In Situ Scatheless Cell Detachment Reveals Correlation between Adhesion Strength and Viability at Single‐Cell Resolution

Single‐cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single‐cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single‐cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single‐cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip‐based live single‐cell extractor (LSCE). We believe that this method will provide a valuable new tool for single‐cell biology.     

In Situ Single‐Cell Western Blot on Adherent Cell Culture

Integrating 2D culture of adherent mammalian cells with single‐cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single‐cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin‐functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one‐step copolymerization process that creates protein‐decorated microwells. After single‐cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell‐based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab‐gel western blot, while revealing cell‐to‐cell heterogeneity in stress responses.     

Chiral and water‐soluble poly(2‐oxazoline)s

We describe the synthesis and characterization of the first water‐soluble and chiral poly(2,4‐disubstituted‐2‐oxazoline)s. While poly(2,4‐dimethyl‐2‐oxazoline)s are water soluble up to 100 °C, aqueous solutions of poly(2‐ethyl‐4‐methly‐2‐oxazoline) exhibit a lower critical solution temperature. This is discussed in context with its constitutional isomers poly(2‐oxazoline)s and poly(2‐oxazine)s. Circular dichroism spectroscopy revealed strong Cotton effects, which are also responsive to temperature in aqueous solution. It is therefore hypothesized that structures, comparable to polyproline helices, are formed in aqueous solution. In contrast to polyproline, poly(2,4‐disubstituted‐2‐oxazoline)s are highly water soluble and therefore represent very interesting pseudo‐polypeptides that may be useful to develop responsive biomimetic biomaterials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Monolithic silica columns functionalized with substituted polyproline‐derived chiral selectors as chiral stationary phases for high‐performance liquid chromatography

In this study, two polyproline‐derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead‐based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.     

Comprehensive and Accurate Ab Initio Energy Surface of Simple Alanine Peptides

Secondary structures for AcAla_nNH₂ (n=2–4) peptides have been analyzed by means of ab initio MP2, CCSD(T), and DFT‐B3PW91 methods using large basis sets and including implicit hydration effects and thermal corrections. In addition to the classical “pure” right‐handed 3₁₀‐ and α‐helices, left‐handed polyproline II, inverse γ‐turn, and fully extended conformations, a large number of “mixed” structures obtained by combining characteristic φ/ψ angles of each residue in every way have been found. All mixed structures are energetically accessible and can be present at significant levels in aqueous solution. Furthermore, they represent the way through which hierarchical unfolding of 3₁₀‐ and α‐helices or nonhierarchical polyproline II to fully extended transitions occur. Computational results are in qualitative agreement with the large amount of experimental data for simpler polyalanines and provide significant insight into their thermodynamic properties and how these can be modulated by interactions with solvent.     

Silaproline Helical Mimetics Selectively Form an All‐trans PPII Helix

The polyproline II helix (PPII) is increasingly recognized as an important element in peptide and protein structures. The discovery of pertinent PPII peptidomimetics is of great interest to tune physical properties of the targeted structure. A series of silaproline oligomers from dimer to pentamer were synthesized. CD studies, NMR spectroscopy and molecular modeling revealed that the ribbon preferentially populates the polyproline type II secondary structure in both [D]chloroform and [D₄]MeOH. The characteristics of this new lipophilic PPII‐like helix were determined.     

Cell penetrating agents based on a polyproline helix scaffold

Cell penetrating agents were designed and synthesized that introduce cationic and hydrophobic moieties along the backbone of a polyproline helix (PPII) in an amphiphilic manner. The CD profile has the features that are expected for a PPII helix, demonstrating that the addition of these groups had little effect on the backbone structure. Dramatic increases in uptake were found with MCF-7 cells when up to six guanidinium groups were positioned on the polyproline helix, whereas only modest increases in cellular uptake were observed with the amine-containing polyproline compounds as compared to their flexible counterparts. Amphiphilicity played a key role in the enhanced cell translocation, as scrambled versions of the designed agents, with hydrophobic and cationic groups on all faces of the helix, were only as effective as their flexible peptide counterparts. Interestingly, the most potent agent, P11LRR, demonstrated almost an order of magnitude more efficient cellular uptake as compared to that of the well-studied Tat peptide, with minimal cytotoxicity.     

