Bacteriophage T5 gene D10 encodes a branch-migration protein

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms

Accelerometers can measure total and activity-specific energy expenditures in free-ranging marine mammals only if linked to time-activity budgets

Peer reviewedPostprin

A long-acting GH receptor antagonist through fusion to GH binding protein.

Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed an alternative technology for generating a long acting potent GHR antagonist through translational fusion of a mutated GH linked to GH binding protein and tested three candidate molecules. All molecules had the amino acid change (G120R), creating a competitive GHR antagonist and we tested the hypothesis that an amino acid change in the GH binding domain (W104A) would increase biological activity. All were antagonists in bioassays. In rats all antagonists had terminal half-lives >20 hours. After subcutaneous administration in rabbits one variant displayed a terminal half-life of 40.5 hours. A single subcutaneous injection of the same variant in rabbits resulted in a 14% fall in IGF-I over 7 days. IN CONCLUSION: we provide proof of concept that a fusion of GHR antagonist to its binding protein generates a long acting GHR antagonist and we confirmed that introducing the W104A amino acid change in the GH binding domain enhances antagonist activity

Antibiotics limit adaptation of drug-resistant Staphylococcus aureus to hypoxia

Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of tetL, encoding the acquired tetracycline-specific efflux pump. In contrast, evolutionary adaptation by S. aureus to hypoxic conditions, which evolved in the absence of antibiotics through mutations affecting gyrB, was impeded by antibiotic treatment. Together, these data suggest that the horizontal acquisition of a new resistance mechanism is merely a starting point for the emergence of high-level resistance under antibiotic selection but that antibiotic treatment constrains pathogen adaptation to other important environmental selective forces such as hypoxia, which in turn could limit the survival of these highly resistant but poorly adapted genotypes after antibiotic treatment is ended

1H, 15N and 13C backbone resonance assignments of flap endonuclease from Plasmodium falciparum

Flap endonuclease (FEN) enzymes are a group of metallonucleases that have essential roles in DNA repair and the maintenance of genomic resilience. They catalyse hydrolytic cleavage of a phosphodiester bond to remove 5′-flaps present on double-stranded DNA molecules formed during DNA replication. FEN locates a target scissile bond through the structural recognition of a pronounced bend in the double-stranded DNA substrate along with the presence of both a 5′-flap and a 1-nucleotide 3′-flap. FEN enzymes share a common structural architecture and are functionally conserved across all living organisms. In this work, we report the 1H, 15N and 13C backbone resonance assignments of residues 2–349 of FEN from Plasmodium falciparum (PfFEN349) in its substrate-free state. Using heteronuclear multidimensional NMR spectroscopy, 90% of all backbone resonances of PfFEN349 were assigned, with 298 backbone amide resonances out of 337 theoretically-detectible resonances (which exclude 10 prolines and the N-terminal glycine) identified in the 1H–15 N TROSY spectrum. Prediction of solution secondary structure content from a chemical shift analysis using the TALOS-N webserver is mostly in good agreement with an AlphaFold model of PfFEN349. The reported assignments provide a pathway for drug discovery as PfFEN349 is a potential target for the development of new inhibitors that could be utilised to control the incidence of malaria across the globe

Schistosoma mansoni secretes a chemokine binding protein with antiinflammatory activity

The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the host's innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been selected to modulate the host to tolerate chronic worm infections, often for decades, without overt morbidity. The modulation of immunity by schistosomes has been shown to prevent a range of immune-mediated diseases, including allergies and autoimmunity. Individual immune-modulating schistosome molecules have, therefore, therapeutic potential as selective manipulators of the immune system to prevent unrelated diseases. Here we show that S. mansoni eggs secrete a protein into host tissues that binds certain chemokines and inhibits their interaction with host chemokine receptors and their biological activity. The purified recombinant S. mansoni chemokine binding protein (smCKBP) suppressed inflammation in several disease models. smCKBP is unrelated to host proteins and is the first described chemokine binding protein encoded by a pathogenic human parasite and may have potential as an antiinflammatory agent

