An autopsy case of sodium nitrite-induced methemoglobinemia with various post-mortem analyses.

The use of sodium nitrite in suicide has become more common among young adults in the Republic of Korea. This report details the case of a 28-year-old man; the man had posted on a social network service detailing his attempt at suicide at 13:45. In the posted article, he stated that he had ingested 84 g of sodium nitrite. A post-mortem (PM) inspection was performed at 21:00, and peripheral blood (PB) was collected. An autopsy was performed approximately 44 h after death. The victim's face was dark brown in color, but the color of his oral mucosa was bright red. Toxicological analyses revealed 33% and 26% methemoglobinemia in the PB collected during PM inspection and autopsy, respectively. The concentration of nitrate in the PB collected during PM inspection, and PB and cardiac blood collected during the autopsy were 220.6 mg/L, 220.0 mg/L, and 218.5 mg/L, respectively. Nitrate was also detected in the pericardial fluid and cerebrospinal fluid at levels of 91.7 mg/L and 50.5 mg/L, respectively. The cause of death was determined to be methemoglobinemia-induced hypoxia due to sodium nitrite ingestion. This intoxication case informs some novel points about nitrite intoxication; the concentration of methemoglobin decreased during the PM period, while the concentration of nitrate was stable. There was no difference in the concentration of nitrate between cardiac and peripheral blood. Nitrate could be detected in the pericardial fluid and cerebrospinal fluid. This new information is helpful for better identifying future cases of nitrite intoxication.

Gelation, phase behavior, and dynamics of ß-lactoglobulin amyloid fibrils at varying concentrations and ionic strengths.

We have investigated the thermodynamic and dynamic behavior of multistranded ß-lactoglobulin protein fibrils in water, by combining static, dynamic, and depolarized dynamic light scattering (SLS, DLS, DDLS), small angle neutron scattering (SANS), rheology, and cryogenic transmission electron microscopy (cryo-TEM). We focus on the region of the phase diagram at which ionic strength and concentration changes induce transitions in gelation and lyotropic liquid crystalline behavior. An increase in ionic strength, induced by NaCl salt, progressively causes the phase transitions from nematic (N) to gel (G) phases; a further increase causes the transition to a translucent phase and to a macroscopic phase separation, respectively. An increase in fibril concentration induces first a phase transition from an isotropic (I) to a nematic phase (N); a further increase induces the formation of a gel phase. The protein gel strength is investigated by rheology measurements. SANS and osmotic compressibility calculated by SLS measurements clearly capture the main features of the IN transition of ß-lactoglobulin protein fibrils. The form and structure factors measured by scattering experiments are analyzed by the polymer reference interaction site model (PRISM). Dynamics of the protein fibrils at different concentrations, measured by polarized and depolarized dynamic light scattering, show both individual and collective diffusion after the isotropic-nematic transition. Above this transition, cryo-TEM images further demonstrate the alignment of the protein fibrils, which is quantified by a 2D order parameter. This work discusses comprehensively, both experimentally and theoretically, the thermodynamics and dynamic features of ß-lactoglobulin amyloid fibrils in a vast region of the concentration-ionic strength phase diagram.

The fabrication of highly ordered silver nanodot patterns by platinum assisted nanoimprint lithography.

Silver has been widely used for optical sensing and imaging applications which benefit from localized surface plasmon resonance (LSPR) in a nanoscale configuration. Many attempts have been made to fabricate and control silver nanostructures in order to improve the high performance in sensing and other applications. However, a fatal mechanical weakness of silver and a lack of durability in oxygen-rich conditions have disrupted the manufacturing of reproducible nanostructures by the top-down lithography approach. In this study, we suggest a steady fabrication strategy to obtain highly ordered silver nanopatterns that are able to provide tunable LSPR characteristics. By using a protecting layer of platinum on a silver surface in the lithography process, we successfully obtained large-area (2.7 × 2.7 mm(2)) silver nanopatterns with high reproducibility. This large-area silver nanopattern was capable of enhancing the low concentration of a Cy3 fluorescence signal (∼10(-10) M) which was labeled with DNA oligomers.

Liquid crystalline phase behavior of protein fibers in water: experiments versus theory.

