Therapeutic potential of mesenchymal stem cells for cerebral small vessel disease.

Cerebral small vessel disease (CSVD), as the most common, chronic and progressive vascular disease on the brain, is a serious neurological disease, whose pathogenesis remains unclear. The disease is a leading cause of stroke and vascular cognitive impairment and dementia, and contributes to about 20% of strokes, including 25% of ischemic strokes and 45% of dementias. Undoubtedly, the high incidence and poor prognosis of CSVD have brought a heavy economic and medical burden to society. The present treatment of CSVD focuses on the management of vascular risk factors. Although vascular risk factors may be important causes or accelerators of CSVD and should always be treated in accordance with best clinical practice, controlling risk factors alone could not curb the progression of CSVD brain injury. Therefore, developing safer and more effective treatment strategies for CSVD is urgently needed. Recently, mesenchymal stem cells (MSCs) therapy has become an emerging therapeutic modality for the treatment of central nervous system disease, given their paracrine properties and immunoregulatory. Herein, we discussed the therapeutic potential of MSCs for CSVD, aiming to enable clinicians and researchers to understand of recent progress and future directions in the field.

Long-Term Imaging of Cys in Cells and Tumor Mice by a Solid-State Fluorescence Probe.

Cysteine is an important biological thiol and is closely related to cancer. It remains a challenge to develop a probe that can provide long-term fluorescence detection and imaging of Cys in cells as well as in living organisms. Here, a solid-state fluorophore HTPQ is combined with an acrylate group to construct a solid-state fluorescent probe HTPQC for Cys recognition. The fluorescence of the probe is quenched when the photoinduced electron transfer (PET) process is turned on and the excited-state intramolecular proton transfer (ESIPT) process is turned off. In the presence of Cys, an obvious solid-state fluorescence signal can be observed. The double quenching mechanism makes the probe HTPQC have the advantages of high sensitivity, good selectivity, and high contrast of biological imaging. Due to low cytotoxicity, the probe HTPQC can be used to detect exogenous and endogenous Cys in living cells and is capable of imaging over long periods of time. By making full use of long wavelengths, the probe can be applied for the detection of Cys levels in tumor mice and equipped with the ability to conduct long-term imaging in vivo.

Case report: MELAS and T3271C mitochondrial mutation in an adult woman.

Introduction: Patients with mitochondrial disorders always show neurological deficits. However, the diversity of clinical manifestations, genetic heterogeneity and threshold effect caused by maternal heredity make its diagnosis very challenging. Case presentation: A 30-year-old female presented to our neurology department with a recurrence of symmetrical weakness proximally in the lower extremities. Seven years ago, the patient had a sudden onset of persistent weakness in bilateral proximal lower extremities, along with elevated creatinine kinase (CK) and CK-MB. Given the diagnosis of Guillain-Barre syndrome, she was treated with high-dose glucocorticoid (GC) therapy at the local hospital and recovered. After admission to our hospital, laboratory analysis revealed elevated CK and alpha-hydroxybutyrate dehydrogenase in serum. Electrocardiography showed sinus tachycardia and left high ventricular voltage. Electromyography (EMG) and evoked potential (EP) suggested peripheral neurogenic damage of the upper and lower extremities with myogenic wear. Chronic inflammatory demyelinating polyneuropathy (CIDP) was initially considered, but neurological symptoms were not significantly improved with glucocorticoid shock therapy. An elevated level of lactate was found. The short-tau inversion recovery (STIR) axial magnetic resonance image (MRI) revealed mild hyperintensities, indicating muscle edema. Meanwhile, muscle biopsies suggested pathological changes in mitochondrial disorders (MIDs) and neuronal damage. Further mitochondrial genome analysis revealed a heteroplasmic m3271 T>C mutation in the mitochondrial tRNA-Leu gene (UUR). Collectively, the patient was finally diagnosed with mitochondrial disorder and apparently improved after the corresponding treatment to regulate energy metabolism. Conclusions: To our knowledge, it's the first report about MELAS with 3271 mutation that have only shown peripheral nerve motion impairment. Proximal weakness is also common in CIDP. In the context of this patient's experience, mitochondrial genome analysis provides an auxiliary criterion for differential diagnosis between MIDs and CIDP. In the meantime, we discussed the clinical effect of GCs on MIDs.

A dual-channel fluorescent nanoprobe for accurate cancer diagnosis by sequential detection of adenosine triphosphate and sulfur dioxide.

