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Background: The “1-3-6-12-day rule” for starting direct oral anticoagulants (DOACs) in patients with nonvalvular atrial fibrillation after acute ischemic stroke or transient ischemic attack recommends timings that may be later than used in clinical practice. We investigated more practical optimal timing of DOAC initiation according to stroke severity. Methods: The combined data of prospective registries in Japan, Stroke Acute Management with Urgent Risk-factor Assessment and Improvement-nonvalvular atrial fibrillation (September 2011 to March 2014) and RELAXED (February 2014 to April 2016) were used. Patients were divided into transient ischemic attack and 3 stroke subgroups by the National Institutes of Health Stroke Scale score: mild (0–7), moderate (8–15), and severe (≥16). The early treatment group was defined as patients starting DOACs earlier than the median initiation day in each subgroup. Outcomes included a composite of recurrent stroke or systemic embolism, ischemic stroke, and severe bleeding within 90 days. Six European prospective registries were used for validation. Results: In the 1797 derivation cohort patients, DOACs were started at median 2 days after transient ischemic attack and 3, 4, and 5 days after mild, moderate, and severe strokes, respectively. Stroke or systemic embolism was less common in Early Group (n=785)—initiating DOACS within 1, 2, 3, and 4 days, respectively—than Late Group (n=1012) (1.9% versus 3.9%; adjusted hazard ratio, 0.50 [95% CI, 0.27–0.89]), as was ischemic stroke (1.7% versus 3.2%, 0.54 [0.27–0.999]). Major bleeding was similarly common in the 2 groups (0.8% versus 1.0%). On validation, both ischemic stroke (2.4% versus 2.2%) and intracranial hemorrhage (0.2% versus 0.6%) were similarly common in Early (n=547) and Late (n=1483) Groups defined using derivation data. Conclusions: In Japanese and European populations, early DOAC initiation within 1, 2, 3, or 4 days according to stroke severity seemed to be feasible to decrease the risk of recurrent stroke or systemic embolism and no increase in major bleeding. These findings support ongoing randomized trials to better establish the optimal timing of DOAC initiation.

Dimethylsulfoniopropionate (DMSP) is an abundant marine organosulfur compound[1] with roles in stress protection[2, 3], chemotaxis[4], nutrient and sulfur cycling[5] and, potentially, climate regulation[6, 7]. Marine algae and bacteria are considered significant DMSP producers, but many diverse representatives lack known DMSP synthesis genes/enzymes[8, 9]. Here, new DMSP biosynthesis enzymes were identified that considerably increase the number and diversity of potential DMSP-producing organisms, inferring new and significant global DMSP producers. A novel bifunctional DMSP biosynthesis enzyme, DsyGD, identified in the rhizobacterium Gynuella sunshinyii, produces DMSP at levels higher than any other bacterium from methylthiohydroxybutyrate (MTHB) via an N-terminal MTHB S-methyltransferase domain (termed DsyG) and a C-terminal dimethylsulfoniohydroxybutyrate (DMSHB) decarboxylase domain (termed DsyD, which is the first reported enzyme with this activity). DsyGD is also found in some filamentous cyanobacteria, not previously known to produce DMSP. Regulation of DMSP production and dsyGD transcription was consistent with their role in osmoprotection. Indeed, cloned dsyGD conferred osmotolerance to bacteria deficient in osmolyte production, something not previously demonstrated for any known DMSP synthesis gene, and which could be exploited for biotechnology e.g., engineering salt tolerance. DsyGD characterisation led to identification of phylogenetically distinct DsyG-like proteins, termed DSYE, with MTHB S-methyltransferase activity, in diverse and environmentally abundant Chlorophyta, Chlorachniophyta, Ochraphyta, Haptophyta and Bacillariophyta algae. These algae comprise a mix of low, high and previously unknown DMSP producers[10]. Algae containing DSYE, particularly bloom-forming Pelagophyceae species, which we showed to accumulate medium-high intracellular DMSP levels, were globally more abundant DMSP producers than Haptophyta, Dinophyta and Bacillariophyta with DSYB and/or TpMMT. This highlights the potential importance of Pelagophyceae and other DSYE containing algae in global DMSP production and sulfur cycling.