We have developed a new method allowing the study of the thermodynamic phase behavior of mesoscopic colloidal systems consisting of amyloid protein fibers in water, obtained by heat denaturation and aggregation of beta-lactoglobulin, a dairy protein. The fibers have a cross section of about 5.2 nm and two groups of polydisperse contour lengths: (i) long fibers of 1-20 microm, showing semiflexible behavior, and (ii) short rods of 100-200 nm long, obtained by cutting the long fibers via high-pressure homogenization. At pH 2 without salt, these fibers are highly charged and stable in water. We have studied the isotropic-nematic phase transition for both systems and compared our results with the theoretical values predicted by Onsager's theory. The experimentally measured isotropic-nematic phase transition was found to occur at 0.4% and at 3% for the long and short fibers, respectively. For both systems, this phase transition occurs at concentrations more than 1 order of magnitude lower than what is expected based on Onsager's theory. Moreover, at low enough pH, no intermediate biphasic region was observed between the isotropic phase and the nematic phase. The phase diagrams of both systems (pH vs concentration) showed similar, yet complex and rich, phase behavior. We discuss the possible physical fundamentals ruling the phase diagram as well as the discrepancy we observe for the isotropic-nematic phase transition between our experimental results and the predicted theoretical results. Our work highlights that systems formed by water-amyloid protein fibers are way too complex to be understood based solely on Onsager's theories. Experimental results are revisited in terms of the Flory's theory (1956) for suspensions of rods, which allows accounting for rod-solvent hydrophobic interactions. This theoretical approach allows explaining, on a semiquantitative basis, most of the discrepancies observed between the experimental results and Onsager's predictions. The sources of protein fibers complex colloidal behavior are analyzed and discussed at length.

Interfacial activity and interfacial shear rheology of native ß-lactoglobulin monomers and their heat-induced fibers.

Interfacial properties of native ß-lactoglobulin monomers and their heat-induced fibers, of two different lengths, were investigated at pH 2, through surface tension measurements at water-air and water-oil interfaces and interfacial shear rheology at the water-oil interface. The applied heat treatment generates a mixed system of fibers with unconverted monomers and hydrolyzed peptides. The surface tension of this system at the water-air interface decreased more rapidly than the surface tension of native monomers, especially at short times (10(-3) to 10(2) s). This behavior was not observed when the unconverted monomers and peptides were removed by dialysis. At the water-oil interface, the adsorption kinetics was much faster than at the water-air interface, with a plateau interfacial pressure value reached after 1 h of adsorption. For all the systems, interfacial shear rheology showed the formation of a highly elastic interface, with solid-like behavior at 1-10(3) s time scales. The highest modulus was observed for the long fibers and the lowest for the native monomers. Creep-compliance curves in the linear regime could be reduced to a single master curve, showing similar spectra of relaxation times for all investigated systems. Upon large deformations, the interfaces formed with long fibers showed the most rigid and fragile behavior. This rigidity was even more pronounced in the presence of unconverted monomers.

Effects of charge double layer and colloidal aggregation on the isotropic-nematic transition of protein fibers in water.

We investigate the effects of variable linear charge density and Debye length on the mesoscopic properties of beta-lactoglobulin fibers in water, by changing the pH and ionic strength, respectively. We determine the isotropic-nematic (I-N) transition by cross-polarized microscopy and quantify by atomic force microscopy the increasing tendency of the fibers to aggregate upon raising ionic strength. We then compare experimental I-N transitions with theoretical expected values based on Onsager theory. Unlike previous reports on lyotropic liquid crystalline behavior of protein fibers, we show that, if double layer effects and aggregation of fibers are correctly included directly in the second virial coefficient and excluded volume, Onsager theory accurately predicts the experimental I-N transition versus pH and ionic strength.

Fabrication of complex patterns with a wide range of feature sizes from a single line prepattern by successive application of capillary force lithography.

The variously shaped gold patterns can be generated from the polydimethylsiloxane (PDMS) mold using line patterns by the capillary force lithography (CFL) process, which is a kind of nanoimprint method following the two-cycle method. After fabrication of micro- or nanosized line patterns at the first cycle, the patterned substrate is used as a substrate for the second cycle of CFL. When the other stamp is placed on the first pattern, rotated by a certain angle with respect to the first stamp, only the overlapped parts remained dot-shaped after the etching process. The various shapes and sizes of patterns can be produced by controlling the CFL conditions such as polymer thickness, reactive ion etching (RIE) time, and degree of rotation angle. The key advantage of the double imprint lithography method is to get the nanosized isolated dot-shaped patterns from microsized line patterns. If we fabricate nanosized isolated dot-shaped patterns directly, we should need predesigned patterns in the form of a master, which is generally prepared by a high-cost and time-consuming process such as E-beam lithography. The successful applications of large-area periodic patterns are nanoelectronic devices, nanoelectromechanical system (NEMS), and biosensors, the template of which is the master of nanoimprint lithography (NIL) and stamp fabrication in soft lithography.

