Discovery and Quantification of Long-Range RNA Base Pairs in Coronavirus Genomes with SEARCH-MaP and SEISMIC-RNA.

RNA molecules perform a diversity of essential functions for which their linear sequences must fold into higher-order structures. Techniques including crystallography and cryogenic electron microscopy have revealed 3D structures of ribosomal, transfer, and other well-structured RNAs; while chemical probing with sequencing facilitates secondary structure modeling of any RNAs of interest, even within cells. Ongoing efforts continue increasing the accuracy, resolution, and ability to distinguish coexisting alternative structures. However, no method can discover and quantify alternative structures with base pairs spanning arbitrarily long distances - an obstacle for studying viral, messenger, and long noncoding RNAs, which may form long-range base pairs. Here, we introduce the method of Structure Ensemble Ablation by Reverse Complement Hybridization with Mutational Profiling (SEARCH-MaP) and software for Structure Ensemble Inference by Sequencing, Mutation Identification, and Clustering of RNA (SEISMIC-RNA). We use SEARCH-MaP and SEISMIC-RNA to discover that the frameshift stimulating element of SARS coronavirus 2 base-pairs with another element 1 kilobase downstream in nearly half of RNA molecules, and that this structure competes with a pseudoknot that stimulates ribosomal frameshifting. Moreover, we identify long-range base pairs involving the frameshift stimulating element in other coronaviruses including SARS coronavirus 1 and transmissible gastroenteritis virus, and model the full genomic secondary structure of the latter. These findings suggest that long-range base pairs are common in coronaviruses and may regulate ribosomal frameshifting, which is essential for viral RNA synthesis. We anticipate that SEARCH-MaP will enable solving many RNA structure ensembles that have eluded characterization, thereby enhancing our general understanding of RNA structures and their functions. SEISMIC-RNA, software for analyzing mutational profiling data at any scale, could power future studies on RNA structure and is available on GitHub and the Python Package Index.

Baked sweetpotato textures and sweetness: An investigation into relationships between physicochemical and cooked attributes.

Sweetpotato varieties vary greatly in perceived textures and sweetness. This study identified physicochemical factors that influence these attributes in cooked sweetpotatoes. Fifteen genotypes grown on three plots were baked and evaluated by a trained descriptive sensory analysis panel for sweetness and 13 texture attributes. Mechanical parameters were measured by texture profile analysis (TPA); and composition (starch, cell wall material, sugar contents), starch properties (thermal, granule type ratios, granule sizes), and amylase activities were characterized. TPA predicted fracturability and firmness well, whereas starch and sugar contents, B-type starch granule ratio, and amylase activities influenced prediction of mouthfeel textures. Sweetness perception was influenced by perceived particle size and sugar contents; and maltose generation during baking was highly correlated with raw sweetpotato starch content. These relationships between physicochemical sweetpotato properties and baked textures and sweetness could benefit breeders and processors in selecting biochemical traits that result in consumer preferred products.

RNA levers and switches controlling viral gene expression.

RNA viruses are diverse and abundant pathogens that are responsible for numerous human diseases. RNA viruses possess relatively compact genomes and have therefore evolved multiple mechanisms to maximize their coding capacities, often by encoding overlapping reading frames. These reading frames are then decoded by mechanisms such as alternative splicing and ribosomal frameshifting to produce multiple distinct proteins. These solutions are enabled by the ability of the RNA genome to fold into 3D structures that can mimic cellular RNAs, hijack host proteins, and expose or occlude regulatory protein-binding motifs to ultimately control key process in the viral life cycle. We highlight recent findings focusing on less conventional mechanisms of gene expression and new discoveries on the role of RNA structures.

3D RNA-scaffolded wireframe origami.

Hybrid RNA:DNA origami, in which a long RNA scaffold strand folds into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of three-dimensional wireframe RNA-scaffolded origami rendered as polyhedra composed of dual-duplex edges. We computationally design, fabricate, and characterize tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds. We characterize secondary and tertiary structures using dimethyl sulfate mutational profiling and cryo-electron microscopy, revealing insight into both global and local, base-level structures of origami. Our top-down sequence design strategy enables the use of long RNAs as functional scaffolds for complex wireframe origami.

