Anti‐human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies.     

Estimation of growth and exopolysaccharide production by two soil cyanobacteria,Scytonema tolypothrichoides and Tolypothrix bouteillei as determined by cultivation in irradiance and temperature crossed gradients

Two filamentous cyanobacteria of the genera Scytonema and Tolypothrix were reported to be effective for stabilizing soil in arid areas due to the production of significant amounts of extracellular polysaccharides (EPS). These EPS may also have applications in the biotechnology industry. Therefore, two cyanobacterial species, Scytonema tolypothrichoides and Tolypothrix bouteillei were examined using crossed gradients of temperature (8–40°C) and irradiance (3–21 W m^?2) to identify their temperature and irradiance optima for maximum biomass and EPS production. According to their reported temperature requirements, both strains were considered mesophilic. The optimum growth range of temperature in S. tolypothrichoides (27 to 34°C) was higher than T. bouteillei (22–32°C). The optimum irradiance range for growth of S. tolypothrichoides (9–13 W m^?2) was slightly lower than T. bouteillei (7–18 W m^?2). Maximum EPS production by S. tolypothrichoides occurred at similar temperatures (28–34°C) as T. bouteillei (27–34°C), both slightly higher than for maximum growth. The optimum irradiance range for EPS production was comparable to that for growth in S. tolypotrichoides (8–13 W m^?2), and slightly lower in T. bouteillei (7–17 W m^?2). The Redundancy Analysis confirmed that temperature was the most important controlling factor and protocols for field applications or for mass cultivation can now be developed.     

Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.     

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

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Interactions among morphotype,nutrition, and temperature impact fitness of an invasive fly

Invasive animals depend on finding a balanced nutritional intake to colonize, survive, and reproduce in new environments. This can be especially challenging during situations of fluctuating cold temperatures and food scarcity, but phenotypic plasticity may offer an adaptive advantage during these periods. We examined how lifespan, fecundity, pre‐oviposition periods, and body nutrient contents were affected by dietary protein and carbohydrate (P:C) ratios at variable low temperatures in two morphs (winter morphs WM and summer morphs SM) of an invasive fly, Drosophila suzukii. The experimental conditions simulated early spring after overwintering and autumn, crucial periods for survival. At lower temperatures, post‐overwintering WM lived longer on carbohydrate‐only diets and had higher fecundity on low‐protein diets, but there was no difference in lifespan or fecundity among diets for SM. As temperatures increased, low‐protein diets resulted in higher fecundity without compromising lifespan, while high‐protein diets reduced lifespan and fecundity for both WM and SM. Both SM and WM receiving high‐protein diets had lower sugar, lipid, and glycogen (but similar protein) body contents compared to flies receiving low‐protein and carbohydrate‐only diets. This suggests that flies spend energy excreting excess dietary protein, thereby affecting lifespan and fecundity. Despite having to recover from nutrient depletion after an overwintering period, WM exhibited longer lifespan and higher fecundity than SM in favorable diets and temperatures. WM exposed to favorable low‐protein diet had higher body sugar, lipid, and protein body contents than SM, which is possibly linked to better performance. Although protein is essential for oogenesis, WM and SM flies receiving low‐protein diets did not have shorter pre‐oviposition periods compared to flies on carbohydrate‐only diets. Finding adequate carbohydrate sources to compensate protein intake is essential for the successful persistence of D. suzukii WM and SM populations during suboptimal temperatures.     

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release

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Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.