete at risk, limitations of conventional screening programmes and contemporary recommendations to identify latent cardiovascular disease in athletic populations.     

Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension

Anorexigens such as aminorex fumarate and dexfenfluramine are associated with the development of severe pulmonary hypertension (PH), which clinically and histopathologically is considered indistinguishable from idiopathic or primary pulmonary hypertension (PPH). For the current study, we asked whether anorexigen-associated PH is characterized by monoclonal pulmonary endothelial cell proliferation (such as in PPH) or, alternatively, is associated with a polyclonal endothelial cell proliferation as found in secondary PH. Analysis of clonality by the human androgen receptor assay was performed in microdissected endothelial cells of plexiform lesions of two patients with anorexigen-associated PH. The four plexiform lesions of Patient 1 and the six of Patient 2 with anorexigen-associated PH exhibited a monoclonal expansion of pulmonary endothelial cells, with a mean clonality ratio of 0.03 +/- 0.01 SE. Our results indicate that appetite suppressant-associated PH is identical to PPH not only in clinical and histopathologic features but also, at a molecular level, in terms of the monoclonal nature of the endothelial cell proliferation. The anorexigens may accelerate the growth of pulmonary endothelial cells in patients with predisposition to develop PPH.     

Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.     

Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation

Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.     

Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity

Small dense low density lipoprotein (LDL) particles have altered apolipoprotein (apo) B conformation and lowered affinity for the LDL receptor (J. Biol. Chem. 1994. 269: 511-519). Herein, we examine the interaction of small dense LDL with cell LDL receptor-independent binding sites. Compared to normal LDL, at low LDL cell media concentrations (<10 microg/ml), small dense LDL had decreased specific binding to the LDL receptor on normal fibroblasts at 4 degrees C, but a 2-fold increased binding to LDL receptor-independent cell sites. At higher LDL concentration (100 microg/ ml), LDL receptor-independent binding of small dense LDL was 4.5-fold that of normal LDL in normal fibroblasts, but greater (2- to 14- fold) in LDL receptor-negative fibroblasts. In LDL receptor-negative fibroblasts at 37 degrees C, small dense LDL had higher (3-fold) cell association than normal size LDL but no effective LDL degradation. At high LDL concentrations (> or =100 microg/ml), LDL binding to normal or LDL receptor-negative fibroblasts was not affected by several anti-apoB monoclonal antibodies or by cell pretreatment with proteases, chondroitinase, or neuraminidase. In contrast, pretreating normal and receptor-negative fibroblasts with heparinase and heparitinase decreased LDL cell binding by 35% and 50%, respectively. Similarly, preincubation of receptor-negative fibroblasts with sodium chlorate, an inhibitor of proteoglycan sulfation, decreased LDL binding by about 45%. We hypothesize that small dense LDL might be more atherogenic than normal size LDL due to decreased hepatic clearance by the LDL receptor, and enhanced anchoring to LDL receptor-independent binding sites in extrahepatic tissues (e.g., the arterial wall), a process mediated, in part, by cell surface proteoglycans.