Dynamics and Evolutionary Conservation of B Complex Protein Recruitment During Spliceosome Activation.

Spliceosome assembly and catalytic site formation (called activation) involve dozens of protein and snRNA binding and unbinding events. The B-complex specific proteins Prp38, Snu23, and Spp381 have critical roles in stabilizing the spliceosome during conformational changes essential for activation. While these proteins are conserved, different mechanisms have been proposed for their recruitment to spliceosomes. To visualize recruitment directly, we used Colocalization Single Molecule Spectroscopy (CoSMoS) to study the dynamics of Prp38, Snu23, and Spp381 during splicing in real time. These proteins bind to and release from spliceosomes simultaneously and are likely associated with one another. We designate the complex of Prp38, Snu23, and Spp381 as the B Complex Protein (BCP) subcomplex. Under splicing conditions, the BCP associates with pre-mRNA after tri-snRNP binding. BCP release predominantly occurs after U4 snRNP dissociation and after NineTeen Complex (NTC) association. Under low concentrations of ATP, the BCP pre-associates with the tri-snRNP resulting in their simultaneous binding to pre-mRNA. Together, our results reveal that the BCP recruitment pathway to the spliceosome is conserved between S. cerevisiae and humans. Binding of the BCP to the tri-snRNP when ATP is limiting may result in formation of unproductive complexes that could be used to regulate splicing.

Low concentrations of methyl jasmonate promote plant growth and mitigate Cd toxicity in Cosmos bipinnatus.

Cadmium (Cd) is a biologically non-essential heavy metal, a major soil pollutant, and extremely harmful to plants. The phytohormone methyl jasmonate (MeJA) plays an important role in plant heavy-metal resistance. However, the understanding of the effects of MeJA supply level on alleviating Cd toxicity in plants is limited. Here, we investigated how MeJA regulated the development of physiological processes and cell wall modification in Cosmos bipinnatus. We found that low concentrations of MeJA increased the dry weight of seedlings under 120 µM Cd stress by reducing the transport of Cd from roots to shoots. Moreover, a threshold concentration of exogenous MeJA increased the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in plant roots, the concentration of Cd in the root cell wall, and the contents of pectin and hemicellulose 1 polysaccharides, through converting Cd into pectin-bound forms. These results suggested that MeJA mitigated Cd toxicity by modulating root cell wall polysaccharide and functional group composition, especially through pectin polysaccharides binding to Cd, with effects on Cd transport capacity, specific chemical forms of Cd, and homeostatic antioxidant systems in C. bipinnatus.

A new active packaging system based on chickpea-based edible coatings added with microcapsules of Cosmos sulphureus Cav. flower extract.

BACKGROUND: The Cosmos sulphureus Cav. plant is studied for its high polyphenolic content with antioxidant properties. Its flowers, rich in phenolic acids, flavonoids, and tannins, hold promise as antioxidants in food preservation. The inclusion of these compounds in chickpea-based coatings with a previously studied preservative effect would be an excellent option as a food preservation method and microencapsulation addresses challenges like dispersion and degradation of polyphenols in the coating. The objective of this research was to evaluate the in vitro antioxidant activity of Cosmos sulphureus leaves, seed, and flower extracts and explore the protective effects of chickpea-based coatings containing microcapsules of flower polyphenolic extract on the chemical quality of stored roasted sunflower seeds during storage. RESULTS: The ethanolic leaf extract exhibited the highest antiradical activity, followed by the aqueous flower extract. After a storage period of 15 days, at 40 °C, the chickpea-based coatings effectively delayed lipid oxidation in the roasted sunflowers seeds, and the inclusion of polyphenolic microcapsules with 0.01% extract (SMC 0.01%) in the coating significantly improved the protective effect. By day 15 of storage, SMC 0.01% showed comparable peroxide value, conjugated dienes, and linoleic acid content to samples containing the synthetic antioxidant BHT (butylated hydroxytoluene). Samples that only contained chickpea-based coating and coating with polyphenolic microcapsules with 0.005% extract exhibited significantly greater reduction in fatty acid content compared to the 0.01% SMC treatment. CONCLUSION: The chickpea-based coating with polyphenolic microcapsules demonstrated antioxidant activity akin to synthetic BHT, offering a promising biopackaging solution for lipid-rich foods like roasted sunflower seeds. © 2024 Society of Chemical Industry.