Nutritional stress exacerbates impact of a novel insecticide on solitary bees' behaviour, reproduction and survival.

Pesticide exposure and food stress are major threats to bees, but their potential synergistic impacts under field-realistic conditions remain poorly understood and are not considered in current pesticide risk assessments. We conducted a semi-field experiment to examine the single and interactive effects of the novel insecticide flupyradifurone (FPF) and nutritional stress on fitness proxies in the solitary bee Osmia bicornis. Individually marked bees were released into flight cages with monocultures of buckwheat, wild mustard or purple tansy, which were assigned to an insecticide treatment (FPF or control) in a crossed design. Nutritional stress, which was high in bees foraging on buckwheat, intermediate on wild mustard and low on purple tansy, modulated the impact of insecticide exposure. Within the first day after application of FPF, mortality of bees feeding on buckwheat was 29 times higher compared with control treatments, while mortality of FPF exposed and control bees was similar in the other two plant species. Moreover, we found negative synergistic impacts of FPF and nutritional stress on offspring production, flight activity, flight duration and flower visitation frequency. These results reveal that environmental policies and risk assessment schemes that ignore interactions among anthropogenic stressors will fail to adequately protect bees and the pollination services they provide.

Variable Effects of Twenty Sugars and Sugar Alcohols on the Retrogradation of Wheat Starch Gels.

Starch retrogradation is desirable for some food textures and nutritional traits but detrimental to sensory and storage qualities of other foods. The objective of this study was to determine the impact of sweetener structure and concentration on the retrogradation of wheat starch gels. The effects of 20 sweeteners selected based on common food usage and stereochemical structures of interest, and ranging in concentration from 10 to 50%w/w, on the retrogradation of wheat starch gels were monitored spectrophotometrically over time. The sweeteners were sucrose, xylose, ribose, glucose, galactose, fructose, mannose, mannitol, L-sorbose, xylitol, tagatose, allulose, maltose, lactose, isomaltulose, isomalt, sorbitol, maltitol, and raffinose. Retrogradation rates and amounts were compared by Avrami equation rate constants (k = 0.1-0.7) and absorbance values measured on day 28 (Abs = 0.1-1.0), respectively. Both sweetener concentration and type significantly affected retrogradation. Gels made with sugar alcohols and high sweetener concentrations (≈≥40%) tended to retrograde more and faster, whereas gels made with sugars and low sweetener concentrations tended to have lower retrogradation rates and amounts. Sweeteners with more equatorial and exocyclic hydroxyl groups (e.g., glucose and maltitol) and those with larger molar volumes (e.g., isomaltulose and raffinose) tended to increase the rate and amount of retrogradation, particularly at higher concentrations. The impact of sweeteners on retrogradation was a balance of factors that promoted retrogradation (intermolecular interactions and residual short-range molecular order) and inhibiting behaviors (interference at crystallization sites), which are influenced by sweetener concentration and structure. Understanding which sweeteners at which concentrations can be used to promote or inhibit retrogradation is useful for product formulation strategies.

Sweetpotato chip texture and fat content: Effects of enzymatic modification of cell wall polymers.

Impacts of cell wall polymers on sweetpotato chip texture and fat content were investigated through enzymatic modification. Covington sweetpotato slices were treated with cellulase, hemicellulase, pectinase, pectin methyl esterase, protease, the enzyme blend Viscozyme, or no enzymes (control) at 40-45°C for 0.5-2 h. Treated slices were blanched, dried, and fried in triplicate per experimental condition. Breaking forces of 20 chips per frying replicate were measured followed by chip fat, moisture, sugar, alcohol insoluble solids, glass transition temperature, and color analyses. Untreated slices from each batch (daily check) were fried and analyzed to account for starting material variability. Viscozyme and protease-treated chips had the greatest reduction in breaking force from untreated chips (-30.9% and -23.7%, respectively), while pectin methyl esterase-treated chips had the lowest reduction in breaking force (-9.0%). Chips treated with Viscozyme for 2 h were 6.7-6.3 percentiles lower in fat than the control. Principal component analysis elucidated that chip breaking force was associated with unfried slice puncture force, alcohol insoluble solids, and chip color, and chip fat content was inversely associated with maltose content and glass transition temperature. Breaking down multiple cell wall polysaccharides or structural proteins weakened chip textures, while strengthening the pectic fraction resulted in harder chips. Chip fat reduction also occurred when multiple cell wall polysaccharides were broken down. Therefore, cell wall polymers impact sweetpotato chip texture and fat contents, and their attributes should be considered when selecting cultivars and processes for sweetpotato chips. PRACTICAL APPLICATION: Sweetpotato chips are an increasingly popular snack, but there is little understanding how cell wall polymers impact chip textures and fat contents. Raw sweetpotato slices were enzymatically treated to selectively modify cell wall polymers before frying. Chip breaking forces were lowered by protease or Viscozyme (cell wall enzyme blend) treatments, while breaking forces were increased with pectin methyl esterase. In addition, chip fat contents were reduced by the Viscozyme treatment. Since cell wall modifications could impact chip texture and fat content, cell wall polymer attributes should be considered in selection and processing of sweetpotatoes for chip manufacturing.

Results of Ring-Testing of a Semifield Study Design to Investigate Potential Impacts of Crop Protection Products on Bumblebees (Hymenoptera, Apidae) and a Proposal of a Potential Test Design.

In Europe, the risk assessment for bees at the European Union or national level has always focussed on potential impacts on honeybees. During the revision of the European Food Safety Authority bee guidance it was explicitly stated that bumblebees and solitary bees should be considered as well and consequently concerns were raised regarding the representativeness of honeybees for these other bee species. These concerns originate from differences in size as well as differences in behavioral and life history traits of other bee species. In response to this concern, the non-Apis working group of the International Commission for Plant-Pollinator Relationships initiated a ring-test of a semifield tunnel study design using the bumblebee Bombus terrestris. Nine laboratories participated, validating and improving the proposed design over a 2-year period. The intention of the ring-test experiments was to develop and if possible, establish a test protocol to conduct more standardized semifield tests with bumblebees. In the present study, the results of the ring-tests are summarized and discussed to give recommendations for a promising experimental design. Environ Toxicol Chem 2022;41:2548-2564. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Indirect COVID-19 health effects and potential mitigating interventions: Cost-effectiveness framework.

BACKGROUND: The COVID-19 pandemic led to important indirect health and social harms in addition to deaths and morbidity due to SARS-CoV-2 infection. These indirect impacts, such as increased depression and substance abuse, can have persistent effects over the life course. Estimated health and cost outcomes of such conditions and mitigation strategies may guide public health responses. METHODS: We developed a cost-effectiveness framework to evaluate societal costs and quality-adjusted life years (QALYs) lost due to six health-related indirect effects of COVID-19 in California. Short- and long-term outcomes were evaluated for the adult population. We identified one evidence-based mitigation strategy for each condition and estimated QALYs gained, intervention costs, and savings from averted health-related harms. Model data were derived from literature review, public data, and expert opinion. RESULTS: Pandemic-associated increases in prevalence across these six conditions were estimated to lead to over 192,000 QALYs lost and to approach $7 billion in societal costs per million population over the life course of adults. The greatest costs and QALYs lost per million adults were due to adult depression. All mitigation strategies assessed saved both QALYs and costs, with five strategies achieving savings within one year. The greatest net savings over 10 years would be achieved by addressing depression ($242 million) and excessive alcohol use ($107 million). DISCUSSION: The COVID-19 pandemic is leading to significant human suffering and societal costs due to its indirect effects. Policymakers have an opportunity to reduce societal costs and health harms by implementing mitigation strategies.

No evidence for impaired solitary bee fitness following pre-flowering sulfoxaflor application alone or in combination with a common fungicide in a semi-field experiment.

