A simple and robust serum-free media for the proliferation of muscle cells.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Protein expression and purification of bioactive growth factors for use in cell culture and cellular agriculture.

Mitogenic growth factors are major cost drivers in serum-free media, contributing up to 95% of the total cost. Here, we present a streamlined workflow detailing cloning, expression testing, protein purification, and bioactivity screening that allows for low-cost production of bioactive growth factors including basic fibroblast growth factor and transforming growth factor ß1. This generalized procedure can be used for multiple families of growth factors with minor modification, and the outputs are bioactive and suitable for cell culture applications. For complete details on the use and execution of this protocol, please refer to Venkatesan, et al.1.

Recombinant production of growth factors for application in cell culture.

Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as "cellular agriculture". These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-ß1 in Escherichia coli. Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.

Molecular analysis and essentiality of Aro1 shikimate biosynthesis multi-enzyme in Candida albicans.

In the human fungal pathogen Candida albicans, ARO1 encodes an essential multi-enzyme that catalyses consecutive steps in the shikimate pathway for biosynthesis of chorismate, a precursor to folate and the aromatic amino acids. We obtained the first molecular image of C. albicans Aro1 that reveals the architecture of all five enzymatic domains and their arrangement in the context of the full-length protein. Aro1 forms a flexible dimer allowing relative autonomy of enzymatic function of the individual domains. Our activity and in cellulo data suggest that only four of Aro1's enzymatic domains are functional and essential for viability of C. albicans, whereas the 3-dehydroquinate dehydratase (DHQase) domain is inactive because of active site substitutions. We further demonstrate that in C. albicans, the type II DHQase Dqd1 can compensate for the inactive DHQase domain of Aro1, suggesting an unrecognized essential role for this enzyme in shikimate biosynthesis. In contrast, in Candida glabrata and Candida parapsilosis, which do not encode a Dqd1 homolog, Aro1 DHQase domains are enzymatically active, highlighting diversity across Candida species.

Identification of a Novel Biosynthetic Gene Cluster in Aspergillus niger Using Comparative Genomics.

Previously, DNA microarrays analysis showed that, in co-culture with Bacillus subtilis, a biosynthetic gene cluster anchored with a nonribosomal peptides synthetase of Aspergillus niger is downregulated. Based on phylogenetic and synteny analyses, we show here that this gene cluster, NRRL3_00036-NRRL3_00042, comprises genes predicted to encode a nonribosomal peptides synthetase, a FAD-binding domain-containing protein, an uncharacterized protein, a transporter, a cytochrome P450 protein, a NAD(P)-binding domain-containing protein and a transcription factor. We overexpressed the in-cluster transcription factor gene NRRL3_00042. The overexpression strain, NRRL3_00042OE, displays reduced growth rate and production of a yellow pigment, which by mass spectrometric analysis corresponds to two compounds with masses of 409.1384 and 425.1331. We deleted the gene encoding the NRRL3_00036 nonribosomal peptides synthetase in the NRRL3_00042OE strain. The resulting strain reverted to the wild-type phenotype. These results suggest that the biosynthetic gene cluster anchored by the NRRL3_00036 nonribosomal peptides synthetase gene is regulated by the in-cluster transcriptional regulator gene NRRL3_00042, and that it is involved in the production of two previously uncharacterized compounds.

Structural characterization of nonstructural protein 1 from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a single-stranded, enveloped RNA virus and the etiological agent of the current coronavirus disease 2019 pandemic. Efficient replication of the virus relies on the activity of nonstructural protein 1 (Nsp1), a major virulence factor shown to facilitate suppression of host gene expression through promotion of host mRNA degradation and interaction with the 40S ribosomal subunit. Here, we report the crystal structure of the globular domain of SARS-CoV-2 Nsp1, encompassing residues 13 to 127, at a resolution of 1.65 Å. Our structure features a six-stranded, capped ß-barrel motif similar to Nsp1 from SARS-CoV and reveals how variations in amino acid sequence manifest as distinct structural features. Combining our high-resolution crystal structure with existing data on the C-terminus of Nsp1 from SARS-CoV-2, we propose a model of the full-length protein. Our results provide insight into the molecular structure of a major pathogenic determinant of SARS-CoV-2.

Structural characterization of aminoglycoside 4'-O-adenylyltransferase ANT(4')-IIb from Pseudomonas aeruginosa.

