Prognostic value of multicenter flow cytometry harmonized assessment of minimal residual disease in acute myeloblastic leukemia.

The assessment of minimal residual disease (MRD) in acute myeloblastic leukemia is of growing interest as a prognostic marker of patients' outcome. Multiparameter flow cytometry (MFC), tracking leukemia-associated immunophenotypic patterns, has been shown in several studies to be a useful tool to investigate MRD. Here, we report a multicenter prospective study which allowed to define a harmonized analysis strategy, as well as the efficacy of MFC MRD to predict outcome. This study included 276 patients, in 10 different MFC centers, of whom 268 had at least 1 MRD check point. The combination of a CD45, CD34, and CD33 backbone, with the addition of CD117, CD13, CD7, and CD15 in 2 five-color tubes allowed to define each patient's multiparameter immunophenotypic characteristics at diagnosis, according to a Boolean combination of gates. The same individual diagnosis gating strategy was then applied at each MRD time point for each patient. MRD levels were stratified according to log by log thresholds, from 5 × 10-2 (the classical morphological threshold to define remission) down to <5 × 10-5 . MRD was found to be constantly negative (<5 × 10-5 ) for 148 patients. Survival analyses significantly associated MRD negativity with a good prognosis and any positive value with poorer outcome. All P values were <0.0001 both for disease-free and overall survival at the earliest time point (post-induction, MRD1) as well as when considering all time points together. Finally, MRD levels were independent of cytogenetics and allowed in fact to further stratify all cytogenetics risk groups. In summary, this multicenter study demonstrates that a simple combination of immunophenotypic markers successfully allows for the detection of MRD in acute myeloblastic leukemia patients, with a strong correlation to outcome.

Construction of a fuzzy and Boolean logic gates based on DNA.

Logic gates are devices that can perform logical operations by transforming a set of inputs into a predictable single detectable output. The hybridization properties, structure, and function of nucleic acids can be used to make DNA-based logic gates. These devices are important modules in molecular computing and biosensing. The ideal logic gate system should provide a wide selection of logical operations, and be integrable in multiple copies into more complex structures. Here we show the successful construction of a small DNA-based logic gate complex that produces fluorescent outputs corresponding to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics.

RNAi-based Boolean gates in the yeast Saccharomyces cerevisiae.

Boolean gates, the fundamental components of digital circuits, have been widely investigated in synthetic biology because they permit the fabrication of biosensors and facilitate biocomputing. This study was conducted to design and construct Boolean gates in the yeast Saccharomyces cerevisiae, the main component of which was the RNA interference pathway (RNAi) that is naturally absent from the budding yeast cells. We tested different expression cassettes for the siRNA precursor (a giant hairpin sequence, a DNA fragment-flanked by one or two introns-between convergent promoters or transcribed separately in the sense and antisense directions) and placed different components under the control of the circuit inputs (i.e., the siRNA precursor or proteins such as the Dicer and the Argonaute). We found that RNAi-based logic gates are highly sensitive to promoter leakage and, for this reason, challenging to implement in vivo. Convergent-promoter architecture turned out to be the most reliable solution, even though the overall best performance was achieved with the most difficult design based on the siRNA precursor as a giant hairpin.

Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells.

Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes. In this study, we combined, in the yeast S. cerevisiae, genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac. S. cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions. We engineered three different (genetic)-enzymatic basic Boolean gates (YES, NOT, and N-IMPLY). In the YES and N-IMPLY gates, human CYP2C9 was expressed under the galactose-inducible GAL1 promoter. The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2- heme prosthetic group in the active site of the human enzyme. Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts. This new approach will help study precise metabolic attributes of human P450s.

Hybrid Boolean gates show that Cas12c controls transcription activation effectively in the yeast S. cerevisiae.

Among CRISPR-Cas systems, type V CRISPR-Cas12c is of significant interest because Cas12c recognizes a very simple PAM (TN) and has the ability to silence gene expression without cleaving the DNA. We studied how new transcription factors for the yeast Saccharomyces cerevisiae can be built on Cas12c. We found that, upon fusion to a strong activation domain, Cas12c is an efficient activator. Its functionality was proved as a component of hybrid Boolean gates, i.e., logic circuits that mix transcriptional and translational control (the latter reached via tetracycline-responsive riboswitches). Moreover, Cas12c activity can be strongly inhibited by the anti-CRISPR AcrVA1 protein. Thus, Cas12c has the potential to be a new tool to control the activation of gene expression within yeast synthetic gene circuits.

Design of Gene Boolean Gates and Circuits with Convergent Promoters.

Gene digital circuits are the subject of many research works due to their various potential applications, from hazard detection to medical diagnostic. Moreover, a remarkable number of techniques, developed in electronics, can be used for the construction of biological digital systems. In our previous works, we showed how to automatize the design and modeling of gene digital circuits whose gates were based on transcription and translation regulation. In this chapter, we illustrate how Boolean gates could be implemented by following a particular architecture, the convergent promoter one, rather diffuse in nature but seldom adopted in Synthetic Biology. Beside gate design, we also explain how to extend our previous modeling approach, based on composable parts and pools of molecules, to quantitatively describe and simulate this particular kind of digital biological devices.

Logic Circuits Based on 2A Peptide Sequences in the Yeast Saccharomyces cerevisiae.