Pesticide exposure is considered a major driver of pollinator decline and the use of neonicotinoid insecticides has been restricted by regulatory authorities due to their risks for pollinators. Impacts of new alternative sulfoximine-based compounds on solitary bees and their potential interactive effects with other commonly applied pesticides in agriculture remain unclear. Here, we conducted a highly replicated full-factorial semi-field experiment with the solitary bee Osmia bicornis, an important pollinator of crops and wild plants in Europe, and Phacelia tanacetifolia as a model crop. We show that spray applications of the insecticide sulfoxaflor (product Closer) and the fungicide azoxystrobin (product Amistar), both alone and combined, had no significant negative impacts on adult female survival or the production, mortality, sex ratio and body size of offspring when sulfoxaflor was applied five days before crop flowering. Our results indicate that for O. bicornis (1) the risk of adverse impacts of sulfoxaflor (Closer) on fitness is small when applied at least five days before crop flowering and (2) that azoxystrobin (Amistar) has a low potential of exacerbating sulfoxaflor effects under field-realistic conditions.

Flowering resources modulate the sensitivity of bumblebees to a common fungicide.

Bees are exposed to various stressors, including pesticides and lack of flowering resources. Despite potential interactions between these stressors, the impacts of pesticides on bees are generally assumed to be consistent across bee-attractive crops, and regulatory risk assessments of pesticides neglect interactions with flowering resources. Furthermore, impacts of fungicides on bees are rarely examined in peer-reviewed studies, although these are often the pesticides that bees are most exposed to. In a full-factorial semi-field experiment with 39 large flight cages, we assessed the single and combined impacts of the globally used azoxystrobin-based fungicide Amistar® and three types of flowering resources (Phacelia, buckwheat, and a floral mix) on Bombus terrestris colonies. Although Amistar is classified as bee-safe, Amistar exposure through Phacelia monocultures reduced adult worker body mass and colony growth (including a 55% decline in workers and an 88% decline in males), while the fungicide had no impact on colonies in buckwheat or the floral mix cages. Furthermore, buckwheat monocultures hampered survival and fecundity irrespective of fungicide exposure. This shows that bumblebees require access to complementary flowering species to gain both fitness and fungicide tolerance and that Amistar impacts are flowering resource-dependent. Our findings call for further research on how different flowering plants affect bees and their pesticide tolerance to improve guidelines for regulatory pesticide risk assessments and inform the choice of plants that are cultivated to safeguard pollinators.

Drug Interactions Affecting Kidney Function: Beware of Health Threats from Triple Whammy.

Triple whammy (TW) is a potentially dangerous drug combination that can lead to acute kidney injury (AKI). This drug interaction (DI) occurs when angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) are used together with diuretics and non-steroidal anti-inflammatory drugs (NSAIDs). One of the most serious consequences of TW DI is an increased risk of developing pre-renal acute kidney injury (pr-AKI). The term TW, in the context of a DI affecting kidney function, is not very widespread. The aim of this article was to gather information on this interaction. Previous knowledge on the mechanism of TW and how to increase patient awareness of this interaction is described. In addition, the specific nature of the acute kidney injury (AKI) caused by triple whammy (AKITW) is presented. On the basis of the current state of knowledge, recommendations on how to manage the TW DI are also demonstrated.

Fungicide and insecticide exposure adversely impacts bumblebees and pollination services under semi-field conditions.

Sulfoximines, the next generation systemic insecticides developed to replace neonicotinoids, have been shown to negatively impact pollinator development and reproduction. However, field-realistic studies on sulfoximines are few and consequences on pollination services unexplored. Moreover, the impacts of other agrochemicals such as fungicides, and their combined effects with insecticides remain poorly investigated. Here, we show in a full factorial semi-field experiment that spray applications of both the product Closer containing the insecticide sulfoxaflor and the product Amistar containing the fungicide azoxystrobin, negatively affected the individual foraging performance of bumblebees (Bombus terrestris). Insecticide exposure further reduced colony growth and size whereas fungicide exposure decreased pollen deposition. We found indications for resource limitation that might have exacerbated pesticide effects on bumblebee colonies. Our work demonstrates that field-realistic exposure to sulfoxaflor can adversely impact bumblebees and that applications before bloom may be insufficient as a mitigation measure to prevent its negative impacts on pollinators. Moreover, fungicide use during bloom could reduce bumblebee foraging performance and pollination services.