Integrated cell wall and transcriptomic analysis revealed the mechanism underlying zinc-induced alleviation of cadmium toxicity in Cosmos bipinnatus.

Plant growth is severely harmed by cadmium (Cd) contamination, while the addition of zinc (Zn) can reduce the toxic effects of Cd. However, the interaction between Cd and Zn on the molecular mechanism and cell wall of Cosmosbipinnatus is unclear. In this study, a transcriptome was constructed using RNA-sequencing. In C. bipinnatus root transcriptome data, the expression of 996, 2765, and 3023 unigenes were significantly affected by Cd, Zn, and Cd + Zn treatments, respectively, indicating different expression patterns of some metal transporters among the Cd, Zn, and Cd + Zn treatments. With the addition of Zn, the damage to the cell wall was reduced, both the proportion and content of polysaccharides in the cell wall were changed, and Cd accumulation was decreased by 32.34%. In addition, we found that Cd and Zn mainly accumulated in pectins, the content of which increased by 30.79% and 61.4% compared to the CK treatment. Thus, Zn could alleviate the toxicity of Cd to C. bipinnatus. This study revealed the interaction between Cd and Zn at the physiological and molecular levels, broadening our understanding of the mechanisms of tolerance to Cd and Zn stress in cosmos.

Effects of cosmos caudatus (Kenikir) antioxidant properties on bone metabolism marker in rat.

Cosmos caudatus leaves are one of around 7500 types of plants that are known to have herbal or medicinal plant properties in Indonesia. This research determines the effectiveness of Cosmos caudatus as an antioxidant agent against cells, biomolecules, and bone density. Forty-three male rat aged 3-4 months were divided into four groups.Group P0 was only given distilled water. Group P1 was given kenikir leaf extract at a dose of 0.91 mg/kg. Group P2 was given kenikir leaf extract at a dose of 1.82 mg/kg. And group P3 was given kenikir leaf extract at 3.64 mg/kg ad libitum once a day for 28 days. The highest average SOD level was in the 1.82 mg/bb P2 conversion dose group (1.09 ± 1.76). The lowest mean CTX level was in the P2 group (8.30 ± 1.10). There was a significant increase in mean trabecular bone in the P2 group (43.33 ± 5.32). The number of osteoblast cells increased significantly at P2 (103.94 (SD 38.14)). The number of osteoclasts decreased from the control group (P0) to 0.60 (SD 0.43) at P2. Indicate that the Cosmos caudatus extract may have advantages as an antioxidant support agent for bone metabolism.

The genomes of Dahlia pinnata, Cosmos bipinnatus, and Bidens alba in tribe Coreopsideae provide insights into polyploid evolution and inulin biosynthesis.

BACKGROUND: The Coreopsideae tribe, a subset of the Asteraceae family, encompasses economically vital genera like Dahlia, Cosmos, and Bidens, which are widely employed in medicine, horticulture, ecology, and food applications. Nevertheless, the lack of reference genomes hinders evolutionary and biological investigations in this tribe. RESULTS: Here, we present 3 haplotype-resolved chromosome-level reference genomes of the tribe Coreopsideae, including 2 popular flowering plants (Dahlia pinnata and Cosmos bipinnatus) and 1 invasive weed plant (Bidens alba), with assembled genome sizes 3.93 G, 1.02 G, and 1.87 G, respectively. We found that Gypsy transposable elements contribute mostly to the larger genome size of D. pinnata, and multiple chromosome rearrangements have occurred in tribe Coreopsideae. Besides the shared whole-genome duplication (WGD-2) in the Heliantheae alliance, our analyses showed that D. pinnata and B. alba each underwent an independent recent WGD-3 event: in D. pinnata, it is more likely to be a self-WGD, while in B. alba, it is from the hybridization of 2 ancestor species. Further, we identified key genes in the inulin metabolic pathway and found that the pseudogenization of 1-FEH1 and 1-FEH2 genes in D. pinnata and the deletion of 3 key residues of 1-FFT proteins in C. bipinnatus and B. alba may probably explain why D. pinnata produces much more inulin than the other 2 plants. CONCLUSIONS: Collectively, the genomic resources for the Coreopsideae tribe will promote phylogenomics in Asteraceae plants, facilitate ornamental molecular breeding improvements and inulin production, and help prevent invasive weeds.

