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Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-l-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C cross-link between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the ß-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.
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A revolution in the field of biocatalysis has enabled scalable access to compounds of high societal values using enzymes. The construction of biocatalytic routes relies on the reservoir of available enzymatic transformations. A review of uncharacterized proteins predicted from genomic sequencing projects shows that a treasure trove of enzyme chemistry awaits to be uncovered. This Review highlights enzymatic transformations discovered through various genome mining methods and showcases their potential future applications in biocatalysis.
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Emergency situations involving the ears, nose, and throat (ENT) area can pose considerable challenges for clinicians and often require an interdisciplinary approach due to the involvement of different organ systems. To avoid damage to highly relevant sensory and perception organs and life-threatening bleeding or respiratory complications, strategies that are as quick and targeted as possible are necessary. This article aims to provide an overview of ENT emergency management strategies. The entire spectrum from simple conservative to highly complex surgical measures plays a role here, both diagnostically and therapeutically. Aspects such as bleeding, respiratory problems, inflammation, hearing disorders, vertigo, facial palsy and injuries to the head and neck area are discussed. In addition, important topics such as preventive measures and possible complications are also addressed to ensure optimal patient care.
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Urgencias Médicas , Nariz , Humanos , Inflamación , Nariz/lesionesRESUMEN
We report an optimized Kalinnikov-Dinh technology for pre-Descemet's endothelial keratoplasty (PDEK) that involves the use of a ring fixator, base, 30G needle connected to a 5-ml syringe with a spring-loaded plunger, and storage media. Our method allows to minimize graft preparation failure and preserves the PDEK graft efficiently, by reducing complications associated with the formation of type 1 big bubbles, including bubble rupture, perforation of Descemet's membrane and endothelium, and formation of type 2 or mixed type of big bubbles, and may contribute to increasing the number of surgeons performing PDEK around the globe.
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Queratoplastia Endotelial de la Lámina Limitante Posterior , Endotelio Corneal , Humanos , Queratoplastia Endotelial de la Lámina Limitante Posterior/métodos , Lámina Limitante Posterior/cirugíaRESUMEN
Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new class involving modifications installed by a cytochrome P450, a multi-nuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes encoded by the BGC were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization with 2D-NMR and Marfey's method on the resulting RiPP demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C crosslink between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid while the methyltransferase acted on the ß-carbon of the α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configurations of the atropisomer that formed upon biaryl crosslinking. The conserved Cys residue in the precursor peptide was not modified as in all other characterized MNIO-containing BGCs; However, mutational analyses demonstrated that it was essential for the MNIO activity on the C-terminal Asp. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to discover new macrocyclic RiPPs and that RiPPs remain a significant source of previously undiscovered enzyme chemistry.
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Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Using mass spectrometry, stable isotope incorporation, and extensive 2D-NMR spectrometry, we report the structural characterization of two unique examples of (C-N) biaryl-linked lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with a Trp-Tyr crosslink, while longipepetin A, from Longimycelium tulufanense, features a Trp-Trp linkage. Besides the unusual bicyclic frame, a Met of longipepetin A undergoes S-methylation to yield a trivalent sulfonium, a heretofore unprecedented RiPP modification. A bioinformatic survey revealed additional lasso peptide BGCs containing P450 enzymes which await future characterization. Lastly, nocapeptin A bioactivity was assessed against a panel of human and bacterial cell lines with modest growth-suppression activity detected towards Micrococcus luteus.
