Jonas Carlsson

An asynchronous wrapper with novel handshake circuits for GALS systems

In this paper, we propose an asynchronous wrapper with novel handshake circuits for data communication to be used in GALS systems. The handshake circuits include two communication ports and a local clock controller. We present two approaches for the implementation of communication ports; one with pure standard cells and the other with Muller C elements. The detailed design methodology is given and the circuits are validated with VHDL and circuits simulation in standard CMOS technology.

Investigation and prediction of the severity of p53 mutants using parameters from structural calculations

A method has been developed to predict the effects of mutations in the p53 cancer suppressor gene. The new method uses novel parameters combined with previously established parameters. The most important parameter is the stability measure of the mutated structure calculated using molecular modelling. For each mutant, a severity score is reported, which can be used for classification into deleterious and nondeleterious. Both structural features and sequence properties are taken into account. The method has a prediction accuracy of 77% on all mutants and 88% on breast cancer mutations affecting WAF1 promoter binding. When compared with earlier methods, using the same dataset, our method clearly performs better. As a result of the severity score calculated for every mutant, valuable knowledge can be gained regarding p53, a protein that is believed to be involved in over 50% of all human cancers.

A Clock Gating Circuit for Globally Asynchronous Locally Synchronous Systems

This paper proposes a circuit that enables the use of off-chip oscillators in a globally asynchronous locally synchronous system. Usually, a local oscillator is used, which can be stopped. However, a local oscillator, e.g., ring oscillator, is not as stable as an off-chip oscillator and is therefore problematic to use for certain applications. The proposed circuit also informs the local synchronous module if the local clock has been stopped for too many of the off-chip oscillators clock cycles, so that it can take appropriate actions, e.g., recalibrate ring oscillators. The circuit has been tested in an Altera Stratix II FPGA and simulated in a 0.35 mum CMOS process

Polypeptide conjugate binders that discriminate between two isoforms of human carbonic anhydrase in human blood.

Two binder candidates 4‐C37L34‐B and 3‐C15L8‐B from a 16‐membered set of 42‐residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence‐based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five‐ to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16‐membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4‐C37L34‐B bound HCAII with a KD of 17 nM and HCAI with a KD of 470 nM, that is, with a 30‐fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3‐C15L8‐B and the two carbonic anhydrases were 60 and 390 nM, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.

Functionally important amino acids in the Arabidopsis thylakoid phosphate transporter: Homology modeling and site-directed mutagenesis

The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acid residues involved in Pi binding and translocation by ANTR1 and in the Na+ dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from E. coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in E. coli and radioactive Pi transport assays and compared with bacterial cells carrying an empty plasmid. The results from Pi- and Na+-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na+ ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters.

A new polymorphism in the coding region of exon four in HSD17B2 in relation to risk of sporadic and hereditary breast cancer

In situ synthesis of oestrogens is of great importance in the development and progression of breast cancer. 17β-hydroxysteroid dehydrogenase (17HSD) type 2 catalyses oxidation from oestradiol to oestrone, and thereby protects the breast epithelial cells from oestradiol. Low expression of 17HSD type 2 has been associated with decreased survival in breast cancer, but no studies have investigated the mechanism behind the low expression. The 17HSD type 2 gene (HSD17B2) was screened for mutations with Single Stranded Conformation Polymorphism (SSCP)-DNA sequencing in 59 sporadic breast cancer cases, 19 hereditary breast cancer cases and seven breast cancer cell lines. DNA samples from 226 healthy individuals were used to identify if changes were previously unknown polymorphisms. No mutation was detected and therefore mutations in HSD17B2 do not explain why some breast tumours exhibit low 17HSD type 2 expression. However, a previously unknown polymorphism was found in exon four (Met226Val). Using molecular modelling, we found that the substituted residue is located at the outer part of the steroid binding site, probably causing minor alterations in the substrate binding. We further studied if the polymorphism contributes to breast cancer susceptibility in a larger material, but did not find an increased risk in the group of 317 sporadic breast cancer patients, 188 breast cancer patients with two close relatives with breast cancer or 122 hereditary breast cancer patients, compared to the healthy control group. We suggest that the detected polymorphism does not contribute to a higher risk of developing breast cancer.

Structural and biochemical investigation of two Arabidopsis shikimate kinases: The heat‐inducible isoform is thermostable

The expression of plant shikimate kinase (SK; EC 2.7.1.71), an intermediate step in the shikimate pathway to aromatic amino acid biosynthesis, is induced under specific conditions of environmental stress and developmental requirements in an isoform‐specific manner. Despite their important physiological role, experimental structures of plant SKs have not been determined and the biochemical nature of plant SK regulation is unknown. The Arabidopsis thaliana genome encodes two SKs, AtSK1 and AtSK2. We demonstrate that AtSK2 is highly unstable and becomes inactivated at 37°C whereas the heat‐induced isoform, AtSK1, is thermostable and fully active under identical conditions at this temperature. We determined the crystal structure of AtSK2, the first SK structure from the plant kingdom, and conducted biophysical characterizations of both AtSK1 and AtSK2 towards understanding this mechanism of thermal regulation. The crystal structure of AtSK2 is generally conserved with bacterial SKs with the addition of a putative regulatory phosphorylation motif forming part of the adenosine triphosphate binding site. The heat‐induced isoform, AtSK1, forms a homodimer in solution, the formation of which facilitates its relative thermostability compared to AtSK2. In silico analyses identified AtSK1 site variants that may contribute to AtSK1 stability. Our findings suggest that AtSK1 performs a unique function under heat stress conditions where AtSK2 could become inactivated. We discuss these findings in the context of regulating metabolic flux to competing downstream pathways through SK‐mediated control of steady state concentrations of shikimate.

Asynchronous data communication with low power for GALS systems

In this paper, we propose an asynchronous wrapper with new handshake circuits and low swing bus drivers for data communication in GALS (globally-asynchronous locally-synchronous) systems with low power. The handshake circuits include two data-ports and a local clock controller. We present two approaches for the implementation of data-ports; one with pure standard cells and the other with Muller C elements. The detailed design methodology is given and the circuits are validated with VHDL and circuits simulation in standard CMOS technology.

Enrichment of ligands with molecular dockings and subsequent characterization for human alcohol dehydrogenase 3.

Alcohol dehydrogenase 3 (ADH3) has been assigned a role in nitric oxide homeostasis due to its function as an S-nitrosoglutathione reductase. As altered S-nitrosoglutathione levels are often associated with disease, compounds that modulate ADH3 activity might be of therapeutic interest. We performed a virtual screening with molecular dockings of more than 40,000 compounds into the active site of human ADH3. A novel knowledge-based scoring method was used to rank compounds, and several compounds that were not known to interact with ADH3 were tested in vitro. Two of these showed substrate activity (9-decen-1-ol and dodecyltetraglycol), where calculated binding scoring energies correlated well with the logarithm of the kcat/Km values for the substrates. Two compounds showed inhibition capacity (deoxycholic acid and doxorubicin), and with these data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs, and cholic acids.

A design approach for GALS based systems-on-chip

In this paper, we present an efficient design flow for GALS systems based on the state-of-art commercial tools, we first designed an asynchronous wrapper equipped with handshake circuits and stoppable clock generator, and then selected a digital FIR filter as a design example. The simulation and verification show our proposed interface circuits is of good performance in terms of speed and area. The design efficiency is also improved by allowing standard tools to better support GALS based designs.