Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections

MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of molecular species in tissue specimens. Numerous methodological improvements have been made to optimize tissue section preparation and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the number of signals observed. We systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize experimental variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and number of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystallization behavior as well as their ability to preserve tissue histology were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma biopsy.     

Splitting (complicated) surfaces is hard

Let be an orientable combinatorial surface. A cycle on is splitting if it has no self-intersections and it partitions into two components, neither of which is homeomorphic to a disk. In other words, splitting cycles are simple, separating, and non-contractible. We prove that finding the shortest splitting cycle on a combinatorial surface is NP-hard but fixed-parameter tractable with respect to the surface genus g and the number of boundary components b of the surface. Specifically, we describe an algorithm to compute the shortest splitting cycle in (g+b)^O(g+b)nlogn time, where n is the complexity of the combinatorial surface.     

AFM studies of solid-supported lipid bilayers formed at a Au(111) electrode surface using vesicle fusion and a combination of Langmuir-Blodgett and Langmuir-Schaefer techniques

Atomic force microscopy (AFM) has been used to characterize the formation of a phospholipid bilayer composed of 1,2-dimyristyl-sn-glycero-3-phosphocholine (DMPC) at a Au(111) electrode surface. The bilayer was formed by one of two methods: fusion of lamellar vesicles or by the combination of Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) deposition. Results indicate that phospholipid vesicles rapidly adsorb and fuse to form a film at the electrode surface. The resulting film undergoes a very slow structural transformation until a characteristic corrugated phase is formed. Force-distance curve measurements reveal that the thickness of the corrugated phase is consistent with the thickness of a bilayer lipid membrane. The formation of the corrugated phase may be explained by considering the elastic properties of the film and taking into account spontaneous curvature induced by the asymmetric environment of the bilayer, in which one side faces the gold substrate and the other side faces the solution. The effect of temperature and electrode potential on the stability of the corrugated phase has also been described.     

Magnetic nanoparticle-peptide conjugates for in vitro and in vivo targeting and extraction of cancer cells

Magnetic cobalt spinel ferrite nanoparticles coated with biocompatible polygalacturonic acid were functionalized with ligands specific for targeting expressed EphA2 receptors on ovarian cancer cells. By using such magnetic nanoparticle-peptide conjugates, targeting and extraction of malignant cells were achieved with a magnetic field. Targeting ovarian cancer cells with receptor specific peptide-modified magnetic nanoparticles resulted in cell capture from a flow stream in vitro and from the peritoneal cavity of mice in vivo. Successful removal of metastatic cancer cells from the abdominal cavity and circulation using magnetic nanoparticle conjugates indicate the feasibility of a dialysis-like treatment and may improve long-term survival rates of ovarian cancer patients. This approach can be applied for fighting other cancers, such as leukemia, once the receptors on malignant cells are identified and the efficacy of targeting ligands is established.     

G-quadruplex DNA assemblies: loop length, cation identity, and multimer formation

G-rich DNA sequences are able to fold into structures called G-quadruplexes. To obtain general trends in the influence of loop length on the structure and stability of G-quadruplex structures, we studied oligodeoxynucleotides with random bases in the loops. Sequences studied are dGGGW(i)GGGW(j)GGGW(k)GGG, with W = thymine or adenine with equal probability, and i, j, and k comprised between 1 and 4. All were studied by circular dichroism, native gel electrophoresis, UV-monitored thermal denaturation, and electrospray mass spectrometry, in the presence of 150 mM potassium, sodium, or ammonium cations. Parallel conformations are favored by sequences with short loops, but we also found that sequences with short loops form very stable multimeric quadruplexes, even at low strand concentration. Mass spectrometry reveals the formation of dimers and trimers. When the loop length increases, preferred quadruplex conformations tend to be more intramolecular and antiparallel. The nature of the cation also has an influence on the adopted structures, with K(+) inducing more parallel multimers than NH4(+) and Na(+). Structural possibilities are discussed for the new quadruplex higher-order assemblies.     