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The article presents the surgical technique of penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK) with femtosecond-laser assistance involving simultaneous implantation of an intracorneal continuous ring (ICCR) or an intracorneal ring segment (ICRS) into the graft. Surgical technique no. 1 - keratoplasty with simultaneous implantation of ICRS. Intrastromal circular tunnel is formed in the central zone of donor cornea using femtosecond laser. Then penetrating trepanation 8.1 mm in diameter is performed symmetrically to the formed tunnel. After preparing penetrating or lamellar recipient bed, suture fixation is placed in the corneal transplant, then the ICRS is implanted into the graft. Surgical technique no. 2 - keratoplasty with simultaneous implantation of ICCR. The donor cornea is dissected from periphery to center using femtosecond laser. Central zone remains untouched. A large diameter full-thickness trepanation is performed and the donor cornea is divided into the anterior and posterior layers. The ICCR is put on the donor cornea while holding the posterior layer with forceps. Penetrating or lamellar recipient bed is prepared, then the corneal graft is fixed with sutures. Transparent corneal graft acceptance does not guarantee high visual acuity due to post-keratoplasty astigmatism. Surgical correction of astigmatism is performed in the long term post-operatively and isn't effective enough. We proposed this new surgical technique of keratoplasty with simultaneous implantation of ICCR and ICRS into the graft as close as possible to the visual axis of the eye, which can help make the postoperative astigmatism minimal both immediately after surgery and in the long term. The study proposes a new approach to intraoperative prevention of post-keratoplasty astigmatism. The technique is simple, safe and effective. Analysis of long-term outcomes is required before recommending this method for widespread use in clinical practice.
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Astigmatismo , Enfermedades de la Córnea , Trasplante de Córnea , Humanos , Trasplante de Córnea/efectos adversos , Córnea/diagnóstico por imagen , Córnea/cirugía , Enfermedades de la Córnea/diagnóstico , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/cirugía , Queratoplastia Penetrante/efectos adversosRESUMEN
Background The coronavirus disease 2019 (COVID-19) pandemic caused changes in surgical practice. For acute appendicitis (AA), measures to control the pandemic might hinder patients from seeking medical care timely, resulting in increasing severity, postoperative complications, and mortality. This study aimed to investigate whether the COVID-19 pandemic had a negative impact on the severity and postoperative outcomes of patients with AA. Methodology We retrospectively reviewed medical records of AA patients treated operatively at Nhan Dan Gia Dinh Hospital hospital from June 1st to September 30th in three consecutive years: pre-pandemic (2019)/Group 1, minor waves (2020)/Group 2, and major wave (2021)/Group 3 (2021). Data were collected focusing on the duration of symptoms, severity of AA, time from admission to operation, postoperative complications, and mortality. Results There were 1,055 patients, including 452 patients in Group 1, 409 in Group 2, and 194 in Group 3. The overall number of patients decreased mainly in non-complicated AA. The percentages of hospital admission after 24 hours gradually increased (20.8%, 27.9%, and 43.8%, p < 0.05). The percentages of complicated AA in Group 2 and Group 3 were statistically higher than in Group 1 (39% and 55% vs. 31%, p < 0.05). Waiting time for operation increased to five hours during the major wave. Laparoscopic appendectomy was performed in 98-99% of AA patients during the pandemic, with an early postoperative complication rate of 5-9% and a mortality rate of 0.2-1%. Conclusions Although the percentages of hospital admission after 24 hours and complicated AA increased, laparoscopic appendectomy was still feasible and effective and should be maintained as the standard management for AA during the COVID-19 pandemic.
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PURPOSE: The study aimed to develop new surgical methods of obtaining and preserving pre-Descemet's endothelial keratoplasty (PDEK) graft in order to reduce the loss of donor material. MATERIAL AND METHODS: The study was conducted on 30 sclerocorneal discs, which were divided into three groups. The first group consisted of 10 sclerocorneal discs to which the standard PDEK graft preparation technique was applied. The second group consisted of 10 sclerocorneal discs, where the PDEK graft preparation was done using the optimized method 1 involving the KD Ring fixator, KD Base for PDEK, KD-30G needle with 5 mL syringe connected to the spring-load plunger, a preservative solution. The third group consisted of 10 sclerocorneal discs with PDEK graft prepared using the optimized method 2, which differed from the optimized method 1 in additional use of the KD artificial chamber for PDEK. When successfully acquiring the type-1 "big bubble", the dissected PDEK graft was preserved for further use in clinical practice using our own technique. RESULTS: In the first group, formation of the type-1 «big bubble¼ was achieved only in 5 out of 10 donor corneas (50%). Bubble ruptures happened in 4 cases (40%) and type-2 «big bubble¼ occurred in 1 case (10%). In the second group (optimized method 1) the type-1 «big bubble¼ was achieved in 9 cases (90%), while in the third group (optimized method 2) the type-1 «big bubble¼ was achieved in 10 cases (100%), proving the effectiveness of our PDEK graft preparation technique. CONCLUSION: We presented a novel surgical approach for acquiring and preserving PDEK graft that minimizes almost all the difficulties that surgeons face, including bubble rupture during pneumodissection and hydrodissection, formation of type-2 and mixed type bubble, and obtaining a PDEK graft of the required size; the details of the surgical technique have been refined. The proposed technique for graft preparation and preservation can be easily implemented in eye banks and is convenient for clinical practice.