Chaos and COSMOS-Considerations on QSM methods with multiple and single orientations and effects from local anisotropy.

PURPOSE: Field-to-susceptibility inversion in quantitative susceptibility mapping (QSM) is ill-posed and needs numerical stabilization through either regularization or oversampling by acquiring data at three or more object orientations. Calculation Of Susceptibility through Multiple Orientations Sampling (COSMOS) is an established oversampling approach and regarded as QSM gold standard. It achieves a well-conditioned inverse problem, requiring rotations by 0°, 60° and 120° in the yz-plane. However, this is impractical in vivo, where head rotations are typically restricted to a range of ±25°. Non-ideal sampling degrades the conditioning with residual streaking artifacts whose mitigation needs further regularization. Moreover, susceptibility anisotropy in white matter is not considered in the COSMOS model, which may introduce additional bias. The current work presents a thorough investigation of these effects in primate brain. METHODS: Gradient-recalled echo (GRE) data of an entire fixed chimpanzee brain were acquired at 7 T (350 µm resolution, 10 orientations) including ideal COSMOS sampling and realistic rotations in vivo. Comparisons of the results included ideal COSMOS, in-vivo feasible acquisitions with 3-8 orientations and single-orientation iLSQR QSM. RESULTS: In-vivo feasible and optimal COSMOS yielded high-quality susceptibility maps with increased SNR resulting from averaging multiple acquisitions. COSMOS reconstructions from non-ideal rotations about a single axis required additional L2-regularization to mitigate residual streaking artifacts. CONCLUSION: In view of unconsidered anisotropy effects, added complexity of the reconstruction, and the general challenge of multi-orientation acquisitions, advantages of sub-optimal COSMOS schemes over regularized single-orientation QSM appear limited in in-vivo settings.

A Sequential Binding Mechanism for 5' Splice Site Recognition and Modulation for the Human U1 snRNP.

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how U1 snRNP recognizes particular 5' splice sites (5'SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5'SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5'SS modulation. We have determined a sequential binding mechanism for modulation of -1A bulged 5'SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged a -1A bulged 5'SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5'SS complexes. Branaplam is a ribonucleoprotein, not RNA duplex alone, targeting drug whose action depends on fundamental properties of 5'SS recognition.

Impact of Pain and Neuropsychiatric Symptoms on Activities in Nursing Home Residents (COSMOS Trial).

OBJECTIVE: This study aims to identify whether pain and dementia-related behavior are associated with different types of activities in nursing home residents, controlled for dementia severity. DESIGN: Cross-sectional baseline data from the multicomponent cluster randomized controlled COSMOS trial (acronym for Communication, Systematic pain treatment, Medication review, Organization of activities, and Safety). SETTING AND PARTICIPANTS: A total of 723 patients from 33 Norwegian nursing homes with 67 units (clusters). Participants aged ≥65 years, with a life expectancy of >6 months, and with valid data on activity were eligible for inclusion. METHODS: Activity was operationalized in time (hours per week) and type (cognitive, social, physical, and no activity). Cognitive function was assessed using the Mini-Mental State Examination (MMSE), pain with the Mobilization-Observation-Behavior-Intensity-Dementia-2 Pain Scale (MOBID-2), and behavior with the Neuropsychiatric Inventory Nursing Home version (NPI-NH). Analyses were performed using linear and logistic regression. Sensitivity analyses for dementia severity were performed to account for effect modification. RESULTS: A total of 289 participants were included (mean age 86.2 [SD 7.6]; 74% female). A higher pain score was associated with less time spent on activity in participants with severe dementia (estimate 0.897, P = .043). A higher score for the NPI-NH mood cluster (depression and anxiety) was associated with a higher likelihood of participation in cognitive activities (odds ratio [OR], 1.073; P = .039). Apathy (OR, 0.884; P = .041) and lack of inhibition (OR, 0.904; P = .042) were associated with a lower likelihood of participation in social activities as well as no engagement in activities (apathy OR, 0.880; P = .042; lack of inhibition OR, 0.894; P = .034). CONCLUSION AND IMPLICATIONS: Pain and dementia-related behavior may influence the participation in activities in the nursing home. There is an urgent need to investigate what type of activity stimulates people in different stages of dementia.