Formation process and solvent-dependent structure of a polyproline self-assembled monolayer on a gold surface

The formation process and structure of a self-assembled monolayer (SAM) of lipoic-acid-terminated polyproline on a gold surface in aqueous solution were investigated by several techniques. The amount of polyproline molecules on the gold surface was determined from the area of the reductive desorption peak, and orientation and thickness of the polyproline SAM were determined in situ by attenuated total reflection infrared (ATR-IR) spectroscopy and ellipsometry. The kinetics of the polyproline SAM formation process were discussed on the basis of these results. The in situ IR study confirmed that the conformation of the polyproline SAM was changed by changing the solvent from water to methanol and methanol to water, as is the case for polyproline dissolved in solution.     

Peptide dendrimers based on polyproline helices

We present a new family of peptide dendrimers based on polyproline helices and cis-4-amino-L-proline as a branching unit. Dendrimers were synthesized by a convergent solid-phase peptide synthesis approach. The conformational transition between polyproline type I helix and polyproline type II helix was observed by circular dichroism in branched polyproline building blocks with more than 14 proline residues and in the resulting dendrimers. Both linear and dendritic polyprolines were found to be actively internalized by rat kidney cells. Preliminary results show that the antibiotic ciprofloxacin form complexes with branched polyproline chains in 99.5% propanol.     

Synthesis of homopolypeptides with PPII structure

Polyprolines are attractive polymers because of their folding property into polyproline II (PPII) structure, their significance in protein/protein interactions, and their potential as new therapeutic targets. Silaproline (Sip) is an analogue of proline, which exhibits similar conformational properties. The presence of dimethylsilyl group confers to Sip a higher lipophilicity as well as an improved resistance to biodegradation. Enantiomerically pure Sip was available in gram quantities from resolution of the enantiomers by chiral high performance liquid chromatography. This study describes the first synthesis of Sip N‐carboxyanhydride (NCA) and shows preliminary results on comparison of polymerization of (l )Pro‐NCA and (d )Sip‐NCA to obtain homopolypeptides with PPII structure, polyproline, and polysilaproline polymers. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3103–3109     

Electrochemical Deposition onto Self‐Assembled Monolayers: New Insights into Micro‐ and Nanofabrication

Pattern transfer with high resolution is a frontier topic in the emerging field of nanotechnologies. Electrochemical molding is a possible route for nanopatterning metal, alloys and oxide surfaces with high resolution in a simple and inexpensive way. This method involves electrodeposition onto a conducting master covered by a self‐assembled alkanethiolate monolayer (SAMs). This molecular film enables direct surface–relief pattern transfer from the conducting master to the inner face of the electrodeposit, and also allows an easy release of the electrodeposited film due their excellent anti‐adherent properties. Replicas of the original conductive master can be also obtained by a simple two‐step procedure. SAM quality and stability under electrodeposition conditions combined with the formation of smooth electrodeposits are crucial to obtain high‐quality pattern transfer with sub‐50 nm resolution.     

Resistance to anticancer drugs permanently alters electrophoretic mobility of cancer cell lines

Electrophoretic mobility is a physical phenomenon defining the mobility of charged particles in a solution under applied electric field. As charged biological systems, living cells including both prokaryotes and eukaryotes have been assessed in terms of electrophoretic mobility to decipher their electrochemical structure. Moreover, determination of electrophoretic mobility of living cancer cells have promoted the advance exploration of the nature of the cancer cells and separation of cancer cells from normal ones under applied electric field. However, electrophoretic mobility of drug‐resistant cells has not yet been examined. In the present study, we determined the electrophoretic mobility of drug‐resistant cancer cell lines for both suspension and adherent cells and compared with those of drug‐sensitive counterparts. We showed that resistance to anticancer drugs alters the electrophoretic mobility in a permanent manner, even lasting without any exposure to anticancer agents for a long time period. We also studied the cellular morphologies of adherent cells where the cellular invaginations and protrusions were increased in drug‐resistant adherent cells, which could be direct cause of altered surface charge and electrophoretic mobility as a result. These findings could be helpful in terms of understanding the electrophysiological and physicochemical background of drug resistance in cancer cells and developing systems to separate drug‐sensitive cells from drug‐resistant ones.     

