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Summary

Background: The transport through a quantum-scale device may be uniquely characterized by its transmission eigenvalues τn. Recently, highly conductive single-molecule junctions (SMJ) with multiple transport channels (i.e., several τn > 0) have been formed from benzene molecules between Pt electrodes. Transport through these multichannel SMJs is a probe of both the bonding properties at the lead–molecule interface and of the molecular symmetry.

Results: We use a many-body theory that properly describes the complementary wave–particle nature of the electron to investigate transport in an ensemble of Pt–benzene–Pt junctions. We utilize an effective-field theory of interacting π-electrons to accurately model the electrostatic influence of the leads, and we develop an ab initio tunneling model to describe the details of the lead–molecule bonding over an ensemble of junction geometries. We also develop a simple decomposition of transmission eigenchannels into molecular resonances based on the isolated resonance approximation, which helps to illustrate the workings of our many-body theory, and facilitates unambiguous interpretation of transmission spectra.

Conclusion: We confirm that Pt–benzene–Pt junctions have two dominant transmission channels, with only a small contribution from a third channel with τn << 1. In addition, we demonstrate that the isolated resonance approximation is extremely accurate and determine that transport occurs predominantly via the HOMO orbital in Pt–benzene–Pt junctions. Finally, we show that the transport occurs in a lead–molecule coupling regime where the charge carriers are both particle-like and wave-like simultaneously, requiring a many-body description.

Summary

We report on an experimental analysis of the charge transport through sulfur-free photochromic molecular junctions. The conductance of individual molecules contacted with gold electrodes and the current–voltage characteristics of these junctions are measured in a mechanically controlled break-junction system at room temperature and in liquid environment. We compare the transport properties of a series of molecules, labeled TSC, MN, and 4Py, with the same switching core but varying side-arms and end-groups designed for providing the mechanical and electrical contact to the gold electrodes. We perform a detailed analysis of the transport properties of TSC in its open and closed states. We find rather broad distributions of conductance values in both states. The analysis, based on the assumption that the current is carried by a single dominating molecular orbital, reveals distinct differences between both states. We discuss the appearance of diode-like behavior for the particular species 4Py that features end-groups, which preferentially couple to the metal electrode by physisorption. We show that the energetic position of the molecular orbital varies as a function of the transmission. Finally, we show for the species MN that the use of two cyano end-groups on each side considerably enhances the coupling strength compared to the typical behavior of a single cyano group.

Inflammation-induced impaired function of vascular endothelium may cause leakage of plasma proteins that can lead to edema. Proteins may leave the vascular lumen through two main paracellular and transcellular pathways. As the first involves endothelial cell (EC) junction proteins and the second caveolae formation, these two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during pathology that causes inflammation. Here we present a newly developed dual-tracer probing method that allows differentiation of transcellular from paracellular transport during pathology. This fluorescence-based method can be used in vitro to test changes in EC layer permeability and in vivo in various animal vascular preparations. The method is based on comparison of low molecular weight molecule (LMWM) transport to that of high molecular weight molecule (HMWM) transport through the EC layer or the vascular wall during physiological and pathological conditions. Since the LMWM will leak through mainly the paracellular and HMWM will move through paracellular (when gaps between the ECs are wide enough) and transcellular pathways, the difference in transport rate (during normal conditions and pathology) of these molecules will indicate the prevailing transport pathway involved in overall protein crossing of vascular wall. Thus, the novel approach of assessing the transport kinetics of different size tracers in vivo by intravital microscopy can clarify questions related to identification of target pathways for drug delivery during various pathologies associated with elevated microvascular permeability.

Summary

We have investigated charge transport in ZnTPPdT–Pyr (TPPdT: 5,15-di(p-thiolphenyl)-10,20-di(p-tolyl)porphyrin) molecular junctions using the lithographic mechanically controllable break-junction (MCBJ) technique at room temperature and cryogenic temperature (6 K). We combined low-bias statistical measurements with spectroscopy of the molecular levels in the form of I(V) characteristics. This combination allows us to characterize the transport in a molecular junction in detail. This complex molecule can form different junction configurations, having an observable effect on the trace histograms and the current–voltage (I(V)) measurements. Both methods show that multiple, stable single-molecule junction configurations can be obtained by modulating the interelectrode distance. In addition we demonstrate that different ZnTPPdT–Pyr junction configurations can lead to completely different spectroscopic features with the same conductance values. We show that statistical low-bias conductance measurements should be interpreted with care, and that the combination with I(V) spectroscopy represents an essential tool for a more detailed characterization of the charge transport in a single molecule.

