Distinct actin–tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes

The effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1 and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections

Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse

Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications

Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae).

Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest's resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects

Mean cuticle thickness (μm ± S.E) of Monheim susceptible (n = 10) and Parramatta resistant (n = 10) <i>Cimex lectularius</i>.

<p>Asterisks (*) indicate statistical significance (p < 0.05).</p

Knockdown (% ± S.E, n = 160) over time (minutes) of Parramatta strain <i>Cimex lectularius</i> upon forced exposure to wet residues of 20 mL/L Demand Insecticide^® (25 g/L lambda-cyhalothrin).

<p>Knockdown (% ± S.E, n = 160) over time (minutes) of Parramatta strain <i>Cimex lectularius</i> upon forced exposure to wet residues of 20 mL/L Demand Insecticide^® (25 g/L lambda-cyhalothrin).</p

Transverse section of <i>Cimex lectularius</i> (Parramatta strain) middle leg tibia and example of twelve point-to-point cuticle measurements methodology.

<p>Letters denote measurements of: a = 9.15 μm, b = 9.82 μm, c = 10.25 μm, d = 7.91 μm, e = 7.16 μm, f = 8.42 μm, g = 8.02 μm, h = 8.41 μm, k = 10.12 μm, m = 8.82 μm, n = 8.31 μm, o = 8.09 μm [i, j and l not included for purpose of legibility].</p

Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors.

Megakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 × 1