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Abstract The engineering of non ribosomal peptide synthetases (NRPS) for new substrate specificity is a potent strategy to incorporate non-canonical amino acids into peptide sequences, thereby creating peptide diversity and broadening applications. The non-ribosomal peptide pyoverdine is the primary siderophore produced by Pseudomonas aeruginosa and holds biomedical promise in diagnosis, bio-imaging and antibiotic vectorization. We engineered the adenylation domain of PvdD, the terminal NRPS in pyoverdine biosynthesis, to accept a functionalized amino acid. Guided by molecular modeling, we rationally designed mutants of P. aeruginosa with mutations at two positions in the active site. A single amino acid change results in the successful incorporation of an azido-l-homoalanine leading to the synthesis of a new pyoverdine analog, functionalized with an azide function. We further demonstrated that copper free click chemistry is efficient on the functionalized pyoverdine and that the conjugated siderophore retains the iron chelation properties and its capacity to be recognized and transported by P. aeruginosa. The production of clickable pyoverdine holds substantial biotechnological significance, paving the way for numerous downstream applications.

End-stage liver diseases have an increasing impact worldwide, exacerbated by the shortage of transplantable organs. Recognized as one of the promising solutions, tissue engineering aims at recreating functional tissues and organs in vitro. The integration of bioprinting technologies with biological 3D models, such as multi-cellular spheroids, has enabled the fabrication of tissue constructs that better mimic complex structures and in vivo functionality of organs. However, the lack of methods for large-scale production of homogeneous spheroids has hindered the upscaling of tissue fabrication. In this work, we introduce a fully automated platform, designed for high-throughput sorting of 3D spheroids based on label-free analysis of brightfield images. The compact platform is compatible with standard biosafety cabinets and includes a custom-made microscope and two fluidic systems that optimize single spheroid handling to enhance sorting speed. We use machine learning to classify spheroids based on their bioprinting compatibility. This approach enables complex morphological analysis, including assessing spheroid viability, without relying on invasive fluorescent labels. Furthermore, we demonstrate the efficacy of transfer learning for biological applications, for which acquiring large datasets remains challenging. Utilizing this platform, we efficiently sort mono-cellular and multi-cellular liver spheroids, the latter being used in bioprinting applications, and confirm that the sorting process preserves viability and functionality of the spheroids. By ensuring spheroid homogeneity, our sorting platform paves the way for standardized and scalable tissue fabrication, advancing regenerative medicine applications.

Abstract Chronic imbalance is a frequent and limiting symptom of patients with chronic unilateral and bilateral vestibulopathy. A full-body kinematic analysis of the movement of patients with vestibulopathy would provide a better understanding of the impact of the pathology on dynamic tasks such as walking. Therefore, this study aimed to investigate the global body movement during walking, its variability (assessed with the GaitSD), and the strategies to stabilise the head (assessed with the head Anchoring Index). The full-body motion capture data of 10 patients with bilateral vestibulopathy (BV), 10 patients with unilateral vestibulopathy (UV), and 10 healthy subjects (HS) walking at several speeds (slow, comfortable, and fast) were analysed in this prospective cohort study. We observed only a few significant differences between groups in parts of the gait cycle (shoulder abduction–adduction, pelvis rotation, and hip flexion–extension) during the analysis of kinematic curves. Only BV patients had significantly higher gait variability (GaitSD) for all three walking speeds. Head stabilisation strategies depended on the plan of motion and walking speed condition, but BV and UV patients tended to stabilise their head in relation to the trunk and HS tended to stabilise their head in space. These results suggest that GaitSD could be a relevant biomarker of chronic instability in BV and that the head Anchoring Index tends to confirm clinical observations of abnormal head-trunk dynamics in patients with vestibulopathy while walking.

ABSTRACT Microbial communities offer considerable potential for tackling environmental challenges by improving the functioning of ecosystems. Top-down community engineering is a promising strategy that could be used to obtain communities of desired function. However, the ecological factors that control the balance between community shaping and propagation are not well understood. Dairy backslopping, which consists of using part of the previous production to inoculate a new one, can be used as a model engineering approach to investigate community dynamics during serial propagations. In this study, 26 raw milk samples were serially propagated 6 times each, giving rise to 26 community lineages. Bacterial community structures were analyzed by metabarcoding, and acidification was recorded by pH monitoring. The results revealed that different types of community lineages could be obtained in terms of taxonomic composition and dynamics. Five lineages reached a repeatable community structure in a few propagation steps, with little variation between the final generations, giving rise to stable acidification kinetics. Moreover, these stabilized communities presented a high variability of structure and diverse acidification properties between community lineages. Besides, the other lineages were characterized by different levels of dynamics leading to parallel or divergent trajectories. The functional properties and dynamics of the communities were mainly related to the relative abundance and the taxonomic composition of lactic acid bacteria within the communities. These findings highlight that short-term schemes of serial fermentation can produce communities with a wide range of dynamics and that the balance between community shaping and propagation is intimately linked to community structure.IMPORTANCEMicrobiome applications require approaches for shaping and propagating microbial communities. Shaping allows the selection of communities with desired taxonomic and functional properties, while propagation allows the production of the biomass required to inoculate the engineered communities in the target ecosystem. In top-down community engineering, where communities are obtained from a pool of mixed microorganisms by acting on environmental variables, a major challenge is to master the balance between shaping and propagation. However, the ecological factors that favor high dynamics of community structure and, conversely, those that favor stability during propagation are not well understood. In this work, short-term dairy backslopping was used to investigate the key role of the taxonomic composition and structure of bacterial communities on their dynamics. The results obtained open up interesting prospects for the biotechnological use of microbiomes, particularly in the field of dairy fermentation, to diversify approaches for injecting microbial biodiversity into cheesemaking processes.

