Proteomics analysis of tumor microenvironment: Implications of metabolic and oxidative stresses in tumorigenesis

Tumorigenesis is always concomitant with microenvironmental alterations. The tumor microenvironment is a heterogeneous and complex milieu, which exerts a variety of stresses on tumor cells for proliferation, survival, or death. Recently, accumulated evidence revealed that metabolic and oxidative stresses both play significant roles in tumor development and progression that converge on a common autophagic pathway. Tumor cells display increased metabolic autonomy, and the hallmark is the exploitation of aerobic glycolysis (termed Warburg effect), which increased glucose consumption and decreased oxidative phosphorylation to support growth and proliferation. This characteristic renders cancer cells more aggressive; they devour tremendous amounts of nutrients from microenvironment to result in an ever‐growing appetite for new tumor vessel formation and the release of more “waste,” including key determinants of cell fate like lactate and reactive oxygen species (ROS). The intracellular ROS level of cancer cells can also be modulated by a variety of stimuli in the tumor microenvironment, such as pro‐growth and pro‐inflammatory factors. The intracellular redox state serves as a double‐edged sword in tumor development and progression: ROS overproduction results in cytotoxic effects and might lead to apoptotic cell death, whereas certain level of ROS can act as a second‐messenger for regulation of such cellular processes as cell survival, proliferation, and metastasis. The molecular mechanisms for cancer cell responses to metabolic and oxidative stresses are complex and are likely to involve multiple molecules or signaling pathways. In addition, the expression and modification of these proteins after metabolic or oxidative stress challenge are diverse in different cancer cells and endow them with different functions. Therefore, MS‐based high‐throughput platforms, such as proteomics, are indispensable in the global analysis of cancer cell responses to metabolic and oxidative stress. Herein, we highlight recent advances in the understanding of the metabolic and oxidative stresses associated with tumor progression with proteomics‐based systems biology approaches. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:267–311, 2013     

Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone

To bypass limitations of ensemble averaging biochemical analysis, microscopy‐based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488‐stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re‐swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.     

LABEL‐FREE BIO‐AFFINITY MASS SPECTROMETRY FOR SCREENING AND LOCATING BIOACTIVE MOLECULES

Despite the recent increase in the development of bioactive molecules in the drug industry, the enormous chemical space and lack of productivity are still important issues. Additional alternative approaches to screen and locate bioactive molecules are urgently needed. Label‐free bio‐affinity mass spectrometry (BA‐MS) provides opportunities for the discovery and development of innovative drugs. This review provides a comprehensive portrayal of BA‐MS techniques and of their applications in screening and locating bioactive molecules. After introducing the basic principles, alongside some application notes, the current state‐of‐the‐art of BA‐MS‐assisted drug discovery is discussed, including native MS, size‐exclusion chromatography‐MS, ultrafiltration‐MS, solid‐phase micro‐extraction‐MS, and cell membrane chromatography‐MS. Finally, several challenges and limitations of the current methods are summarized, with a view to potential future directions for BA‐MS‐assisted drug discovery. © 2019 John Wiley & Sons Ltd. Mass Spec Rev     

Terminal protein-induced stretching of bacteriophage φ29 DNA

Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA–protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double‐stranded DNA genome of bacteriophage φ29. Necessary for this stretching is a protein covalently bound at both 5′ termini of φ29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from ～50% overstretched to ～50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some φ29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single‐molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.     

TectoRNA and 'kissing-loop' RNA: atomic force microscopy of self-assembling RNA structures

RNA molecules have been much less studied by atomic force microscopy (AFM) than have DNA molecules. In this paper, AFM imaging is presented for two different RNA molecules able to self‐assemble into complex supramolecular architectures. The first one is a molecular dimer of a 230‐nt RNA fragment coming from the RNA genome of a murine leukaemia virus. The monomeric RNA fragment, which appears by AFM as an elongated structure with a mean aspect ratio of 1.4, assembles into a dimer of elongated structures through the formation of a ‘kissing‐loop’ RNA interaction. The second one is a large supramolecular fibre formed of artificial self‐assembling RNA molecular units called tectoRNA. The fibre lengths by AFM suggest that there are 50–70 tectoRNA units per fibre. Some methods and limitations are presented for measuring molecular volumes from AFM images.     