Improvement in protein HSQC spectra from addition of betaine

Addition of glycine betaine up to 1 M gave rise to increased intensity for some weak signals in the HSQC spectra of barnase and Plasmodium falciparum flap endonuclease. The signals that were enhanced were low intensity signals, often from amide groups with rapid internal motion (low order parameter). The majority of signals are however somewhat weaker because of the increased viscosity. Addition of betaine is shown to cause a small but significant overall increase in order parameter, no consistent effect on conformational change on the µs-ms timescale, and a reduction in amide exchange rates, by a factor of about 3. The results are consistent with a model in which betaine leads to a reduction in fluctuations within the bulk water, which in turn produces a reduction in internal fluctuations of the protein

Establishing SARS-CoV-2 membrane protein-specific antibodies as a valuable serological target via high-content microscopy

The prevalence and strength of serological responses mounted toward SARS-CoV-2 proteins other than nucleocapsid (N) and spike (S), which may be of use as additional serological markers, remains underexplored. Using high-content microscopy to assess antibody responses against full-length StrepTagged SARS-CoV-2 proteins, we found that 85% (166/196) of unvaccinated individuals with RT-PCR confirmed SARS-CoV-2 infections and 74% (31/42) of individuals infected after being vaccinated developed detectable IgG against the structural protein M, which is higher than previous estimates. Compared with N antibodies, M IgG displayed a shallower time-dependent decay and greater specificity. Sensitivity for SARS-CoV-2 seroprevalence was enhanced when N and M IgG detection was combined. These findings indicate that screening for M seroconversion may be a good approach for detecting additional vaccine breakthrough infections and highlight the potential to use HCM as a rapidly deployable method to identify the most immunogenic targets of newly emergent pathogens

Risk factors for SARS-CoV-2 seroprevalence following the first pandemic wave in UK healthcare workers in a large NHS Foundation Trust [version 1; peer review: awaiting peer review]

Background: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. Methods: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust were prospectively enrolled and sampled at two time points. SARS-CoV-2 antibodies were tested using an in-house assay for IgG and IgA reactivity against Spike and Nucleoprotein (sensitivity 99·47%, specificity 99·56%). Data were analysed using three statistical models: a seroprevalence model, an antibody kinetics model, and a heterogeneous sensitivity model. Results: As of 12th June 2020, 24·4% (n=311/1275) of HCWs were seropositive. Of these, 39·2% (n=122/311) were asymptomatic. The highest adjusted seroprevalence was measured in HCWs on the Acute Medical Unit (41·1%, 95% CrI 30·0–52·9) and in Physiotherapists and Occupational Therapists (39·2%, 95% CrI 24·4–56·5). Older age groups showed overall higher median antibody titres. Further modelling suggests that, for a serological assay with an overall sensitivity of 80%, antibody titres may be markedly affected by differences in age, with sensitivity estimates of 89% in those over 60 years but 61% in those ≤30 years. Conclusions: HCWs in acute medical units working closely with COVID-19 patients were at highest risk of infection, though whether these are infections acquired from patients or other staff is unknown. Current serological assays may underestimate seroprevalence in younger age groups if validated using sera from older and/or more symptomatic individuals

High SARS-CoV-2 incidence and asymptomatic fraction during Delta and Omicron BA.1 waves in The Gambia

Little is known about SARS-CoV-2 infection risk in African countries with high levels of infection-driven immunity and low vaccine coverage. We conducted a prospective cohort study of 349 participants from 52 households in The Gambia between March 2021 and June 2022, with routine weekly SARS-CoV-2 RT-PCR and 6-monthly SARS-CoV-2 serology. Attack rates of 45% and 57% were seen during Delta and Omicron BA.1 waves respectively. Eighty-four percent of RT-PCR-positive infections were asymptomatic. Children under 5-years had a lower incidence of infection than 18-49-year-olds. One prior SARS-CoV-2 infection reduced infection risk during the Delta wave only, with immunity from ≥2 prior infections required to reduce the risk of infection with early Omicron lineage viruses. In an African population with high levels of infection-driven immunity and low vaccine coverage, we find high attack rates during SARS-CoV-2 waves, with a high proportion of asymptomatic infections and young children remaining relatively protected from infection