Abstract Background Microbial communities harbor important biotechnological potential in diverse domains, however, the engineering and propagation of such communities still face both knowledge and know-how gaps. More specifically, culturing tools are needed to propagate and shape microbial communities, to obtain desired properties, and to exploit them. Previous work suggested that micro-confinement and segregation of microorganisms using invert (water-in-oil, w/o) emulsion broth can shape communities during propagation, by alleviating biotic interactions and inducing physiological changes in cultured bacteria. The present work aimed at evaluating invert emulsion and simple broth monophasic cultures for the propagation and shaping of bacterial communities derived from raw milk in a serial propagation design. Results The monophasic setup resulted in stable community structures during serial propagation, whereas the invert emulsion system resulted in only transiently stable structures. In addition, different communities with different taxonomic compositions could be obtained from a single inoculum. Furthermore, the implementation of invert emulsion systems has allowed for the enrichment of less abundant microorganisms and consequently facilitated their isolation on culture agar plates. Conclusions The monophasic system enables communities to be propagated in a stable manner, whereas the invert emulsion system allowed for the isolation of less abundant microorganisms and the generation of diverse taxonomic compositions from a single inoculum.

This contribution looks at occidental geomancy practices called « geobiology », based on a survey initiated in 2020 in the Alpine region around the Grenoble urban region. Geobiology refers to a set of ways of feeling that are attentive to terrestrial, telluric and cosmic energies, mobilized for the purpose of caring for humans and animals or harmonizing the habitat. Based on a few examples of how we come into contact with these phenomena mobilizing different modalities of internal senses and somatic experience, we’ll show that they involve cosmologies that situate human beings between sky and earth, and manifest themselves in sensitive relationships with living environments.

Aquaculture is expected to play a vital role in solving the challenge of sustainably providing the growing world population with healthy and nutritious food. Pathogen outbreaks are a major risk for the sector, so early detection and a timely response are crucial. This can be enabled by monitoring the pathogen levels in aquaculture facilities. This paper describes a photonic biosensing platform based on silicon nitride waveguide technology with integrated active components, which could be used for such applications. Compared to the state of the art, the current system presents improvements in terms of miniaturization of the Photonic Integrated Circuit (PIC) and the development of wafer-level processes for hybrid integration of active components and for material-selective chemical and biological surface modification. Furthermore, scalable processes for integrating the PIC in a microfluidic cartridge were developed, as well as a prototype desktop readout instrument. Three bacterial aquaculture pathogens (Aeromonas salmonicida, Vagococcus salmoninarum, and Yersinia ruckeri) were selected for assay development. DNA biomarkers were identified, corresponding primer-probe sets designed, and qPCR assays developed. The biomarker for Aeromonas was also detected using the hybrid PIC platform. This is the first successful demonstration of biosensing on the hybrid PIC platform.

Color is one of the first criteria to assess the quality of cheese. However, very limited data are available on the color heterogeneity of the rind and its relationship with microbial community structure. In this study, the color of a wide range of smear-ripened Munster cheeses from various origins was monitored during storage by photographic imaging and data analysis in the CIELAB color space using luminance, chroma, and hue angle as descriptors. Different levels of inter- and intra-cheese heterogeneity were observed. The most heterogeneous Munster cheeses were the darkest with orange-red colors. The most homogeneous were the brightest with yellow-orange. K-means clustering revealed three clusters distinguished by their color heterogeneity. Color analysis coupled with metabarcoding showed that rinds with heterogeneous color exhibited higher microbial diversity associated with important changes in their microbial community structure during storage. In addition, intra-cheese community structure fluctuations were associated with heterogeneity in rind color. The species Glutamicibacter arilaitensis and Psychrobacter nivimaris/piscatorii were found to be positively associated with the presence of undesirable brown patches. This study highlights the close relationship between the heterogeneity of the cheese rind and its microbiota.