Molecular dynamics study of the interfacial slip phenomenon in ultrathin lubricating films

The tribological performance of thin films of a liquid alkane has been studied through a molecular dynamics simulation with particular attention being paid to the phenomenon of interfacial slip. The model system for the simulation consists of two solid walls, with the lubricant molecules confined between them. Molecules of n‐decane (C₁₀H₂₂) were chosen to represent the lubricant molecules. The results of the simulation show: the average velocity of decane molecules in a Couette flow exhibits largely a linear distribution, but with a slip velocity at the solid‐liquid interface; when the simulations are performed at different temperatures, the slip ratios were found to vary with temperature; slip behaviour depends strongly on the solid‐fluid interaction; and slip ratios increase with decreasing film thickness, suggesting that slip in the thin films is a confinement‐related phenomenon.     

Quantitative optical lock‐in detection for quantitative imaging of switchable and non‐switchable components

Reversible photoswitching has been proposed as a way to identify molecules that are present in small numbers over a large, non‐switching, background. This approach, called optical‐lock‐in‐detection (OLID) requires the deterministic control of the fluorescence of a photochromic emitter through optical modulation between a bright (on) and a dark state (off). OLID yields a high‐contrast map where the switching molecules are pinpointed, but the fractional intensities of the emitters are not returned. The present work presents a modified OLID approach (quantitative OLID or qOLID) that yields quantitative information of the switching (f_SW) and non‐switching (f_NS) components. After the validation of the method with a sample dataset and image sequence, we apply qOLID to measurements in cells that transiently express the photochromic protein EYQ1. We show that qOLID is efficient in separating the modulated from the non‐modulated signal, the latter deriving from background/autofluorescence or fluorophores emitting in the same spectral region. Finally, we apply qOLID to Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging. We here demonstrate that qOLID is able to highlight the distribution of FRET intensity in a sample by using a photochromic donor and a non‐photochromic acceptor.     

Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best‐known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second‐harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second‐harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second‐harmonic generation, as a second‐order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser‐scanning microscope. In this work, we combine the axial resolving power of second‐harmonic generation and chiral sensitivity of second‐harmonic generation circular dichroism to realize three‐dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second‐harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second‐harmonic generation circular dichroism response in complicated three‐dimensional biological systems. The sample we use is starch granules whose second‐harmonic generation‐active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second‐harmonic generation for right‐handed circularly polarized excitation is significantly different from second‐harmonic generation for left‐handed one, offering excellent second‐harmonic generation circular dichroism contrast that approaches 100%. In addition, three‐dimensional visualization of second‐harmonic generation circular dichroism distribution with sub‐micrometer spatial resolution is realized. We observed second‐harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second‐harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three‐dimensional structures of starch granules. The second‐harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.     

Simultaneous multiplane imaging‐based localization encoded (SMILE) microscopy for super‐resolution volume imaging

We propose a widefield‐based rapid super‐resolution volume imaging technique. This technique requires encoding single molecules to their respective planes and subsequent identification of the locus of individual molecule (both in the focal plane and off‐focal planes). Experimentally, this is achieved by precise calibration of system PSF size and its natural spread in the off‐focal planes using sub‐diffraction fluorescent beads. The specimen plane touching the coverslip is chosen as the focal plane whereas planes far from coverslip (situated at large penetration depths) represent off‐focal planes. The identification and sorting of single molecules are carried out by setting multiple cut‐offs to the respective PSFs and a 3D super‐resolved volume image is reconstructed. SMILE microscopy technique eliminates the need for multiple z‐plane scanning, minimizes radiation‐dose and enables rapid super‐resolution volume imaging.     

Nanometre localization of single ReAsH molecules

ReAsH is a red‐emitting dye that binds to the unique sequence Cys‐Cys‐Xaa‐Xaa‐Cys‐Cys (where Xaa is a noncysteine amino acid) in the protein. We attached a single ReAsH to a calmodulin with an inserted tetracysteine motif and immobilized individual calmodulins to a glass surface at low density. Total internal reflection fluorescence microscopy was used to image individual ReAsH molecules. We determined the centre of the distribution of photons in the image of a single molecule in order to determine the position of the dye within 5 nm precision and with an image integration time of 0.5 s. The photostability of ReAsH was also characterized and observation times ranging from several seconds to over a minute were observed. We found that 2‐mercaptoethanesulphonic acid increased the number of collected photons from ReAsH molecules by a factor of two. Individual ReAsH molecules were then moved via a nanometric stage in 25 or 40 nm steps, either at a constant rate or at a Poisson‐distributed rate. Individual steps were clearly seen, indicating that the observation of translational motion on this scale, which is relevant for many biomolecular motors, is possible with ReAsH.     

Morphological and functional characterization of circulating hemocytes using microscopy techniques

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi‐granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule‐containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid–Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell‐mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase‐mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme‐agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.     