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.     

Influence of an Unnatural Amino Acid Side Chain on the Conformational Dynamics of Peptides

In this work, a non‐natural amino acid, H‐propargylglycine‐OH (Pra), is chosen to examine the side‐chain effect on the backbone conformation of small peptides. The conformations of two synthesized Pra‐containing tripeptides, Ac‐Pra‐Pra‐NH₂ (PPTP) and Ac‐Pra‐Ala‐NH₂ (PATP), are examined by infrared (IR) spectroscopy in combination with molecular dynamics (MD) simulations and quantum chemical computations. By analyzing the joint distributions of backbone torsional angles, several significant conformations can be identified for the two tripeptides solvated in D₂O. At room temperature, 44 % of PPTP exists in the α‐α conformation and 33 % of PATP exists in the α‐polyproline‐II conformation. Larger structural inhomogeneity is seen in both cases by MD simulations at elevated temperatures. Thus even a small side chain, such as the propargyl group can significantly alter the peptide backbone conformations. The results suggest that there is no overwhelming conformational propensity of the Pra residue in short peptides. IR spectra simulated in the amide‐I region using two different methods, reasonably reproduce the experimental IR spectra and their temperature dependence.     

Influence of Sequential Modifications and Carbohydrate Variations in Synthetic AFGP Analogues on Conformation and Antifreeze Activity

Certain Arctic and Antarctic ectotherm species have developed strategies for survival under low temperature conditions that, among others, consist of antifreeze glycopeptides (AFGP). AFGP form a class of biological antifreeze agents that exhibit the ability to inhibit ice growth in vitro and in vivo and, hence, enable life at temperatures below the freezing point. AFGP usually consist of a varying number of (Ala‐Ala‐Thr)_n units (n=4–55) with the disaccharide β‐D ‐galactosyl‐(1→3)‐α‐N‐acetyl‐D ‐galactosamine glycosidically attached to every threonine side chain hydroxyl group. AFGP have been shown to adopt polyproline II helical conformation. Although this pattern is highly conserved among different species, microheterogeneity concerning the amino acid composition usually occurs; for example, alanine is occasionally replaced by proline in smaller AFGP. The influence of minor and major sequence mutations on conformation and antifreeze activity of AFGP analogues was investigated by replacement of alanine by proline and glycosylated threonine by glycosylated hydroxyproline. The target compounds were prepared by using microwave‐enhanced solid phase peptide synthesis. Furthermore, artificial analogues were obtained by copper‐catalyzed azide–alkyne cycloaddition (CuAAC): propargyl glycosides were treated with polyproline helix II‐forming peptides comprising (Pro‐Azp‐Pro)_n units (n=2–4) that contained 4‐azidoproline (Azp). The conformations of all analogues were examined by circular dichroism (CD). In addition, microphysical analysis was performed to provide information on their inhibitory effect on ice recrystallization.     

'Living cantilever arrays' for characterization of mass of single live cells in fluids

The size of a cell is a fundamental physiological property and is closely regulated by various environmental and genetic factors. Optical or confocal microscopy can be used to measure the dimensions of adherent cells, and Coulter counter or flow cytometry (forward scattering light intensity) can be used to estimate the volume of single cells in a flow. Although these methods could be used to obtain the mass of single live cells, no method suitable for directly measuring the mass of single adherent cells without detaching them from the surface is currently available. We report the design, fabrication, and testing of 'living cantilever arrays', an approach to measure the mass of single adherent live cells in fluid using silicon cantilever mass sensor. HeLa cells were injected into microfluidic channels with a linear array of functionalized silicon cantilevers and the cells were subsequently captured on the cantilevers with positive dielectrophoresis. The captured cells were then cultured on the cantilevers in a microfluidic environment and the resonant frequencies of the cantilevers were measured. The mass of a single HeLa cell was extracted from the resonance frequency shift of the cantilever and was found to be close to the mass value calculated from the cell density from the literature and the cell volume obtained from confocal microscopy. This approach can provide a new method for mass measurement of a single adherent cell in its physiological condition in a non-invasive manner, as well as optical observations of the same cell. We believe this technology would be very valuable for single cell time-course studies of adherent live cells.     