Relationships between isolated sweetpotato starch properties and textural attributes of sweetpotato French fries.

Sweetpotato French fry (SPFF) textures have been associated with dry matter and starch contents, but these do not fully account for all textural differences. This study investigated the relationships between the physicochemical properties of sweetpotato starch and textural attributes of sweetpotato fries. Starches from 16 sweetpotato genotypes that varied in dry matter content were isolated and analyzed. The amylose content, pasting temperatures and viscosities, and textural properties of equilibrated starch gels were measured. Correlational analysis was performed with the respective SPFF mechanical and sensory texture attributes. Sweetpotato starch amylose content ranged from 17.3% to 21.1%, and the pasting and gel textural properties varied significantly between starches. Starch from orange-fleshed sweetpotatoes had lower pasting temperatures than starches from yellow/cream-fleshed genotypes, 72.2 ± 2.0 and 75.5 ± 1.1 °C, respectively. Notable inverse correlations were observed between the starch pasting temperature and perceived moistness (r = -0.63) and fibrousness (r = -0.70) of fries, whereas SPFF denseness was positively associated with starch pasting viscosity (r = 0.60) and nonstarch alcohol-insoluble solids content. Fry textures were likely affected by cooked starch properties, which should be considered when selecting varieties for sweetpotato fries. PRACTICAL APPLICATION: Without the aid of a batter, sweetpotato French fries (SPFFs) tend to be soft and limp-undesirable attributes in a fried food. The physiochemical properties of starch, the most abundant component in sweetpotato fries, were further explored in this study to better understand the properties of sweetpotato starch that influence SPFF textures. These findings can be used by sweetpotato processors and breeders for developing new sweetpotato varieties that are designed for production of fried products with desirable textures.

Sulfoxaflor insecticide and azoxystrobin fungicide have no major impact on honeybees in a realistic-exposure semi-field experiment.

Exposure to pesticides is considered a major threat to bees and several neonicotinoid insecticides were recently banned in cropland within the European Union in light of evidence of their potential detrimental effects. Nonetheless, bees remain exposed to many pesticides whose effects are poorly understood. Recent evidence suggests that one of the most prominent replacements of the banned neonicotinoids - the insecticide sulfoxaflor - harms bees and that fungicides may have been overlooked as a driver of bee declines. Realistic-exposure studies are, however, lacking. Here, we assess the impact of the insecticide Closer (active ingredient: sulfoxaflor) and the widely used fungicide Amistar (a.i.: azoxystrobin) on honeybees in a semi-field study (10 flight cages containing a honeybee colony, for each of three treatments: Closer, Amistar, control). The products were applied according to label instructions either before (Closer) or during (Amistar) the bloom of purple tansy. We found no significant effects of Closer or Amistar on honeybee colony development or foraging activity. Our study suggests that these pesticides pose no notable risk to honeybees when applied in isolation, following stringent label instructions. The findings on Closer indicate that a safety-period of 5-6 days between application and bloom, which is only prescribed in a few EU member states, may prevent its impacts on honeybees. However, to conclude whether Closer and Amistar can safely be applied, further realistic-exposure studies should examine their effects in combination with other chemical or biological stressors on various pollinator species.

Effects of Sugars and Sugar Alcohols on the Gelatinization Temperatures of Wheat, Potato, and Corn Starches.

