Topological analysis of 3D digital ovules identifies cellular patterns associated with ovule shape diversity.

Tissue morphogenesis remains poorly understood. In plants, a central problem is how the 3D cellular architecture of a developing organ contributes to its final shape. We address this question through a comparative analysis of ovule morphogenesis, taking advantage of the diversity in ovule shape across angiosperms. Here, we provide a 3D digital atlas of Cardamine hirsuta ovule development at single cell resolution and compare it to an equivalent atlas of Arabidopsis thaliana. We introduce nerve-based topological analysis as a tool for unbiased detection of differences in cellular architectures and corroborate identified topological differences between two homologous tissues by comparative morphometrics and visual inspection. We find that differences in topology, cell volume variation and tissue growth patterns in the sheet-like integuments and the bulbous chalaza, are associated with differences in ovule curvature. In contrast, the radialized, conical ovule primordia and nucelli exhibit similar shapes despite differences in internal cellular topology and tissue growth patterns. Our results support the notion that the structural organization of a tissue is associated with its susceptibility to shape changes during evolutionary shifts in 3D cellular architecture.

Cost awareness among intensivists in their daily clinical practice: a prospective multicentre study.

BACKGROUND: Better cost-awareness is a prerogative in achieving the best benefit/risk/cost ratio in the care. We aimed to assess the cost-awareness of intensivists in their daily clinical practice and to identify factors associated with accurate estimate of cost (50-150% of the real cost). METHODS: We performed a prospective observational study in seven French ICUs. We compared the estimate of intensivists of the daily costs of caring with the real costs on a given day. The estimates covered five categories (drugs, laboratory tests, imaging modalities, medical devices, and waste) whose sum represented the overall cost. RESULTS: Of the 234 estimates made by 65 intensivists, 70 (29.9%) were accurate. The median overall cost estimate (€330 [170; 620]) was significantly higher than the real cost (€178 [124; 239], p < 0.001). This overestimation was found in four categories, in particular for waste (€40 [15; 100] vs. €1.1 [0.6; 2.3], p < 0.001). Only the laboratory tests were underestimated (€65 [30; 120] vs. €106 [79; 138], p < 0.001). Being aware of the financial impact of prescriptions was factor associated with accurate estimate (OR: 5.05, 95%CI:1.47-17.4, p = 0.01). However, feeling able to accurately perform estimation was factor negatively associated with accurate estimate (OR: 0.11, 95%CI: 0.02-0.71, p = 0.02). CONCLUSION: French intensivists have a poor awareness of costs in their daily clinical practice. Raising awareness of the financial impact of prescriptions, and of the cost of laboratory tests and waste are the main areas for improvement that could help achieve the objective of the best care at the best cost.

Isolation and comparison of neural stem cells from the adult rat brain and spinal cord canonical neurogenic niches.

Here, we present a unified protocol for the extraction, culture, and basic characterization of rat neural stem cells (NSCs) from all three canonical neurogenic niches in the brain and spinal cord. We describe tissue dissection and dissociation, cell culture, followed by EdU labeling and characterization of NSCs. By yielding considerable numbers of viable cells per animal, this protocol enables the establishment of substantial, long-term cell banks, thus offering cost and labor efficiency while significantly reducing the numbers of animals used.

Validation of Functional Connectivity of Engineered Neuromuscular Junction With Recombinant Monosynaptic Pseudotyped ΔG-Rabies Virus Tracing.

