Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

Bio-organic Synthesi

Application of a highly selective cathepsin S two-step activity-based probe in multicolor bio-orthogonal correlative light-electron microscopy

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.Bio-organic Synthesi

Application of a highly selective Cathepsin S two-step activity-based probe in multicolor bio-orthogonal correlative light-electron microscopy

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.Microscopic imaging and technolog

The Click-On gamma probe, a second-generation tethered robotic gamma probe that improves dexterity and surgical decision-making

Purpose Decision-making and dexterity, features that become increasingly relevant in (robot-assisted) minimally invasive surgery, are considered key components in improving the surgical accuracy. Recently, DROP-IN gamma probes were introduced to facilitate radioguided robotic surgery. We now studied if robotic DROP-IN radioguidance can be further improved using tethered Click-On designs that integrate gamma detection onto the robotic instruments themselves. Methods Using computer-assisted drawing software, 3D printing and precision machining, we created a Click-On probe containing two press-fit connections and an additional grasping moiety for a ProGrasp instrument combined with fiducials that could be video tracked using the Firefly laparoscope. Using a dexterity phantom, the duration of the specific tasks and the path traveled could be compared between use of the Click-On or DROP-IN probe. To study the impact on surgical decision-making, we performed a blinded study, in porcine models, wherein surgeons had to identify a hidden Co-57-source using either palpation or Click-On radioguidance. Results When assembled onto a ProGrasp instrument, while preserving grasping function and rotational freedom, the fully functional prototype could be inserted through a 12-mm trocar. In dexterity assessments, the Click-On provided a 40% reduction in movements compared to the DROP-IN, which converted into a reduction in time, path length, and increase in straightness index. Radioguidance also improved decision-making; task-completion rate increased by 60%, procedural time was reduced, and movements became more focused. Conclusion The Click-On gamma probe provides a step toward full integration of radioguidance in minimal invasive surgery. The value of this concept was underlined by its impact on surgical dexterity and decision-making.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ

Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling.Bio-organic Synthesi

Blue laser cooling transitions in Tm I

We have studied possible candidates for laser cooling transitions in $^{169}$Tm in the spectral region 410 -- 420 nm. By means of saturation absorption spectroscopy we have measured the hyperfine structure and rates of two nearly closed cycling transitions from the ground state $4\textrm{f}^{13}6\textrm{s}^2(^2\textrm{F}_0)(J_g=7/2)$ to upper states $4\textrm{f}^{12}(^3\textrm{H}_5)5\textrm{d}_{3/2}6\textrm{s}^2(J_e=9/2)$ at 410.6 nm and $4\textrm{f}^{12}(^3\textrm{F}_4)5\textrm{d}_{5/2}6\textrm{s}^2(J_e=9/2)$ at 420.4 nm and evaluated the life times of the excited levels as 15.9(8) ns and 48(6) ns respectively. Decay rates from these levels to neighboring opposite-parity levels are evaluated by means of Hartree-Fock calculations. We conclude, that the strong transition at 410.6 nm has an optical leak rate of less then $2\cdot10^{-5}$ and can be used for efficient laser cooling of $^{169}$Tm from a thermal atomic beam. The hyperfine structure of two other even-parity levels which can be excited from the ground state at 409.5 nm and 418.9 nm is also measured by the same technique. In addition we give a calculated value of $7(2)$ s$^{-1}$ for the rate of magnetic-dipole transition at 1.14 $\mu$m between the fine structure levels $(J_g=7/2)\leftrightarrow(J'_g=5/2)$ of the ground state which can be considered as a candidate for applications in atomic clocks.Comment: 8 pages, 5 figure

A lysosome-targeted tetrazine for organelle-specific click-to-release chemistry in antigen presenting cells

Bioorthogonal deprotectionsare readily used to control biologicalfunction in a cell-specific manner. To further improve the spatialresolution of these reactions, we here present a lysosome-targetedtetrazine for an organelle-specific deprotection reaction. We showthat trans-cyclooctene deprotection with this reagentcan be used to control the biological activity of ligands for invariantnatural killer T cells in the lysosome to shed light on the processingpathway in antigen presenting cells. We then use the lysosome-targetedtetrazine to show that long peptide antigens used for CD8(+) T cell activation do not pass through this organelle, suggestinga role for the earlier endosomal compartments for their processing.Bio-organic Synthesi

Radio precursors to neutron star binary mergings

We discuss a possible generation of radio bursts preceding final stages of binary neutron star mergings which can be accompanied by short gamma-ray bursts. Detection of such bursts appear to be advantageous in the low-frequency radio band due to a time delay of ten to several hundred seconds required for radio signal to propagate in the ionized intergalactic medium. This delay makes it possible to use short gamma-ray burst alerts to promptly monitor specific regions on the sky by low-frequency radio facilities, especially by LOFAR. To estimate the strength of the radio signal, we assume a power-law dependence of the radio luminosity on the total energy release in a magnetically dominated outflow, as found in millisecond pulsars. Based on the planned LOFAR sensitivity at 120 MHz, we estimate that the LOFAR detection rate of such radio transients could be about several events per month from redshifts up to $z\sim1.3$ in the most optimistic scenario. The LOFAR ability to detect such events would crucially depend on exact efficiency of low-frequency radio emission mechanism.Comment: 6 pages, 2 figures, Accepted for publication in Astrophysics & Space Science. Largely extended version of ArXiv:0912.521

Anti-topoisomerase, but not anti-centromere B cell responses in systemic sclerosis display active, Ig-secreting cells associated with lung fibrosis

Objectives Almost all patients with systemic sclerosis (SSc) harbour autoantibodies. Anti-topoisomerase antibodies (ATA) and anti-centromere antibodies (ACA) are most prevalent and associate with distinct clinical phenotypes. B cell responses underlying these phenotypes are ill-defined. To understand how B cell autoreactivity and disease pathology connect, we determined phenotypic and functional characteristics of autoreactive B cells in ATA-positive and ACA-positive patients.Methods Levels and isotypes of autoantibodies secreted by ex vivo cultured peripheral blood mononuclear cells from patients with ATA-positive (n=22) and ACA-positive (n=20) SSc were determined. Antibody secreting cells (ASCs) were isolated by cell sorting and cultured separately. Correlations were studied between the degree of spontaneous autoantibody production and the presence and degree of interstitial lung disease (ILD).Results Circulating B cells secreting either ATA-immunoglobulin G (IgG) or ACA-IgG on stimulation was readily detectable in patients. The ATA response, but not the ACA response, showed additional secretion of autoreactive IgA. ATA-IgG and ATA-IgA were also secreted spontaneously. Additional cell sorting confirmed the presence of ATA-secreting plasmablasts. The degree of spontaneous ATA-secretion was higher in patients with ILD than in those without (pConclusion In contrast to ACA-positive patients, ATA-positive patients show signs of recent activation of the B cell response that hallmarks this disease. The degree of activation correlates with the presence and severity of ILD, the most deleterious disease manifestation. This could explain differential responsiveness to B cell depleting therapy. The abundant and spontaneous secretion of ATA-IgG and ATA-IgA may point toward a continuously activating trigger.</div

Bioorthogonal correlative light-electron microscopy of mycobacterium tuberculosis in macrophages reveals the effect of antituberculosis drugs on subcellular bacterial distribution

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis (Mtb) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (ex cellula). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution.Bio-organic SynthesisMedical Biochemistr