Aminoglycosides were one of the first classes of broad-spectrum antibacterial drugs clinically used to effectively combat infections. The rise of resistance to these drugs, mediated by enzymatic modification, has since compromised their utility as a treatment option, prompting intensive research into the molecular function of resistance enzymes. Here, we report the crystal structure of aminoglycoside nucleotidyltransferase ANT(4')-IIb in apo and tobramycin-bound forms at a resolution of 1.6 and 2.15 Å, respectively. ANT(4')-IIb was discovered in the opportunistic pathogen Pseudomonas aeruginosa and conferred resistance to amikacin and tobramycin. Analysis of the ANT(4')-IIb structures revealed a two-domain organization featuring a mixed ß-sheet and an α-helical bundle. ANT(4')-IIb monomers form a dimer required for its enzymatic activity, as coordination of the aminoglycoside substrate relies on residues contributed by both monomers. Despite harbouring appreciable primary sequence diversity compared to previously characterized homologues, the ANT(4')-IIb structure demonstrates a surprising level of structural conservation highlighting the high plasticity of this general protein fold. Site-directed mutagenesis of active site residues and kinetic analysis provides support for a catalytic mechanism similar to those of other nucleotidyltransferases. Using the molecular insights provided into this ANT(4')-IIb-represented enzymatic group, we provide a hypothesis for the potential evolutionary origin of these aminoglycoside resistance determinants.

Group II introns: versatile ribozymes and retroelements.

Group II introns are catalytic RNAs (ribozymes) and retroelements found in the genomes of bacteria, archaebacteria, and organelles of some eukaryotes. The prototypical retroelement form consists of a structurally conserved RNA and a multidomain reverse transcriptase protein, which interact with each other to mediate splicing and mobility reactions. A wealth of biochemical, cross-linking, and X-ray crystal structure studies have helped to reveal how the two components cooperate to carry out the splicing and mobility reactions. In addition to the standard retroelement form, group II introns have evolved into derivative forms by either losing specific splicing or mobility characteristics, or becoming functionally specialized. Of particular interest are the eukaryotic derivatives-the spliceosome, spliceosomal introns, and non-LTR retroelements-which together make up approximately half of the human genome. On a practical level, the properties of group II introns have been exploited to develop group II intron-based biotechnological tools. WIREs RNA 2016, 7:341-355. doi: 10.1002/wrna.1339 For further resources related to this article, please visit the WIREs website.

Evolution of group II introns.

Present in the genomes of bacteria and eukaryotic organelles, group II introns are an ancient class of ribozymes and retroelements that are believed to have been the ancestors of nuclear pre-mRNA introns. Despite long-standing speculation, there is limited understanding about the actual pathway by which group II introns evolved into eukaryotic introns. In this review, we focus on the evolution of group II introns themselves. We describe the different forms of group II introns known to exist in nature and then address how these forms may have evolved to give rise to spliceosomal introns and other genetic elements. Finally, we summarize the structural and biochemical parallels between group II introns and the spliceosome, including recent data that strongly support their hypothesized evolutionary relationship.

A pipeline of programs for collecting and analyzing group II intron retroelement sequences from GenBank.

BACKGROUND: Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated. RESULTS: Here we present a pipeline of 11 programs that collect and analyze group II intron sequences from GenBank. The pipeline begins with a BLAST search of GenBank using a set of representative group II IEPs as queries. Subsequent steps download the corresponding genomic sequences and flanks, filter out non-group II introns, assign introns to phylogenetic subclasses, filter out incomplete and/or non-functional introns, and assign IEP sequences and RNA boundaries to the full-length introns. In the final step, the redundancy in the data set is reduced by grouping introns into sets of ≥95% identity, with one example sequence chosen to be the representative. CONCLUSIONS: These programs should be useful for comprehensive identification of group II introns in sequence databases as data continue to rapidly accumulate.

A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro.

Reverse transcriptases (RTs) are RNA-dependent DNA polymerases that usually function in the replication of selfish DNAs such as retrotransposons and retroviruses. Here, we have biochemically characterized a RT-related protein, AbiK, which is required for abortive phage infection in the Gram-positive bacterium Lactococcus lactis. In vitro, AbiK does not exhibit the properties expected for an RT, but polymerizes long DNAs of 'random' sequence, analogous to a terminal transferase. Moreover, the polymerized DNAs appear to be covalently attached to the AbiK protein, presumably because an amino acid serves as a primer. Mutagenesis experiments indicate that the polymerase activity resides in the RT motifs and is essential for phage resistance in vivo. These results establish a novel biochemical property and a non-replicative biological role for a polymerase.