Dimethylsulfoxonium propionate (DMSOP) is a recently identified and abundant marine organosulfur compound with purported roles in oxidative stress protection, global carbon and sulfur cycling1. Diverse algae and bacteria synthesise DMSOP from dimethylsulfoniopropionate (DMSP), which potentially limits the production of climate-active gases e.g., dimethylsulfide (DMS) generated from microbial DMSP cleavage1. Here, DMSOP was found at mM levels in saltmarsh sediment, >10-fold higher than DMSP, and orders of magnitude higher than DMSOP levels previously reported in seawater1. Moreover, we showed bacteria could utilise DMSOP as an osmoprotectant. Some bacteria also cleave DMSOP liberating dimethyl sulfoxide (DMSO), an ubiquitous marine metabolite and acrylate1, but the enzymes responsible and their environmental importance were unknown. Here, we elucidated the DMSOP cleavage mechanism/s in diverse heterotrophic bacteria, e.g., SAR11 clade and Roseobacters, and fungi and phototrophic algae, e.g., Emiliania huxleyi, not previously known to have this activity. All these diverse organisms utilised their DMSP lyase ‘Ddd’ or ‘Alma1’ enzymes, that span five protein families, to cleave DMSOP with similar specific activities to DMSP. Bacteria with DMSP lyases that used DMSP as a carbon source likewise used DMSOP. Furthermore, ddd gene transcription in these bacteria was induced by DMSOP, including dddK in SAR11 strain HTCC1062. We determined the structure of DddK bound to DMSOP and concluded that the catalytic mechanisms of DMSOP cleavage by Ddd enzymes were like those for DMSP. Given the predicted teragram DMSOP production budget1, its newly found abundance in marine sediments, and the abundance of microbial DMSP lyase genes and transcripts in marine environments, DMSOP cleavage is likely a globally significant process influencing global carbon and sulfur fluxes and marine ecological interactions.

Despite extensive use of micelles in materials and colloidal science, their supramolecular organization as well as host-guest interactions within these dynamic assemblies are poorly understood. Small guest molecules in the presence of micelles undergo constant exchange between a micellar aggregate and the surrounding solution, posing a considerable challenge for their molecular level characterisation. In this work we reveal the interaction maps between small guest molecules and surfactants forming micelles via novel applications of NMR techniques supported with state-of-the-art analytical methods used in colloidal science. Model micelles composed of structurally distinct surfactants (block non-ionic polymer Pluronic® F-127, non-ionic surfactant Tween 20 or Tween 80 and ionic surfactant SLS, sodium lauryl sulphate) were selected and loaded with model small molecules of biochemical relevance (i.e. the drugs fluconazole, FLU or indomethacin, IMC) known to have different partition coefficients. Molecular level organization of FLU or IMC within hydrophilic and hydrophobic domains of micellar aggregates was established using combination of NMR methods (1D 1H NMR, 1D 19F NMR, 2D 1H-1H NOESY and 2D 1H-19F HOESY, and the multifrequency-STD NMR) and corroborated with molecular dynamics (MD) simulations. This is the first application of multifrequency-STD NMR to colloidal systems, enabling us to elucidate intricately detailed patterns of drug/micelle interactions in a single NMR experiment within minutes. Importantly, our results indicate that flexible surfactants, such as block copolymers and polysorbates, form micellar aggregates with a surface composed of both hydrophilic and hydrophobic domains and do not follow the classical core-shell model of the micelle. We propose that the magnitude of the changes in 1H chemical shifts corroborated with interaction maps obtained from DEEP-STD NMR and 2D NMR experiments can be used as an indicator of the strength of the guest-surfactant interactions. This NMR toolbox can be adopted for the analysis of broad range colloidal host-guest systems from soft materials to biological systems.

Weak protein–ligand interactions have been demonstrated to play key roles in biological processes, particularly in those involving quick cellular responses after certain stimuli (e.g. signal transduction). Although powerful biophysical techniques are available to gain high-resolution structural information of protein–ligand complexes of high affinity, NMR spectroscopy has been demonstrated to stand out among them for protein–ligand studies within the limit of weak affinity. In particular, ligand-based NMR techniques allow the detection and quantification of weak biomolecular binding processes, where saturation transfer difference (STD) NMR techniques have demonstrated through the years their strong ability to not only detect binding processes but also to provide structural information about the ligand mode of binding in the receptor-binding pocket, a highly valuable piece of information for the further development of enhanced binders along the process of drug discovery. This is particularly useful in fragment-based drug-discovery approaches, where the binding of the identified initial small fragments tends to fall within the low affinity range of the spectrum. In this chapter we briefly introduce the different classical STD NMR approaches, and later focus in detail on novel developments based on multi-frequency STD NMR experiments, which expand further the analytical capabilities of STD NMR, exemplified by the DEEP-STD NMR protocol, by providing also some key information on the nature of the protein residues in contact with the ligands in the bond state.

