Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion

Serine proteases in the nervous system have functional roles in neural plasticity. Among them, urokinase-type plasminogen activator (uPA) exerts a variety of functions during development, and is involved in learning and memory. Furthermore, psychostimulants strongly induce uPA expression in the mesolimbic dopaminergic pathway. In this study, doxycycline-regulatable lentiviruses expressing either uPA, a dominant-negative form of uPA, or non-regulatable lentiviruses expressing small interfering RNAs (siRNAs) targeted against uPA have been prepared and injected into the ventral tegmental area (VTA) of rat brains. Over-expression of uPA in the VTA induces doxycycline-dependent expression of its receptor, uPAR, but not its inhibitor, plasminogen activator inhibitor-1 (PAI-1). uPAR expression in the VTA is repressed upon silencing of uPA with lentiviruses expressing siRNAs. In addition, over-expression of uPA in the VTA promotes a 15-fold increase in locomotion activity upon cocaine delivery. Animals expressing the dominant-negative form of uPA did not display such hyperlocomotor activity. These cocaine-induced behavioural changes, associated with uPA expression, could be suppressed in the presence of doxycycline or uPA-specific siRNAs expressing lentiviruses. These data strongly support the major role of urokinase in cocaine-mediated plasticity changes.     

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super‐resolution imaging, we revealed the nanoscale organization and dynamics of branched F‐actin regulators in spines. Branched F‐actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger‐like protrusions. This spatial segregation differs from lamellipodia where both branched F‐actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin‐like protein‐2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F‐actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free‐diffusion on the membrane. Enhanced Rac1 activation and Shank3 over‐expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F‐actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.     

BMP10 Signaling Promotes the Development of Endocardial Cells from Human Pluripotent Stem Cell-Derived Cardiovascular Progenitors

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Distribution of the Rubredoxin Gene Among the Clostridium butyricum Species

With PCR methods, the rubredoxin gene was systematically identified among 11 strains of Clostridium butyricum; this ubiquity means major functions in the metabolism of the Clostridia. The 11 PCR products allowed deduction of a sequence of 26 amino acids corresponding to positions 11–36 of the rubredoxin. They all contained the tyrosines at positions 11 and 13 and the phenylalanine at position 30 characteristic of the rubredoxin, but differed at positions 14–17, 20, 25, 29, and 31, allowing determination of three types of rubredoxins among these 11 strains of C. butyricum. Received: 13 October 1998 / Accepted: 23 November 1998     

Role of acetate and butyrate in the induction of NADH: rubredoxin oxidoreductase in Clostridium acetobutylicum

Study of the biosynthesis of NADH: rubredoxin oxidoreductase in resting cells of Clostridium acetobutylicum shows that this enzyme is synthesized at a maximal rate in the presence of acetic acid at a concentration of 3 g . l-1 and at pH 4.8. Protons do not play any role in this biosynthesis since no induction is observed in a medium without acetate for the same values of pH. Butyric acid at a concentration of 0.5 g . l-1 gives 50% induction and formic acid, isobutyric acid and propionic acid have no inductive action on NADH: rubredoxin oxidoreductase. These results are confirmed by studies using a dialysis bag. Only a culture against acetic acid at an initial concentration of 2 g . l-1 gives maximal biosynthesis of the enzyme, whereas a culture in which all products of metabolism are eliminated gives an activity which is 80% lower.     

Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.Subject terms: Microbiome, Microbial ecology