Power Density Titration of Reversible Photoisomerization of a Fluorescent Protein Chromophore in the Presence of Thermally Driven Barrier Crossing Shown by Quantitative Millisecond Serial Synchrotron X-ray Crystallography.

We present millisecond quantitative serial X-ray crystallography at 1.7 Å resolution demonstrating precise optical control of reversible population transfer from Trans-Cis and Cis-Trans photoisomerization of a reversibly switchable fluorescent protein, rsKiiro. Quantitative results from the analysis of electron density differences, extrapolated structure factors, and occupancy refinements are shown to correspond to optical measurements of photoinduced population transfer and have sensitivity to a few percent in concentration differences. Millisecond time-resolved concentration differences are precisely and reversibly controlled through intense continuous wave laser illuminations at 405 and 473 nm for the Trans-to-Cis and Cis-to-Trans reactions, respectively, while the X-ray crystallographic measurement and laser illumination of the metastable Trans chromophore conformation causes partial thermally driven reconversion across a 91.5 kJ/mol thermal barrier from which a temperature jump between 112 and 128 K is extracted.

Coexistence of Incoherent and Ultrafast Coherent Exciton Transport in a Two-Dimensional Superatomic Semiconductor.

Fully leveraging the remarkable properties of low-dimensional semiconductors requires developing a deep understanding of how their structure and disorder affect the flow of electronic energy. Here, we study exciton transport in single crystals of the two-dimensional superatomic semiconductor CsRe6Se8I3, which straddles a photophysically rich yet elusive intermediate electronic-coupling regime. Using femtosecond scattering microscopy to directly image exciton transport in CsRe6Se8I3, we reveal the rare coexistence of coherent and incoherent exciton transport, leading to either persistent or transient electronic delocalization depending on temperature. Notably, coherent excitons exhibit ballistic transport at speeds approaching an extraordinary 1600 km/s over 300 fs. Such fast transport is mediated by J-aggregate-like superradiance, owing to the anisotropic structure and long-range order of CsRe6Se8I3. Our results establish superatomic crystals as ideal platforms for studying the intermediate electronic-coupling regime in highly ordered environments, in this case displaying long-range electronic delocalization, ultrafast energy flow, and a tunable dual transport regime.

Optical Imaging of Ultrafast Phonon-Polariton Propagation through an Excitonic Sensor.

Hexagonal boron nitride (hBN) hosts phonon polaritons (PhP), hybrid light-matter states that facilitate electromagnetic field confinement and exhibit long-range ballistic transport. Extracting the spatiotemporal dynamics of PhPs usually requires "tour de force" experimental methods such as ultrafast near-field infrared microscopy. Here, we leverage the remarkable environmental sensitivity of excitons in two-dimensional transition metal dichalcogenides to image PhP propagation in adjacent hBN slabs. Using ultrafast optical microscopy on monolayer WSe2/hBN heterostructures, we image propagating PhPs from 3.5 K to room temperature with subpicosecond and few-nanometer precision. Excitons in WSe2 act as transducers between visible light pulses and infrared PhPs, enabling visible-light imaging of PhP transport with far-field microscopy. We also report evidence of excitons in WSe2 copropagating with hBN PhPs over several micrometers. Our results provide new avenues for imaging polar excitations over a large frequency range with extreme spatiotemporal precision and new mechanisms to realize ballistic exciton transport at room temperature.

Understanding the leading indicators of hospital admissions from COVID-19 across successive waves in the UK.

