Psilocybin Efficacy and Mechanisms of Action in Major Depressive Disorder: a Review.

PURPOSE OF THE REVIEW: We aim to provide an overview of the current state of knowledge about the efficacy of psilocybin in the treatment of depression, as well as its mechanisms of action. RECENT FINDINGS: Psilocybin has a large, rapid, and persistent clinical effect in the treatment of resistant or end-of-life depression. Tolerance is good, with mild side effects limited to a few hours after dosing. The studies conducted to date have had small sample sizes. One clinical trial has been conducted against a reference treatment (escitalopram) without showing a significant superiority of psilocybin in the main outcome. The neurobiological mechanisms, mostly unknown, differ from those of SSRI antidepressants. Psilocybin represents a promising alternative in the treatment of depression. Further research with larger sample sizes, particularly against reference treatments, is needed to better understand the neurobiological factors of its effects and to investigate its potential for use in everyday practice.

Loss of syndecan-1 induces a pro-inflammatory phenotype in endothelial cells with a dysregulated response to atheroprotective flow.

Fluid shear stresses are potent regulators of vascular homeostasis and powerful determinants of vascular disease progression. The glycocalyx is a layer of glycoaminoglycans, proteoglycans, and glycoproteins that lines the luminal surface of arteries. The glycocalyx interacts directly with hemodynamic forces from blood flow and, consequently, is a prime candidate for the mechanosensing of fluidic shear stresses. Here, we investigated the role of the glycocalyx component syndecan-1 (sdc-1) in controlling the shear stress-induced signaling and flow-mediated phenotypic modulation in endothelial cells. We found that knock-out of sdc-1 abolished several key early signaling events of endothelial cells in response to shear stress including the phosphorylation of Akt, the formation of a spatial gradient in paxillin phosphorylation, and the activation of RhoA. After exposure to atheroprotective flow, we found that sdc-1 knock-out endothelial cells had a phenotypic shift to an inflammatory/pro-atherosclerotic phenotype in contrast to the atheroprotective phenotype of wild type cells. Consistent with these findings, we found increased leukocyte adhesion to sdc-1 knock-out endothelial cells in vitro that was reduced by re-expression of sdc-1. In vivo, we found increased leukocyte recruitment and vascular permeability/inflammation in sdc-1 knock-out mice. Taken together, our studies support a key role for sdc-1 in endothelial mechanosensing and regulation of endothelial phenotype.

Evaluating and mitigating clinical samples matrix effects on TX-TL cell-free performance.

Cell-free biosensors are promising tools for medical diagnostics, yet their performance can be affected by matrix effects arising from the sample itself or from external components. Here we systematically evaluate the performance and robustness of cell-free systems in serum, plasma, urine, and saliva using two reporter systems, sfGFP and luciferase. In all cases, clinical samples have a strong inhibitory effect. Of the different inhibitors, only RNase inhibitor mitigated matrix effects. However, we found that the recovery potential of RNase inhibitor was partially muted by interference from glycerol contained in the commercial buffer. We solved this issue by designing a strain producing an RNase inhibitor protein requiring no additional step in extract preparation. Furthermore, our new extract yielded higher reporter levels than previous conditions and tempered interpatient variability associated with matrix effects. This systematic evaluation and improvements of cell-free system robustness unified across many types of clinical samples is a significant step towards developing cell-free diagnostics for a wide range of conditions.

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts.

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

PeroxiHUB: A Modular Cell-Free Biosensing Platform Using H2O2 as Signal Integrator.

