Leveraging wastewater surveillance to detect viral diseases in livestock settings.

Wastewater surveillance has evolved into a powerful tool for monitoring public health-relevant analytes. Recent applications in tracking severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection highlight its potential. Beyond humans, it can be extended to livestock settings where there is increasing demand for livestock products, posing risks of disease emergence. Wastewater surveillance may offer non-invasive, cost-effective means to detect potential outbreaks among animals. This approach aligns with the "One Health" paradigm, emphasizing the interconnectedness of animal, human, and ecosystem health. By monitoring viruses in livestock wastewater, early detection, prevention, and control strategies can be employed, safeguarding both animal and human health, economic stability, and international trade. This integrated "One Health" approach enhances collaboration and a comprehensive understanding of disease dynamics, supporting proactive measures in the Anthropocene era where animal and human diseases are on the rise.

Escherichia coli "TatExpress" strains export several g/L human growth hormone to the periplasm by the Tat pathway.

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 "TatExpress" strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.

Exploitation of the Escherichia coli lac operon promoter for controlled recombinant protein production.

The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here, we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give a controlled expression that is suitable for diverse biotechnology applications.

Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.

Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

Combined ligand-observe (19)F and protein-observe (15)N,(1)H-HSQC NMR suggests phenylalanine as the key Δ-somatostatin residue recognized by human protein disulfide isomerase.

Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand Δ-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using (19)F ligand-observe and (15)N,(1)H-HSQC protein-observe NMR methods. Phe residues in Δ-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103 + 47 µM until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of Δ-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of (19)F ligand-observe and complimentary (15)N,(1)H-HSQC titrations to monitor interactions from the protein's perspective. (19)F ligand-observe NMR was ratified as mirroring (15)N protein-observe but highlighted the advantage that (19)F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity.

Sharks and people: insight into the global practices of tourism operators and their attitudes to shark behaviour.

Shark tourism is a popular but controversial activity. We obtained insights into this industry via a global e-mailed questionnaire completed by 45 diving/snorkelling operators who advertised shark experiences (shark operators) and 49 who did not (non-shark operators). 42% of shark operators used an attractant to lure sharks and 93% stated they had a formal code of conduct which 86% enforced "very strictly". While sharks were reported to normally ignore people, 9 operators had experienced troublesome behaviour from them. Whilst our research corroborates previous studies indicating minimal risk to humans from most shark encounters, a precautionary approach to provisioning is required to avoid potential ecological and societal effects of shark tourism. Codes of conduct should always stipulate acceptable diver behaviour and appropriate diver numbers and shark operators should have a moral responsibility to educate their customers about the need for shark conservation.

19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI).

We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with Δ-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 µM.