Headache in the international cohort study of mobile phone use and health (COSMOS) in the Netherlands and the United Kingdom.

Headache is a common condition with a substantial burden of disease worldwide. Concerns have been raised over the potential impact of long-term mobile phone use on headache due to radiofrequency electromagnetic fields (RF-EMFs). We explored prospectively the association between mobile phone use at baseline (2009-2012) and headache at follow-up (2015-2018) by analysing pooled data consisting of the Dutch and UK cohorts of the Cohort Study of Mobile Phone Use and Health (COSMOS) (N = 78,437). Frequency of headache, migraine, and information on mobile phone use, including use of hands-free devices and frequency of texting, were self-reported. We collected objective operator data to obtain regression calibrated estimates of voice call duration. In the model mutually adjusted for call-time and text messaging, participants in the high category of call-time showed an adjusted odds ratio (OR) of 1.04 (95 % CI: 0.94-1.15), with no clear trend of reporting headache with increasing call-time. However, we found an increased risk of weekly headache (OR = 1.40, 95 % CI: 1.25-1.56) in the high category of text messaging, with a clear increase in reporting headache with increasing texting. Due to the negligible exposure to RF-EMFs from texting, our results suggest that mechanisms other than RF-EMFs are responsible for the increased risk of headache that we found among mobile phone users.

Phenotype Systemic Lupus Erythematosus Patients from EPIC Cosmos.

Systemic Lupus Erythematosus (SLE) is a widespread autoimmune disease for which early diagnosis is paramount in improving clinical outcomes. In this project, we used the de-identified patients from Epic Cosmos to retrieve the ICD code for SLE, checked data quality based on the EULAR/ACR classification systems, created an approach to determine the SLE patients, and performed statistical analyses on lab tests and clinical characteristics. Our preliminary results showed that clinical notes must be reviewed to improve the completeness, as structured EHR data fields provide limited information in determining if a patient meets the established classification criteria.

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos. In this study, flower buds with sizes of 5-7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension. The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

Abscisic acid (ABA) alleviates cadmium toxicity by enhancing the adsorption of cadmium to root cell walls and inducing antioxidant defense system of Cosmos bipinnatus.

Cadmium (Cd) pollution is a global problem affecting soil ecology and plant growth. Abscisic acid (ABA) acts as a growth and stress hormone, regulates cell wall synthesis, and plays an important role in plant responses to stress. There are few studies on the mechanisms behind abscisic acid alleviation of cadmium stress in Cosmos bipinnatus, especially in regards to regulation of the root cell wall. This study examined the effects of different concentrations of abscisic acid at different concentrations of cadmium stress. Through adding 5 µmol/L and 30 µmol/L cadmium, followed by spraying 10 µmol/L and 40 µmol/L ABA in a hydroponic experiment, it was found that under two concentrations of cadmium stress, low concentration of ABA improved root cell wall polysaccharide, Cd, and uronic acid content. Especially in pectin, after the application of low concentration ABA, the cadmium concentration was significantly increased by 1.5 times and 1.2 times compared with the Cd concentration under Cd5 and Cd30 treatment alone, respectively. Fourier-Transform Infrared spectroscopy (FTIR) demonstrated that cell wall functional groups such as -OH and -COOH were increased with exposure to ABA. Additionally, the exogenous ABA also increased expression of three kinds of antioxidant enzymes and plant antioxidants. The results of this study suggest that ABA could reduce Cd stress by increasing Cd accumulation, promoting Cd adsorption on the root cell wall, and activating protective mechanisms. This result could help promote application of C. bipinnatus for phytostabilization of cadmium-contaminated soil.

Rethinking nucleic acids from their origins to their applications.