The gelatinization temperature (Tgel) of starch increases in the presence of sweeteners due to sweetener-starch intermolecular interactions in the amorphous regions of starch. Different starch botanical sources contain different starch architectures, which may alter sweetener-starch interactions and the effects of sweeteners on Tgels. To document these effects, the Tgels of wheat, potato, waxy corn, dent corn, and 50% and 70% high amylose corn starches were determined in the presence of eleven different sweeteners and varying sweetener concentrations. Tgels of 2:1 sweetener solution:starch slurries were measured using differential scanning calorimetry. The extent of Tgel elevation was affected by both starch and sweetener type. Tgels of wheat and dent corn starches increased the most, while Tgels of high amylose corn starches were the least affected. Fructose increased Tgels the least, and isomalt and isomaltulose increased Tgels the most. Overall, starch Tgels increased more with increasing sweetener concentration, molar volume, molecular weight, and number of equatorial and exocyclic hydroxyl groups. Starches containing more short amylopectin chains, fewer amylopectin chains that span through multiple clusters, higher number of building blocks per cluster, and shorter inter-block chain lengths exhibited the largest Tgel increases in sweetener solutions, attributed to less stable crystalline regions.

Relative humidity-temperature transition boundaries for anhydrous ß-caffeine and caffeine hydrate crystalline forms.

Caffeine is a hydrate-forming polymorphic crystalline compound that can exist in α, ß, and hydrate forms. Phase transitions between hydrate and anhydrous forms of a crystalline ingredient, and related water migration, can create product quality challenges. The objective of this study was to determine the relative humidity (RH)-temperature phase boundary between anhydrous ß-caffeine and caffeine hydrate. The ß-caffeine→caffeine hydrate and caffeine hydrate→ß-caffeine RH-temperature transition boundaries were determined from 20 to 45 °C using a combination of water activity (aw ) controlled solution and vapor-mediated equilibration, moisture sorption, powder X-ray diffraction, and Fourier-transform infrared spectroscopy techniques. Two transition boundaries were measured: the ß-caffeine→caffeine hydrate transition boundary (0.835 ± 0.027 aw at 25 °C) was higher than the caffeine hydrate→ß-caffeine transition boundary (0.625 ± 0.003 aw at 25 °C). Moisture sorption rates for ß-caffeine, even at high RHs (>84% RH), were slow. However, caffeine hydrate rapidly dehydrated at low RHs (<30% RH) into a metastable transitional anhydrous state with a similar X-ray diffraction pattern to metastable α-caffeine. Exposing this dehydrated hydrate to higher RHs (>65% RH) at lower temperatures (20 to 30 °C) resulted in full restoration to a 4/5 caffeine hydrate. This transitional anhydrous state was unstable and converted to a less hygroscopic state after annealing at 50 °C and 0% RH for 1 day. It was postulated that the caffeine hydrate→ß-caffeine was the true ß-caffeine↔caffeine hydrate phase boundary and that ß-caffeine could be metastable above the caffeine hydrate→ß-caffeine transition boundary. These caffeine RH-temperature transition boundaries could be used for selecting formulation and storage conditions to maintain the desired caffeine crystalline form. PRACTICAL APPLICATION: Caffeine can exist as either an anhydrous (without water) or hydrate (internalized water) crystalline state. The stability of each caffeine crystalline form is dictated by humidity (or water activity) and temperature, and these environmental stability boundaries for the caffeine crystalline forms are reported in this manuscript. Conversions between the two crystalline states can lead to deleterious effects; for example, the presence of caffeine hydrate crystals in a low water activity food (e.g., powder) could lead to the relocation of the water in caffeine to other ingredients in the food system, leading to unwanted water-solid interactions that could cause clumping and/or degradation.

RH-temperature stability diagram of α- and ß-anhydrous and monohydrate lactose crystalline forms.