Following the end of universal testing in the UK, hospital admissions are a key measure of COVID-19 pandemic pressure. Understanding leading indicators of admissions at the National Health Service (NHS) Trust, regional and national geographies help health services plan for ongoing pressures. We explored the spatio-temporal relationships of leading indicators of hospitalisations across SARS-CoV-2 waves in England. This analysis includes an evaluation of internet search volumes from Google Trends, NHS triage calls and online queries, the NHS COVID-19 app, lateral flow devices (LFDs), and the ZOE app. Data sources were analysed for their feasibility as leading indicators using Granger causality, cross-correlation, and dynamic time warping at fine spatial scales. Google Trends and NHS triages consistently temporally led admissions in most locations, with lead times ranging from 5 to 20 days, whereas an inconsistent relationship was found for the ZOE app, NHS COVID-19 app, and LFD testing, which diminished with spatial resolution, showing cross-correlation of leads between -7 and 7 days. The results indicate that novel surveillance sources can be used effectively to understand the expected healthcare burden within hospital administrative areas though the temporal and spatial heterogeneity of these relationships is a key determinant of their operational public health utility.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Ultrafast imaging of polariton propagation and interactions.

Semiconductor excitations can hybridize with cavity photons to form exciton-polaritons (EPs) with remarkable properties, including light-like energy flow combined with matter-like interactions. To fully harness these properties, EPs must retain ballistic, coherent transport despite matter-mediated interactions with lattice phonons. Here we develop a nonlinear momentum-resolved optical approach that directly images EPs in real space on femtosecond scales in a range of polaritonic architectures. We focus our analysis on EP propagation in layered halide perovskite microcavities. We reveal that EP-phonon interactions lead to a large renormalization of EP velocities at high excitonic fractions at room temperature. Despite these strong EP-phonon interactions, ballistic transport is maintained for up to half-exciton EPs, in agreement with quantum simulations of dynamic disorder shielding through light-matter hybridization. Above 50% excitonic character, rapid decoherence leads to diffusive transport. Our work provides a general framework to precisely balance EP coherence, velocity, and nonlinear interactions.

NMR Fingerprints of Formyl Phloroglucinol Meroterpenoids and Their Application to the Investigation of Eucalyptus gittinsii subsp. gittinsii.

NMR fingerprints provide powerful tools to identify natural products in complex mixtures. Principal component analysis and machine learning using 1H and 13C NMR data, alongside structural information from 180 published formyl phloroglucinols, have generated diagnostic NMR fingerprints to categorize subclasses within this group. This resulted in the reassignment of 167 NMR chemical shifts ascribed to 44 compounds. Three pyrano-diformyl phloroglucinols, euglobal In-1 and psiguadiols E and G, contained 1H and 13C NMR data inconsistent with their predicted phloroglucinol subclass. Subsequent reinterpretation of their 2D NMR data combined with DFT 13C NMR chemical shift and ECD calculations led to their structure revisions. Direct covariance processing of HMBC data permitted 1H resonances for individual compounds in mixtures to be associated, and analysis of their 1H/13C HMBC correlations using the fingerprint tool further classified components into phloroglucinol subclasses. NMR fingerprinting HMBC data obtained for six eucalypt flower extracts identified three subclasses of pyrano-acyl-formyl phloroglucinols from Eucalyptus gittinsii subsp. gittinsii. New, eucalteretial F and (+)-eucalteretial B, and known, (-)-euglobal VII and eucalrobusone C, compounds, each belonging to predicted subclasses, were isolated and characterized. Staphylococcus aureus and Plasmodium falciparum screening revealed eucalrobusone C as the most potent antiplasmodial formyl phloroglucinol to date.

Inferring the multiplicity of founder variants initiating HIV-1 infection: a systematic review and individual patient data meta-analysis.