Reviewed are three decades of synthetic biology research in our laboratory that has generated alternatives to standard DNA and RNA as possible informational systems to support Darwinian evolution, and therefore life, and to understand their natural history, on Earth and throughout the cosmos. From this, we have learned that: • the core structure of nucleic acids appears to be a natural outcome of non-biological chemical processes probably in constrained, intermittently irrigated, sub-aerial aquifers on the surfaces of rocky planets like Earth and/or Mars approximately 4.36 ± 0.05 billion years ago; • however, this core is not unique. Synthetic biology has generated many different molecular systems able to support the evolution of molecular information; • these alternatives to standard DNA and RNA support biotechnology, including DNA synthesis, human diagnostics, biomedical research and medicine; • in particular, they support laboratory in vitro evolution (LIVE) with performance to generate catalysts at least 104-105 fold better than standard DNA libraries, enhancing access to receptors and catalysts on demand. Coupling nanostructures to the products of LIVE with expanded DNA offers new approaches for disease therapy; and • nevertheless, a polyelectrolyte structure and size regular building blocks are required for any informational polymer to support Darwinian evolution. These features serve as universal and agnostic biosignatures, useful for seeking life throughout the Solar System. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Development on potential skin anti-aging agents of Cosmos caudatus Kunth via inhibition of collagenase, MMP-1 and MMP-3 activities.

BACKGROUND: Skin aging is associated with degradation of collagen by matrix metalloproteinases (MMPs), which leads to loss of skin elasticity and formation of wrinkles. Cosmos caudatus Kunth (CC) has been traditionally claimed as an anti-aging agent in Malaysia. Despite its well-known antioxidant activity, the anti-aging properties of CC was not validated. PURPOSE: This study aimed to investigate the anti-aging potential of CC extracts and fractions, particularly their inhibition of collagenase, MMP-1 and MMP-3 activities in human dermal fibroblasts CCD-966SK, followed by isolation, identification and analysis of their bioactive constituents. STUDY DESIGN AND METHODS: DPPH assay was firstly used to evaluate the antioxidant activity throughout the bioactivity-guided fractionation. Cell viability was determined using MTS assay. Collagenase activity was examined, while MMP-1 and MMP-3 expression were measured using qRT-PCR and western blotting. Then, chemical identification of pure compounds isolated from CC fractions was done by using ESIMS, 1H and 13C NMR spectroscopies. HPLC analyses were carried out for bioactive fractions to quantify the major components. RESULTS: Throughout the antioxidant activity-guided fractionation, fractions CC-E2 and CC-E3 with antioxidant activity and no toxicity towards CCD-966SK cells were obtained from CC 75% ethanol partitioned layer (CC-E). Both fractions inhibited collagenase activity, MMP-1 and MMP-3 mRNA and protein expression, as well as NF-κB activation induced by TNF-α in CCD-966SK cells. 14 compounds, which mainly consists of flavonoids and their glycosides, were isolated. Quercitrin (14.79% w/w) and quercetin (11.20% w/w) were major compounds in CC-E2 and CC-E3, respectively, as quantified by HPLC. Interestingly, both fractions also inhibited the MMP-3 protein expression synergistically, compared with treatment alone. CONCLUSION: The quantified CC fractions rich in flavonoid glycosides exhibited skin anti-aging effects via the inhibition of collagenase, MMP-1 and MMP-3 activities, probably through NF-κB pathway. This is the first study reported on MMP-1 and MMP-3 inhibitory activity of CC with its chemical profile, which revealed its potential to be developed as anti-aging products in the future.

Effect of chromium stress on metal accumulation and cell wall fractions in Cosmos bipinnatus.

As one of the major pollutants in the environment, chromium (Cr), a heavy metal, poses a serious threat to urban green spaces and human life and health. Cosmos bipinnatus is considered a potential accumulator of Cr, and the differences in cellular Cr distribution and compartmentalization may uncover the mechanisms involved in its tolerance to Cr. To elucidate the effects of Cr stress on C. bipinnatus and determine the mechanism of Cr tolerance in C. bipinnatus, we investigated the physiological indicators, subcellular distribution and chemical forms, cell wall fractions and their Cr contents, uronic acid content in the cell wall fractions, and Fourier transform infrared spectroscopy (FTIR) of the cell wall. The results showed that the antioxidant enzyme activities in C. bipinnatus under Cr stress and most of the Cr were fixed in the cell wall. Notably, changes in the content of pectin fractions in the cell wall affected the accumulation of Cr in the cell wall of C. bipinnatus and the stability of negatively charged groups. In addition, the carboxyl and hydroxyl groups played a role in fixing metal in various parts of the C. bipinnatus cell wall.

Jasmonates and salicylic acid as enigmatic orchestrators of capitula senescence in Cosmos sulphureus Cav.

