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Time-resolved crystallography reveals how bacteriorhodopsin uses light to drive chemistry Absorption of a photon in the visible region of the solar spectrum is the fundamental step in the photochemical reactions that drive biological processes such as imaging, photosynthesis, and sensory perception. All known photoreceptors are proteins that contain a chromophore: a small organic molecule that absorbs in the visible region of the spectrum ( 1 ). The bacterial integral membrane protein bacteriorhodopsin (bR) uses retinal as its chromophore (see the figure), capturing the energy in a photon to drive retinal isomerization and pump protons across the membrane. bR is a readily accessible analog to the visual rhodopsins in, for example, the human eye. On page 145 of this issue, Nogly et al. ( 2 ) used time-resolved x-ray crystallography ( 3 , 4 ) to determine intermediate structures of bR at near-atomic resolution on the femtosecond to picosecond time scales at which isomerization occurs.

Background A common challenge for randomised controlled trials (RCTs) is recruiting enough participants to be adequately powered to answer the research question. Recruitment has been set as a priority research area in trials to improve recruitment and thereby reduce wasted resources in conducted trials that fail to recruit sufficiently. Methods We conducted a systematic mixed studies review to identify the factors associated with recruitment to RCTs in general practice. On September 8, 2020, English language studies were identified from MEDLINE, EMBASE, Cochrane Database of Systematic Reviews and CENTRAL databases for published studies. NTIS and OpenGrey were searched for grey literature, and BMC Trials was hand searched. A narrative synthesis was conducted for qualitative studies and a thematic synthesis for qualitative studies. Results Thirty-seven studies met the inclusion criteria. These were of different study types (10 cross-sectional, 5 non-randomised studies of interventions, 2 RCTs, 10 qualitative and 10 mixed methods). The highest proportion was conducted in the UK (48%). The study quality was generally poor with 24 (65%) studies having major concerns. A complex combination of patient, practitioner or practice factors, and patient, practitioner or practice recruitment were assessed to determine the possible associations. There were more studies of patients than of practices or practitioners. Conclusions For practitioners and patients alike, a trial that is clinically relevant is critical in influencing participation. Competing demands are given as an important reason for declining participation. There are concerns about randomisation relating to its impact on shared decision-making and not knowing which treatment will be assigned. Patients make decisions about whether they are a candidate for the trial even when they objectively fulfil the eligibility criteria. General practice processes, such as difficulties arranging appointments, can hinder recruitment, and a strong pre-existing doctor-patient relationship can improve recruitment. For clinicians, the wish to contribute to the research enterprise itself is seldom an important reason for participating, though clinicians reported being motivated to participate when the research could improve their clinical practice. One of the few experimental findings was that opportunistic recruitment resulted in significantly faster recruitment compared to systematic recruitment. These factors have clear implications for trial design. Methodologically, recruitment research of practices and practitioners should have increased priority. Higher quality studies of recruitment are required to find out what actually works rather than what might work. Trial registration PROSPERO CRD42018100695. Registered on 03 July 2018.

The management of people with multiple chronic conditions challenges health-care systems designed around single conditions. There is international consensus that care for multimorbidity should be patient-centred, focus on quality of life, and promote self-management towards agreed goals. However, there is little evidence about the effectiveness of this approach. Our hypothesis was that the patient-centred, so-called 3D approach (based on dimensions of health, depression, and drugs) for patients with multimorbidity would improve their health-related quality of life, which is the ultimate aim of the 3D intervention. We did this pragmatic cluster-randomised trial in general practices in England and Scotland. Practices were randomly allocated to continue usual care (17 practices) or to provide 6-monthly comprehensive 3D reviews, incorporating patient-centred strategies that reflected international consensus on best care (16 practices). Randomisation was computer-generated, stratified by area, and minimised by practice deprivation and list size. Adults with three or more chronic conditions were recruited. The primary outcome was quality of life (assessed with EQ-5D-5L) after 15 months' follow-up. Participants were not masked to group assignment, but analysis of outcomes was blinded. We analysed the primary outcome in the intention-to-treat population, with missing data being multiply imputed. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN06180958. Between May 20, 2015, and Dec 31, 2015, we recruited 1546 patients from 33 practices and randomly assigned them to receive the intervention (n=797) or usual care (n=749). In our intention-to-treat analysis, there was no difference between trial groups in the primary outcome of quality of life (adjusted difference in mean EQ-5D-5L 0·00, 95% CI −0·02 to 0·02; p=0·93). 78 patients died, and the deaths were not considered as related to the intervention. To our knowledge, this trial is the largest investigation of the international consensus about optimal management of multimorbidity. The 3D intervention did not improve patients' quality of life. National Institute for Health Research.

How bacteria see red Phytochromes are photoreceptors that regulate light responses in plants, fungi and bacteria through reversible photoconversion between red and far-red light-absorbing states. Using temperature-scanning cryocrystallography, Yang et al . obtain structural information about three intermediates and key conformational changes that occur during the photoreaction of a bacteriophytochrome from Pseudomonas aeruginosa . Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria 1 . They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses 2 . Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states 3 , 4 , 5 , 6 , 7 . However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E -to- Z isomerization about the C 15 = C 16 double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C 15 methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 Å resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an \"arm\" structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common and may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.

An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an α-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical \"allosteric lever arm\" is a general scheme for coupling the function of two proteins.

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecuies. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a \"flip-and-rotate\" model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.

ObjectiveTo synthesise international evidence for demand, use and outcomes of primary care out-of-hours health services (OOHS).DesignSystematic scoping review.Data sourcesCINAHL; Medline; PsyARTICLES; PsycINFO; SocINDEX; and Embase from 1995 to 2019.Study selectionEnglish language studies in UK or similar international settings, focused on services in or directly impacting primary care.Results105 studies included: 54% from mainland Europe/Republic of Ireland; 37% from UK. Most focused on general practitioner-led out-of-hours cooperatives. Evidence for increasing patient demand over time was weak due to data heterogeneity, infrequent reporting of population denominators and little adjustment for population sociodemographics. There was consistent evidence of higher OOHS use in the evening compared with overnight, at weekends and by certain groups (children aged <5, adults aged >65, women, those from socioeconomically deprived areas, with chronic diseases or mental health problems). Contact with OOHS was driven by problems perceived as urgent by patients. Respiratory, musculoskeletal, skin and abdominal symptoms were the most common reasons for contact in adults; fever and gastrointestinal symptoms were the most common in the under-5s. Frequent users of daytime services were also frequent OOHS users; difficulty accessing daytime services was also associated with OOHS use. There is some evidence to suggest that OOHS colocated in emergency departments (ED) can reduce demand in EDs.ConclusionsPolicy changes have impacted on OOHS over the past two decades. While there are generalisable lessons, a lack of comparable data makes it difficult to judge how demand has changed over time. Agreement on collection of OOHS data would allow robust comparisons within and across countries and across new models of care. Future developments in OOHS should also pay more attention to the relationship with daytime primary care and other services.PROSPERO registration numberCRD42015029741.

Trans -to- cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p -coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (I T ) resembles a highly strained transition state in which the torsion angle is located halfway between the trans - and cis- isomers. The reaction trajectory of I T bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway. Time-resolved X-ray crystallography on photoactive yellow protein shows the existence of a short-lived, highly distorted intermediate whose reaction trajectory bifurcates along ‘bicycle-pedal’ and ‘hula-twist’ pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue, which switches off the bicycle-pedal pathway.