Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Morphology, behavior, and phylogenomics of Oxytoxum lohmannii, Dinoflagellata.

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair.

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.

Doppler Endoscopic Probe Monitoring of Blood Flow Improves Risk Stratification and Outcomes of Patients With Severe Nonvariceal Upper Gastrointestinal Hemorrhage.

BACKGROUND & AIMS: For 4 decades, stigmata of recent hemorrhage in patients with nonvariceal lesions have been used for risk stratification and endoscopic hemostasis. The arterial blood flow that underlies the stigmata rarely is monitored, but can be used to determine risk for rebleeding. We performed a randomized controlled trial to determine whether Doppler endoscopic probe monitoring of blood flow improves risk stratification and outcomes in patients with severe nonvariceal upper gastrointestinal hemorrhage. METHODS: In a single-blind study performed at 2 referral centers we assigned 148 patients with severe nonvariceal upper gastrointestinal bleeding (125 with ulcers, 19 with Dieulafoy's lesions, and 4 with Mallory Weiss tears) to groups that underwent standard, visually guided endoscopic hemostasis (control, n = 76), or endoscopic hemostasis assisted by Doppler monitoring of blood flow under the stigmata (n = 72). The primary outcome was the rate of rebleeding after 30 days; secondary outcomes were complications, death, and need for transfusions, surgery, or angiography. RESULTS: There was a significant difference in the rates of lesion rebleeding within 30 days of endoscopic hemostasis in the control group (26.3%) vs the Doppler group (11.1%) (P = .0214). The odds ratio for rebleeding with Doppler monitoring was 0.35 (95% confidence interval, 0.143-0.8565) and the number needed to treat was 7. CONCLUSIONS: In a randomized controlled trial of patients with severe upper gastrointestinal hemorrhage from ulcers or other lesions, Doppler probe guided endoscopic hemostasis significantly reduced 30-day rates of rebleeding compared with standard, visually guided hemostasis. Guidelines for nonvariceal gastrointestinal bleeding should incorporate these results. ClinicalTrials.gov no: NCT00732212 (CLIN-013-07F).

Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.

Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.

Natural history of definitive diverticular hemorrhage based on stigmata of recent hemorrhage and colonoscopic Doppler blood flow monitoring for risk stratification and definitive hemostasis.

BACKGROUND AND AIMS: Few prospective reports describe the short-term natural history of colon diverticular hemorrhage based on stigmata of recent hemorrhage, and none include blood flow detection for risk stratification or as a guide to definitive hemostasis. Our purposes were to report the 30-day natural history of definitive diverticular hemorrhage based on stigmata and to describe Doppler probe blood flow detection as a guide to definitive hemostasis. METHODS: Different cohorts of patients with severe diverticular bleeding and stigmata on urgent colonoscopy are reported. For 30-day natural history, patients were treated medically. If severe rebleeding occurred, they had surgical or angiographic treatment. We report natural history with major stigmata (active bleeding, visible vessel, or adherent clot) and no stigmata or flat spots after clots were washed away. We also report Doppler probe detection of arterial blood flow underneath stigmata before and after hemostasis in a recent cohort. RESULTS: For natural history, patients with major stigmata treated medically had 65.8% (25/38) rebleeding rates, and 44.7% (17/38) had intervention for hemostasis. Patients with spots or clean bases had no rebleeding. A Doppler probe detected arterial blood flow in 92% of major stigmata--none after hemostasis--and there was no rebleeding. CONCLUSIONS: (1) Patients with major stigmata treated medically had high rates of rebleeding and intervention for hemostasis. (2) Patients with clean diverticula or only flat spots had no rebleeding. (3) High rates of arterial blood flow were detected under major stigmata with a Doppler probe, but with obliteration by hemostasis no rebleeding occurred.

Doppler endoscopic probe as a guide to risk stratification and definitive hemostasis of peptic ulcer bleeding.

