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Abstract Cancer cells in acidic tumor regions are aggressive and a key therapeutic target, but distinguishing between acid-exposed and hypoxic cells is challenging. Here, we use carbonic anhydrase 9 (CA9) antibodies to mark acidic areas in both hypoxic and respiring tumor areas, along with an HRE-GFP reporter for hypoxia, to isolate distinct cell populations from 3D tumor spheroids. Transcriptomic analysis of CA9-positive, hypoxia-negative cells highlights enriched fatty acid desaturase activity. Inhibiting or silencing stearoyl-CoA desaturase-1 (SCD1) induces ferroptosis in CA9-positive acidic cancer cells and delays mouse tumor growth, an effect enhanced by omega-3 fatty acid supplementation. Using acid-exposed cancer cells and patient-derived tumor organoids, we show that SCD1 inhibition increases acidic cancer cell reliance on external mono-unsaturated fatty acids, depriving hypoxic cells of essential resources. This bystander effect provides unbiased evidence for a lack of full overlap between hypoxic and acidic tumor compartments, highlighting a rationale for targeting desaturase activity in cancer.

Background Immunogenic cell death (ICD) and ferroptosis have recently emerged as key factors in the anticancer immune response. Among the treatments able to induce ICD and the associated release of danger signals is photodynamic therapy (PDT). Ferroptosis for its part results from lipid peroxidation and is induced by CD8+ T cells to kill nearby cancer cells on IFN-γ production. We aimed to combine the two concepts, that is, to evaluate whether the strong pro-oxidant effects of PDT may promote ferroptosis and antigen release and to develop a procedure for in situ PDT to prepare the soil for highly endocytotic immature dendritic cell (iDC) adoptive transfer. This approach was implemented for managing peritoneal carcinomatosis, a lesion often associated with poor outcomes.Methods We used three-dimensional (3D) heterotypic spheroids made of cancer cells, exposed them to a white light-activated OR141 photosensitizer (PS), and subsequently complexified them by adding iDC and naive lymphocytes. We next used a model of mouse peritoneal carcinomatosis and administered PDT using laparoscopy to locally induce photoactivation using the endoscope light. The immune response following adoptive transfer of iDC was tracked both in vivo and ex vivo using isolated immune cells from in situ vaccinated mice.Results Cancer cells undergoing PDT-induced cell death significantly increased ICD markers and the infiltration of iDCs in spheroids, relying on ferroptosis. These actions induced the sequential activation of CD8+ and CD4+ T cells as revealed by a significant spheroid 3D structure deterioration and, remarkably, were not recapitulated by conventional ferroptosis inducer RSL3. Using LED light from an endoscope for in situ photoactivation of PS enabled us to apply the vaccination modality in mice with peritoneal tumors. Consecutive intraperitoneal injection of iDCs resulted in delayed tumor growth, increased survival rates, and prevented tumor relapse on rechallenge. CD8+ T cell response was supported by depletion experiments, nodal detection of early activated T cells, and ex vivo T cell-induced cytotoxicity toward spheroids.Conclusions The combination of in situ PDT locally delivered by an endoscope light and iDC administration induces a durable memory immune response against peritoneal carcinomatosis thereby opening new perspectives for the treatment of a life-threatening condition.

Abstract Predatory bacteria feed upon other bacteria in various environments. Bdellovibrio exovorus is an obligate epibiotic predator that attaches on the prey cell surface, where it grows and proliferates. Although the mechanisms allowing feeding through the prey cell envelope are unknown, it has been proposed that the prey’s proteinaceous S-layer may act as a defensive structure against predation. Here, we use time-lapse and cryo-electron microscopy to image the lifecycle of B. exovorus feeding on Caulobacter crescentus. We show that B. exovorus proliferates by non-binary division, primarily generating three daughter cells. Moreover, the predator feeds on C. crescentus regardless of the presence of an S-layer, challenging its assumed protective role against predators. Finally, we show that apparently secure junctions are established between prey and predator outer membranes.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Abstract Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker's yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.