BACKGROUND: HIV-1 infections initiated by multiple founder variants are characterised by a higher viral load and a worse clinical prognosis than those initiated with single founder variants, yet little is known about the routes of exposure through which transmission of multiple founder variants is most probable. Here we used individual patient data to calculate the probability of multiple founders stratified by route of HIV exposure and study methodology. METHODS: We conducted a systematic review and meta-analysis of studies that estimated founder variant multiplicity in HIV-1 infection, searching MEDLINE, Embase, and Global Health databases for papers published between Jan 1, 1990, and Sept 14, 2020. Eligible studies must have reported original estimates of founder variant multiplicity in people with acute or early HIV-1 infections, have clearly detailed the methods used, and reported the route of exposure. Studies were excluded if they reported data concerning people living with HIV-1 who had known or suspected superinfection, who were documented as having received pre-exposure prophylaxis, or if the transmitting partner was known to be receiving antiretroviral treatment. Individual patient data were collated from all studies, with authors contacted if these data were not publicly available. We applied logistic meta-regression to these data to estimate the probability that an HIV infection is initiated by multiple founder variants. We calculated a pooled estimate using a random effects model, subsequently stratifying this estimate across exposure routes in a univariable analysis. We then extended our model to adjust for different study methods in a multivariable analysis, recalculating estimates across the exposure routes. This study is registered with PROSPERO, CRD42020202672. FINDINGS: We included 70 publications in our analysis, comprising 1657 individual patients. Our pooled estimate of the probability that an infection is initiated by multiple founder variants was 0·25 (95% CI 0·21-0·29), with moderate heterogeneity (Q=132·3, p<0·0001, I2=64·2%). Our multivariable analysis uncovered differences in the probability of multiple variant infection by exposure route. Relative to a baseline of male-to-female transmission, the predicted probability for female-to-male multiple variant transmission was significantly lower at 0·13 (95% CI 0·08-0·20), and the probabilities were significantly higher for transmissions in people who inject drugs (0·37 [0·24-0·53]) and men who have sex with men (0·30 [0·33-0·40]). There was no significant difference in the probability of multiple variant transmission between male-to-female transmission (0·21 [0·14-0·31]), post-partum transmission (0·18 [0·03-0·57]), pre-partum transmission (0·17 [0·08-0·33]), and intra-partum transmission (0·27 [0·14-0·45]). INTERPRETATION: We identified that transmissions in people who inject drugs and men who have sex with men are significantly more likely to result in an infection initiated by multiple founder variants, and female-to-male infections are significantly less probable. Quantifying how the routes of HIV infection affect the transmission of multiple variants allows us to better understand how the evolution and epidemiology of HIV-1 determine clinical outcomes. FUNDING: Medical Research Council Precision Medicine Doctoral Training Programme and a European Research Council Starting Grant.

Observation of Cation Chromophore Photoisomerization of a Fluorescent Protein Using Millisecond Synchrotron Serial Crystallography and Infrared Vibrational and Visible Spectroscopy.

The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm-1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.

Light activation of Orange Carotenoid Protein reveals bicycle-pedal single-bond isomerization.

Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9'-C8' and C7'-C6' single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.

Using phylogenetics to infer HIV-1 transmission direction between known transmission pairs.

Inferring the transmission direction between linked individuals living with HIV provides unparalleled power to understand the epidemiology that determines transmission. Phylogenetic ancestral-state reconstruction approaches infer the transmission direction by identifying the individual in whom the most recent common ancestor of the virus populations originated. While these methods vary in accuracy, it is unclear why. To evaluate the performance of phylogenetic ancestral-state reconstruction to determine the transmission direction of HIV-1 infection, we inferred the transmission direction for 112 transmission pairs where transmission direction and detailed additional information were available. We then fit a statistical model to evaluate the extent to which epidemiological, sampling, genetic, and phylogenetic factors influenced the outcome of the inference. Finally, we repeated the analysis under real-life conditions with only routinely available data. We found that whether ancestral-state reconstruction correctly infers the transmission direction depends principally on the phylogeny's topology. For example, under real-life conditions, the probability of identifying the correct transmission direction increases from 32%-when a monophyletic-monophyletic or paraphyletic-polyphyletic tree topology is observed and when the tip closest to the root does not agree with the state at the root-to 93% when a paraphyletic-monophyletic topology is observed and when the tip closest to the root agrees with the root state. Our results suggest that documenting larger differences in relative intrahost diversity increases our confidence in the transmission direction inference of linked pairs for population-level studies of HIV. These findings provide a practical starting point to determine our confidence in transmission direction inference from ancestral-state reconstruction.

