Charting the sequence-activity landscape of peptide inhibitors of translation termination.

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Recent structural advances in constrained helical peptides.

Given the ubiquity of the ⍺-helix in the proteome, there has been much research in developing mimics of ⍺-helices, and most of this study has been toward developing protein-protein interaction inhibitors. A common strategy for mimicking ⍺-helices has been through the use of constrained, helical peptides. The addition of a constraint typically provides for conformational and proteolytic stability and, in some cases, cell permeability. Some of the most well-known strategies included are lactam formation and hydrocarbon "stapling." Beyond those strategies, there have been many recent advances in developing constrained peptides. The purpose of this review is to highlight recent advances in the development of new helix-stabilizing technologies, constraint diversification strategies, tether diversification strategies, and combination strategies that create new bicyclic helical peptides.

Model-Based Autonomous Navigation with Moment of Inertia Estimation for Unmanned Aerial Vehicles.

The dominant navigation system for low-cost, mass-market Unmanned Aerial Vehicles (UAVs) is based on an Inertial Navigation System (INS) coupled with a Global Navigation Satellite System (GNSS). However, problems tend to arise during periods of GNSS outage where the navigation solution degrades rapidly. Therefore, this paper details a model-based integration approach for fixed wing UAVs, using the Vehicle Dynamics Model (VDM) as the main process model aided by low-cost Micro-Electro-Mechanical Systems (MEMS) inertial sensors and GNSS measurements with moment of inertia calibration using an Unscented Kalman Filter (UKF). Results show that the position error does not exceed 14.5 m in all directions after 140 s of GNSS outage. Roll and pitch errors are bounded to 0.06 degrees and the error in yaw grows slowly to 0.65 degrees after 140 s of GNSS outage. The filter is able to estimate model parameters and even the moment of inertia terms even with significant coupling between them. Pitch and yaw moment coefficient terms present significant cross coupling while roll moment terms seem to be decorrelated from all of the other terms, whilst more dynamic manoeuvres could help to improve the overall observability of the parameters.

Time-Gated Luminescence Detection of Enzymatically Produced Hydrogen Sulfide: Design, Synthesis, and Application of a Lanthanide-Based Probe.

Hydrogen sulfide (H2S) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for H2S, LP2-Cu(II)-Ln(III), a heterobinuclear metal complex that uses Cu(II) decomplexation to control millisecond-scale-lifetime-Tb(III)- or Eu(III)-emission intensity. LP2-Cu(II)-Ln(III) responded rapidly, selectively, and with high sensitivity to aqueous H2S. The probe's potential for biological applications was verified by measuring the H2S generated by the slow-releasing chemical-sulfide-donor GYY4147, by cystathionine γ-lyase (CSE), and by Na2S-stimulated HeLa cells.

A "cross-stitched" peptide with improved helicity and proteolytic stability.

A new computational approach to obtain quantitative energy profiles for helix folding was used in the design of orthogonal hydrocarbon and lactam bicyclic peptides. The proteolytically stable, "cross-stitched" peptide SRC2-BCP1 shows nanomolar affinity for estrogen receptor α and X-ray crystallography confirms a helical binding pose.

Analysing the Zenith Tropospheric Delay Estimates in On-line Precise Point Positioning (PPP) Services and PPP Software Packages.

As Global Navigation Satellite System (GNSS) signals travel through the troposphere, a tropospheric delay occurs due to a change in the refractive index of the medium. The Precise Point Positioning (PPP) technique can achieve centimeter/millimeter positioning accuracy with only one GNSS receiver. The Zenith Tropospheric Delay (ZTD) is estimated alongside with the position unknowns in PPP. Estimated ZTD can be very useful for meteorological applications, an example is the estimation of water vapor content in the atmosphere from the estimated ZTD. PPP is implemented with different algorithms and models in online services and software packages. In this study, a performance assessment with analysis of ZTD estimates from three PPP online services and three software packages is presented. The main contribution of this paper is to show the accuracy of ZTD estimation achievable in PPP. The analysis also provides the GNSS users and researchers the insight of the processing algorithm dependence and impact on PPP ZTD estimation. Observation data of eight whole days from a total of nine International GNSS Service (IGS) tracking stations spread in the northern hemisphere, the equatorial region and the southern hemisphere is used in this analysis. The PPP ZTD estimates are compared with the ZTD obtained from the IGS tropospheric product of the same days. The estimates of two of the three online PPP services show good agreement (<1 cm) with the IGS ZTD values at the northern and southern hemisphere stations. The results also show that the online PPP services perform better than the selected PPP software packages at all stations.

GNSS Trajectory Anomaly Detection Using Similarity Comparison Methods for Pedestrian Navigation.

The urban setting is a challenging environment for GNSS receivers. Multipath and other anomalies typically increase the positioning error of the receiver. Moreover, the error estimate of the position is often unreliable. In this study, we detect GNSS trajectory anomalies by using similarity comparison methods between a pedestrian dead reckoning trajectory, recorded using a foot-mounted inertial measurement unit, and the corresponding GNSS trajectory. During a normal walk, the foot-mounted inertial dead reckoning setup is trustworthy up to a few tens of meters. Thus, the differing GNSS trajectory can be detected using form similarity comparison methods. Of the eight tested methods, the Hausdorff distance (HD) and the accumulated distance difference (ADD) give slightly more consistent detection results compared to the rest.

FcγRIIIa-Syk Co-signal Modulates CD4+ T-cell Response and Up-regulates Toll-like Receptor (TLR) Expression.

CD4(+) T-cells in systemic lupus erythematosus (SLE) patients show altered T-cell receptor signaling, which utilizes Fc-receptor γ-chain FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4(+) T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4(+) T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4(+) T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A(+) and IFN-γ(high) CD4(+) T-cells in vitro. Cytokines IL-1ß, IL-6, TGF-ß1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4(+) T-cells that expressed CD25, CD69, and CD98 bound to ICs showed pSyk and produced IFN-γ and IL-17A. This FcγRIIIa-mediated co-signal differentially up-regulated the expression of IFN pathway genes compared with CD28 co-signal. FcγRIIIa-pSyk up-regulated several toll-like receptor genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these toll-like receptor pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses.

Time-Gated Detection of Cystathionine γ-Lyase Activity and Inhibition with a Selective, Luminogenic Hydrogen Sulfide Sensor.

Herein, we report the design, synthesis, and characterization of a lanthanideIII complex-based probe for the time-gated luminescence detection of hydrogen sulfide (H2 S) in aqueous media. The probe's unique sensing mechanism relies on the selective reduction of azide to amine by sulfide, followed by intramolecular cyclization to form a quinolinone. The quinolinone is a sensitizer that absorbs near-UV light and transfers excitation energy to coordinated TbIII or EuIII ions to trigger a strong "turn-on" luminescence response with ms-scale lifetimes characteristic of lanthanide complexes. Using this probe, we developed a robust, high throughput screening (HTS) assay for detecting H2 S generated by cystathionine γ-lyase (CSE), one of the main producers of H2 S in mammalian cells. In a 240-compound screen to identify potential CSE inhibitors, the EuIII analogue of the sensor showed a low false-positive rate and high Z'-factor (>0.7).