Long range oxidative damage as a result of charge transport is shown to occur through single crossover junctions assembled from four semi-complementary strands of DNA. When a rhodium complex is tethered to one of the arms of the four-way junction assembly, thereby restricting its intercalation into the π-stack, photo-induced oxidative damage occurs to varying degrees at all guanine doublets in the assembly, though direct strand scission only occurs at the predicted site of intercalation. In studies where the Mg2+ concentration was varied, so as to perturb base stacking at the junction, charge transport was found to be enhanced but not to be strongly localized to the arms that preferentially stack on each other. These data suggest that the conformations of four-way junctions can be relatively mobile. Certainly, in four-way junctions charge transport is less discriminate than in the more rigidly stacked DNA double crossover assemblies.

Summary

π-Conjugation plays an important role in charge transport through single molecular junctions. We describe in this paper the construction of a mechanically controlled break-junction setup (MCBJ) equipped with a highly sensitive log I–V converter in order to measure ultralow conductances of molecular rods trapped between two gold leads. The current resolution of the setup reaches down to 10 fA. We report single-molecule conductance measurements of an anthracene-based linearly conjugated molecule (AC), of an anthraquinone-based cross-conjugated molecule (AQ), and of a dihydroanthracene-based molecule (AH) with a broken conjugation. The quantitative analysis of complementary current–distance and current–voltage measurements revealed details of the influence of π-conjugation on the single-molecule conductance.

We fabricate and characterize carbon-fiber tips for their use in combined scanning tunneling and force microscopy based on piezoelectric quartz tuning fork force sensors. An electrochemical fabrication procedure to etch the tips is used to yield reproducible sub-100-nm apex. We also study electron transport through single-molecule junctions formed by a single octanethiol molecule bonded by the thiol anchoring group to a gold electrode and linked to a carbon tip by the methyl group. We observe the presence of conductance plateaus during the stretching of the molecular bridge, which is the signature of the formation of a molecular junction.

The barrier integrity of the corneal endothelium, which is conferred by its tight and adherens junctions, is critical for the maintenance of deturgescence of the corneal stroma. Although characteristically leaky, the barrier integrity restricts fluid leakage into the stroma such that the rate of leak does not exceed the rate of the endothelial active fluid transport directed toward the aqueous humor. At a molecular level, the barrier integrity is influenced by the actin cytoskeleton and microtubules, which are coupled to tight and adherens junctions via a variety of linker proteins. Since the cytoskeleton is affected by Rho family small GTPases and p38 MAP kinase, among others, many pathophysiological stimuli induce plasticity to the cytoskeleton and thereby elicit dynamic regulation of the barrier integrity. This review presents an overview of the impact of several bioactive factors on the barrier integrity of the corneal endothelium through altered actin cytoskeleton and/or disassembly of microtubules. The main focus is on the effect of TNF-α (tumor necrosis factor-α) which is a pro-inflammatory molecule found in the intraocular milieu during allograft rejection and anterior uveitis. This cytokine elicits acute activation of p38 MAP kinase, induces disassembly of microtubules, disrupts the peri-junctional actomyosin ring, and concomitantly breaks down the barrier integrity. These effects of TNF-α could be inhibited by stabilizing the microtubules, co-treating with a selective p38 MAP kinase inhibitor, and elevating intracellular cAMP via A2B receptors or direct exposure to forskolin. Overall, the corneal edema following a potential breakdown of the endothelial barrier integrity can be rescued pharmacologically by inhibiting specific cell-signaling mechanisms.

Purpose of review

An enormous body of research has been focused on exploring the mechanisms through which epithelial cells establish their characteristic polarity. It is clear that under normal circumstances cell–cell contacts mediated by the calcium-dependent adhesion proteins of the intercellular adhesion junctions are required to initiate complete polarization. Furthermore, formation of the tight, or occluding, junctions that limit paracellular permeability has long been thought to help to establish polarity by preventing the diffusion of membrane proteins between the two plasmalemmal domains. This review will discuss several selected kinases and protein complexes and highlight their relevance to transporting epithelial cell polarization.

Recent findings

Recent work has shed new light on the roles of junctional complexes in establishing and maintaining epithelial cell polarity. In addition, work from several laboratories, suggests that the formation of these junctions is tied to processes that regulate cellular energy metabolism.

Summary

Junctional complexes and energy sensing kinases constitute a novel class of machinery whose capacity to generate and modulate epithelial cell polarity is likely to have wide ranging and important physiological ramifications.

