Qua Re qui possum non esse popularis: The representation of Populares in the Late Roman Republic.

The terms popularis and optimate have been employed in both ancient and modern literature to interpret late Roman Republican politics. The purpose of this work is to express the diversity and change of the popularis label from 133 to 88 B.C. as a consequence of developing political practices. A chronological assessment of five key popularis tribunes in this period; Ti. Sempronius Gracchus, G. Sempronius Gracchus, L. Appuleius Saturninus, M. Livius Drusus and P. Sulpicius Rufus determines the variation in political methodologies exploited by these men in response to an optimate opposition. An assessment of Cicero’s works then considers how the discrepancies exhibited by these politicians could be manipulated for oratorical advantage. This subsequently reveals the perception of pre-Sullan populares in the time of Cicero, a generation later. This work ultimately aims to demonstrate the individualistic nature of late Republican politicians, the evolution of political practice in the period and the diverse employment of political labels in contemporary sources

Single-molecule and Single-cell Approaches in Molecular Bioengineering

Protein sequences inhabit a discrete set in macromolecular space with incredible capacity to treat human disease. Despite our ability to program and manipulate protein sequences, the vast majority of protein development efforts are still done heuristically without a unified set of guiding principles. This article highlights work in understanding biophysical stability and function of proteins, developing new biophysical measurement tools and building high-throughput screening platforms to explore functional protein sequences. We highlight two primary areas. First, molecular biomechanics is a subfield concerned with the response of proteins to mechanical forces, and how we can leverage mechanical force to control protein function. The second subfield investigates the use of polymers and hydrogels in protein engineering and directed evolution in pursuit of new molecular systems with therapeutic applications. These two subdisciplines complement each other by shedding light onto sequence and structural features that can be used to impart stability into therapeutic proteins

Removal of a Conserved Disulfide Bond Does Not Compromise Mechanical Stability of a VHH Antibody Complex

Single-domain VHH antibodies are promising reagents for medical therapy. A conserved disulfide bond within the VHH framework region is known to be critical for thermal stability, however, no prior studies have investigated its influence on the stability of VHH antibody–antigen complexes under mechanical load. Here, we used single-molecule force spectroscopy to test the influence of a VHH domain’s conserved disulfide bond on the mechanical strength of the interaction with its antigen mCherry. We found that although removal of the disulfide bond through cysteine-to-alanine mutagenesis significantly lowered VHH domain denaturation temperature, it had no significant impact on the mechanical strength of the VHH:mCherry interaction with complex rupture occurring at ∼60 pN at 103–104 pN/sec regardless of disulfide bond state. These results demonstrate that mechanostable binding interactions can be built on molecular scaffolds that may be thermodynamically compromised at equilibrium

Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening

Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of D -Amino acid oxidase from Rhodotorula gracilis ( Rg DAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between Rg DAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled library screening by fluorescence activated cell sorting (FACS). We screened a Rg DAAOx variant library containing ∼10 6 clones while lowering the D -Ala substrate concentration over three sorting rounds in order to isolate variants with low K m . After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower K m than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the Rg DAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes

Ophyiulus in Victoria: results of millipede surveys from south-eastern Australia

The composition and ecology of the millipede fauna of Victoria remain poorly understood. We collected millipedes as part of a series of ecological arthropod surveys across south-eastern Australia, focusing mainly on Victoria. These samples almost exclusively contained millipedes from the introduced order Julida. We pursued species identification of the julids when it became apparent there were species other than the well-recorded Ommatoiulus moreleti (Lucas, 1860) (Portuguese millipede) in the samples. The majority of specimens were O. moreleti, but we also detected at least one species of Cylindroiulus Verhoeff, 1894, as well as an Ophyiulus Berlese, 1884, species, specimens of which have been identified as Ophyiulus cf. targionii. These are the first Ophyiulus records for Victoria to our knowledge. We present preliminary data on the abundance through the year of Ophyiulus. This is the first study to examine this species in Victoria and little is currently known about its likely impact on agriculture or on native species. Monitoring and research of the species in the future is therefore warranted

Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin

Staphylococcus aureus (S. aureus) is an invasive and life-threatening pathogen that has undergone extensive coevolution with its mammalian hosts. Its molecular adaptations include elaborate mechanisms for immune escape and hijacking of the coagulation and fibrinolytic pathways. These capabilities are enacted by virulence factors including microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the plasminogen-activating enzyme staphylokinase (SAK). Despite the ability of S. aureus to modulate coagulation, until now the sensitivity of S. aureus virulence factors to digestion by proteases of the coagulation system was unknown. Here, we used protein engineering, biophysical assays, and mass spectrometry to study the susceptibility of S. aureus MSCRAMMs to proteolytic digestion by human thrombin, plasmin, and plasmin/SAK complexes. We found that MSCRAMMs were highly resistant to proteolysis, and that SAK binding to plasmin enhanced this resistance. We mapped thrombin, plasmin, and plasmin/SAK cleavage sites of nine MSCRAMMs and performed biophysical, bioinformatic, and stability analysis to understand structural and sequence features common to protease-susceptible sites. Overall, our study offers comprehensive digestion patterns of S. aureus MSCRAMMs by thrombin, plasmin, and plasmin/SAK complexes and paves the way for new studies into this resistance and virulence mechanism

Biasing effects of receptor-ligand complexes on protein-unfolding statistics

Protein receptor-ligand pairs are increasingly used as specific molecular handles in single-molecule protein-unfolding experiments. Further, known marker domains, also referred to as fingerprints, provide unique unfolding signatures to identify specific single-molecule interactions, when receptor-ligand pairs themselves are investigated. We show here that in cases where there is an overlap between the probability distribution associated with fingerprint domain unfolding and that associated with receptor-ligand dissociation, the experimentally measured force distributions are mutually biased. This biasing effect masks the true parameters of the underlying free energy landscape. To address this, we present a model-free theoretical framework that corrects for the biasing effect caused by such overlapping distributions

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials