Biology subjects

Dupin, A.

Publications and source records attributed to Dupin, A..

5 recordsLinked to original sources

bioRxiv2024

Autonomous biogenesis of the entire protein translation machinery excluding ribosomes

Recreating the conditions for autonomous biogenesis of the protein translation machinery is fundamental to our understanding of living systems and is essential for the assembly of minimal cell models. All components of the translation machinery, including the ribosomes, translation factors and aminoacyl tRNA synthetases, are made of proteins and are therefore involved in their own synthesis, posing a unique challenge for self-biogenesis. Here, we created physicochemical conditions for autonomous biogenesis of all the translation machinery, excluding the ribosome. We surface-immobilized synthetic genes coding for all thirty components as densely packed DNA brushes forming a reaction center that localizes, concentrates and catalyzes their simultaneous synthesis. To demonstrate their activity, we first determined empirically the minimal critical concentration of each translation protein required to initiate its own self-biogenesis in bulk solution. We then assembled a minimal gene expression reaction with all translation proteins at their critical concentrations. Under these dilute conditions, reporter proteins were not synthesized unless the DNA brushes encoded all the translation proteins, thereby demonstrating their co-synthesis, functionality and engagement in their own synthesis. This scenario of a complex biochemical process that amplifies itself can be generalized and extended to impact our understanding toward the design of autonomous self-replicating biological system.

synthetic biology↗

bioRxiv2024

Cell-free immuno-profiling on a genetically programmed biochip

Emerging cell-free synthetic biology approaches provide biosafe, cheap, and versatile genetic tools to advance therapeutic research and development. Combined with micro-fabrication technology, we developed a platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies and human receptors in miniaturized compartments on a silicon chip. Photolithographic surface patterning of protein traps and on chip expression from high density gene brushes generated a continuous surface density gradient of fluorescently labeled antigens. Antibodies binding to the antigen gradient generate a full binding curve in each single compartment for affinity determination. We used the SARS-CoV-2 antigens as a model to profile the specificity and affinity of monoclonal antibodies to > 30 viral epitopes synthesized simultaneously on one chip in a genotype-phenotype linked compartments. We further profiled polyclonal antibodies in minute volumes of human sera, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. Cell-free co-synthesis of the human ACE2 receptor with the viral Receptor-Binding-Domain yielded relative binding affinities to different SARS-CoV-2 variants. This rapid, quantitative, and on-chip genetically programmed approach allows to study complex protein-protein interactions independent of protein purification steps for human immuno-profiling with a fast response time for combating emerging pathogens.

bioengineering↗

bioRxiv2024

Dissecting the contribution of vagal subcircuits in sepsis-induced brain dysfunctions.

Sepsis, a life-threatening syndrome caused by a dysregulated host response to infection, induces a range of acute effects on the brain, including sickness behaviour and sepsis-associated encephalopathy. In addition, sepsis can lead to durable changes in neuronal circuits, resulting in long-term impairments such as post-traumatic stress disorder (PTSD). These brain dysfunctions are not directly caused by brain infection but result from peripheral inflammatory signals relayed to the brain via neural and humoral pathways. The vagal complex in the brainstem, composed of the nucleus of the solitary tract (NTS) and the area postrema, plays a crucial role in sensing and relaying these signals. Notably, the activation of the vagal complex triggers neurovegetative, neuroendocrine, and behavioural responses to infection. Chronic electrical vagus nerve stimulation has been used clinically to treat various brain disorders and is being investigated for its potential to alleviate inflammation and immune diseases through the anti-inflammatory reflex. However, a deeper understanding of the involvement of the vagus nerve and downstream brain circuits in sepsis-induced brain activation and dysfunction is needed to optimize therapeutic strategies. To investigate the role of the vagal complex in sepsis-induced brain dysfunction, various techniques were employed to manipulate vagus nerve activity and downstream circuits in a rodent model of sepsis by caecal ligation and puncture. Subdiaphragmatic vagotomy and pharmacogenetic manipulation of NTS and nodose (i.e. vagus sensory neurons) were implemented, revealing that vagotomy effectively reduced acute brain activation, inflammatory responses, and sickness behaviour triggered by sepsis. Additionally, transient activation of NTS neurons had a significant impact on inflammatory responses, sickness behaviour, and long-term PTSD-like consequences. This study underscores the complex interplay among the vagus nerve, brain circuits, and systemic inflammation during sepsis, emphasizing the critical importance of understanding these interactions in the development of targeted therapeutic interventions.

neuroscience↗

bioRxiv2021

Synthetic cell-based materials extract positional information from morphogen gradients

Dynamic biomaterials composed of synthetic cellular structures have the potential to adapt and functionally differentiate guided by physical and chemical cues from their environment. Inspired by developing biological systems, which efficiently extract positional information from chemical morphogen gradients in the presence of environmental uncertainties, we here investigate the analogous question: how well can a synthetic cell determine its position within a synthetic multicellular structure? In order to calculate positional information in such systems, we created and analyzed a large number of replicas of synthetic cellular assemblies, which were composed of emulsion droplets connected via lipid bilayer membranes. The droplets contained cell-free two-node feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. We found that in our system, simple anterior-posterior differentiation is possible, but positional information is limited by gene expression noise, and is also critically affected by the temporal evolution of the morphogen gradient and the life-time of the cell-free expression system contained in the synthetic cells. Using a 3D printing approach, we demonstrate morphogen-based differentiation also in larger tissue-like assemblies.

synthetic biology↗

bioRxiv2019

A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independent of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably detected less than 10 nM fluorescein concentrations (with a lower limit of detection of {approx}6.8 nM), which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs in the nanomolar range using a Cas13a-based fluorescence assay. Cas13a is a RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device architecture could be a valuable educational tool that integrates hardware, software and biochemistry concepts.

bioengineering↗