Zonula occludens toxin (Zot) is an enterotoxin obtained from the bacterium vibrio cholerae that has been shown to reversibly and safely open the tight junctions and enhance paracellular transport. AT1002 is a novel synthetic hexapeptide derived from Zot. The hypothesis to be tested in this study is that AT1002 enhances the oral absorption of ardeparin, a low molecular weight heparin (LMWH). To test this hypothesis, drug transport through Caco-2 cell monolayers was monitored in the presence and absence of AT1002. Regional permeability studies using rat intestine were performed. Cell viability in the presence of various concentrations of enhancer was determined. The absorption of ardeparin after oral administration in rats was measured by anti-factor Xa assay. Furthermore, the eventual mucosal and epithelial damage was histologically evaluated. Higher ardeparin permeability (~2-fold) compared to control was observed in the presence of 0.025% of AT1002. Regional permeability studies revealed that the permeability of ardeparin across the duodenal membrane was improved by the AT1002. Cell viability studies showed no significant cytotoxicity below 0.0028% of AT1002. In the presence of 100 μg/kg of AT1002, ardeparin oral bioavailability was significantly increased (Frelative/s.c ~ 20.5%). Furthermore, AT1002 at a dose of 100 μg/kg did not induce any observable morphological damage on gastrointestinal (GI) tissues in vivo. These in vivo and in vitro results suggest that the co-administration of LMWH with AT1002 may be a useful delivery strategy to increase its permeability and hence oral absorption.

We report a first-principles study of electron ballistic transport through a molecular junction containing deoxycytidine-monophosphate (dCMP) connected to metal electrodes. A guanidinium ion and guanine nucleobase are tethered to gold electrodes on opposite sides to form hydrogen bonds with the dCMP molecule providing an electric circuit. The circuit mimics a component of a potential device for sequencing unmodified single-stranded DNA. The molecular conductance is obtained from DFT Green’s function scattering methods and is compared to estimates from the electron tunneling decay constant obtained from the complex band structure. The result is that a complete molecular dCMP circuit of ‘linker((CH2)2)–guanidinium–phosphate–deoxyribose–cytosine–guanine’ has a very low conductance (of the order of fS) while the hydrogen-bonded guanine–cytosine base-pair has a moderate conductance (of the order of tens to hundreds of nS). Thus, while the transverse electron transfer through base-pairing is moderately conductive, electron transfer through a complete molecular dCMP circuit is not. The gold Fermi level is found to be aligned very close to the HOMO for both the guanine–cytosine base-pair and the complete molecular dCMP circuit. Results for two different plausible geometries of the hydrogen-bonded dCMP molecule reveal that the conductance varies from fS for an extended structure to pS for a slightly compressed structure.

The tight junction, or zonula occludens, forms an intercellular barrier between epithelial cells within the gastrointestinal tract and liver and, by limiting the movement of water and solutes through the intercellular space, maintains the physicochemical separation of tissue compartments. The paracellular barrier properties of junctions are regulated and quite different among epithelia. The junction also forms an intramembrane barrier between the apical and basolateral membrane domains, contributing to segregation of biochemically distinct components of these plasma membrane surfaces. Here we briefly review three rapidly developing areas of medically relevant basic knowledge about the tight junction. First, we describe the presently incomplete knowledge of the molecular structure of the tight junction as a framework for understanding its functional properties. Second, we consider experimental evidence defining how the barrier properties of junctions are physiologically regulated and, third, how barrier properties are specifically altered in, and contribute to, pathologic processes affecting epithelia.

Images

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.

Hg2+ is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg2+ affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg2+ decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg2+-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg2+ treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg2+-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren’s syndrome, and possibly other disorders of fluid transport.

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL.

Electrons are allowed to pass through a single atom connected to two electrodes without being scattered as the characteristic size is much smaller than the inelastic mean free path. In this quasi-ballistic regime, it is difficult to predict where and how power dissipation occurs in such current-carrying atomic system. Here, we report direct assessment of electrical heating in a metallic nanocontact. We find asymmetric electrical heating effects in the essentially symmetric single-atom contact. We simultaneously identified the voltage polarity independent onset of the local heating by conducting the inelastic noise spectroscopy. As a result, we revealed significant heat dissipation by hot electrons transmitting ballistically through the junction that creates a hot spot at the current downstream. This technique can be used as a platform for studying heat dissipation and transport in atomic/molecular systems.