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Enfermedades de la Córnea , Queratoplastia Endotelial de la Lámina Limitante Posterior , Humanos , Lámina Limitante Posterior/cirugía , Queratoplastia Endotelial de la Lámina Limitante Posterior/métodos , Enfermedades de la Córnea/cirugía , Córnea/cirugía , Endotelio CornealRESUMEN
Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.
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Venoms of predatory marine cone snails are intensely studied because of the biomedical applications of the neuropeptides that they contain, termed conotoxins. Meanwhile some gastropod lineages have independently acquired secretory glands strikingly similar to the venom gland of cone snails, suggesting that they possess similar venoms. Here we focus on the most diversified of these clades, the genus Vexillum. Based on the analysis of a multi-species proteo-transcriptomic dataset, we show that Vexillum species indeed produce complex venoms dominated by highly diversified short cysteine-rich peptides, vexitoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show sequence similarity to conotoxins and adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine knot motif. The Vexillum envenomation gland (gL) is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between vexitoxin genes, and their ancestral 'somatic' counterparts compared to that in conotoxins, and we find support for this hypothesis in the evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform the origin of conotoxins, and how they may help to address outstanding questions in venom evolution.
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Conotoxinas , Caracol Conus , Animales , Conotoxinas/genética , Caracol Conus/química , Caracol Conus/genética , Cisteína , Péptidos/química , Caracoles , PonzoñasRESUMEN
Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.
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Productos Biológicos , Péptidos , Productos Biológicos/química , Vías Biosintéticas , Péptidos/química , Péptidos Cíclicos/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , PiridinasRESUMEN
A new surgical technique of transscleral-intracorneal suture fixation of intraocular lenses with suture ends located within the transparent corneal or corneal-limbal paracentesis is reported. This new technique significantly reduces scleral and conjunctival trauma, is minimally invasive, is simple to perform, is not associated with complicated intraocular surgical maneuvers, and precludes suture knot, flange, and suture-end erosion. The internal part of the corneal stroma or corneal-limbal area serves as a secure depot for suture-end fixation with durable resistance. The technique enables visualization of the exact position of suture ends over time.
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Lentes Intraoculares , Técnicas de Sutura , Humanos , Implantación de Lentes Intraoculares/métodos , Esclerótica/cirugía , SuturasRESUMEN
Fermentation of cocoa is a key process to obtain aromatic chocolate products from raw cocoa beans. Hitherto, the levels of fermentation in cocoa are determined using destructive techniques, for example by a cut-test to manually observe the colour inside the beans, or by quantifying ammonia nitrogen (NH3) in the cocoa powder. In this paper, we present the use of Terahertz hyperspectral imaging as a new way to non-destructively analyse and detect fermented cocoa beans. The study analysed two sets of twenty-two cocoa bean samples with different levels of fermentation from two producers in Brazil. A correlation between fermentation conditions and the outcome results of their THz measurements was observed.