The accurate distribution of genetic material is crucial for all organisms. In most bacteria, chromosome segregation is achieved by the ParABS system, in which the ParB-bound parS sequence is actively partitioned by ParA. While this system is highly conserved, its adaptation in organisms with unique lifestyles and its regulation between developmental stages remain largely unexplored. Bdellovibrio bacteriovorus is a predatory bacterium proliferating through polyploid replication and non-binary division inside other bacteria. Our study reveals the subcellular dynamics and multi-layered regulation of the ParABS system, coupled to the cell cycle of B. bacteriovorus. We found that ParA:ParB ratios fluctuate between predation stages, their balance being critical for cell cycle progression. Moreover, the parS chromosomal context in non-replicative cells, combined with ParB depletion at cell division, critically contribute to the unique cell cycle-dependent organization of the centromere in this bacterium, highlighting new levels of complexity in chromosome segregation and cell cycle control.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Abstract Several observational studies have found a link between the long-term use of benzodiazepines and dementia, which remains controversial. Our study was designed to assess (i) whether the long-term use of benzodiazepines, at two different doses, has an irreversible effect on cognition, (ii) and whether there is an age-dependent effect. One hundred and five C57Bl/6 male mice were randomly assigned to the 15 mg/kg/day, the 30 mg/kg/day diazepam-supplemented pellets, or the control group. Each group comprised mice aged 6 or 12 months at the beginning of the experiments and treated for 16 weeks. Two sessions of behavioral assessment were conducted: after 8 weeks of treatment and after treatment completion following a 1-week wash-out period. The mid-treatment test battery included the elevated plus maze test, the Y maze spontaneous alternation test, and the open field test. The post-treatment battery was upgraded with three additional tests: the novel object recognition task, the Barnes maze test, and the touchscreen-based paired-associated learning task. At mid-treatment, working memory was impaired in the 15 mg/kg diazepam group compared to the control group (p = 0.005). No age effect was evidenced. The post-treatment assessment of cognitive functions (working memory, visual recognition memory, spatial reference learning and memory, and visuospatial memory) did not significantly differ between groups. Despite a cognitive impact during treatment, the lack of cognitive impairment after long-term treatment discontinuation suggests that benzodiazepines alone do not cause irreversible deleterious effects on cognitive functions and supports the interest of discontinuation in chronically treated patients.

The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication.

This article aims to examine the effect of intra-institutional conflicts in the European Commission on the extent of changes made to legislative proposals in trilogue negotiations. We develop and test three hypotheses related to how conflicts within the Commission, namely that intra-institutional disagreements during policy formulation (h1), and potential conflicts with previous (h2) or subsequent (h3) colleges of commissioners, increase the number of amendments to the Commission’s proposal adopted in trilogues. To test our hypotheses, we use a new dataset measuring the number of changes between Commission proposals and adopted legislation for 216 legislative acts negotiated between 2012 and 2019 by means of text-mining techniques. It is important to note that we control for differences between the Commission’s proposals and the co-legislators’ positions in order to distinguish between an effect on preferences anticipation and on the negotiations proper. Our results indicate that intra-institutional conflicts affect the Commission’s anticipation of the co-legislators’ positions. The effect on its behaviour in trilogues, that is, after the legislative proposal has been tabled, is less clear. Regarding the latter, only the number of Directorates-General involved is significantly linked with the number of amendments tabled. These findings suggest that while intra-institutional disagreements affect the Commission’s role in trilogues, the range of preferences is more important than the intensity of conflicts.

For policies to be legitimate, both the policy process and the underlying reasons must be transparent to the public. In the EU, the lion’s share of legislation is nowadays negotiated in informal secluded meeting called trilogues. Therefore, presentation of the trilogues compromise by the rapporteur to the European Parliament (EP) plenary is, arguably, one of the few formal occasions for ‘transparency in process,’ i.e., public access to the details of actual interactions between policymakers. The aim of this article is thus to examine the extent to which rapporteurs are transparent about trilogue negotiations when presenting legislative compromises to the EP during plenary sessions, and to assess whether the extent of transparency is linked to the extent of conflict between legislative actors and to elements of the political context related to rapporteurs. To this purpose, we coded 176 rapporteur speeches and, on this basis, concluded that these speeches poorly discuss the trilogue negotiations. Interinstitutional negotiations are discussed in only 64% of cases, and even when they are, the extent of information about trilogues is generally small. While we do not find support for an effect of political conflicts, some characteristics linked with rapporteurs are significantly related to transparency in process of their speeches. This is the case for their political affiliation and their national culture of transparence.