Cell junctions are sites of intercellular adhesion that maintain the integrity of epithelial tissue and regulate signalling between cells. These adhesive junctions are comprised of protein complexes that serve to establish an intercellular cytoskeletal network for anchoring cells, in addition to regulating cell polarity, molecular transport and communication. The expression of cell adhesion molecules is tightly controlled and their downregulation is essential for epithelial-mesenchymal transition (EMT), a process that facilitates the generation of morphologically and functionally diverse cell types during embryogenesis. The characteristics of EMT are a loss of cell adhesion and increased cellular mobility. Hence, in addition to its normal role in development, dysregulated EMT has been linked to cancer progression and metastasis, the process whereby primary tumors migrate to invasive secondary sites in the body. This paper will review the current understanding of cell junctions and their role in cancer, with reference to the abnormal regulation of junction protein genes. The potential use of cell junction molecules as diagnostic and prognostic markers will also be discussed, as well as possible therapies for adhesive dysregulation.

Summary

Epithelial monolayers are major determinants of three-dimensional tissue organization and provide the structural foundation for the body plan and all of its component organs. Epithelial cells are connected by junctional complexes containing the cell adhesion molecule E-cadherin. These adherens junctions mediate stable cohesion between cells but must be actively reorganized to allow tissue remodeling during development. Recent studies demonstrate that junctional proteins are dynamically turned over at the cell surface, even in cells that do not appear to be moving. The redistribution of E-cadherin through spatially regulated endocytosis and exocytosis contributes to cell adhesion, cell polarity, and is also involved in cell rearrangement. Here we describe recent progress in understanding the roles of the vesicle transport machinery in regulating cell adhesion and junctional dynamics during epithelial morphogenesis in vivo.

The blood–brain barrier (BBB) is the most significant obstacle to effective CNS drug delivery. It possesses structural and biochemical features (i.e., tight-junction protein complexes and, influx and efflux transporters) that restrict xenobiotic permeation. Pathophysiological stressors (i.e., peripheral inflammatory pain) can alter BBB tight junctions and transporters, which leads to drug-permeation changes. This is especially critical for opioids, which require precise CNS concentrations to be safe and effective analgesics. Recent studies have identified molecular targets (i.e., endogenous transporters and intracellular signaling systems) that can be exploited for optimization of CNS drug delivery. This article summarizes current knowledge in this area and emphasizes those targets that present the greatest opportunity for controlling drug permeation and/or drug transport across the BBB in an effort to achieve optimal CNS opioid delivery.

In order to verify the existence of a blood-thymus barrier to circulating macromolecules, the permeability of the vessels of the thymus was analyzed in young adult mice using electron opaque tracers of different molecular dimensions (horseradish peroxidase, cytochrome c, catalase, ferritin, colloidal lanthanum). Results show that although blood-borne macromolecules do penetrate the thymus, their parenchyma] distribution is limited to the medulla of the lobe by several factors: (a) the differential permeability of the various segments of the vascular tree; (b) the spatial segregation of these segments within the lobe; (c) the strategic location of parenchymal macrophages along the vessels. The cortex is exclusively supplied by capillaries, which have impermeable endothelial junctions. Although a small amount of tracer is transported by plasmalemmal vesicles through the capillary endothelium, this tracer is promptly sequestrated by macrophages stretched out in a continuous row along the cortical capillaries and it does not reach the intercellular clefts between cortical lymphocytes and reticular cells. The medulla contains all the leaky vessels, namely postcapillary venules and arterioles. Across the walls of the venules, large quantities of all injected tracers escape through the clefts between migrating lymphocytes and endothelial cells; also the arterioles have a small number of endothelial junctions which are permeable to peroxidase, but do not allow passage of tracers of higher molecular weight. The tracers released by the leaky vessels penetrate the intercellular clefts of the medulla, but they never reach the cortical parenchyma, even at long time intervals after the injection. Therefore, a blood-thymus barrier to circulating macromolecules does exist, but is limited to the cortex. Medullary lymphocytes are freely exposed to blood-borne substances.