Biomedical application of MALDI mass spectrometry for small‐molecule analysis

Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is an emerging analytical tool for the analysis of molecules with molar masses below 1,000 Da; that is, small molecules. This technique offers rapid analysis, high sensitivity, low sample consumption, a relative high tolerance towards salts and buffers, and the possibility to store sample on the target plate. The successful application of the technique is, however, hampered by low molecular weight (LMW) matrix‐derived interference signals and by poor reproducibility of signal intensities during quantitative analyses. In this review, we focus on the biomedical application of MALDI‐MS for the analysis of small molecules and discuss its favorable properties and its challenges as well as strategies to improve the performance of the technique. Furthermore, practical aspects and applications are presented. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:101–120, 2011     

Self‐assembled monolayers of DNA on cysteamine modified Au(111) surface: Atomic force microscopy study

The λ‐DNA molecules self‐assemble on cysteamine‐modified gold (111) surface to form flat‐lying self‐assembled monolayers (SAMs). The formation kinetics of such DNA SAMs is studied by atomic force microscopy (AFM). AFM results show that DNA molecules do not arrange themselves on cysteamine‐modified gold (111) surface into a well‐ordered monolayer. It is also found that the surface density of DNA monolayer does not increase as the DNA concentration increases. The high temperature of DNA solution and the immersing in ultrapure water produce some obvious DNA bundles. Whereas divalent cations in DNA solution result in the formation of more compact DNA films. The obtained information may be useful for practical application of the DNA films and further theoretical studies. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

The forgotten science: reviving morphology

Are modern science and clinicians forgetting or ignoring the importance of morphology and microscopy in studying disease and disease patterns? Here we ponder that current science research over‐emphasizes the value of molecules and disease modelling, or rather under‐estimate the usefulness of microscopy and morphology. We debate the usefulness of morphology in contemporary research and wonder whether our techniques are too old‐fashioned or whether our field is seen as redundant.     

Ab‐initio investigation of chemical‐bond formation at the diamond‐like carbon surface

Diamond‐like carbon (DLC) is a promising material for tribology‐based applications. We investigate the susceptibility of the DLC surface to some characteristic molecules that are potentially present in various lubricants by means of ab‐initio calculations. We demonstrate that the strongest bond is formed between the oxygen atoms from the molecule and the metallic dopants present in the DLC. We present the first experimental evidence that proves the theoretical hypothesis. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultraviolet photofragmentation of biomolecular ions

Mass spectrometric identification of all types of molecules relies on the observation and interpretation of ion fragmentation patterns. Peptides, proteins, carbohydrates, and nucleic acids that are often found as components of complex biological samples represent particularly important challenges. The most common strategies for fragmenting biomolecular ions include low‐ and high‐energy collisional activation, post‐source decay, and electron capture or transfer dissociation. Each of these methods has its own idiosyncrasies and advantages but encounters problems with some types of samples. Novel fragmentation methods that can offer improvements are always desirable. One approach that has been under study for years but is not yet incorporated into a commercial instrument is ultraviolet photofragmentation. This review discusses experimental results on various biological molecules that have been generated by several research groups using different light wavelengths and mass analyzers. Work involving short‐wavelength vacuum ultraviolet light is particularly emphasized. The characteristics of photofragmentation are examined and its advantages summarized. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:425–447, 2009     

An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy

The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein‐tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report, the construction, properties and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.     

High‐throughput continuous flow femtosecond laser‐assisted cell optoporation and transfection

We present a femtosecond‐laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro‐flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam‐shaping generates a focal geometry along a line which is scanned across the micro‐flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell‐light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high‐throughput laser‐assisted cell‐poration system which could be integrated in lab‐on‐a‐chip devices. Microsc. Res. Tech. 77:974–979, 2014. © 2014 Wiley Periodicals, Inc.     

A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences

We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two‐motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state‐of‐the‐art tracking methods, especially for sequences with high spot density.     

Physical and chemical modification of zinc carboxylate‐containing lubricants by molecular structure changes

Twenty‐two samples of zinc carboxylates were prepared from saturated, non‐cyclic carboxylic acids with systematically altered hydrocarbon chain structures under well‐defined conditions. Essential temperature‐ and solvent‐induced changes concerning the coordination of the carboxylate group are shown using selected prepared samples, and general rules for systems containing zinc carboxylates in nonpolar media are presented. It is shown that there are three possible structural forms for zinc carboxylates: polymeric sheet, polymeric chain, and closed tetranuclear oxo complex molecules. These forms are closely related through their physical and chemical transitions. The results show that the spatial structure of the hydrocarbon chain is a crucial factor, besides physical and chemical conditions, limiting the possible forms of zinc carboxylates present in the system. The principles presented enable us to predict and design the behaviour of a system containing zinc carboxylate additives.