Structure of heat-induced beta-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate.

We report on the conformation of heat-induced bovine beta-lactoglobulin (betalg) aggregates prepared at different pH conditions, and their complexes with model anionic surfactants such as sodium dodecyl sulfate (SDS). The investigation was carried out by combining a wide range of techniques such as ultra small angle light scattering, static and dynamic light scattering, small angle neutron scattering, small-angle X-ray scattering, electrophoretic mobility, isothermal titration calorimetry (ITC) and transmission electron microscopy. Three types of aggregates were generated upon heating betalg aqueous dispersions at increasing pH from 2.0 to 5.8 to 7.0: rod-like aggregates, spherical aggregates, and worm-like primary aggregates, respectively. These aggregates were shown not only to differ for their sizes and morphologies, but also for their internal structures and fractal dimensions. The main differences between aggregates are discussed in terms of the ionic charge and conformational changes arising for betalg at different pHs. The formation of complexes between SDS and the various protein aggregates at pH 3.0 was shown to occur by two main mechanisms: at low concentration of SDS, the complex formation occurs essentially by ionic binding between the positive residues of the protein and the negative sulfate heads of the surfactant. At complete neutralization of charges, precipitation of the complexes is observed. Upon further increase in SDS concentration, complex formation of SDS and the protein aggregates occurs primarily by hydrophobic interactions, leading to (i) the formation of an SDS double layer around the protein aggregates, (ii) the inversion of the total ionic charge of each individual protein aggregate, and (iii) the complete redispersion of the protein aggregate-SDS complexes in water. Remarkably, the SDS double layer around the protein aggregates provides an efficient protective shield, preventing precipitation of the aggregates at any possible pH values, including those values corresponding to the isoelectric pH of the aggregates.

Enhanced sensitivity of surface plasmon resonance (SPR) immunoassays using a peroxidase-catalyzed precipitation reaction and its application to a protein microarray.

We describe a method to improve the sensitivity of surface plasmon resonance (SPR) immunoassays using a horseradish peroxidase (HRP)-catalyzed precipitation reaction. The precipitation reaction catalyzed by HRP bound to the SPR biosensor surface via a sandwich immunoassay induced a shift in the SPR angle. Human interferon (IFN)-gamma at concentrations ranging from 0.01 to 100 ng/ml was detectable by this method. We also show that this biocatalytic signal amplification method can be applied to SPR imaging (SPRI), in an immunoassay of multiple proteins on a protein microarray format.

High Density of Line Pattern by Using De-Wetting During Soft-Lithography.

Here we prepared highly reliable and high density of pattern by using de-wetting induced soft-lithography. Additional line pattern arising from de-wetting of PS is generated in the protrusion of poly-dimethyl-siloxane (PDMS) mold by controlling thermal annealing time and molecular weight of PS. We found that such de-wetting and pattern formation is not dependent on the PS film thickness, but strongly influenced by molecular weight and annealing time of PS. These results indicate that high molecular weight of PS with such circumstance suppresses the mobility of the polymer chain, enhancing the surface tension of the polymer. We demonstrate that these de-wetting induced patterns attributed to the change of the mobility and the surface tension of PS chains, creating a high density of patterns with high reproducibility.

Distribution of cyanide in heart blood, peripheral blood and gastric contents in 21 cyanide related fatalities.

This paper presents 21 cases related to cyanide intoxication by oral ingestion. Cyanide concentrations in biological specimens are especially different from the type of postmortem specimens, and very important in interpreting the cause of death in postmortem forensic toxicology. Besides the detection of cyanide in autopsy specimens, the autopsy findings were unremarkable. Biological samples (0.2mL or equal to less than 10µg of cyanide) were analyzed colorimetrically for cyanide. In a series of 21 cyanide fatalities, the concentration ranges (mean±SD) of cyanide in heart blood, peripheral blood and gastric contents were 0.1-248.6mg/L (38.1±56.6mg/L), 0.3-212.4mg/L (17.1±45.1mg/L) and 2.0-6398.0mg/kg (859.0±1486.2mg/kg), respectively. The ranges of the heart/peripheral blood concentration ratio and gastric contents/peripheral blood concentration ratio were 0.3-10.6 (mean 3.4) and 3.4-402.4 (mean 86.0), respectively. From the difference of cyanide concentration and the concentration ratio of cyanide in different types of postmortem specimens, the possibility of the postmortem redistribution of cyanide and death by oral ingestion of cyanide could be confirmed. We reported cyanide fatal cases along with a review of literature.

