Integrated electronic detection of biomolecules

Electronics offers several unique opportunities for the detection and characterization of biomolecules such as oligonucleotides and proteins. Solid-state microfabrication technology, similar to that used to make integrated circuits, can be employed to make integrated electronic sensing systems that are capable of simultaneously detecting multiple molecules. Here, we review some of the capabilities afforded by electronics for rapid and sensitive detection of biomolecules and discuss a recent demonstration of a multi-marker electronic sensing system for detection of uropathogens in clinical samples.     

Molecular geometry,vibrations and electrode potentials of 2-(4,5-dihydroxy-2-methylphenyl)-2-phenyl-2H-indene-1,3-dione; experimental and theoretical attempts

The electrode potential of 2-(4,5-dihydroxy-2-methylphenyl)-2-phenyl-2H-indene-1,3-dione (DMPID) in acetonitrile has been calculated. The calculations were performed using ab initio molecular orbital calculations (HF), and density functional theory (DFT) with the inclusion of entropic and thermochemical corrections to yield free energies of redox reactions. The electrode potential of DMPID was also obtained experimentally with the aid of an electrochemical technique (cyclic voltammetry). The values for geometric parameters and the vibrational frequencies of DMPID and 2-(6-methyl-3,4-dioxocyclohexa-1,5-dienyl)-2-phenyl-2H-indene-1,3-dione (MDPID) were also computed using the same levels with the basis set of 6-31G(d). The calculated IR spectrum of DMPID used for the assignment of IR frequencies was observed in the experimental FT-IR spectrum and the calculated IR and FT-IR observed spectra of DMPID were compared with correlation factor of 0.996. It should be mentioned that the present work is the first research on coagulant derivative molecules in which the electrode potential of a molecule is calculated. Optimized structures of 2-(6-methyl-3,4-dioxocyclohexa-1,5-dienyl)-2-phenyl-2H-indene-1,3-dione (MDPID)     

HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway

Human heat-shock protein (HSP)70 activates innate immune cells and hence requires no additional adjuvants to render bound peptides immunogenic. Here we tested the assumption that endogenous HSP70 activates the Toll/IL-1 receptor signal pathway similar to HSP60 and pathogen-derived molecular patterns. We show that HSP70 induces interleukin-12 (IL-12) and endothelial cell-leukocyte adhesion molecule-1 (ELAM-1) promoters in macrophages and that this is controlled by MyD88 and TRAF6. Furthermore, HSP70 causes MyD88 relocalization and MyD88-deficient dendritic cells do not respond to HSP70 with proinflammatory cytokine production. Using the system of genetic complementation with Toll-like receptors (TLR) we found that TLR2 and TLR4 confer responsiveness to HSP70 in 293T fibroblasts. The expanding list of endogenous ligands able to activate the ancient Toll/IL-1 receptor signal pathway is in line with the "danger hypothesis" proposing that the innate immune system senses danger signals even if they originate from self.     

Accelerated design of bioconversion processes using automated microscale processing techniques

Microscale processing techniques are rapidly emerging as a means to increase the speed of bioprocess design and reduce material requirements. Automation of these techniques can reduce labour intensity and enable a wider range of process variables to be examined. This article examines recent research on various individual microscale unit operations including microbial fermentation, bioconversion and product recovery techniques. It also explores the potential of automated whole process sequences operated in microwell formats. The power of the whole process approach is illustrated by reference to a particular bioconversion, namely the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one for the production of optically pure lactones.     

Successful adoptive immunotherapy with vaccine-sensitized T cells,despite no effect with vaccination alone in a weakly immunogenic tumor model

Tumor cell vaccines have been successful at inducing immunity in naïve mice, but only in a few reports has vaccination alone induced regression of established tumors and, generally, only when they are very small. Clinically, vaccinations alone may not be able to cause regression of established human cancers, which tend to be weakly immunogenic. We hypothesized that pharmacologic ex vivo amplification of a vaccination-induced immune response with subsequent adoptive immunotherapy (AIT) to tumor-bearing animals would be more effective in treatment of these animals than vaccination alone. The 4T1 and 4T07 mammary carcinomas are derived from the same parental cell line, but 4T1 is much less immunogenic and more aggressive than 4T07. Vaccination with either 4T1, 4T1-IL-2, or 4T07-IL-2 was not effective as treatment for established 4T1 tumors. However, 4T1 or 4T07-IL-2-vaccine-sensitized draining lymph node (DLN) cells, activated ex vivo with bryostatin 1 and ionomycin and expanded in culture, induced complete tumor regressions when adoptively transferred to 4T1 tumor-bearing animals. This was effective against small tumors as well as more advanced tumors, 10 days after tumor cell inoculation. Furthermore, as would be required for this approach to be used clinically, vaccine-DLN cells obtained from mice with established progressive 4T1 tumors (inoculated 10 days before vaccination) also induced regression of 4T1 tumors in an adoptive host. In none of these experiments was exogenous IL-2 required to induce tumor regression. The response to tumor cell vaccine can be amplified by ex vivo pharmacologic activation of sensitized T cells, which can then cure an established, weakly immunogenic and highly aggressive tumor that was resistant to vaccination alone.     

Inhibition of human hemoglobin autoxidation by sodium n-dodecyl sulphate

The effect of sodium n-dodecyl sulphate (SDS) on hemoglobin autoxidation was studied in the presence of a 100 mM phosphate buffer (pH 7.0) by different methods. These included spectrophotometry, fluorescence technique, cyclic voltametry, differential scanning calorimetry, and densitometry. Spectroscopic studies showed that SDS concentrations up to 1 mM increased deoxy-, decreases oxy-, and had no significant effect on the met- conformation of hemoglobin. Therefore, a SDS concentration up to 1 mM increased the deoxy form of hemoglobin as the folded, compact state and decreases the oxy conformation. The turbidity measurements and differential scanning calorimetry techniques indicated a more stable conformation for hemoglobin in the presence of SDS up to 1 mM. Electrochemical studies also confirmed a more difficult oxidation under these conditions. The induction of the deoxy form in the presence of SDS was confirmed by densitometry techniques. The compact structure of deoxyhemoglobin blocks the formation of met-conformation in low SDS concentrations.     

Investigation on the effect of static magnetic field up to 15 mT on the viability and proliferation rate of rat bone marrow stem cells

This investigation was performed to evaluate the influence of the static magnetic field up to 15 mT on the viability and proliferation rate of rat bone marrow stem cells. Cells from passage 5 were trypsinized, and a cell suspension was prepared. The cells were counted and cultured in 25-cm² flasks. They were incubated for 1 d, washed with phosphate-buffered saline, and then exposed with different intensities of static magnetic field (4, 7, and 15 mT) at different exposure times (24, 48, 72, and 96 h). Cells were then washed with phosphate-buffered saline, trypsinized, and a cell suspension was prepared separately from each flask. To investigate the viability and proliferation rates of treated cells, staining with Trypan blue and counting were performed with an optical microscope. The mean number of whole cells and living cells was considered as proliferation and survival rates, respectively. Increasing of intensity and time of static magnetic field exposure decreased the viability percent and proliferation rate in treated groups compared with corresponded control. However, reduced cell viability, where this occurred, is exclusively due to apoptosis since necrosis is never observed by others.     

Protein psychrophilicity: role of residual structural properties in adaptation of proteins to low temperatures

In order to investigate the structural distribution responsible for protein psychrophilicity, a systematic comparative analysis of 13 pairs of psychrophilic and mesophilic proteins is reported. Three kinds of residue structural states such as exposed, intermediate and buried were considered for analyzing the structural patterns of single amino acids and amino acids in different groups. The statistical test revealed that higher frequency in exposed state of Ala, higher frequency in intermediate state of His, lower frequency in buried state of Lys, lower frequency in exposed state of Gln, higher frequency in exposed state and in intermediate state of Thr, higher frequency in exposed and intermediate state of tiny and small amino acids groups could be critical factors related with protein psychrophilicity. Such structure-based differences of residual properties would help to develop a strategy for designing psychrophilic proteins.     

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.