Current preclinical models of neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS), can significantly benefit from in vitro neuroengineering approaches that enable the selective study and manipulation of neurons, networks, and functional units of interest. Custom-designed compartmentalized microfluidic culture systems enable the co-culture of different relevant cell types in interconnected but fluidically isolated microenvironments. Such systems can thus be applied for ALS disease modeling, as they enable the recapitulation and study of neuromuscular junctions (NMJ) through co-culturing of motor neurons and muscle cells in separate, but interconnected compartments. These in vitro systems are particularly relevant for investigations of mechanistic aspects of the ALS pathological cascade in engineered NMJ, as progressive loss of NMJ functionality may constitute one of the hallmarks of disease related pathology at early onset, in line with the dying back hypothesis. In such models, ability to test whether motor neuron degeneration in ALS starts at the nerve terminal or at the NMJ and retrogradely progresses to the motor neuron cell body largely relies on robust methods for verification of engineered NMJ functionality. In this study, we demonstrate the functionality of engineered NMJs within a microfluidic chip with a differentially perturbable microenvironment using a designer pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing.

[Evidence-based primary prevention and health promotion: methods and procedures in 5 research consortia]. / Evidenzbasierung in Primärprävention und Gesundheitsförderung: Methoden und Vorgehensweisen in 5 Forschungsverbünden.

Between 2014 and 2022, the 5 German research networks AEQUIPA, CAPITAL4HEALTH, HLCA, PartKommPlus, and SMARTACT are investigating topics of primary prevention and health promotion with the aim of further deepening the evidence base in these areas. The work of the 5 research networks for primary prevention and health promotion is presented, analysed, and discussed from an internal perspective. A model of evidence-based public health serves as a structuring framework.The 5 research networks use a variety of access routes for the generation of evidence with regard to the participation of nonacademic, civil society actors and users. There is a wide range of study designs - from randomised controlled trials and systematic reviews to diverse qualitative designs. The use of models and theories supports the evidence base. Beyond evidence generation, all research networks focus on at least exemplary implementation of new evidence.Due to the diversity of methods, a diversified evidence-based approach can be realised, taking into account network-specific aspects. Structural circumstances limit the further systematic strengthening of the evidence base. In particular, the involvement of nonacademic, civil society actors for the work with hard-to-reach target groups often cannot be financed or is considered too time consuming under the given circumstances. The COVID-19 pandemic highlights the importance of a flexible spectrum of methods, employing both digital and analogue methods in a meaningful way.

Transcultural adaptation and French validation of the Pain Sensitivity Questionnaire.

PURPOSE: To validate a French translation of the Pain Sensitivity Questionnaire (PSQ), which is a valuable tool to predict an individual's natural disposition to feel pain that could be used after surgery. METHODS: We studied content validity, internal consistency, convergent validity (anxiety, depression and catastrophism) and test-retest reliability of the French version of the PSQ (PSQ-F) in 146 patients either before scheduled surgery or during pregnancy; then, convergent and concurrent validity in 85 healthy volunteers submitted to nociceptive tests. RESULTS: Internal consistency of the PSQ-F was found to be excellent, with Cronbach's α at 0.866, 0.886, and 0.927, respectively for its "minor", "moderate" and "total" scores. Test-retest reliability was significant, with intraclass correlation coefficients at 0.629, 0.629, and 0.635, respectively for the above- mentioned scores. These three scores correlated with anxiety, depression and catastrophizing scores in patients, but not in healthy volunteers, possibly because of low and few variant psychometric scores in this group. They were inversely correlated to the temperature needed to evoke heat pain rated 6 out of 10, but not to the mechanical pain threshold (electronic von Frey), nor to the heat pain threshold. Finally, they directly correlated to the pain induced by the cold pressor test (minor and total scores only). DISCUSSION: This validated version can now be used by French-speaking researchers and physicians. TRIAL REGISTRATION: www.ClinicalTrials.gov (NCT03113903); 14 April, 2017.

