Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell.

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template - encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage - expressed in the 'protein synthesis using recombinant elements' (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Tethered Ribosomes: Toward the Synthesis of Nonproteinogenic Polymers in Bacteria.

The ribosome is the core element of the translational apparatus and displays unrivaled fidelity and efficiency in the synthesis of long polymers with defined sequences and diverse compositions. Repurposing ribosomes for the assembly of nonproteinogenic (bio)polymers is an enticing prospect with implications for fundamental science, bioengineering and synthetic biology alike. Here, we review tethered ribosomes, which feature inseparable large and small subunits that can be evolved for novel function without interfering with native translation. Following a tutorial summary of ribosome structure, function, and biogenesis, we introduce design and optimization strategies for the creation of orthogonal and tethered ribosomes. We also highlight studies, in which (rational) engineering efforts of these designer ribosomes enabled the evolution of new functions. Lastly, we discuss future prospects and challenges that remain for the ribosomal synthesis of tailor-made (bio)polymers.

Preoperative C-reactive protein is related with renal cell tumor dimension? Preliminary results.

INTRODUCTION: Renal tumors do not benefit from an unanimously accepted tumor marker. We tried to evaluate the advantages of preoperative C-reactive protein (CRP) values and monitor the dynamic of CRP values from the perspective of the evolution of patients diagnosed with Grawitz tumors. PATIENTS, MATERIALS AND METHODS: We researched the medical records of patients with renal parenchymal tumors admitted to the Urological Clinic in Iasi, Romania, between 01.01.2018 and 01.08.2022. Data were collected regarding age, environment, comorbidities, paraclinical data, tumor characteristics, and treatment performed. Ninety-six patients were included. The data on the inflammatory syndrome pre- and postoperatively were evaluated comparatively. All patients were diagnosed with clear cell renal cell carcinoma (RCC). RESULTS: We found that the renal tumor dimension correlates with an increased preoperative CRP level. For other variables, the correlations regarding age, sex, tumor, node, metastasis (TNM) stage, and size in relation to the increase or decrease of CRP had no statistical significance. CONCLUSIONS: The analysis of preoperative CRP and CRP dynamics could predict the tumor's aggressiveness and the treatment's effectiveness. A clear association between CRP levels and RCC pathogenesis is not yet defined, thus, further studies are necessary.

Correlations between stones composition, dietary and comorbidities context of the lithiasic patient.

The incidence of nephrolithiasis is in full expansion, its etiology being frequently associated with lifestyle changes. The objective of this retrospective study, carried out between April and December 2019, was to identify the correlations of the known lithogenic factors with the chemical structure of the calculi in the patients from the North-Eastern region of Romania. The results obtained after the data analysis of our LAMPA questionnaire (L - liquids, A - antecedents, M - medication, P - associated pathologies, A - aliments) made in evidence a statistically relevant relationship between the heredocollateral history of lithiasis and calcium oxalate dihydrate (COD) calculi, hypertension and calcium oxalate monohydrate (COM) stones, uric lithiasis and diabetes, COD, and obesity, between predominantly uric lithiasis and meat or meat-derived products consumption, between frequent potato consumption and COD stones and the frequently consume of dairy products and predominantly COM calculi. The authors concluded that the use of a complex questionnaire, like LAMPA, together with Fourier-transform infrared (FTIR) spectroscopic and morphological analysis are essential steps for developing an efficient metaphylaxis.