Antifreeze protein mimetic metallohelices with potent ice recrystallization inhibition activity

Antifreeze proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure-property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze protein mimetics and provides insight into the origins of ice-growth inhibition

Understanding the reactogenicity of 4CMenB vaccine: Comparison of a novel and conventional method of assessing post-immunisation fever and correlation with pre-release in vitro pyrogen testing

BACKGROUND: Better understanding of vaccine reactogenicity is crucial given its potential impact upon vaccine safety and acceptance. Here we report a comparison between conventional and novel (continuous) methods of monitoring temperature and evaluate any association between reactogenicity and the monocyte activation test (MAT) employed for testing four-component capsular group B meningococcal vaccine (4CMenB) batches prior to release for clinical use in Europe. METHODS: Healthy 7-12-week-old infants were randomised in two groups: group PCV13 2 + 1 (received pneumococcal conjugate vaccine 13 valent (PCV13) at 2, 4 and 12 months) and group PCV13 1 + 1 (received reduced schedule at 3 and 12 months). In both, infants received the remaining immunisations as per UK national schedule (including 4CMenB at 2, 4 and 12 months of age). Fever was measured for the first 24 h after immunisations using an axillary thermometer and with a wireless continuous temperature monitoring device (iButton®). To measure the relative pyrogenicity of individual 4CMenB batches, MAT was performed according to Ph. Eu. chapter 2.6.30 method C using PBMCs with IL-6 readout. RESULTS: Fever rates detected by the iButton® ranged from 28.7% to 76.5% and from 46.6% to 71.1% in group PCV13 2 + 1 and PCV13 1 + 1 respectively, across all study visits. The iButton® recorded a higher number of fever episodes when compared with axillary measurements in both groups (range of axillary temperature fevers; group PCV13 2 + 1: 6.7%-38%; group PCV13 1 + 1: 11.4%-37.1%). An agreement between the two methods was between 0.39 and 0.36 (p < 0.001) at 8 h' time-point post primary immunisations. No correlation was found between MAT scores and fever rates, or other reported adverse events. CONCLUSIONS: It is likely that conventional, intermittent, fever measurements underestimates fever rates following immunisation. 4CMenB MAT scores didn't predict reactogenicity, providing reassurance that vaccine batches with the highest acceptable pyrogen level are not associated with an increase in adverse events. Clinicaltrials.gov identifier: NCT02482636

Development and Assessment of a Diagnostic DNA Oligonucleotide Microarray for Detection and Typing of Meningitis-Associated Bacterial Species.

Meningitis is commonly caused by infection with a variety of bacterial or viral pathogens. Acute bacterial meningitis (ABM) can cause severe disease, which can progress rapidly to a critical life-threatening condition. Rapid diagnosis of ABM is critical, as this is most commonly associated with severe sequelae with associated high mortality and morbidity rates compared to viral meningitis, which is less severe and self-limiting. We have designed a microarray for detection and diagnosis of ABM. This has been validated using randomly amplified DNA targets (RADT), comparing buffers with or without formamide, in glass slide format or on the Alere ArrayTubeTM (Alere Technologies GmbH) microarray platform. Pathogen-specific signals were observed using purified bacterial nucleic acids and to a lesser extent using patient cerebral spinal fluid (CSF) samples, with some technical issues observed using RADT and glass slides. Repurposing the array onto the Alere ArrayTubeTM platform and using a targeted amplification system increased specific and reduced nonspecific hybridization signals using both pathogen nucleic and patient CSF DNA targets, better revealing pathogen-specific signals although sensitivity was still reduced in the latter. This diagnostic microarray is useful as a laboratory diagnostic tool for species and strain designation for ABM, rather than for primary diagnosis

Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease

The BcgI endonuclease exemplifies a subset of restriction enzymes, the Type IIB class, which make two double-strand breaks (DSBs) at each copy of their recognition sequence, one either side of the site, to excise the sequence from the remainder of the DNA. In this study, we show that BcgI is essentially inactive when bound to a single site and that to cleave a DNA with one copy of its recognition sequence, it has to act in trans, bridging two separate DNA molecules. We also show that BcgI makes the two DSBs at an individual site in a highly concerted manner. Intermediates cut on one side of the site do not accumulate during the course of the reaction: instead, the DNA is converted straight to the final products cut on both sides. On DNA with two sites, BcgI bridges the sites in cis and then generally proceeds to cut both strands on both sides of both sites without leaving the DNA. The BcgI restriction enzyme can thus excise two DNA segments together, by cleaving eight phosphodiester bonds within a single-DNA binding event

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer

The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in four infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein with corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ and/or TNF-ɑ producing T cells is comparable between infants and adults. On principal component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19

Hate crimes against trans people: assessing emotions, behaviors and attitudes towards criminal justice agencies

Based on a survey of 593 lesbian, gay, bisexual, and transgender (LGBT) people in the United Kingdom, this study shows that direct anti-LGBT hate crimes (measured by direct experiences of victimization) and indirect anti-LGBT hate crimes (measured by personally knowing other victims of hate crime) are highly prolific and frequent experiences for LGBT people. Our findings show that trans people are particularly susceptible to hate crimes, both in terms of prevalence and frequency. This article additionally highlights the negative emotional and (intended) behavioral reactions that were correlated with an imagined hate crime scenario, showing that trans people are more likely to experience heightened levels of threat, vulnerability, and anxiety compared with non-trans LGB people. The study found that trans people are also more likely to feel unsupported by family, friends, and society for being LGBT, which was correlated with the frequency of direct (verbal) abuse they had previously endured. The final part of this study explores trans people’s confidence levels in the Government, the police, and the Crown Prosecution Service (CPS) in relation to addressing hate crime. In general, trans people felt that the police are not effective at policing anti-LGBT hate crime, and they are not respectful toward them as victims; this was especially true where individuals had previous contact with the police. Respondents were also less confident in the CPS to prosecute anti-LGBT hate crimes, though the level of confidence was slightly higher when respondents had direct experience with the CPS. The empirical evidence presented here supports the assertion that all LGBT people, but particularly trans individuals, continue to be denied equal participation in society due to individual, social, and structural experiences of prejudice. The article concludes by arguing for a renewed policy focus that must address this issue as a public health problem

Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping

Many enzymes acting on DNA require Mg2+ ions not only for catalysis but also to bind DNA. Binding studies often employ Ca2+ as a substitute for Mg2+, to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca2+ mimics Mg2+, Ca2+ causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg2+ present as the DNA is cleaved so, to study the effect of Mg2+ on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca2+ or Mg2+ but, unlike wild-type SfiI with Ca2+, the binding was reversible. With both mutants, dissociation was slow with Ca2+ but was in one case much faster with Mg2+. Hence, Ca2+ can affect DNA binding differently from Mg2+. Moreover, SfiI is an archetypal system for DNA looping; on DNA with two recognition sites, it binds to both sites and loops out the intervening DNA. While the dynamics of looping cannot be measured with wild-type SfiI and Ca2+, it becomes accessible with the mutant and Mg2+

Increased reports of severe myocarditis associated with enterovirus infection in neonates, United Kingdom, 27 June 2022 to 26 April 2023

Enteroviruses are a common cause of seasonal childhood infections. The vast majority of enterovirus infections are mild and self-limiting, although neonates can sometimes develop severe disease. Myocarditis is a rare complication of enterovirus infection. Between June 2022 and April 2023, twenty cases of severe neonatal enteroviral myocarditis caused by coxsackie B viruses were reported in the United Kingdom. Sixteen required critical care support and two died. Enterovirus PCR on whole blood was the most sensitive diagnostic test. We describe the initial public health investigation into this cluster and aim to raise awareness among paediatricians, laboratories and public health specialists

Dissecting protein-induced DNA looping dynamics in real time

Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA–protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA–protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA–protein dynamics in real time