Direct investigation of intracellular presence of gold nanoparticles via photothermal heterodyne imaging.

Nanotechnology as well as advanced microscopy can play a fundamental role in understanding biological mechanisms. Here we present a study that combines a new type of nanomaterial with a new type of microscopy and highlights the potential for gathering novel information about cell membrane penetration and cytosol local viscosity. On the material side, we used gold nanoparticles that have an ordered stripe-like arrangement of domains. These "striped" nanoparticles are able to penetrate cell membranes directly without porating them. On the microscopy side, we used photothermal heterodyne imaging which allows detection of individual nanometer-sized gold particles in complex media. We showed that we can probe cytosolic presence as well as dynamics of these nanoparticles even at very low concentrations. We used the fluctuations of the photothermal signal from particles diffusing in the detection volume to estimate local cytosol viscosity which is about 20 times larger than that of water. This work opens new perspectives for mapping local diffusion properties of nano-objects inside living cells.

Understanding amyloid aggregation by statistical analysis of atomic force microscopy images.

The aggregation of proteins is central to many aspects of daily life, including food processing, blood coagulation, eye cataract formation disease and prion-related neurodegenerative infections. However, the physical mechanisms responsible for amyloidosis-the irreversible fibril formation of various proteins that is linked to disorders such as Alzheimer's, Creutzfeldt-Jakob and Huntington's diseases-have not yet been fully elucidated. Here, we show that different stages of amyloid aggregation can be examined by performing a statistical polymer physics analysis of single-molecule atomic force microscopy images of heat-denatured beta-lactoglobulin fibrils. The atomic force microscopy analysis, supported by theoretical arguments, reveals that the fibrils have a multistranded helical shape with twisted ribbon-like structures. Our results also indicate a possible general model for amyloid fibril assembly and illustrate the potential of this approach for investigating fibrillar systems.

Application of supramolecular nanostamping to the replication of DNA nanoarrays.

The rapid development of molecular biology is creating a pressing need for arrays of biomolecules that are able to detect smaller and smaller volumes of analytes. This goal can be achieved by shrinking the average size and spacing of the arrays' constituent features. While bioarrays with dot size and spacing on the nanometer scale have been successfully fabricated via scanning probe microscopy-based techniques, such fabrication methods are serial in nature and consequently slow and expensive. Additionally, the development of truly small arrays able to analyze scarce volumes of liquids is hindered by the present use of optical detection, which sets the minimum dot spacing on the order of roughly half the excitation wavelength. Here, we show that supramolecular nanostamping, a recently introduced truly parallel method for the stamping of DNA features, can efficiently reproduce DNA arrays with features as small as 14 +/- 2 nm spaced 77 +/- 10 nm. Moreover, we demonstrate that hybridization of these nanoarrays can be detected using atomic force microscopy in a simple and scaleable way that additionally does not require labeling of the DNA strands.

Patterned arrays of au rings for localized surface plasmon resonance.

In this paper, we examined the characteristic behavior of localized surface plasmon resonances (LSPR) of Au dot and ring arrays in response to the selective binding of biomolecules. To do this, patterned arrays of Au rings and dots with various feature scales were fabricated over large areas by an imprint lithography technique. Our results showed that the LSPR spectra of the Au nanorings exhibited a blue shift with increase in the ring widths and asymptotically converged to those for Au nanodots. This clearly implies that the LSPR spectra can be tuned over an extended wavelength range by varying the ring width. For an illustrative purpose, the patterned Au structures were used to detect the binding of streptavidin to biotin. In doing this, the Au patterns were chemically modified with G4 dendrimers of amine terminated poly(amidoamine), which facilitated the tethering of biotin onto the Au pattern. Exposure of the biotinylated Au nanorings to aqueous streptavidin solution induced both red-shifts of the LSPR spectra and changes in the peak intensities. The sensitivity of the LSPR spectra to the binding of the biomolecules was enhanced as the ring width of Au rings was decreased.

A fusion protein expression analysis using surface plasmon resonance imaging.

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.