Electrogenerated chemiluminescence: an oxidative-reductive mechanism between quinolone antibiotics and tris(2,2'-bipyridyl)ruthenium(II)

The cyclic voltammetry and electrogenerated chemiluminescent (ECL) reactions of a series of quinolone and fluoroquinolone antibiotics were investigated in a flow injection analysis (FIA) system. 7-Piperazinyl fluoroquinolone antibiotics were found to participate as a coreactant in an oxidative-reductive ECL mechanism with tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) as the luminescent reagent. The reaction mechanism was investigated in order to understand and optimize the processes leading to light emission. The optimal conditions included a solution pH ∼7 at a flow rate of 3.0 mL min⁻¹ with no added organic modifier and application of 1.2 V vs. a Pt quasi-reference electrode (QRE). Fluoroquinolones containing a tertiary distal nitrogen on the piperazine ring, such as enrofloxacin and ofloxacin, reacted to produce more intense ECL than those with a secondary nitrogen, such as ciprofloxacin and norfloxacin. The method linear range, precision, detection limits, and sensitivity for the detection of enrofloxacin and ciprofloxacin were compared to that of tripropylamine. The method was applied to the determination of the ciprofloxacin content in a pharmaceutical preparation. The assay is discussed in terms of its analytical figures of merit, ease of use, speed, accuracy and application to pharmaceutical samples.     

Birefringence and scattering of light in colloidal solutions of magnetite in kerosene

The birefringence and dynamic and static scattering of light in colloidal solutions of magnetite nanoparticles in kerosene with different concentrations of the solid phase have been investigated. It is shown that these solutions contain both individual colloidal particles about 12 nm in diameter and their aggregates up to 100?600 nm in diameter. The largest aggregates are formed in solutions with the lowest concentration (on the order of 0.001 vol % or lower). The presence of relatively large aggregates makes it possible to observe specific features of optical anisotropy relaxation in these solutions, which are related to the non-Rayleigh character of light scattering from magnetite-particle aggregates.     

Prediction of the structures of the plant-specific regions of vascular plant cellulose synthases and correlated functional analysis

Seed plants express cellulose synthase (CESA) protein isoforms with non-redundant functions, but how the isoforms function differently is unknown. Compared to bacterial cellulose synthases, CESAs have two insertions in the large cytosolic loop: the relatively well-conserved Plant Conserved Region (P-CR) and a Class Specific Region (CSR) that varies between CESAs. Absent any atomic structure of a plant CESA, we used ab initio protein structure prediction and molecular modeling to explore how these plant-specific regions may modulate CESA function. We modeled P-CR and CSR peptides from Arabidopsis thaliana CESAs representing the six clades of seed plant CESAs. As expected, the predicted wild type P-CR structures were similar. Modeling of the mutant P-CR of Atcesa8 ^R362K (fra6) suggested that changes in local structural stability and surface electrostatics may cause the mutant phenotype. Among CSRs within CESAs required for primary wall cellulose synthesis, the amino sequence and the modeled arrangement of helices was most similar in AtCESA1 and AtCESA3. Genetic complementation of known Arabidopsis mutants showed that the CSRs of AtCESA1 and AtCESA3 can function interchangeably in vivo. Analysis of protein surface electrostatics led to ideas about how the surface charges on CSRs may mediate protein–protein interactions. Refined modeling of the P-CR and CSR regions of GhCESA1 from cotton modified their tertiary structures, spatial relationships to the catalytic domain, and preliminary predictions about CESA oligomer formation. Cumulatively, the results provide structural clues about the function of plant-specific regions of CESA.     

Lattice-type self-similar sets with pluriphase generators fail to be Minkowski measurable

A long-standing conjecture of Lapidus states that under certain conditions, self-similar fractal sets fail to be Minkowski measurable if and only if they are of lattice type. It was shown by Falconer and Lapidus (working independently but both using renewal theory) that nonlattice self-similar subsets of \({\mathbb {R}}\) are Minkowski measurable, and the converse was shown by Lapidus and v. Frankenhuijsen a few years later, using complex dimensions. Around that time, Gatzouras used renewal theory to show that nonlattice self-similar subsets of \({\mathbb {R}}^d\) that satisfy the open set condition are Minkowski measurable for \(d \ge 1\). Since then, much effort has been made to prove the converse. In this paper, we prove a partial converse by means of renewal theory. Our proof allows us to recover several previous results in this regard, but is much shorter and extends to a more general setting; several technical conditions appearing in previous work have been removed.