Cell-free systems have great potential for delivering robust, inexpensive, and field-deployable biosensors. Many cell-free biosensors rely on transcription factors responding to small molecules, but their discovery and implementation still remain challenging. Here we report the engineering of PeroxiHUB, an optimized H2O2-centered sensing platform supporting cell-free detection of different metabolites. H2O2 is a central metabolite and a byproduct of numerous enzymatic reactions. PeroxiHUB uses enzymatic transducers to convert metabolites of interest into H2O2, enabling rapid reprogramming of sensor specificity using alternative transducers. We first screen several transcription factors and optimize OxyR for the transcriptional response to H2O2 in a cell-free system, highlighting the need for preincubation steps to obtain suitable signal-to-noise ratios. We then demonstrate modular detection of metabolites of clinical interest─lactate, sarcosine, and choline─using different transducers mined via a custom retrosynthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the preincubation step is crucial for optimal sensor operation. We then show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Finally, we demonstrate that a peroxiHUB lactate biosensor can detect endogenous levels of this metabolite in clinical samples. Given the wide range of enzymatic reactions producing H2O2, the PeroxiHUB platform will support cell-free detection of a large number of metabolites in a modular and scalable fashion.

Molecular tension in syndecan-1 is regulated by extracellular mechanical cues and fluidic shear stress.

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 has higher mechanical tension in regions of cell adhesion, on and in response to nanotopographical cues. In addition, we found that substrate stiffness also regulated the mechanical tension of syndecan-1 and altered its binding to actin, myosin iiB and signaling intermediates including Src, PKA and FAK. Application of fluidic shear stress created a gradient in tension in syndecan-1 and led to enhanced association with actin, Src, myosin IIb and other cytoskeleton related molecules. Overall, our studies support that syndecan-1 is responsive to the mechanical environment of the cells and alters its association with actin and signaling intermediates in response to mechanical stimuli.

Programmable receptors enable bacterial biosensors to detect pathological biomarkers in clinical samples.

Bacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

Metabolic perceptrons for neural computing in biological systems.

Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene expression regulation for information processing using digital logic. Here, we present a different approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input perceptrons for desired binary classification of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.

Plug-and-play metabolic transducers expand the chemical detection space of cell-free biosensors.

Cell-free transcription-translation systems have great potential for biosensing, yet the range of detectable chemicals is limited. Here we provide a workflow to expand the range of molecules detectable by cell-free biosensors through combining synthetic metabolic cascades with transcription factor-based networks. These hybrid cell-free biosensors have a fast response time, strong signal response, and a high dynamic range. In addition, they are capable of functioning in a variety of complex media, including commercial beverages and human urine, in which they can be used to detect clinically relevant concentrations of small molecules. This work provides a foundation to engineer modular cell-free biosensors tailored for many applications.

Microbially derived biosensors for diagnosis, monitoring and epidemiology.

Living cells have evolved to detect and process various signals and can self-replicate, presenting an attractive platform for engineering scalable and affordable biosensing devices. Microbes are perfect candidates: they are inexpensive and easy to manipulate and store. Recent advances in synthetic biology promise to streamline the engineering of microbial biosensors with unprecedented capabilities. Here we review the applications of microbially-derived biosensors with a focus on environmental monitoring and healthcare applications. We also identify critical challenges that need to be addressed in order to translate the potential of synthetic microbial biosensors into large-scale, real-world applications.

A multichannel dampened flow system for studies on shear stress-mediated mechanotransduction.

Shear stresses are powerful regulators of cellular function and potent mediators of the development of vascular disease. We have designed and optimized a system allowing the application of flow to cultured cells in a multichannel format. By using a multichannel peristaltic pump, flow can be driven continuously in the system for long-term studies in multiple isolated flow loops. A key component of the system is a dual-chamber pulse dampener that removes the pulsatility of the flow without the need for having an open system or elevated reservoir. We optimized the design parameters of the pulse dampening chambers for the maximum reduction in flow pulsation while minimizing the fluid needed for each isolated flow channel. Human umbilical vein endothelial cells (HUVECs) were exposed to steady and pulsatile shear stress using the system. We found that cells under steady flow had a marked increased production of eNOS and formation of actin stress fibers in comparison to those under pulsatile flow conditions. Overall, the results confirm the utility of the device as a practical means to apply shear stress to cultured cells in the multichannel format and provide steady, long term flow to microfluidic devices.