BACKGROUND AND AIMS: For more than 4 decades endoscopists have relied on ulcer stigmata for risk stratification and as a guide to hemostasis. None used arterial blood flow underneath stigmata to predict outcomes. For patients with severe peptic ulcer bleeding (PUB), we used a Doppler endoscopic probe (DEP) for (1) detection of blood flow underlying stigmata of recent hemorrhage (SRH), (2) quantitating rates of residual arterial blood flow under SRH after visually directed standard endoscopic treatment, and (3) comparing risks of rebleeding and actual 30-day rebleed rates for spurting arterial bleeding (Forrest [F] IA) and oozing bleeding (F IB). METHODS: Prospective cohort study of 163 consecutive patients with severe PUB and different SRH. RESULTS: All blood flow detected by the DEP was arterial. Detection rates were 87.4% in major SRH-spurting arterial bleeding (F IA), non-bleeding visible vessel (F IIA), clot (F IIB)-and were significantly lower at 42.3% (P < .0001) for an intermediate group of oozing bleeding (F IB) or flat spot (F IIC). For spurting bleeding (F IA) versus oozing (F IB), baseline DEP arterial flow was 100% versus 46.7%, residual blood flow detected after endoscopic hemostasis was 35.7% versus 0%, and 30-day rebleed rates were 28.6% versus 0% (all P < .05). CONCLUSIONS: (1) For major SRH versus oozing or spot, the arterial blood flow detection rate by the DEP was significantly higher, indicating a higher rebleed risk. (2) Before and after endoscopic treatment, spurting (F IA) PUB had significantly higher rates of blood flow detection than oozing (F IB) PUB and a significantly higher 30-day rebleed rate. (3) The DEP is recommended as a new endoscopic guide with SRH to improve risk stratification and potentially definitive hemostasis for PUB.

Feast or flee: bioelectrical regulation of feeding and predator evasion behaviors in the planktonic alveolate Favella sp. (Spirotrichia).

Alveolate (ciliates and dinoflagellates) grazers are integral components of the marine food web and must therefore be able to sense a range of mechanical and chemical signals produced by prey and predators, integrating them via signal transduction mechanisms to respond with effective prey capture and predator evasion behaviors. However, the sensory biology of alveolate grazers is poorly understood. Using novel techniques that combine electrophysiological measurements and high-speed videomicroscopy, we investigated the sensory biology of Favella sp., a model alveolate grazer, in the context of its trophic ecology. Favella sp. produced frequent rhythmic depolarizations (∼500 ms long) that caused backward swimming and are responsible for endogenous swimming patterns relevant to foraging. Contact of both prey cells and non-prey polystyrene microspheres at the cilia produced immediate mechanostimulated depolarizations (∼500 ms long) that caused backward swimming, and likely underlie aggregative swimming patterns of Favella sp. in response to patches of prey. Contact of particles at the peristomal cavity that were not suitable for ingestion resulted in depolarizations after a lag of ∼600 ms, allowing time for particles to be processed before rejection. Ingestion of preferred prey particles was accompanied by transient hyperpolarizations (∼1 s) that likely regulate this step of the feeding process. Predation attempts by the copepod Acartia tonsa elicited fast (∼20 ms) animal-like action potentials accompanied by rapid contraction of the cell to avoid predation. We have shown that the sensory mechanisms of Favella sp. are finely tuned to the type, location, and intensity of stimuli from prey and predators.

Comparison of Three Risk Scores to Predict Outcomes of Severe Lower Gastrointestinal Bleeding.

BACKGROUND AND AIMS: Improved medical decisions by using a score at the initial patient triage level may lead to improvements in patient management, outcomes, and resource utilization. There is no validated score for management of lower gastrointestinal bleeding (LGIB) unlike for upper gastrointestinal bleeding. The aim of our study was to compare the accuracies of 3 different prognostic scores [Center for Ulcer Research and Education Hemostasis prognosis score, Charlson index, and American Society of Anesthesiologists (ASA) score] for the prediction of 30-day rebleeding, surgery, and death in severe LGIB. METHODS: Data on consecutive patients hospitalized with severe gastrointestinal bleeding from January 2006 to October 2011 in our 2 tertiary academic referral centers were prospectively collected. Sensitivities, specificities, accuracies, and area under the receiver operator characteristic curve were computed for 3 scores for predictions of rebleeding, surgery, and mortality at 30 days. RESULTS: Two hundred thirty-five consecutive patients with LGIB were included between 2006 and 2011. Twenty-three percent of patients rebled, 6% had surgery, and 7.7% of patients died. The accuracies of each score never reached 70% for predicting rebleeding or surgery in either. The ASA score had a highest accuracy for predicting mortality within 30 days (83.5%), whereas the Center for Ulcer Research and Education Hemostasis prognosis score and the Charlson index both had accuracies <75% for the prediction of death within 30 days. CONCLUSIONS: ASA score could be useful to predict death within 30 days. However, a new score is still warranted to predict all 30 days outcomes (rebleeding, surgery, and death) in LGIB.