Cancer is one of the major diseases that seriously endanger the health of all mankind. Accurate diagnosis of early cancer is the most promising way to reduce cancer harm and improve patient survival. However, many developed fluorescent probes for cancer imaging only have the function of identifying one marker, which cannot meet the needs of accurate diagnosis. Here, a fluorescent nanoprobe (CPH@ZIF-90) utilizing ZIF-90 to encapsulate SO2-sensitive dye (CPH) is synthesized for the sequential detection of ATP and SO2. The nanoprobe first interacts with ATP to release CPH, thus increasing the fluorescence at 685 nm and realizing the near-infrared (NIR) fluorescence detection of ATP. Then, SO2 acts on the released CPH through nucleophilic addition, affecting the π-conjugated structure of CPH and resulting in enhanced fluorescence at 580 nm. CPH@ZIF-90 exhibits satisfactory sensitivity and selectivity for sequential detection of ATP and SO2. Excitedly, CPH@ZIF-90 can sequentially image the endogenous ATP and SO2 in cells, showing sensitive fluorescence changes in dual channels (red and green). Due to the NIR emission properties of CPH@ZIF-90 and its ability to enrich in tumor, it is applied to monitor ATP and SO2 in mice and distinguish normal mice from tumor mice. The ability of CPH@ZIF-90 to sequentially detect two cancer-related biomarkers makes it provide meaningful assistance in accurate early diagnosis of cancer.

A near-infrared fluorescent probe for monitoring abnormal mitochondrial viscosity in cancer and fatty-liver mice model.

Viscosity is an important component of cell microenvironment, and abnormal mitochondrial viscosity is associated with many diseases such as tumor and fatty liver. Herein, a near-infrared fluorescence probe (QX-V) based on quinoline-xanthene dye for detecting viscosity is constructed. In high viscosity medium, the free rotation of single bond is inhibited and the fluorescence is released. The probe shows high sensitivity together with good selectivity. Notably, QX-V has a long excitation wavelength (710 nm) and emission wavelength (786 nm). At the same time, the probe is a positively charged molecule that can target mitochondria. QX-V can not only distinguish cancer cells from normal cells, but also make a distinction between normal cells and fatty hepatocytes. In addition, QX-V is used to image viscosity abnormality in tumor-bearing mice. The probe also has a good ability to image viscosity abnormality caused by liver injury in fatty-liver mice.

Joint Power and Time Allocation in Hybrid NoMA/OMA IoT Networks for Two-Way Communications.

This article investigates two-way communications between an access point (AP) and multiple terminals in low-cost Internet of Things (IoT) networks. The main issues considered are the asymmetric transmission traffic on the uplink (UL) and downlink (DL), and the unbalanced receivers processing capability at the AP and the terminals. As a solution, a hybrid non-orthogonal multiple access/orthogonal multiple access (NoMA/OMA) scheme together with a joint power and time allocation method is proposed to address these issues. For the system design, we formulated the optimization problem with the aim of minimizing the system power and satisfying the UL and DL transmission rate constraints. Due to the coupling of power and time variables in the objective function and the multi-user interference (MUI) in the UL transmission rate constraints, the formulated problem is shown to be non-linear and non-convex and thus is hard to solve. To obtain a numerical, efficient solution, the original problem is first reformulated to be a convex one relying on the successive convex approximation (SCA) method, and then a numerical efficient solution is thus obtained by using an iterative routine. The proposed transmission scheme is shown to be not only physically feasible but also power-efficient.

A nitrobenzoxadiazole-based near-infrared fluorescent probe for the specific imaging of H2S in inflammatory and tumor mice.

As a common gaseous signaling molecule, hydrogen sulfide (H2S) plays a vital role in physiology and pathology. The development of fluorescent probes for detecting H2S has attracted widespread attention. However, most of the reported fluorescent probes with nitrobenzoxadiazole (NBD) as the recognition group have been widely used to simultaneously detect biothiols and H2S, instead of specifically detecting H2S. Herein, a novel NBD-based near-infrared (NIR) fluorescent probe named CX-N for the detection of H2S is synthesized. The selectivity of CX-N for H2S is significantly higher than that for biothiols and other potential interferences. After reacting with H2S, CX-N shows a significant increase in NIR fluorescence (75-fold), large Stokes shift (155 nm) and fast response (4 min). And the possible response mechanism of CX-N to H2S is given and confirmed by HPLC and HRMS. Based on the low cytotoxicity of CX-N, it has been used for H2S imaging in live cells and zebrafish. More importantly, CX-N has also been successfully applied for the real-time imaging of H2S in inflammatory and tumor mice based on its NIR emission, which provides a reliable platform for the specific recognition of H2S in complex biological systems.

Cryo-Electron Microscopy Structure and Interactions of the Human Cytomegalovirus gHgLgO Trimer with Platelet-Derived Growth Factor Receptor Alpha.