Background: In patients with atrial fibrillation who suffered an ischemic stroke while on treatment with nonvitamin K antagonist oral anticoagulants, rates and determinants of recurrent ischemic events and major bleedings remain uncertain. Methods: This prospective multicenter observational study aimed to estimate the rates of ischemic and bleeding events and their determinants in the follow-up of consecutive patients with atrial fibrillation who suffered an acute cerebrovascular ischemic event while on nonvitamin K antagonist oral anticoagulant treatment. Afterwards, we compared the estimated risks of ischemic and bleeding events between the patients in whom anticoagulant therapy was changed to those who continued the original treatment. Results: After a mean follow-up time of 15.0±10.9 months, 192 out of 1240 patients (15.5%) had 207 ischemic or bleeding events corresponding to an annual rate of 13.4%. Among the events, 111 were ischemic strokes, 15 systemic embolisms, 24 intracranial bleedings, and 57 major extracranial bleedings. Predictive factors of recurrent ischemic events (strokes and systemic embolisms) included CHA 2 DS 2 -VASc score after the index event (odds ratio [OR], 1.2 [95% CI, 1.0–1.3] for each point increase; P =0.05) and hypertension (OR, 2.3 [95% CI, 1.0–5.1]; P =0.04). Predictive factors of bleeding events (intracranial and major extracranial bleedings) included age (OR, 1.1 [95% CI, 1.0–1.2] for each year increase; P =0.002), history of major bleeding (OR, 6.9 [95% CI, 3.4–14.2]; P =0.0001) and the concomitant administration of an antiplatelet agent (OR, 2.8 [95% CI, 1.4–5.5]; P =0.003). Rates of ischemic and bleeding events were no different in patients who changed or not changed the original nonvitamin K antagonist oral anticoagulants treatment (OR, 1.2 [95% CI, 0.8–1.7]). Conclusions: Patients suffering a stroke despite being on nonvitamin K antagonist oral anticoagulant therapy are at high risk of recurrent ischemic stroke and bleeding. In these patients, further research is needed to improve secondary prevention by investigating the mechanisms of recurrent ischemic stroke and bleeding.

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development., This project has received funding from the European Union’s Horizon 2020 Research and Innovation program under grant agreement 681137 (to K.J.D. and I.H.), the Medical Research Council (MRC) (to K.J.D. [MR/K024426/1]), The Rosetrees Trust (to K.J.D. [M686]) and Fondation Dormeur, Vaduz (to K.J.D). This research was funded or supported by the National Institute for Health Research Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London and/or the NIHR Clinical Research Facility. The views expressed are those of the authors and not necessarily those of the National Health Service (NHS), the National Institute for Health Research (NIHR), or the Department of Health. N.R. acknowledges funding from Ministry of Science and Education grants CTQ2017-90039-R, RTC-2017-6126-1, and CTQ2011-27874 (fellowship to K.B.) and the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (grant MDM-2017-0720). F.A. was funded by the Wellcome Trust (104958/Z/14/Z). J.A. was supported by the Spanish Ministry of Science, Innovation and Universities through the grant PID2019-109395GB-I00. J.A. and S.M. acknowledge support of BBSRC (grant BB/P010660/1). T.H. and S.W. were funded by Biotechnology and Biological Sciences Research Council (BBSRC) Norwich Research Park Doctoral Training Grant BB/M011216/1. IAVI’s work is made possible by generous support from many donors, including the Bill & Melinda Gates Foundation, the Ministry of Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan in partnership with The World Bank, the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency for Development Cooperation, the United Kingdom Department for International Development (DFID), and the United States Agency for International Development. The full list of IAVI donors is available at www.iavi.org. Brendan McAtarsney and Jonathan Hare from the IAVI Human Immunology Lab (HIL) for coordinating the samples transfers and shipments. Monica Agromayor and the KCL Nikon Centre for assistance and advice on confocal microscopy. NMRI strain Schistosoma mansoni-infected Biomphalaria glabrata snails were provided by the NIAID Schistosomiasis Resource Center, Rockville, USA.