The permeability of the alveolar-capillary membrane to a small molecular weight protein, horseradish peroxidase (HRP), was investigated by means of ultrastructural cytochemistry. Mice were injected intravenously with HRP and sacrificed at varying intervals. Experiments with intranasally instilled HRP were also carried out. The tissue was fixed in formal-dehyde-glutaraldehyde fixative. Frozen sections were cut, incubated in Graham and Karnovsky's medium for demonstrating HRP activity, postfixed in OsO4, and processed for electron microscopy. 90 sec after injection, HRP had passed through endothelial junctions into underlying basement membranes, but was stopped from entering the alveolar space by zonulae occludentes between epithelial cells. HRP was demonstrated in pinocytotic vesicles of both endothelial and epithelial cells, but the role of these vesicles in net protein transport appeared to be minimal. Intranasally instilled HRP was similarly prevented from permeating the underlying basement membrane by epithelial zonulae occludentes. Pulmonary endothelial intercellular clefts stained with uranyl acetate appeared to contain maculae occludentes rather than zonulae occludentes. HRP did not alter the ultrastructure of these junctions.

The blood-brain barrier (BBB) maintains brain homeostasis and limits the entry of toxins and pathogens into the brain. Despite its importance, little is known about the molecular mechanisms regulating the development and function of this crucial barrier. In this study we have developed methods to highly purify and gene profile endothelial cells from different tissues, and by comparing the transcriptional profile of brain endothelial cells with those purified from the liver and lung, we have generated a comprehensive resource of transcripts that are enriched in the BBB forming endothelial cells of the brain. Through this comparison we have identified novel tight junction proteins, transporters, metabolic enzymes, signaling components, and unknown transcripts whose expression is enriched in central nervous system (CNS) endothelial cells. This analysis has identified that RXRalpha signaling cascade is specifically enriched at the BBB, implicating this pathway in regulating this vital barrier. This dataset provides a resource for understanding CNS endothelial cells and their interaction with neural and hematogenous cells.

Auditory transduction, amplification, and hair cell survival depend on the regulation of extracellular [K+] in the cochlea. K+ is removed from the vicinity of sensory hair cells by epithelial cells, and may be distributed through the epithelial cell syncytium, reminiscent of “spatial buffering” in glia. Hypothetically, K+ is then transferred from the epithelial syncytium into the connective tissue syncytium within the cochlear lateral wall, enabling recirculation of K+ back into endolymph. This may involve secretion of K+ from epithelial root cells, and its re-uptake via transporters into spiral ligament fibrocytes. The molecular basis of this secretion is not known. Using a combination of approaches we demonstrated that the resting conductance in guinea pig root cells was dominated by K+ channels, most likely composed of the Kir4.1 subunit. Dye injections revealed extensive intercellular gap junctional coupling, and delineated the root cell processes that penetrated the spiral ligament. Following uncoupling using 1-octanol, individual cells had Ba2+-sensitive weakly rectifying currents. In the basal (high-frequency encoding) cochlear region K+ loads are predicted to be the highest, and root cells in this region had the largest surface area and the highest current density, consistent with their role in K+ secretion. Kir4.1 was localized within root cells by immunofluorescence, and specifically to root cell process membranes by immunogold labeling. These results support a role for root cells in cochlear K+ regulation, and suggest that channels composed of Kir4.1 subunits may mediate K+ secretion from the epithelial gap junction network.

Hydrogen bonding has a ubiquitous role in electron transport1,2 and in molecular recognition, with DNA base-pairing being the best known example.3 Scanning tunneling microscope (STM) images4 and measurements of the decay of tunnel-current as a molecular junction is pulled apart by the STM tip, 5 are sensitive to hydrogen-bonded interactions. Here we show that these tunnel-decay signals can be used to measure the strength of hydrogen bonding in DNA basepairs. Junctions that are held together by three hydrogen bonds per basepair (e.g., guanine-cytosine interactions) are stiffer than junctions held together by two hydrogen bonds per basepair (e.g., adenine-thymine interactions). Similar, but less-pronounced, effects are observed on the approach of the tunneling probe, implying that hydrogen-bond dependent attractive forces also have a role in determining the rise of current. These effects provide new mechanisms for making sensors that transduce a molecular recognition event into an electronic signal.

The manufacture of integrated circuits with single-molecule building blocks is a goal of molecular electronics. While research in the past has been limited to bulk experiments on self-assembled monolayers, advances in technology have now enabled us to fabricate single-molecule junctions. This has led to significant progress in understanding electron transport in molecular systems at the single-molecule level and the concomitant emergence of new device concepts. Here, we review recent developments in this field. We summarize the methods currently used to form metal-molecule-metal structures and some single-molecule techniques essential for characterizing molecular junctions such as inelastic electron tunnelling spectroscopy. We then highlight several important achievements, including demonstration of single-molecule diodes, transistors, and switches that make use of electrical, photo, and mechanical stimulation to control the electron transport. We also discuss intriguing issues to be addressed further in the future such as heat and thermoelectric transport in an individual molecule.