Origin, Clinical Characteristics and 30-Day Outcomes of Severe Hematochezia in Cirrhotics and Non-cirrhotics.

BACKGROUND: The sites of origin, causes and outcomes of severe hematochezia have not been compared between cirrhotics and non-cirrhotics. In cirrhotics versus non-cirrhotics presenting with severe hematochezia, we aimed at (1) identifying the site and etiology of gastro-intestinal bleeding and independent predictors of bleeding from the upper gastrointestinal tract versus small bowel or the colon, (2) comparing 30-day clinical outcomes, and (3) proposing an algorithm for management of severe hematochezia. METHODS: In this cohort study from two university-based medical centers, 860 consecutive patients with severe hematochezia admitted from 1995 to 2011 were prospectively enrolled with 160 (18.6 %) cirrhotics. We studied (a) general clinical and laboratory characteristics of cirrhotics versus non-cirrhotics, (b) predictors of bleeding sites in each patient group by multiple variable regression analysis, and compared (c) 30-day outcomes, including rebleeding, surgery and deaths. RESULTS: Cirrhosis independently predicted an upper gastrointestinal source of bleeding (OR 3.47; 95 % CI 2.01-5.96) as well as history of hematemesis, melena in the past 30 days, positive nasogastric aspirate, prior upper gastrointestinal bleeding or use of aspirin or non-steroidal anti-inflammatory. The most prevalent diagnoses were esophageal varices (20 %) in cirrhotics and colon diverticular bleeding (27.1 %) in non-cirrhotics. Thirty-day rates of rebleeding, surgical interventions and deaths were 23.1 versus 15 % (P = 0.01), 14.4 versus 6.4 % (P < 0.001), and 17.5 versus 4.1 % (P < 0.001), in cirrhotics versus non-cirrhotics, respectively. CONCLUSIONS: Cirrhosis predicted an upper gastrointestinal site of bleeding in patients presenting with severe hematochezia. The 30-day rates of rebleeding, surgery, and death were significantly higher in cirrhotics than in non-cirrhotics.

Report from the 17th Annual Western Canadian Gastrointestinal Cancer Consensus Conference; Edmonton, Alberta; 11-12 September 2015.

The 17th annual Western Canadian Gastrointestinal Cancer Consensus Conference (wcgccc) was held in Edmonton, Alberta, 11-12 September 2015. The wcgccc is an interactive multidisciplinary conference attended by health care professionals from across Western Canada (British Columbia, Alberta, Saskatchewan, and Manitoba) who are involved in the care of patients with gastrointestinal cancer. Surgical, medical, and radiation oncologists; pathologists; radiologists; and allied health care professionals participated in presentation and discussion sessions for the purposes of developing the recommendations presented here. This consensus statement addresses current issues in the management of gastric cancer.

The Food Web of Boiling Springs Lake Appears Dominated by the Heterolobosean Tetramitus thermacidophilus Strain BSL.

We studied the protist grazers of Boiling Springs Lake (BSL), an acid geothermal feature in Lassen Volcanic National Park, using a combination of culture and genetic approaches. The major predator in BSL is a vahlkampfiid ameba closely related (95% 18S+ITS rRNA identity) to Tetramitus thermacidophilus, a heterolobose ameboflagellate recently isolated from volcanic geothermal acidic sites in Europe and Russia, as well as an uncultured heterolobosean from the nearby Iron Mountain acid mine drainage site. Tetramitus thermacidophilus strain BSL is capable of surviving the physical extremes of BSL, with optimal growth at 38-50 °C and pH 2-5. This bacterivore also ingested conidiospores of the ascomycete Phialophora sp., but ultrastructural observations reveal the latter may not be readily digested, and conidia were not separable from the ameoboflagellate culture, suggesting a possible symbiosis. DGGE fingerprint transects studies showed the organism is restricted to near-lake environs, and we detected an average of ~500 viable cysts/cm(3) sediment on the shoreline. Other grazing protists were isolated from lakeshore environments, including the lobose amebae Acanthamoeba sp. and Hartmannella sp., and the kinetoplastid flagellate Bodo sp., but none could tolerate both low pH and high temperature. These appear to be restricted to cooler near lake geothermal features, which also contain other potential grazer morphotypes observed but not successfully cultured, including ciliates, euglenids, testate amebae, and possible cercozoans. We compare the food web of BSL with other acidic or geothermal sites, and discuss the impact of protists in this unique environment.