The fine-tuning of the intricate network of plant growth hormones empowers the balanced responses of plants to environmental and developmental signals. Salicylic acid and jasmonates are emerging as advanced hormones that provide plants with resistance to environmental stresses. Senescence is characterized by coordinated and systematic crosstalk between phytohormones that remodels the biochemical and physiological mechanisms in plants, resulting in cell death. The present investigation examines the role of jasmonates (methyl jasmonate and jasmonic acid) and salicylic acid (SA) in regulating the petal senescence of detached stalks of Cosmos sulphureus. Based on our results, it was revealed that SA and jasmonic acid (JA) at 40 µM and methyl jasmonate (MJ) at 0.75 µM concentration delayed the senescence of detached flowers of C. sulphureus considerably. These growth regulators improved the membrane stability, reinforced the antioxidant enzyme activities and averted the upsurge of hydrogen peroxide (H2O2) content in the petals. Additionally, SA and jasmonates preserved higher content of total phenols, reducing sugars and soluble proteins in the petals, besides impeding the bacterial growth in testing solutions which corroborated with the maximum solution uptake. The elevated soluble protein content was found to be associated with low specific protease activity (SPA) and α-amino acid content in the petal tissues. Our study concluded that SA and jasmonates delayed flower senescence by averting oxidative stress and maintaining the nutritional status of the petals.

Identification of transient intermediates during spliceosome activation by single molecule fluorescence microscopy.

Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA 3'-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of Saccharomyces cerevisiae spliceosomes in vitro. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both ([Formula: see text]) and provide a kinetic framework for its formation and transformation during activation. Prior to [Formula: see text] assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing-dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. We propose that this interaction may play a role in formation of transient spliceosome intermediates formed during activation.

Phenotypic prevalence of obesity and metabolic syndrome among an underdiagnosed and underscreened population of over 50 million children and adults.

Background: Metabolic syndrome is a phenotypic condition associated with a variety of genotypes. Studies of rare genotypes can be made more difficult by clinical underscreening of the population for the phenotypic traits that define metabolic syndrome to clinicians. Studies have demonstrated underdiagnosis of pediatric obesity, as well as reduced rates of pediatric screening for obesity related conditions, including conditions leading to a diagnosis of metabolic syndrome. If true, there may be a significant underdiagnosis of metabolic syndrome among the pediatric population compared to the adult population. Methods: Using Epic's Cosmos Data Network aggregated, de-identified patient data collected from healthcare organizations using the Epic electronic health record (EHR), we examined obesity and metabolic syndrome rates among adult and pediatric patients. We also examined screening rates for obesity related conditions and metabolic syndrome among adult and pediatric patients across the United States. We also sought to compare rates between subgroups within the population including age, sex, and race. Results: In our population, 45% of adults and 27% of pediatric population were obese by age and gender specific BMI criteria. 38% of the obese adult population had an ICD-10 code associated with the diagnosis vs. 52% of the pediatric population. Of adults meeting obesity criteria, 36% had results for appropriate, guideline-based blood laboratory testing for insulin resistance, 40-42% for dyslipidemia, and 55% for hepatic steatosis. 36% of obese adult patients had none of the recommended blood laboratory testing. 31% of the adult population met diagnostic criteria for metabolic syndrome. Of pediatric patients meeting obesity criteria, 27% had results for appropriate blood laboratory testing for insulin resistance, 28% for dyslipidemia, and 33% for hepatic steatosis. 59% of obese pediatric patients had none of the recommended blood laboratory testing. 3% of the pediatric population met criteria for diagnosis of metabolic syndrome. Discussion: This study represents one of the largest multicenter national cohorts assembled for studying metabolic syndrome (over 50 million patients) and demonstrates the power of emerging aggregated EHR tools for research. Although obesity is better diagnosed in pediatric patients than in adult patients, significantly lower screening rates for obesity related conditions occurred in pediatric patients compared to adults. Statistically significant, but clinically negligible differences in screening rates were found by race and gender. These results support smaller prior studies that suggest that obesity is under-diagnosed and obesity related conditions underscreened in pediatric and adult populations, and additionally suggests underdiagnosis of metabolic syndrome among United States pediatric and adult patients.

Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP.

In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used colocalization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.