Human cytomegalovirus (HCMV) is a herpesvirus that produces disease in transplant patients and newborn children. Entry of HCMV into cells relies on gH/gL trimer (gHgLgO) and pentamer (gHgLUL128-131) complexes that bind cellular receptors. Here, we studied the structure and interactions of the HCMV trimer, formed by AD169 strain gH and gL and TR strain gO proteins, with the human platelet-derived growth factor receptor alpha (PDGFRα). Three trimer surfaces make extensive contacts with three PDGFRα N-terminal domains, causing PDGFRα to wrap around gO in a structure similar to a human hand, explaining the high-affinity interaction. gO is among the least conserved HCMV proteins, with 8 distinct genotypes. We observed high conservation of residues mediating gO-gL interactions but more extensive gO variability in the PDGFRα interface. Comparisons between our trimer structure and a previously determined structure composed of different subunit genotypes indicate that gO variability is accommodated by adjustments in the gO-PDGFRα interface. We identified two loops within gO that were disordered and apparently glycosylated, which could be deleted without disrupting PDGFRα binding. We also identified four gO residues that contact PDGFRα, which when mutated produced markedly reduced receptor binding. These residues fall within conserved contact sites of gO with PDGFRα and may represent key targets for anti-trimer neutralizing antibodies and HCMV vaccines. Finally, we observe that gO mutations distant from the gL interaction site impact trimer expression, suggesting that the intrinsic folding or stability of gO can impact the efficiency of trimer assembly. IMPORTANCE HCMV is a herpesvirus that infects a large percentage of the adult population and causes significant levels of disease in immunocompromised individuals and birth defects in the developing fetus. The virus encodes a complex protein machinery that coordinates infection of different cell types in the body, including a trimer formed of gH, gL, and gO subunits. Here, we studied the interactions of the HCMV trimer with its receptor on cells, the platelet derived growth factor receptor α (PDGFRα), to better understand how HCMV coordinates virus entry into cells. Our results add to our understanding of HCMV strain-specific differences and identify sites on the trimer that represent potential targets for therapeutic antibodies or vaccine development.

Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera.

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Šfrom a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Šlevel to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.

High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs.

Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

Ultra-high-frequency radio-frequency acoustic molecular imaging with saline nanodroplets in living subjects.

Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.

Mediator structure and conformation change.

Mediator is a universal adaptor for transcription control. It serves as an interface between gene-specific activator or repressor proteins and the general RNA polymerase II (pol II) transcription machinery. Previous structural studies revealed a relatively small part of Mediator and none of the gene activator-binding regions. We have determined the cryo-EM structure of the Mediator at near-atomic resolution. The structure reveals almost all amino acid residues in ordered regions, including the major targets of activator proteins, the Tail module, and the Med1 subunit of the Middle module. Comparison of Mediator structures with and without pol II reveals conformational changes that propagate across the entire Mediator, from Head to Tail, coupling activator- and pol II-interacting regions.

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion.

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.

The LRR-RLK Protein HSL3 Regulates Stomatal Closure and the Drought Stress Response by Modulating Hydrogen Peroxide Homeostasis.

Guard cells shrink in response to drought stress and abscisic acid (ABA) signaling, thereby reducing stomatal aperture. Hydrogen peroxide (H2O2) is an important signaling molecule acting to induce stomatal closure. As yet, the molecular basis of control over the level of H2O2 in the guard cells remains largely unknown. Here, the leucine-rich repeat (LRR)-receptor-like kinase (RLK) protein HSL3 has been shown to have the ability to negatively regulate stomatal closure by modulating the level of H2O2 in the guard cells. HSL3 was markedly up-regulated by treating plants with either ABA or H2O2, as well as by dehydration. In the loss-of-function hsl3 mutant, both stomatal closure and the activation of anion currents proved to be hypersensitive to ABA treatment, and the mutant was more tolerant than the wild type to moisture deficit; the overexpression of HSL3 had the opposite effect. In the hsl3 mutant, the transcription of NADPH oxidase gene RbohF involved in H2O2 production showed marked up-regulation, as well as the level of catalase activity was weakly inducible by ABA, allowing H2O2 to accumulate in the guard cells. HSL3 was concluded to participate in the regulation of the response to moisture deficit through ABA-induced stomatal closure triggered by the accumulation of H2O2 in the guard cells.

Plasmonic and Electrostatic Interactions Enable Uniformly Enhanced Liquid Bacterial Surface-Enhanced Raman Scattering (SERS).