Structural Properties and Stereochemically Distinct Folding Preferences of 4,5‐cis and trans‐Methano‐L‐Proline Oligomers: The Shortest Crystalline PPII‐Type Helical Proline‐Derived Tetramer

The synthesis, structural properties, and folding patterns of a series of L ‐proline methanologues represented by cis‐ and trans‐4,5‐methano‐L ‐proline amides and their oligomers are reported as revealed by X‐ray crystallography, circular dichroism measurements, and DFT calculations. We disclose the first example of a crystalline tetrameric proline congener to exhibit a polyproline II helical conformation. Experimental evidence of PPII‐type helical arrangement (both in solution and in the solid state) of cis‐4,5‐methano‐L ‐proline oligomers is supported by theoretical calculations reflecting the extent of n→π* stabilization of the trans‐amide conformation.     

Coordination‐Driven Folding and Assembly of a Short Peptide into a Protein‐like Two‐Nanometer‐Sized Channel

Short peptide helices have attracted attention as suitable building blocks for soft functional materials, but they are rarely seen in crystalline materials. A new artificial nanoassembly of short peptide helices in the crystalline state is presented in which peptide helices are arranged three‐dimensionally by metal coordination. The folding and assembly processes of a short peptide ligand containing the Gly‐Pro‐Pro sequence were induced by silver(I) coordination in aqueous alcohol, and gave rise to a single crystal composed of polyproline II helices. Crystallographic studies revealed that this material possesses two types of unique helical nanochannel; the larger channel measures more than 2 nm in diameter. Guest uptake properties were investigated by soaking the crystals in polar solutions of guest molecules; anions, organic chiral molecules, and bio‐oligomers are effectively encapsulated by this peptide‐folded porous crystal, with moderate to high chiral recognition for chiral molecules.     

Stable conformations of tripeptides in aqueous solution studied by UV circular dichroism spectroscopy

Determination of the precise solution structure of peptides is of utmost importance to the understanding of protein folding and peptide drugs. Herein, we have measured the UV circular dichroism (UVCD) spectra of tri-alanine dissolved in D(2)O, H(2)O, and glycerol. The results clearly show the coexistence of a polyproline II or 3(1)-helix and a somewhat disordered flat beta-strand conformation, in complete agreement with recent predictions from spectroscopic data (Eker et al. J. Am. Chem. Soc. 2002, 124, 14 330-14 341). A thermodynamic analysis revealed that enthalpic contributions of about 11 and 17 kJ/mol stabilize polyproline II in D(2)O and H(2)O, respectively, but at room temperature they are counterbalanced by entropic contributions, which clearly favor the more disordered beta-strand conformation. It is hypothesized that this delicate balance is the reason for the variety of structural propensities of amino acid residues in the absence of nonlocal interactions. The isotope effect yielding a higher occupation of polyproline II in H(2)O with respect to D(2)O strongly suggests that a hydrogen-bonding network involving the peptide and water molecules in the hydration shell plays a major role in stabilizing this conformation. The equilibrium between polyproline II and beta-strand is practically maintained in glycerol, which suggests that glycerol can substitute water as stabilizing solvent for the polyproline II conformation. We also measured the UVCD spectra of tri-valine and tri-lysine (both at acidic pD) in D(2)O and found them to adopt a flat beta-strand and left-handed turn structure, respectively, in accordance with recent analyses of vibrational spectroscopy data. Generally, the present study adds substantial evidence to the notion that the so-called random coil state of peptides is much more structured than generally assumed.     

An oligomeric ser-pro dipeptide mimetic assuming the polyproline II helix conformation

A hexapeptide assembled from dipeptide building blocks assumes the secondary structure of the polyproline II (PPII) helix. Side chain-to-backbone hydrogen bonds stabilize peptide torsions next to the fused ring systems. The solution structure of the polyhydroxylated peptide was studied by spectroscopic methods.