The smfBox is an open-source platform for single-molecule FRET.

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET's broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist.

We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.

Functional expression of α7-nicotinic acetylcholine receptors by muscle afferent neurons.

The exercise pressor reflex (EPR) is generated by group III and IV muscle afferents during exercise to increase cardiovascular function. Muscle contraction is triggered by ACh, which is metabolized into choline that could serve as a signal of exercise-induced activity. We demonstrate that ACh can induce current in muscle afferents neurons isolated from male Sprague-Dawley rats. The nicotinic ACh receptors (nAChRs) appear to be expressed by some group III-IV neurons since capsaicin (TRPV1) and/or ATP (P2X) induced current in 56% of ACh-responsive neurons. α7- And α4ß2-nAChRs have been shown to be expressed in sensory neurons. An α7-nAChR antibody stained 83% of muscle afferent neurons. Functional expression was demonstrated by using the specific α7-nAChR blockers α-conotoxin ImI (IMI) and methyllycaconitine (MLA). MLA inhibited ACh responses in 100% of muscle afferent neurons, whereas IMI inhibited ACh responses in 54% of neurons. Dihydro-ß-erythroidine, an α4ß2-nAChR blocker, inhibited ACh responses in 50% of muscle afferent neurons, but recovery from block was not observed. Choline, an α7-nAChR agonist, elicited a response in 60% of ACh-responsive neurons. Finally, we demonstrated the expression of α7-nAChR by peripherin labeled (group IV) afferent fibers within gastrocnemius muscles. Some of these α7-nAChR-positive fibers were also positive for P2X3 receptors. Thus choline could serve as an activator of the EPR by opening α7-nAChR expressed by group IV (and possible group III) afferents. nAChRs could become pharmacological targets for suppressing the excessive EPR activation in patients with peripheral vascular disease.

NaV1.8 channels are expressed in large, as well as small, diameter sensory afferent neurons.

Sensory neurons in the dorsal root ganglia (DRG) express a subset of voltage dependent sodium channels (NaV) including NaV1.1, 1.6, 1.7, 1.8 and 1.9. Previous work supported preferential localization of NaV1.8 channels to small-medium diameter, nociceptive afferent neurons. However, we recently published evidence that NaV1.8 was the dominant NaV channel expressed in the somas of small, medium and large diameter muscle afferent neurons, which is consistent with other reports. Here, we extend those results to show that NaV1.8 expression is not correlated with afferent neuron diameter. Using immunocytochemistry, we found NaV1.8 expression in ~50% of sensory afferent neurons with diameters ranging from 20 to 70 µm. In addition, electrophysiological analysis shows that the kinetic and inactivation properties of NaV1.8 current are invariant with neuron size. These data add further support to the idea that NaV1.8 contributes to the electrical excitability of both nociceptive and non-nociceptive sensory neurons.

Tetrodotoxin-resistant voltage-dependent sodium channels in identified muscle afferent neurons.

Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (Na(V)) channels. In patch-clamp recordings, we found that the dominant Na(V) current in the majority of identified neurons was insensitive to tetrodotoxin (TTX-R), with Na(V) current in only a few (14%) neurons showing substantial (>50%) TTX sensitivity (TTX-S). The TTX-R current was sensitive to a Na(V)1.8 channel blocker, A803467. Immunocytochemistry demonstrated labeling of muscle afferent neurons by a Na(V)1.8 antibody, which further supported expression of these channels. A portion of the TTX-R Na(V) current appeared to be noninactivating during our 25-ms voltage steps, which suggested activity of Na(V)1.9 channels. The majority of the noninactivating current was insensitive to A803467 but sensitive to extracellular sodium. Immunocytochemistry showed labeling of muscle afferent neurons by a Na(V)1.9 channel antibody, which supports expression of these channels. Further examination of the muscle afferent neurons showed that functional TTX-S channels were expressed, but were largely inactivated at physiological membrane potentials. Immunocytochemistry showed expression of the TTX-S channels Na(V)1.6 and Na(V)1.7 but not Na(V)1.1. Na(V)1.8 and Na(V)1.9 appear to be the dominant functional sodium channels in small- to medium-diameter muscle afferent neurons. The expression of these channels is consistent with the identification of these neurons as Group III and IV, which mediate the exercise pressor reflex.

