Common mitochondrial deletions in RNA-Seq: evaluation of bulk, single-cell, and spatial transcriptomic datasets.

Common mitochondrial DNA (mtDNA) deletions are large structural variants in the mitochondrial genome that accumulate in metabolically active tissues with age and have been investigated in various diseases. We applied the Splice-Break2 pipeline (designed for high-throughput quantification of mtDNA deletions) to human RNA-Seq datasets and describe the methodological considerations for evaluating common deletions in bulk, single-cell, and spatial transcriptomics datasets. A robust evaluation of 1570 samples from 14 RNA-Seq studies showed: (i) the abundance of some common deletions detected in PCR-amplified mtDNA correlates with levels observed in RNA-Seq data; (ii) RNA-Seq library preparation method has a strong effect on deletion detection; (iii) deletions had a significant, positive correlation with age in brain and muscle; (iv) deletions were enriched in cortical grey matter, specifically in layers 3 and 5; and (v) brain regions with dopaminergic neurons (i.e., substantia nigra, ventral tegmental area, and caudate nucleus) had remarkable enrichment of common mtDNA deletions.

Synthetic development: building mammalian multicellular structures with artificial genetic programs.

Synthetic biology efforts began in simple single-cell systems, which were relatively easy to manipulate genetically (Cameron et al., 2014). The field grew exponentially in the last two decades, and one of the latest frontiers are synthetic developmental programs for multicellular mammalian systems (Black et al., 2017; Wieland and Fussenegger, 2012) to genetically control features such as patterning or morphogenesis. These programs rely on engineered cell-cell communications, multicellular gene regulatory networks and effector genes. Here, we contextualize the first of these synthetic developmental programs, examine molecular and computational tools that can be used to generate next generation versions, and present the general logic that underpins these approaches. These advances are exciting as they represent a novel way to address both control and understanding in the field of developmental biology and tissue development (Elowitz and Lim, 2010; Velazquez et al., 2018; White et al., 2018; Morsut, 2017). This field is just at the beginning, and it promises to be of major interest in the upcoming years of biomedical research.

Quantitative Characterization of α-Synuclein Aggregation in Living Cells through Automated Microfluidics Feedback Control.

Aggregation of α-synuclein and formation of inclusions are hallmarks of Parkinson's disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed α-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either wild-type (WT) α-synuclein or the disease-associated A53T mutant from the galactose (Gal)-inducible promoter. A computer-controlled microfluidics device regulated α-synuclein in cells by means of closed-loop feedback control. We demonstrated that inclusion formation is strictly concentration dependent and that the aggregation threshold of the A53T mutant is about half of the WT α-synuclein (56%). We chemically modulated the proteasomal and autophagic pathways and demonstrated that autophagy is the main determinant of A53T α-synuclein inclusions' clearance. In addition to proposing a technology to overcome current limitations in dynamically regulating protein expression levels, our results contribute to the biology of PD and have relevance for therapeutic applications.

Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach.

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified "hybrid" versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

Regulation of Gene Expression and Signaling Pathway Activity in Mammalian Cells by Automated Microfluidics Feedback Control.

Gene networks and signaling pathways display complex topologies and, as a result, complex nonlinear behaviors. Accumulating evidence shows that both static (concentration) and dynamical (rate-of-change) features of transcription factors, ligands and environmental stimuli control downstream processes and ultimately cellular functions. Currently, however, methods to generate stimuli with the desired features to probe cell response are still lacking. Here, combining tools from Control Engineering and Synthetic Biology (cybergenetics), we propose a simple and cost-effective microfluidics-based platform to precisely regulate gene expression and signaling pathway activity in mammalian cells by means of real-time feedback control. We show that this platform allows (i) to automatically regulate gene expression from inducible promoters in different cell types, including mouse embryonic stem cells; (ii) to precisely regulate the activity of the mTOR signaling pathway in single cells; (iii) to build a biohybrid oscillator in single embryonic stem cells by interfacing biological parts with virtual in silico counterparts. Ultimately, this platform can be used to probe gene networks and signaling pathways to understand how they process static and dynamic features of specific stimuli, as well as for the rapid prototyping of synthetic circuits for biotechnology and biomedical purposes.

Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica.

An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0kDa and a peak of 54.0kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI-MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55°C, pH 8.5 and 2M NaCl, and high thermostability was showed at 55°C and 3M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n-hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena.