StarSync: An extendable standard-cell mesochronous synchronizer

StarSync, a mesochronous synchronizer, enables low latency and full throughput crossing of clock domain boundaries having same frequency but different phases. Full back pressure is supported, where the receiver can start and stop accepting words without any data loss. Variable depth buffering is provided, supporting a wide range of short and long range communications and accommodating multi-cycle wire delays. Burst data can also be accommodated thanks to buffering. Dynamic phase shifting due to varying voltage and temperature are mitigated by increasing the separation between write and read pointers. The synchronizer is exposed to metastability risk only during reset. It is suitable for implementation using standard cell design and requires neither delay lines nor other full custom circuits. It is shown that a minimum of four buffer stages are required, to mitigate skew in reset synchronization, in contrast with previous proposals for three stages.     

An automated noncontact deposition interface for liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS). This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface. The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths. Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis. The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic. The latter, exemplified by alpha-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps. Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation. Experimental results show that there is no significant decrease in chromatographic resolution using this device. To assess the behavior of the apparatus for complex mixtures, 5 microg of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13,500 unique analytes were detected in a single LC/MS analysis.     

Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry

Protein tyrosine phosphorylation cascades are difficult to analyze and are critical for cell signaling in higher eukaryotes. Methodology for profiling tyrosine phosphorylation, considered herein as the assignment of multiple protein tyrosine phosphorylation sites in single analyses, was reported recently (Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443-448). The technology platform included the use of immunoprecipitation, immobilized metal affinity chromatography (IMAC), liquid chromatography, and tandem mass spectrometry. In the present report, we show that when using complex mixtures of peptides from human cells, methylation improved the selectivity of IMAC for phosphopeptides and eliminated the acidic bias that occurred with unmethylated peptides. The IMAC procedure was significantly improved by desalting methylated peptides, followed by gradient elution of the peptides to a larger IMAC column. These improvements resulted in assignment of approximately 3-fold more tyrosine phosphorylation sites, from human cell lysates, than the previous methodology. Nearly 70 tyrosine-phosphorylated peptides from proteins in human T cells were assigned in single analyses. These proteins had unknown functions or were associated with a plethora of fundamental cellular processes. This robust technology platform should be broadly applicable to profiling the dynamics of tyrosine phosphorylation.     

Selective estrogen-receptor modulators (SERMs) in the cyclopentadienylrhenium tricarbonyl series: synthesis and biological behaviour

A series of organometallic antiestrogens based on the OH-tamoxifen (OH-Tam) skeleton and bearing the (eta(5)-C(5)H(4))Re(I)(CO)(3) unit has been prepared by using McMurry coupling for the purpose of studying their biological behaviour. The cyclopentadienylrhenium tricarbonyl moiety is indeed stable in biological media, compact, lipophilic and easy to handle. Furthermore, this study allowed us to select the best candidates for subsequent use as radiopharmaceuticals either for imaging or therapy by using appropriate radionucleides, namely (99m)Tc and (188)Re. In these molecules the beta-phenyl group of OH-Tam has been replaced by the (eta(5)-C(5)H(4))Re(CO)(3) moiety, and the length of the dimethylamino side chain --O(CH(2))(n)N(CH(3))(2) was varied (n=2, 3, 4, 5 and 8). The compounds 7 a-7 e were obtained as mixtures of their Z and E isomers, which could be separated by semipreparative HPLC. Unlike their ferrocene homologues, the compounds do not isomerise in solution. Structural identification was carried out with NMR spectroscopy by using the HMBC and NOE techniques and was confirmed by the X-ray structural determination of (E)-7 a (n=2). These molecules were more lipophilic than OH-Tam (log P(o/w)=4.5-6.3) and they were all reasonably well recognized by the two forms of the estrogen receptor (ERalpha and ERbeta). For example, (Z)-7 b (n=3) has high relative binding affinity (RBA) values of 31 % for ERalpha and 16.8 % for ERbeta. The antiproliferative effects of two pairs of isomers, (Z)- and (E)-7 b (n=3) and (Z)- and (E)-7 d (n=5), were studied at a molarity of 1 microM on two breast-cancer cell lines, MCF7 (ERalpha positive) and MDA-MB231 (ERalpha negative). These molecules had an antiproliferative effect on MCF7 cells slightly higher than that of OH-Tam and no effect on MDA-MB231 cells. Thus, the antiproliferative effect observed on the MCF7 cells seemed essentially to be linked to an antiestrogenic effect. Molecular modelling studies have allowed us to rationalise these effects and select the best compounds for future development of a radioactive series.     

