A Standardized Nomenclature Design for Systematic Referencing and Identification of Animal Cellular Material.

The documentation, preservation and rescue of biological diversity increasingly uses living biological samples. Persistent associations between species, biosamples, such as tissues and cell lines, and the accompanying data are indispensable for using, exchanging and benefiting from these valuable materials. Explicit authentication of such biosamples by assigning unique and robust identifiers is therefore required to allow for unambiguous referencing, avoid identification conflicts and maintain reproducibility in research. A predefined nomenclature based on uniform rules would facilitate this process. However, such a nomenclature is currently lacking for animal biological material. We here present a first, standardized, human-readable nomenclature design, which is sufficient to generate unique and stable identifying names for animal cellular material with a focus on wildlife species. A species-specific human- and machine-readable syntax is included in the proposed standard naming scheme, allowing for the traceability of donated material and cultured cells, as well as data FAIRification. Only when it is consistently applied in the public domain, as publications and inter-institutional samples and data are exchanged, distributed and stored centrally, can the risks of misidentification and loss of traceability be mitigated. This innovative globally applicable identification system provides a standard for a sustainable structure for the long-term storage of animal bio-samples in cryobanks and hence facilitates current as well as future species conservation and biomedical research.

Comprehensive quantitative modeling of translation efficiency in a genome-reduced bacterium.

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

Relationships between Clinical and Non-Clinical Variables concerning Traumatic Dental Injuries in Deciduous Teeth Attended in a Children's Hospital.

This study aimed to describe Traumatic Dental Injuries (TDI) in a child population, with a discussion focused on the impact of non-clinical variables on TDI. A cross-sectional, descriptive, and relational study about TDI in deciduous dentition in a children's hospital was performed. A total of 166 patients were included, of which 51.8% were male and 48.2% were female. Subluxation was the most observed injury (37.5%), and high-severity lesions predominated (60.2%). Regarding non-clinical variables, 89.2% of the patients attended urgent care centers within 24 h, and 43.4% within the first 3 h. Pointed objects were the leading cause of TDI (47%). Most TDIs were concentrated between the ages of 2 and 4 (53.5%). Concerning the place of TDI occurrence, the school (41.6%) was associated with faster urgent dental care attendance, and the home (37.3%) was associated with TDI occurrence in children under 2 years of age. Previous TDI experience (24.1% of patients) did not generate differences in the time interval between the TDI and arrival at the hospital, compared with children without a TDI history. While the behavior of clinical variables agrees with the literature reviewed, several non-clinical variables show wide differences. There is a need to identify the non-clinical variables that can significantly interact with phenomena specific to the study population (social, demographic, and cultural). The study of these variables can be useful in applying health policies. In the group studied, the non-clinical data reveals the need to educate parents or guardians on the importance of timely care in TDI, the long-term consequences of traumatism affecting deciduous dentition, and the implication of the maturation of the child's motor skills in TDI.

Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae.

Protein degradation is a crucial cellular process in all-living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP-dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA-seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half-life measurements also suggest a role for Lon-modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.

Mycoplasma pneumoniae Genome Editing Based on Oligo Recombineering and Cas9-Mediated Counterselection.

Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains.

Determination of the Gene Regulatory Network of a Genome-Reduced Bacterium Highlights Alternative Regulation Independent of Transcription Factors.

Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria.

Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae.

DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.

Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium.

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.

Impact of genome reduction on bacterial metabolism and its regulation.

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.