Using robotic devices might improve recovery post-stroke, but the optimal way to apply robotic assistance has yet to be determined. The current study aimed to investigate whether training under the robotic active-assisted mode improves bimanual motor skill learning (biMSkL) more than training under the active mode in stroke patients. Twenty-six healthy individuals (HI) and 23 chronic hemiparetic stroke patients with a detectable lesion on MRI or CT scan, who demonstrated motor deficits in the upper limb, were randomly allocated to two parallel groups. The protocol included a two-day training on a new bimanual cooperative task, LIFT-THE-TRAY, under either the active or active-assisted modes (where assistance decreased in a pre-determined stepwise fashion) with the bimanual version of the REAplan® robotic device. The hypothesis was that the active-assisted mode would result in greater biMSkL than the active mode. The biMSkL was quantified by a speed-accuracy trade-off (SAT) before (T1) and immediately after (T2) training on days 1 and 2 (T3 and T4). The change in SAT after 2 days of training (T4/T1) indicated that both HI and stroke patients learned and retained the bimanual cooperative task. After 2 days of training, the active-assisted mode did not improve biMSkL more than the active mode (T4/T1) in HI nor stroke patients. Whereas HI generalized the learned bimanual skill to different execution speeds in both the active and active-assisted subgroups, the stroke patients generalized the learned skill only in the active subgroup. Taken together, the active-assisted mode, applied in a pre-determined stepwise decreasing fashion, did not improve biMSkL more than the active mode in HI and stroke subjects. Stroke subjects might benefit more from robotic assistance when applied “as-needed.” This study was approved by the local ethical committee (Comité d’éthique médicale, CHU UCL Namur, Mont-Godinne, Yvoir, Belgium; Internal number: 54/2010, EudraCT number: NUB B039201317382) on July 14, 2016 and was registered with ClinicalTrials.gov (Identifier: NCT03974750) on June 5, 2019.

Abstract Aim The aim of the study was to describe and compare feeling of competence regarding humanistic caring in Registered Nurses (RN) and nursing students (NS). Design A quantitative comparative cross‐sectional research design was used. Methods A convenience sample of 196 RN and 47 NS in a teaching hospital in Belgium completed a self‐administered questionnaire composed of a sociodemographic survey and the Caring Nurse‐Patient Interactions Scale (CNPI‐23) developed by Cossette et al. Results The four dimensions of the CNPI‐23 were compared using the Skillings–Mack test. Both groups scored higher on “humanistic” and “comforting” than on “clinical” and “relational” care and both scored lowest on this last dimension. Linear regressions showed that none of the variables had a statistically significant influence on the CNPI‐23 scores, except for NS “state of health,” which influenced their feeling of competence regarding “relational care.”

Summary: The predatory bacterium Bdellovibrio bacteriovorus invades and proliferates inside other bacteria by non-binary division. Here we describe a fluorescence-based technique for the immediate evaluation of predator density independently of plaque formation, an optimized setup to monitor predation in microplates, and the CuRveR package to quantify both prey killing and predator proliferation dynamics. This protocol allows to assess the impact of mutations or chemicals on predation. CuRveR also constitutes a user-friendly tool to analyze growth or decay data unrelated to predation.For complete details on the use and execution of this profile, please refer to Kaljević et al., 2021.

Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. Arabidopsis PIP2;7 trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, a plasma membrane Qa-SNARE involved in membrane fusion. To better understand the interaction mechanism, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in Nicotiana benthamiana. SYP121 consists of four regions, N, H, Q, and C, and sequential deletions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE functional domains, interact with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or Q regions alone could fully restore the interaction with PIP2;7, suggesting that the interacting motif depends on the conformation taken by the HQ region. When investigating the interacting motif(s) in PIP2;7, we observed that deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that at the N-terminal amino acid residues 18–26 were involved in the interaction. Domain swapping experiments between PIP2;7 and PIP2;6, a PIP isoform that does not interact with SYP121, showed that PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal, suggesting that, as it is the case for SYP121, the interaction motif(s) in PIP2;7 depend on the protein conformation. Finally, we also showed that PIP2;7 physically interacted with other Arabidopsis SYP1s and SYP121 orthologs.

Background: Continuous compensation of dopamine represents an ideal symptomatic treatment for Parkinson's disease (PD). The feasibility in intracerebroventricular administration (i.c.v.) of dopamine previously failed because of unresolved dopamine oxidation. Objectives: We aim to test the feasibility, safety margins and efficacy of continuous i.c.v. of anaerobic-dopamine (A-dopamine) with a pilot translational study in a non-human primate model of PD. Methods: Continuous and circadian i.c.v. of A-dopamine was administered through a micro-pump connected to a subcutaneous catheter implanted into the right frontal horn of 8 non-human primates treated with 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP). A-dopamine was assessed at acute doses previously reported for dopamine as well as evaluating the long term therapeutic index of A-dopamine in comparison to anaerobically prepared L-dopa or methyl ester L-dopa. Results: Over 60 days of a continuous circadian i.c.v. of A-dopamine improved motor symptoms (therapeutic index from 30 to 70 mg/day) without tachyphylaxia. No dyskinesia was observed even with very high doses. Death after 1 to 10 days (without neuronal alteration) was only observed with doses in excess of 160 mg whereas L-dopa i.c.v. was not effective at any dose. The technical feasibility of the administration regimen was confirmed for an anaerobic preparation of dopamine and for administration of a minimal infusion volume by micro-pump at a constant flow that prevented obstruction. Conclusion: Continuous circadian i.c.v. of A-dopamine appears to be feasible and shows efficacy without dyskinesia with a safe therapeutic index.

Bdellovibrio bacteriovorus is a predatory bacterium that feeds upon and proliferates inside other Gram-negative bacteria. Upon entry into the periplasmic space of the prey envelope, B. bacteriovorus initiates an exquisite developmental program in which it digests the host resources and grows as a filament, which eventually divides in a non-binary manner, releasing a variable number of daughter cells. The progeny then escape from the prey ghost to encounter new victims and resume the predation cycle. Owing to its unique biology, B. bacteriovorus undoubtedly represents an attractive model to unravel novel mechanisms of bacterial cell cycle control and cellular organization. Yet, the molecular factors behind the sophisticated lifestyle of this micro-predator are still mysterious. In particular, the spatiotemporal dynamics of proteins that control key cellular processes such as transmission of the genetic information, cell growth and division remain largely unexplored. In this Perspective article, I highlight outstanding fundamental questions related to these aspects and arising from the original biology of this bacterium. I also discuss available insights and potential cell biology approaches based on quantitative live imaging techniques, in combination with bacterial genetics and biochemistry, to shed light on the intracellular organization of B. bacteriovorus in space and time.

Le bassin du Niger représente un important potentiel de développement régional, en particulier s’agissant de l’irrigation et de l’hydroélectricité. Un enjeu majeur pour l’Autorité du Bassin du Niger (ABN) et ses neuf États membres est la cohérence globale de l’aménagement du bassin et de sa gestion. Les grands barrages structurants existants sont Sélingué au Mali, Kandadji au Niger, Kainji, Jebba, Shiroro, Dadin Kowa au Nigeria et Lagdo au Cameroun. Les projets en cours sont ceux de Fomi et Taoussa en Guinée et au Mali. L’Annexe 2 à la Charte de l’eau de l’ABN relative au Règlement d’eau pour la gestion coordonnée des barrages structurants a été élaborée en 2019 par le groupement CACG – OIEau – Nodalis - ISL et validée fin 2019 par le Conseil des Ministres de l’ABN. Sa mise en application repose sur une actualisation du modèle d’allocation des ressources en eau de l’ABN et sur un outil de gestion tactique. Outre le recueil des données et l’opérationnalisation des prévisions, les améliorations possibles de la gestion coordonnée des barrages du bassin du Niger concernent en particulier la mise en oeuvre du Comité Technique Permanent de l’ABN, en charge de l’application du Règlement de gestion coordonnée. Plus généralement une organisation institutionnelle cohérente est nécessaire, ainsi qu’une reconnaissance justifiée du rôle de l’ABN.