RéSUMé: OBJECTIF: Valider une version en langue française du Pain Sensitivity Questionnaire (PSQ), qui permet d'identifier la sensibilité naturelle d'un individu à la douleur, ce qui pourrait être applicable après une chirurgie. MéTHODE: Nous avons étudié la validité de structure interne, la validité convergente (anxiété, dépression et catastrophisme) et la reproductibilité par test-retest de la version française du PSQ (PSQ-F) chez 146 patients en situation préopératoire ou en cours de grossesse, puis la validité convergente et de structure contre critère externe chez 85 sujets volontaires sains soumis à des tests nociceptifs. RéSULTATS: La consistance interne du PSQ-F était excellente avec des α de Cronbach égaux à 0,866, 0,886 et 0,927, respectivement pour ses scores « mineur ¼, « modéré ¼ et « total ¼. La reproductibilité était satisfaisante, avec des coefficients de corrélation intra-classe, respectivement à 0,629, 0,629 et 0,635. Ces trois scores étaient corrélés à l'anxiété, la dépression et le catastrophisme, mais pas chez les volontaires sains qui avaient des scores psychométriques bas et peu variables. Ils étaient anti-corrélés au seuil de nociception thermique chaud en épreuve supra-liminale, mais pas au seuil de nociception mécanique ponctuelle, ni au seuil de nociception thermique chaud en épreuve liminale. Enfin, les scores « mineur ¼ et « total ¼ étaient corrélés à la douleur moyenne ressentie à l'immersion du pied en eau froide. CONCLUSION: Cette version validée peut être utilisée par les chercheurs et cliniciens francophones. ENREGISTREMENT DE L'éTUDE: www.ClinicalTrials.gov (NCT03113903); le 14 avril 2017.

Structuring a multi-nodal neural network in vitro within a novel design microfluidic chip.

Neural network formation is a complex process involving axon outgrowth and guidance. Axon guidance is facilitated by structural and molecular cues from the surrounding microenvironment. Micro-fabrication techniques can be employed to produce microfluidic chips with a highly controlled microenvironment for neural cells enabling longitudinal studies of complex processes associated with network formation. In this work, we demonstrate a novel open microfluidic chip design that encompasses a freely variable number of nodes interconnected by axon-permissible tunnels, enabling structuring of multi-nodal neural networks in vitro. The chip employs a partially open design to allow high level of control and reproducibility of cell seeding, while reducing shear stress on the cells. We show that by culturing dorsal root ganglion cells (DRGs) in our microfluidic chip, we were able to structure a neural network in vitro. These neurons were compartmentalized within six nodes interconnected through axon growth tunnels. Furthermore, we demonstrate the additional benefit of open top design by establishing a 3D neural culture in matrigel and a neuronal aggregate 3D culture within the chips. In conclusion, our results demonstrate a novel microfluidic chip design applicable to structuring complex neural networks in vitro, thus providing a versatile, highly relevant platform for the study of neural network dynamics applicable to developmental and regenerative neuroscience.

[Subjective Needs of Support in Families with a Mentally Ill Parent ­ A Literature Review]. / Subjektive Hilfebedarfe in Familien mit psychisch erkranktem Elternteil - Eine Literaturübersicht.

Mentally ill parents are often sceptical about professional help for their children although these children face an increased risk to develop a mental disease themselves. To get a better understanding of needs and help-seeking behaviour in those families a systematic literature review was conducted. Four databases (FIS, PsycINFO, PSYNDEX, PubPsych) were scanned for international and national research literature. Out of 18,057 articles 56 were included which report quantitative or qualitative studies taking the children's and parents' perspectives into account. A thematic synthesis was done to categorize the needs. Results concerning the help-seeking behaviour and the influence of demographic variables were extracted and summarized. Our results were limited by the aspect that no evaluation of study quality had been made and influences on the categorizing process by the authors' subjective perceptions are likely. There were a lot of hints regarding the needs of the families, but little report was found about help-seeking behaviour and demographic variables. The "health literacy" concept was discussed as a basis for further research in this area.

Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies.

Despite their favorable pharmacokinetic properties, single-chain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 x 10(-9) to 1.5 x 10(-11) m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 x 10(-10) to <1.8 x 10(-12) m. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86 degrees C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.

CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation.

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of > or =95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from approximately 20 nM to 3.7 microM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida.

A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (-)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l-limonene in the 7 position to produce enantiopure (-)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.