Lactose crystals exhibit polymorphic, deliquescent, and hydrate-forming traits and can exist in monohydrate, ß-anhydrate, stable α-anhydrate, and hygroscopic α-anhydrate (isomorphic desolvate) forms. The objective of this study was to identify the relative humidity (RH) and temperature boundaries at which anhydrate-hydrate transitions and deliquescence occur for these lactose crystal forms. The deliquescence point (RH0) of lactose monohydrate was determined by measuring the water activity (aw) of a saturated solution, and the RH0s of the anhydrates were determined using dynamic vapor sorption measurement techniques. Increasing temperatures from 20 to 50 °C resulted in decreases in RH0 from 99 to 98% RH for the monohydrate, 89 to 82% RH for the ß-anhydrate, and 87 to 82% RH for the stable α-anhydrate. The effects of temperature on the anhydrate-hydrate RH boundaries were determined using a combination of controlled aw equilibration, powder X-ray diffraction, and Fourier-transform infrared spectroscopy techniques. Increasing temperature from 20 to 50 °C resulted in increasing RHs of the anhydrate-to-hydrate boundaries: the ß-anhydrate-to-monohydrate boundary increased from 77 to 79% RH, the stable α-anhydrate-to-monohydrate from 63 to 79% RH, and the unstable α-anhydrate-to-monohydrate from 10 to 13% RH. This is the first report of an RH-temperature stability map for lactose crystalline forms.

RH-Temperature Stability Diagram of the Dihydrate, ß-Anhydrate, and α-Anhydrate Forms of Crystalline Trehalose.

Trehalose crystals exhibit polymorphic, deliquescent, and hydrate-forming traits and can exist in dihydrate, ß-anhydrate, or α-anhydrate (isomorphic desolvate) forms. The objective of this study was to identify the relative humidity (RH) and temperature boundaries for phase changes of these different trehalose crystal forms. The deliquescence points (RH0 s) of the anhydrate and dihydrate trehalose crystals were determined from 20 to 50 °C using a combination of water activity and dynamic vapor sorption measurement techniques. Increasing temperatures from 20 to 50 °C resulted in decreases in RH0 from 95.5% to 90.9% RH for the dihydrate and 69.9% to 62.0% RH for the ß-anhydrate. The effects of temperature on the anhydrate-hydrate RH boundaries were also determined, using a combination of equilibration in controlled water activity solutions, powder X-ray diffraction, and Fourier-transform infrared spectroscopy techniques. Increasing temperatures resulted in increases in the anhydrate-hydrate RH boundaries. The irreversible ß-anhydrate to dihydrate boundary increased from 44.9% to 57.8% RH, and the reversible α-anhydrate to dihydrate boundary increased from 10% to 25% RH, as temperature increased from 20 to 50 °C. This is the first report of an RH-temperautre stability map for crystalline trehalose. PRACTICAL APPLICATION: The manuscript addresses the issue of the physical stability and phase transformations of crystalline trehalose stored in different temperature and relative humidity environments. Unwanted hydrate formation or dehydration of crystal hydrates can lead to other undesirable water-solid interactions and/or physical modifications that have the potential to influence product quality and delivery traits. Therefore, this study identified relative humidity and temperature stability boundaries of the different trehalose crystal forms, using a variety of established and novel techniques to create a relative humidity-temperature stability map of crystalline trehalose from 20 to 50 °C.

Rotational Atherectomy Combined with Drug Coated-Balloons for in-Stent Restenosis.

BACKGROUND: Refractory coronary in-stent restenosis remains a clinically relevant problem in interventional cardiology despite the use of drug coated balloon angioplasty and further drug eluting stent deployment. In this study, we investigated whether the novel approach of lesion debulking with rotational atherectomy prior to drug coated balloon angioplasty for challenging coronary in-stent restenosis is safe and effective. METHODS: Procedural and registry data was retrospectively analysed for 26 patients who underwent rotational atherectomy immediately followed by drug coated balloon angioplasty to 43 coronary in-stent restenosis lesions with mean follow up of 19 months. RESULTS: Lesion success was achieved in all cases with no major procedural complications. There were no instances of death or myocardial infarction in the follow up period. Target lesion revascularisation occurred in six patients and target vessel revascularisation occurred in eight patients. All target lesion revascularisation occurred in lesions that had already failed drug coated balloon angioplasty without debulking previously while four such lesions were free of lesion failure in the follow up period. CONCLUSIONS: Lesion debulking with rotational atherectomy followed by drug coated balloon angioplasty is a feasible treatment option for selected cases of in-stent restenosis. Further study is needed to fully assess its efficacy in comparison to conventional treatment.