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Cacao , Chocolate , Brasil , Fermentación , Imágenes HiperespectralesRESUMEN
Treatment of vestibular dysfunction remains a challenge for many otolaryngologists. This is true not only for establishing the differential neurotologic diagnosis, but also for treatment and treatment monitoring in vestibular dysfunction patients. Particularly quality control of therapeutic measures is generally poorly documented. The validated German version of the Dizziness Handicap Inventory (DHI) represents a viable option for evaluation and monitoring of treatment outcomes. In this study, patients who were treated at the Otorhinolaryngology Department of the University Hospital Aachen because of unilateral peripheral vestibular disorders were asked to complete the DHI before and after treatment. Posttherapeutic DHI scores were collected by telephone. Evaluation of the DHI scores underlined the significant benefit of treatment in 92%. Furthermore, the DHI prove to be useful for documentation of patients' disorders and treatment and thus for quality assurance.
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Mareo , Enfermedades Vestibulares , Evaluación de la Discapacidad , Mareo/diagnóstico , Mareo/terapia , Humanos , Control de Calidad , Vértigo/diagnóstico , Vértigo/terapia , Enfermedades Vestibulares/diagnóstico , Enfermedades Vestibulares/terapiaRESUMEN
BACKGROUND: The genus Charaea Baly is distributed in the eastern Palaearctic, Himalayas, China and adjacent countries of the Oriental Region. Currently, 59 species of the genus Charaea have been recorded. The species of Charaea is characterised with a robust tubular aedeagus that terminates with a more or less distinct apical process with the Charaeacoomani group having an internal sac with long sharp lateral sclerites. Up to now, 13 species of this group have been described in the Oriental Region, four of which are found in Vietnam. NEW INFORMATION: Charaeadinhcuongi sp. nov. is described as a new species, based on specimens collected from Phu Quoc Island in southern Vietnam. Colour photographs of habitus and body details and DNA barcode sequences are presented. An identification key is provided for all Vietnamese species from the Charaeacoomani group.
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JC virus (JCV) causes progressive multifocal leukoencephalopathy in immunocompromised patients. The prevalence and genotype patterns of JCV vary between different geographical regions. This study was done to investigate the prevalence and genotype distribution of JCV in patients with hematological malignancies in Vietnam. A total of 48 urine samples were collected from patients with hematological malignancies. DNA was extracted and detection of JCV was by nested-polymerase chain reaction. Sequence analysis was obtained and a phylogenetic tree was constructed for genotyping of JCV. Twenty-seven (56.25%) urine samples tested positive for JCV. JCV genotype 7 was only observed in this study. Subtype analysis showed that JCV subtype 7A was the most commonly prevalent, followed by 7B1 and 7C1. Other subtypes were not detected in this population. There were no significant differences associated with age, gender, and biochemical parameters between patients with JCV and without JCV excretion in urine. The present study showed a high prevalence of JCV in the urine of patients with hematologic malignancies. The most common genotype found in this population was JCV subtype 7A.
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Neoplasias Hematológicas/virología , Virus JC/genética , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Adulto , Anciano , ADN Viral/genética , ADN Viral/orina , Femenino , Genotipo , Neoplasias Hematológicas/epidemiología , Neoplasias Hematológicas/orina , Humanos , Virus JC/aislamiento & purificación , Masculino , Persona de Mediana Edad , Filogenia , Infecciones por Polyomavirus/epidemiología , Infecciones por Polyomavirus/orina , Prevalencia , Infecciones Tumorales por Virus/epidemiología , Infecciones Tumorales por Virus/orina , Vietnam/epidemiología , Carga ViralRESUMEN