Protein-A-mediated targeting of bacteriochlorophyll-IgG to Staphylococcus aureus: a model for enhanced site-specific photocytotoxicity

A model for studying the efficiency of photodynamic action with a photosensitizer placed exclusively on the bacterial cell wall has been used. Bacteriochlorophyllide molecules, conjugated to rabbit immunoglobulin G (IgG), were synthesized. The conjugated pigment bacteriochlorophyll (Bchl)-IgG bound with high specificity to protein-A residues naturally exposed on the cell wall of the bacterium Staphylococcus aureus Cowan I. In bacterial suspensions the phototoxicity of the targeted conjugates (0.5-2.5 pigment per IgG molecule) was dose dependent (LD50 = 1.7 microM) in the presence of light (lambda > 550 nm) and inhibited by native IgG but not by ovalbumin, suggesting selective interaction with protein-A on the bacterial cell wall. No dark toxicity was noticed even with the highest conjugate concentration tested. In contrast, the photocytotoxicity of bacteriochlorophyll-serine (Bchl-Ser, LD50 = 0.07 microM) used as a nontargeted control was not inhibited by IgG. In spite of its lower apparent potency, Bchl-IgG was found to be 30 times more efficacious than Bchl-Ser: At LD50, only 66,000 Bchl-IgG molecules were bound per bacterium compared to 1,900,000 molecules of Bchl-Ser. The higher efficacy of Bchl-IgG is explained by its exclusive position on the bacterial cell wall. Consequently, photogeneration of oxidative species is confined to the cell wall and its vicinity, a seemingly highly susceptible domain for photodynamic action. In considering the design of cell-specific sensitizers for bacterial and cancer therapies, it would be beneficial to identify the more discretely sensitive subcellular domains as targets.     

LINEAR PROGRAMMING, SIMULATED ANNEALING AND TABU SEARCH HEURISTICS FOR LOTSIZING IN BOTTLENECK ASSEMBLY SYSTEMS

Multilevel lotsizing is one of the most challenging subjects in production planning, especially in the presence of capacity constraints. In this paper we investigate lotsizing heuristics for assembly production systems with a bottleneck. More specifically, we discuss heuristics based on Linear Programming (LP), and compare the performance of these heuristics with the performance of approaches based on simulated annealing and tabu search techniques.A comparison of the three heuristics on a set of test problems shows that simulated annealing and tabu search perform well compared to pure LP-based heuristics, but the effectiveness of the latter heuristics can be improved by combining them with elements from simulated annealing and tabu search.     

Frequency of silent ischemic heart disease in patients with diabetes mellitus

Early detection of silent ischaemia plays an important role in prevention of sudden cardiac death and acute myocardial infarction. More frequent occurrence of silent ischaemia in patients with diabetes mellitus and manifestations of ischaemic heart disease has been relayed in several studies. No studies aimed at frequency of occurrence of silent ischaemia in diabetic patients without clinical symptoms of ischaemic heart disease have been performed yet. Objectives of this study were the examination of the latter case. This study involved 67 patients with diabetes mellitus without clinical symptoms of ischaemic heart disease. The average duration time of diabetes mellitus was 11 years. The patients were divided in two groups. The first group included 26 patients with insulin dependent diabetes mellitus. The second group included 41 patients with non insulin dependent diabetes mellitus. The first control group consisted of 35 non diabetic patients with ischaemic heart disease, and the second control group consisted of 22 healthy volunteers. 24-hours ambulatory Holter monitoring and ECG exercise test were performed in all subjects. The diagnosis of silent ischaemia was established in patients with positive results of both examinations in ECG-records without any following pain. In case of only one positive results the dipyridamole stress echocardiography test with ECG was carried out to prove the diagnosis. It was proved, that silent ischaemia occurs in 19.2% of patients with insulin dependent diabetes mellitus and in 22% non insulin diabetic patients. No statistic differences between frequency of silent ischaemia occurrence in both groups were revealed. The application of 24-hours Holter monitoring combined with ECG-exercise stress test seems to be the best method in early recognition of silent ischaemia in diabetic patients.