Abstract Background 3-methylmethcathinone (3-MMC) has been available on the European drug market for several years, but an increase in its availability seems to have occurred around 2020, associated with reports of harm and death. We aimed to analyze the composition of the supposed 3-MMC samples purchased and its concordance with the assumed composition of the drug. Methods A prospective multicenter (n = 6) study was conducted between February 2021 and September 2021 in Auvergne-Rhone-Alpes, France. The inclusion criteria were: 3-MMC users over 18 years of age in contact with a community-based organization (CBO) called AIDES. Consumption was evaluated with an anonymized questionnaire and samples of 3-MMC powder were analyzed with a combination of qualitative (GC–MS) and quantitative methods (UPLC-MS/MS), to compare the assumed and real compositions of the products purchased. Results We studied 45 samples provided by 33 users. The study population was predominantly male (91%), with a median age of 40 years, most were university graduates and regular users of 3-MMC. Intravenous drug use was reported by 15.2% of the population. Most of the users bought their 3-MMC online via the Clear Web. Drug testing was requested by 86% of the users, highlighting the need for this type of harm reduction strategy. The purity of the 3-MMC powder samples tested ranged from 21 to 98%. Other NPS drugs, such as 4-CEC (4-chloroethcathinone), 4-MMC, and 2-fluorodeschloroketamine (2-FDCK), supplied as methoxphenidine (MXP), were also detected. Conclusion This prospective study shows that 3-MMC purity and dose vary considerably. It also describes the characteristics of 3-MMC users and their expectations of a drug-checking program. Our data suggest that drug-checking services may be useful in this population. Health associations and laboratories should work together to help increase access to such programs.

Antimicrobial resistance poses an urgent and formidable global public health threat. The escalation of bacterial multidrug resistance to antibiotics has the potential to become a leading cause of global mortality if there is no substantial improvement in antimicrobial development and therapy protocols. In light of this, it is imperative to identify the molecular determinants responsible for the reduced antibiotic activity associated with RND efflux pumps. This comprehensive study meticulously examines Minimum Inhibitory Concentration (MIC) data obtained from in vitro tests for various antibiotic families and non-active dye compounds, sourced from diverse literature references. The primary focus of this study is to assess the susceptibility of these agents to efflux-resistant Escherichia coli strains, integrating both MIC data and relevant physicochemical properties. The central objective is to unveil the specific substituents that significantly influence the uptake process mediated by the AcrAB-TolC efflux system. This exploration seeks to reveal the consequences of these substituents on pharmacodynamic responses, providing valuable insights into Structure-Activity Relationships. It is noteworthy that this analysis represents a pioneering effort, with prospective implications for RND efflux pump-producing strains. Ultimately, deciphering efflux markers is crucial to effectively mitigate the emergence of specific resistance and to better monitor the role of this primary resistance mechanism in Gram-negative bacteria, particularly as observed in clinical antibiotic therapy practice.

IntroductionEarlier studies exploring the value of executive functioning (EF) indices for assessing treatment effectiveness and predicting treatment response in attention-deficit/hyperactivity disorder (ADHD) mainly focused on pharmacological treatment options and revealed rather heterogeneous results. Envisioning the long-term goal of personalized treatment selection and intervention planning, this study comparing methylphenidate treatment (MPH) and a home-based neurofeedback intervention (NF@Home) aimed to expand previous findings by assessing objective as well as subjectively reported EF indices and by analyzing their value as treatment and predictive markers.MethodsChildren and adolescents (n = 146 in the per protocol sample) aged 7–13 years with a formal diagnosis of an inattentive or combined presentation of ADHD were examined. We explored the EF performance profile using the Conners Continuous Performance Task (CPT) and the BRIEF self-report questionnaire within our prospective, multicenter, randomized, reference drug-controlled NEWROFEED study with sites in five European countries (France, Spain, Switzerland, Germany, and Belgium). As primary outcome for treatment response, the clinician-rated ADHD Rating Scale-IV was used. Patients participating in this non-inferiority trial were randomized to either NF@home (34–40 sessions of TBR or SMR NF depending on the pre-assessed individual alpha peak frequency) or MPH treatment (ratio: 3:2). Within a mixed-effects model framework, analyses of change were calculated to explore the predictive value of neurocognitive indices for ADHD symptom-related treatment response.ResultsFor a variety of neurocognitive indices, we found a significant pre-post change during treatment, mainly in the MPH group. However, the results of the current study reveal a rather limited prognostic value of neurocognitive indices for treatment response to either NF@Home or MPH treatment. Some significant effects emerged for parent-ratings only.DiscussionCurrent findings indicate a potential value of self-report (BRIEF global score) and some objectively measured neurocognitive indices (CPT commission errors and hit reaction time variability) as treatment markers (of change) for MPH. However, we found a rather limited prognostic value with regard to predicting treatment response not (yet) allowing recommendation for clinical use. Baseline symptom severity was revealed as the most relevant predictor, replicating robust findings from previous studies.