Interrogative pressure in simulated forensic interviews: the effects of negative feedback.

Much experimental research on interrogative pressure has concentrated on the effects of leading questions, and the role of feedback in influencing responses in the absence of leading questions has been neglected by comparison. This study assessed the effect of negative feedback and the presence of a second interviewer on interviewee responding in simulated forensic interviews. Participants viewed a videotape of a crime, answered questions about the clip and were requestioned after receiving feedback. Compared with neutral feedback, negative feedback resulted in more response changes, higher reported state anxiety and higher ratings of interview difficulty. These results are consistent with Gudjonsson and Clark's (1986) model of interrogative suggestibility. The presence and involvement of a second interviewer did not significantly affect interviewee responding, although trait anxiety scores were elevated when a second interviewer was present. The theoretical and applied implications of these findings are considered.

Phosphorus source alters host plant response to ectomycorrhizal diversity.

We examined the influence of phosphorus source and availability on host plant (Pinus rigida) response to ectomycorrhizal diversity under contrasting P conditions. An ectomycorrhizal richness gradient was established with equimolar P supplied as either inorganic phosphate or organic inositol hexaphosphate. We measured growth and N and P uptake of individual P. rigida seedlings inoculated with one, two, or four species of ectomycorrhizal fungi simultaneously and without mycorrhizas in axenic culture. Whereas colonization of P. rigida by individual species of ectomycorrhizal fungi decreased with increasing fungal richness, colonization of all species combined increased. Plant biomass and N content increased across the ectomycorrhizal richness gradient in the organic but not the inorganic P treatment. Plants grown under organic P conditions had higher N concentration than those grown under inorganic P conditions, but there was no effect of richness. Phosphorus content of plants grown in the organic P treatment increased with increasing ectomycorrhizal richness, but there was no response in the inorganic P treatment. Phosphorus concentration was higher in plants grown at the four-species richness level in the organic P treatment, but there was no effect of diversity under inorganic P conditions. Overall, few ectomycorrhizal composition effects were found on plant growth or nutrient status. Phosphatase activities of individual ectomycorrhizal fungi differed under organic P conditions, but there was no difference in total root system phosphatase expression between the inorganic or organic P treatments or across richness levels. Our results provide evidence that plant response to ectomycorrhizal diversity is dependent on the source and availability of P.

Otitis media and hearing loss among 12-16-year-old Inuit of Inukjuak, Quebec, Canada.

OBJECTIVES: Chronic otitis media (COM) and associated hearing loss is a frequent problem for many Inuit children in Canada. In this study, we evaluated individuals aged 12-16 years living in Inukjuak, to determine the prevalence of middle ear disease and hearing loss, and the effect of hearing loss on academic performance. METHODS: Otological examination, hearing test, medical and school file review were performed in November 1997. 88 individuals were seen. RESULTS: Otological examination revealed maximal scarring in 1.8%, minimal scarring in 34.9%, normal eardrums in 49.1% and chronic otitis media in 16.9%. There were 62 individuals whose ear exams could be directly compared with a previous exam done in 1987. Of those, there were three ears that had developed COM and 4/13 ears with COM in 1987 that had healed. Hearing tests found bilateral normal hearing in 80% (PTA <20dB), unilateral loss in 15% and bilateral loss in 5%. Hearing loss was associated with poorer academic performance in Language (p<.05). A similar trend was found in Mathematics but not in Inuttitut. CONCLUSION: Chronic otitis media remains a significant problem among the Inuit, with a prevalence of 16.9% in individuals aged 12-16 years. One in five in this age group has hearing loss, and this hearing loss impacts on academic performance.