Specific adhesion of membranes simultaneously supports dual heterogeneities in lipids and proteins.

Membrane adhesion is a vital component of many biological processes. Heterogeneities in lipid and protein composition are often associated with the adhesion site. These heterogeneities are thought to play functional roles in facilitating signalling. Here we experimentally examine this phenomenon using model membranes made of a mixture of lipids that is near a phase boundary at room temperature. Non-adherent model membranes are in a well-mixed, disordered-fluid lipid phase indicated by homogeneous distribution of a fluorescent dye that is a marker for the fluid-disordered (Ld) phase. We specifically adhere membranes to a flat substrate bilayer using biotin-avidin binding. Adhesion produces two types of coexisting heterogeneities: an ordered lipid phase that excludes binding proteins and the fluorescent membrane dye, and a disordered lipid phase that is enriched in both binding proteins and membrane dye compared with the non-adhered portion of the same membrane. Thus, a single type of adhesion interaction (biotin-avidin binding), in an initially-homogeneous system, simultaneously stabilizes both ordered-phase and disordered-phase heterogeneities that are compositionally distinct from the non-adhered portion of the vesicle. These heterogeneities are long-lived and unchanged upon increased temperature.

Membrane adhesion and the formation of heterogeneities: biology, biophysics, and biotechnology.

Membrane adhesion is essential to many vital biological processes. Sites of membrane adhesion are often associated with heterogeneities in the lipid and protein composition of the membrane. These heterogeneities are thought to play functional roles by facilitating interactions between proteins. However, the causal links between membrane adhesion and membrane heterogeneities are not known. Here we survey the state of the field and indicate what we think are understudied areas ripe for development.

Consensus statement: the 16th Annual Western Canadian Gastrointestinal Cancer Consensus Conference; Saskatoon, Saskatchewan; September 5-6, 2014.

The 16th annual Western Canadian Gastrointestinal Cancer Consensus Conference was held in Saskatoon, Saskatchewan, September 4-5, 2014. The Consensus Conference is an interactive, multidisciplinary event attended by health care professionals from across western Canada (British Columbia, Alberta, Saskatchewan, and Manitoba) involved in the care of gastrointestinal cancer. Surgical, medical, and radiation oncologists; pathologists; radiologists; and allied health care professionals participated in presentation and discussion sessions for the purposes of developing the recommendations presented here. This consensus statement addresses current issues in the management of colorectal cancer.

Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells.

The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.

Recent innovations in long-term care coverage and financing: a rapid scoping review.

OBJECTIVES: To identify, chart and analyse the literature on recent initiatives to improve long-term care (LTC) coverage, financial protection and financial sustainability for persons aged 60 and older. DESIGN: Rapid scoping review. DATA SOURCES: Four databases and four sources of grey literature were searched for reports published between 2017 and 2022. After using a supervised machine learning tool to rank titles and abstracts, two reviewers independently screened sources against inclusion criteria. ELIGIBILITY CRITERIA: Studies published from 2017-2022 in any language that captured recent LTC initiatives for people aged 60 and older, involved evaluation and directly addressed financing were included. DATA EXTRACTION AND ANALYSIS: Data were extracted using a form designed to answer the review questions and analysed using descriptive qualitative content analysis, with data categorised according to a prespecified framework to capture the outcomes of interest. RESULTS: Of 24 reports, 22 were published in peer-reviewed journals, and two were grey literature sources. Study designs included quasi-experimental study, policy analysis or comparison, qualitative description, comparative case study, cross-sectional study, systematic literature review, economic evaluation and survey. Studies addressed coverage based on the level of disability, income, rural/urban residence, employment and citizenship. Studies also addressed financial protection, including out-of-pocket (OOP) expenditures, copayments and risk of poverty related to costs of care. The reports addressed challenges to financial sustainability such as lack of service coordination and system integration, insufficient economic development and inadequate funding models. CONCLUSIONS: Initiatives where LTC insurance is mandatory and accompanied by commensurate funding are situated to facilitate ageing in place. Efforts to expand population coverage are common across the initiatives, with the potential for wider economic benefits. Initiatives that enable older people to access the services needed while avoiding OOP-induced poverty contribute to improved health and well-being. Preserving health in older people longer may alleviate downstream costs and contribute to financial sustainability.

Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings.

Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E. coli and mammalian cells.