Surface-enhanced Raman spectroscopy (SERS) is a promising cellular identification and drug susceptibility testing platform, provided it can be performed in a controlled liquid environment that maintains cell viability. We investigate bacterial liquid-SERS, studying plasmonic and electrostatic interactions between gold nanorods and bacteria that enable uniformly enhanced SERS. We synthesize five nanorod sizes with longitudinal plasmon resonances ranging from 670 to 860 nm and characterize SERS signatures of Gram-negative Escherichia coli and Serratia marcescens and Gram-positive Staphylococcus aureus and Staphylococcus epidermidis bacteria in water. Varying the concentration of bacteria and nanorods, we achieve large-area SERS enhancement that is independent of nanorod resonance and bacteria type; however, bacteria with higher surface charge density exhibit significantly higher SERS signal. Using cryo-electron microscopy and zeta potential measurements, we show that the higher signal results from attraction between positively charged nanorods and negatively charged bacteria. Our robust liquid-SERS measurements provide a foundation for bacterial identification and drug testing in biological fluids.

Publisher Correction: A glycoprotein B-neutralizing antibody structure at 2.8 Å uncovers a critical domain for herpesvirus fusion initiation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A glycoprotein B-neutralizing antibody structure at 2.8 Å uncovers a critical domain for herpesvirus fusion initiation.

Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8 Å cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies.

Distinct Conformational States Underlie Pausing during Initiation of HIV-1 Reverse Transcription.

A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have identified specific sites along the viral RNA genomic template in which reverse transcriptase (RT) stalls. These stalling points, which occur after the addition of three and five template dNTPs, may serve as checkpoints to regulate the precise timing of HIV-1 reverse transcription following viral entry. Structural studies of reverse transcriptase initiation complexes (RTICs) have revealed unique conformations that may explain the slow rate of incorporation; however, questions remain about the temporal evolution of the complex and features that contribute to strong pausing during initiation. Here we present cryo-electron microscopy and single-molecule characterization of an RTIC after three rounds of dNTP incorporation (+3), the first major pausing point during reverse transcription initiation. Cryo-electron microscopy structures of a +3 extended RTIC reveal conformational heterogeneity within the RTIC core. Three distinct conformations were identified, two of which adopt unique, likely off-pathway, intermediates in the canonical polymerization cycle. Single-molecule Förster resonance energy transfer experiments confirm that the +3 RTIC is more structurally dynamic than earlier-stage RTICs. These alternative conformations were selectively disrupted through structure-guided point mutations to shift single-molecule Förster resonance energy transfer populations back toward the on-pathway conformation. Our results support the hypothesis that conformational heterogeneity within the HIV-1 RTIC during pausing serves as an additional means of regulating HIV-1 replication.

Limited dishevelled/Axin oligomerization determines efficiency of Wnt/ß-catenin signal transduction.

In Wnt/ß-catenin signaling, the transcriptional coactivator ß-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of ß-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of ß-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.

Stem cells can give rise to many types of specialized cells through a process called differentiation, which is partly regulated by changes in the levels of a protein known as ß-catenin. On one hand, a 'destruction complex' can keep ß-catenin levels low; this complex includes a protein called Axin and an enzyme known as GSK-3, which can tag ß-catenin for degradation. On the other hand, when ß-catenin levels need to increase, another protein called Dishevelled is activated. By binding to Axin, Dishevelled can bring the destruction complex in contact with other proteins, which leads to the deactivation of GSK-3. Dishevelled and Axin interact via a region that is similar in the two proteins, called DIX in Dishevelled and DAX in Axin. Studies of DIX and DAX have shown that both regions can form polymers ­ that is, a high number of similar units can bind together to form larger structures. However, these experiments were at higher concentrations than would be found in the cell. It was thought that, when combined, DIX and DAX might form these long chains together, preventing Axin from carrying out its role in destroying ß-catenin. Kan et al. set out to better understand this process by studying how DIX and DAX behave separately, and how they interact. The proteins were examined using a technique called cryo-electron microscopy, which allows scientists to dissect the structure of large proteins. When there was a high concentration of DIX in the sample, the molecules attached to one another to form long double-stranded helices. Similarly, DAX also formed helices, but these were shorter and only single-stranded. When the two proteins were combined, DAX bound only to the ends of short DIX chains, so that there are not more than four DAX chains attached to each DIX double helix. To see if this behaviour happens naturally, Kan et al. attached fluorescent tags to Dishevelled proteins and followed them in living cells: this showed that Dishevelled forms smaller chains with fewer than ten molecules. Together these results highlight how Dishevelled binds to Axin to deactivate GSK-3, to prevent the enzyme from promoting ß-catenin destruction. Mutations in the genes that encode ß-catenin or its regulators are associated with cancer. Ultimately, a better understanding of how ß-catenin is regulated could help to identify new opportunities for drug development.

Dynamics of the context-specific translation arrest by chloramphenicol and linezolid.

Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide-bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not substantially alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site transfer RNA to undergo repeated rounds of dissociation and nonproductive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.