Robust set-point regulation of gene expression using resource competition couplings in mammalian cells

Gene expression depends on the cellular con-text. One major contributor to gene expression variability is competition for limited transcriptional and translational re-sources, which may induce indirect couplings among otherwise independently-regulated genes. Here, we apply control theoretical concepts and tools to design an incoherent feedforward loop (iFFL) biomolecular controller operating in mammalian cells using translational-resource competition couplings. Harnessing a resource-aware mathematical model, we demonstrate analytically and computationally that our resource-aware design can achieve near-constant set-point regulation of gene expression whilst ensuring robustness to plasmid uptake variation. We also provide an analytical condition on the model parameters to guide the design of the resource-aware iFFL controller ensuring robustness and performance in set-point regulation. Our theoretical design based on translational-resource competition couplings represents a promising approach to build more sophisticated resource-aware control circuits operating at the host-cell level

Oxidative potential associated with urban aerosol deposited into the respiratory system and relevant elemental and ionic fraction contributions

Size-segregated aerosol measurements were carried out at an urban and at an industrial site. Soluble and insoluble fractions of elements and inorganic ions were determined. Oxidative potential (OP) was assessed on the soluble fraction of Particulate Matter (PM) by ascorbic acid (AA), dichlorofluorescein (DCFH) and dithiothreitol (DTT) assays. Size resolved elemental, ion and OP doses in the head (H), tracheobronchial (TB) and alveolar (Al) regions were estimated using the Multiple-Path Particle Dosimetry (MPPD) model. The total aerosol respiratory doses due to brake and soil resuspension emissions were higher at the urban than at the industrial site. On the contrary, the doses of anthropic combustion tracers were generally higher at the industrial site. In general, the insoluble fraction was more abundantly distributed in the coarse than in the fine mode and vice versa for the soluble fraction. Consequently, for the latter, the percent of the total respiratory dose deposited in TB and Al regions increased. Oxidative potential assay (OPAA) doses were distributed in the coarse region; therefore, their major contribution was in the H region. The contribution in the TB and Al regions increased for OPDTT and OPDCFH

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

ATLAS RPC Quality Assurance results at INFN Lecce

The main results of the quality assurance tests performed on the Resistive Plate Chamber used by the ATLAS experiment at LHC as muon trigger chambers are reported and discussed. Since July 2004, about 270 RPC units has been certified at INFN Lecce site and delivered to CERN, for being integrated in the final muon station of the ATLAS barrel region. We show the key RPC characteristics which qualify the performance of this detector technology as muon trigger chamber in the harsh LHC enviroments. These are dark current, chamber efficiency, noise rate, gas volume tomography, and gas leakage.Comment: Comments: 6 pages, 1 table, 9 figures Proceedings of XXV Physics in Collision-Prague, Czech Republic, 6-9 July 200

Direct cross section measurement for the 18O(p,γ)19F reaction at astrophysical energies at LUNA

18 O ( p, γ ) 19 F plays an important role in the AGB star scenarios. The low energy cross section could be influenced by a hypothetical low energy resonance at 95 keV and by the tails of the higher energy broad states. The 95 keV resonance lies in the energy window corresponding to the relevant stellar temperature range of 40-50 MK.Measurements of the direct cross section were performed at the Laboratory for Underground Nuclear Astrophysics (LUNA), including the unobserved low energy resonance, the higher energy resonances and the non-resonant component, taking advantage of the extremely low environmental background. Here we report on the experimental setup and the status of the analysis

Resource-aware whole-cell model of division of labour in a two-strain consortium for complex substrate degradation

Background Low-cost sustainable feedstocks are essential for commercially viable biotechnologies. These feedstocks, often derived from plant or food waste, contain a multitude of different complex biomolecules which require multiple enzymes to hydrolyse and metabolise. Current standard biotechnology uses monocultures in which a single host expresses all the proteins required for the consolidated bioprocess. However, these hosts have limited capacity for expressing proteins before growth is impacted. This limitation may be overcome by utilising division of labour (DOL) in a consortium, where each member expresses a single protein of a longer degradation pathway. Results Here, we model a two-strain consortium, with one strain expressing an endohydrolase and a second strain expressing an exohydrolase, for cooperative degradation of a complex substrate. Our results suggest that there is a balance between increasing expression to enhance degradation versus the burden that higher expression causes. Once a threshold of burden is reached, the consortium will consistently perform better than an equivalent single-cell monoculture. Conclusions We demonstrate that resource-aware whole-cell models can be used to predict the benefits and limitations of using consortia systems to overcome burden. Our model predicts the region of expression where DOL would be beneficial for growth on starch, which will assist in making informed design choices for this, and other, complex-substrate degradation pathways

Cervical Laminoplasty with Polypropylene Mesh: Our Experience

Cervical laminoplasty was made to reduce the complications of the simple laminectomy. The main indication is degenerative cervical myelopathy with posterior compression to two or more levels. To simplify this procedure with more effectiveness, we conceived a cervical laminoplastica with polypropylene mesh, generally used in abdominoplasty, to provide more resistance during mechanisms of flexion-extension. MATERIALS AND METHODS: We analyzed 35 patients (27 males, 8 females) with degenerative cervical myelopathy who underwent surgical tretament at our institution between 2010 and 2015. RESULTS AND DISCUSSION: The main duration of surgery was about 90 minutes without intra or post-operative complications. Clinical and radiological follow-up was done at 1, 3 and 6 months, with cervical dynamic Rx and cervical RM at 1one year. There were no complications and 70% of the patients experienced a stable clinical improvement, while in 30% of patients stabilization of clinical deficits. The duration of the superior clinical to one year and the age gt of 70 years the more meaningful negative prognostic indicators have appeared. CONCLUSIONS: A clinical history more than one year and the age elder than 70 years appear the main negative prognostic factors. The advantage of the laminoplasty is a smaller anatomical damage of the posterior elements without cervical instability, and use of polypropylene mesh provides more resistance during flexion-extension

MIRELLA: a mathematical model explains the effect of microRNA-mediated synthetic genes regulation on intracellular resource allocation

Competition for intracellular resources, also known as gene expression burden, induces coupling between independently co-expressed genes, a detrimental effect on predictability and reliability of gene circuits in mammalian cells. We recently showed that microRNA (miRNA)-mediated target downregulation correlates with the upregulation of a co-expressed gene, and by exploiting miRNAs-based incoherent-feed-forward loops (iFFLs) we stabilise a gene of interest against burden. Considering these findings, we speculate that miRNA-mediated gene downregulation causes cellular resource redistribution. Despite the extensive use of miRNA in synthetic circuits regulation, this indirect effect was never reported before. Here we developed a synthetic genetic system that embeds miRNA regulation, and a mathematical model, MIRELLA, to unravel the miRNA (MI) RolE on intracellular resource aLLocAtion. We report that the link between miRNA-gene downregulation and independent genes upregulation is a result of the concerted action of ribosome redistribution and ‘queueing-effect’ on the RNA degradation pathway. Taken together, our results provide for the first time insights into the hidden regulatory interaction of miRNA-based synthetic networks, potentially relevant also in endogenous gene regulation. Our observations allow to define rules for complexity- and context-aware design of genetic circuits, in which transgenes co-expression can be modulated by tuning resource availability via number and location of miRNA target sites