Gene digital circuits are the subject of many studies in Synthetic Biology due to their various applications from pollutant detection to medical diagnostics and biocomputing. Complex logic functions are calculated via small genetic components that mimic Boolean gates, i.e., they implement basic logic operations. Gates interact by exchanging proteins or noncoding RNAs. To carry out logic operations in the yeast Saccharomyces cerevisiae, we chose three bacterial repressors commonly used for proofs of concept in Synthetic Biology, namely, TetR, LexA, and LacI. We coexpressed them via synthetic polycistronic cassettes based on 2A peptide sequences. Our initial results highlighted the successful application of four 2A peptides─from Equine rhinitis B virus-1 (ERBV-1 2A), Operophtera brumata cypovirus 18 (OpbuCPV18 2A), Ljungan virus (LV2A), and Thosea asigna virus (T2A)─to the construction of single and two-input Boolean gates. In order to improve protein coexpression, we modified the original 2A peptides with the addition of the glycine-serine-glycine (GSG) prefix or by using two different 2As sequences in tandem. Remarkably, we finally realized a well-working tri-cistronic vector that carried LexA-HBD(hER), TetR, and LacI separated, in the order, by GSG-T2A and ERBV-1 2A. This plasmid led to the implementation of three-input circuits containing AND and OR gates. Taken together, polycistronic constructs simplify the cloning and coexpression of multiple proteins with a dramatic reduction in the complexity of gene digital circuits.

NOT Gates Based on Protein Degradation as a Case Study for a New Modular Modeling via SBML Level 3-Comp Package.

In 2008, we were among the first to propose a method for the visual design and modular modeling of synthetic gene circuits, mimicking the way electronic circuits are realized in silico. Basic components were DNA sequences that could be composed, first, into transcription units (TUs) and, then, circuits by exchanging fluxes of molecules, such as PoPS (polymerase per second) and RiPS (ribosomes per seconds) as suggested by Drew Endy. However, it became clear soon that such fluxes were not measurable, which highlighted the limit of using some concepts from electronics to represent biological systems. SBML Level 3 with the comp package permitted us to revise circuit modularity, especially for the modeling of eukaryotic networks. By using the libSBML Python API, TUs-rather than single parts-are encoded in SBML Level 3 files that contain species, reactions, and ports, i.e., the interfaces that permit to wire TUs into circuits. A circuit model consists of a collection of SBML Level 3 files associated with the different TUs plus a "main" file that delineates the circuit structure. Within this framework, there is no more need for any flux of molecules. Here, we present the SBML Level 3-based models and the wet-lab implementations of Boolean NOT gates that make use, in the yeast Saccharomyces cerevisiae, of the bacterial ClpX-ClpP system for protein degradation. This work is the starting point towards a new piece of software for the modular design of eukaryotic gene circuits and shows an alternative way to build genetic Boolean gates.

On electrical gates on fungal colony.

Mycelium networks are promising substrates for designing unconventional computing devices providing rich topologies and geometries where signals propagate and interact. Fulfilling our long-term objectives of prototyping electrical analog computers from living mycelium networks, including networks hybridised with nanoparticles, we explore the possibility of implementing Boolean logical gates based on electrical properties of fungal colonies. We converted a 3D image-data stack of Aspergillus niger fungal colony to an Euclidean graph and modelled the colony as resistive and capacitive (RC) networks, where electrical parameters of edges were functions of the edges' lengths. We found that and, or and and-not gates are implementable in RC networks derived from the geometrical structure of the real fungal colony.

On Boolean gates in fungal colony.

A fungal colony maintains its integrity via flow of cytoplasm along mycelium network. This flow, together with possible coordination of mycelium tips propagation, is controlled by calcium waves and associated waves of electrical potential changes. We propose that these excitation waves can be employed to implement a computation in the mycelium networks. We use FitzHugh-Nagumo model to imitate propagation of excitation in a single colony of Aspergillus niger. Boolean values are encoded by spikes of extracellular potential. We represent binary inputs by electrical impulses on a pair of selected electrodes and we record responses of the colony from sixteen electrodes. We derive sets of two-inputs-on-output logical gates implementable the fungal colony and analyse distributions of the gates.

Plant leaf computing.

Action potentials are multi-functional signals in plants. While most plant cells are conductive, and electrically coupled with each other, the action potentials are channelled faster along the plants' vascular network. Conductivity of the networks is geometrically constrained, thus allow for selective propagation and interaction between the impulses. Using FitzHugh-Nagumo model we show that it is possible to realise a functionally complete set of Boolean functions by selecting locations of stimulating and recording electrodes. The results pave theoretical grounds for further experimental studies of plant-based computing.

Parts & pools: a framework for modular design of synthetic gene circuits.

Published in 2008, Parts & Pools represents one of the first attempts to conceptualize the modular design of bacterial synthetic gene circuits with Standard Biological Parts (DNA segments) and Pools of molecules referred to as common signal carriers (e.g., RNA polymerases and ribosomes). The original framework for modeling bacterial components and designing prokaryotic circuits evolved over the last years and brought, first, to the development of an algorithm for the automatic design of Boolean gene circuits. This is a remarkable achievement since gene digital circuits have a broad range of applications that goes from biosensors for health and environment care to computational devices. More recently, Parts & Pools was enabled to give a proper formal description of eukaryotic biological circuit components. This was possible by employing a rule-based modeling approach, a technique that permits a faithful calculation of all the species and reactions involved in complex systems such as eukaryotic cells and compartments. In this way, Parts & Pools is currently suitable for the visual and modular design of synthetic gene circuits in yeast and mammalian cells too.