Summary: Although the brain accounts for only 2% of the total body mass, it consumes the most energy. Neuronal metabolism is tightly controlled, but it remains poorly understood how neurons meet their energy demands to sustain synaptic transmission. Here we provide evidence that AMP-activated protein kinase (AMPK) is pivotal to sustain neuronal energy levels upon synaptic activation by adapting the rate of glycolysis and mitochondrial respiration. Furthermore, this metabolic plasticity is required for the expression of immediate-early genes, synaptic plasticity, and memory formation. Important in this context, in neurodegenerative disorders such as Alzheimer disease, dysregulation of AMPK impairs the metabolic response to synaptic activation and processes that are central to neuronal plasticity. Altogether, our data provide proof of concept that AMPK is an essential player in the regulation of neuroenergetic metabolic plasticity induced in response to synaptic activation and that its deregulation might lead to cognitive impairments. : Neuroscience; Molecular Neuroscience; Cellular Neuroscience Subject Areas: Neuroscience, Molecular Neuroscience, Cellular Neuroscience

In Parkinson's disease (PD) depletion of dopamine in the nigro-striatal pathway is a main pathological hallmark that requires continuous and focal restoration. Current predominant treatment with intermittent oral administration of its precursor, Levodopa (l-dopa), remains the gold standard but pharmacological drawbacks trigger motor fluctuations and dyskinesia. Continuous intracerebroventricular (i.c.v.) administration of dopamine previously failed as a therapy because of an inability to resolve the accelerated dopamine oxidation and tachyphylaxia. We aim to overcome prior challenges by demonstrating treatment feasibility and efficacy of continuous i.c.v. of dopamine close to the striatum. Dopamine prepared either anaerobically (A-dopamine) or aerobically (O-dopamine) in the presence or absence of a conservator (sodium metabisulfite, SMBS) was assessed upon acute MPTP and chronic 6-OHDA lesioning and compared to peripheral l-dopa treatment. A-dopamine restored motor function and induced a dose dependent increase of nigro-striatal tyrosine hydroxylase positive neurons in mice after 7 days of MPTP insult that was not evident with either O-dopamine or l-dopa. In the 6-OHDA rat model, continuous circadian i.c.v. injection of A-dopamine over 30 days also improved motor activity without occurrence of tachyphylaxia. This safety profile was highly favorable as A-dopamine did not induce dyskinesia or behavioral sensitization as observed with peripheral l-dopa treatment. Indicative of a new therapeutic strategy for patients suffering from l-dopa related complications with dyskinesia, continuous i.c.v. of A-dopamine has greater efficacy in mediating motor impairment over a large therapeutic index without inducing dyskinesia and tachyphylaxia.

The envelope of Gram-negative bacteria is critical for survival across a wide range of environmental conditions. The inner membrane, the periplasm and the outer membrane form a complex compartment, home to many essential processes. Hence, constant monitoring by envelope stress response systems ensure correct biogenesis of the envelope and maintain its homeostasis. Inside the periplasm, the cell wall, made of peptidoglycan, has been under the spotlight for its critical role in bacterial growth as well as being the target of many antibiotics. While much research is centered around understanding the role of the many enzymes involved in synthesizing the cell wall, much less is known about how the cell can detect perturbations of this assembly process, and how it is regulated during stress. In this review, we explore the current knowledge of cell wall defects sensing by stress response systems, mainly in the model bacterium Escherichia coli. We also discuss how these systems can respond to cell wall perturbations to increase fitness, and what implications this has on cell wall regulation.