For large-scale analysis of complex protein mixtures, liquid chromatography - tandem mass spectrometry (LC-MS/MS) has been proven to be one of the most versatile tools due to its high sensitivity and ability to both identify and quantify thousands of proteins in a single measurement. Sample preparation typically comprises site-specific cleavage of proteins into peptides, followed by desalting and concomitant peptide enrichment, which is commonly performed by solid phase extraction. Desalting workflows may include multiple liquid handling steps and are thus error prone and labour intensive. To improve the reproducibility of sample preparation for low amounts of protein, we present a centrifugal microfluidic disk that automates all liquid handling steps required for peptide desalting by solid phase extraction (DesaltingDisk). Microfluidic implementation was enabled by a novel centrifugal microfluidic dosing on demand structure that enabled mapping multiple washing steps onto a microfluidic disk. Evaluation of the microfluidic disk was performed by LC-MS/MS analysis of tryptic HEK-293 eukaryotic cell peptide mixtures desalted either using the microfluidic disk or a manual workflow. A comparable number of peptides were identified in the disk and manual set with 19 775 and 20 212 identifications, respectively. For a core set of 10 444 peptides that could be quantified in all injections, intensity coefficients of variation were calculated based on label-free quantitation intensities. The disk set featured smaller variability with a median CV of 9.3% compared to the median CV of 12.6% for the manual approach. Intensity CVs on protein level were lowered from 5.8% to 4.2% when using the LabDisk. Interday reproducibility for both workflows was assessed by LC-SRM/MS analysis of samples that were spiked with 11 synthetic peptides of varying hydrophobicity. Except for the most hydrophilic and hydrophobic peptides, the average CV was lowered to 3.6% for the samples processed with the disk compared to 7.2% for the manual workflow. The presented centrifugal microfluidic DesaltingDisk demonstrates the potential to improve reproducibility in the sample preparation workflow for proteomic mass spectrometry, especially for application with limited amount of sample material.
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Proteómica , Espectrometría de Masas en Tándem , Automatización , Cromatografía Liquida , Células HEK293 , Humanos , Microfluídica , Péptidos , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The current standard of care for acute atrial fibrillation (AF) focuses primarily on immediate restoration of sinus rhythm by cardioversion, although AF often terminates spontaneously. OBJECTIVE: To identify determinants of early spontaneous conversion (SCV) in patients presenting at the emergency department (ED) because of AF. METHODS: An observational study was performed of patients who visited the ED with documented AF between July 2014 and December 2016. The clinical characteristics and demographics of patients with and without SCV were compared. RESULTS: We enrolled 943 patients (age 69⯱ 12 years, 47% female). SCV occurred within 3â¯h of presentation in 158 patients (16.8%). Logistic regression analysis showed that duration of AF <24â¯h [odds ratio (OR) 7.7, 95% confidence interval (CI) 3.5-17.2, pâ¯< 0.001], left atrial volume index <42â¯ml/m2 (OR 1.8, 95% CI 1.2-2.8, pâ¯= 0.010), symptoms of near-collapse at presentation (OR 2.4, 95% CI 1.2-5.1, pâ¯= 0.018), a lower body mass index (BMI) (OR 0.9, 95% CI 0.91-0.99, pâ¯= 0.028), a longer QTc time during AF (OR 1.01, 95% CI 1.0-1.02, pâ¯= 0.002) and first-detected AF (OR 2.5, 95% CI 1.6-3.9, pâ¯< 0.001) were independent determinants of early SCV. CONCLUSION: Early spontaneous conversion of acute AF occurs in almost one-sixth of admitted patients during a short initial observation in the ED. Spontaneous conversion is most likely to occur in patients with first-onset, short-duration AF episodes, lower BMI, and normal left atrial size.
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New asterosaponin, acanthaglycoside G (1), along with three previously known steroidal oligoglycosides (2â4), were isolated from the ethanolic extract of the starfish Acanthaster planci, collected off the coast of Vietnam. The structure of 1 was mainly elucidated by extensive NMR and ESIMS techniques as sodium 6-O-{ß-D-fucopyranosyl-(1â2)-ß-D-quinovopyranosyl-(1â4)-[ß-D-quinovopyranosyl-(1â2)]-ß-D-quinovopyranosyl-(1â3)-ß-D-quinovopyranosyl}-6α-hydroxy-5α-pregn-9(11)-en-20-one-3ß-yl sulfate. Compounds 3 and 4 showed slight cytotoxic activities against cancer RPMI-7951, HT-29, and MDA-MB-231 cell lines, but effectively inhibited in non-toxic concentrations colony formation of HT-29 and MDA-MB-231 cells and cell migration of MDA-MB-231 cells. Compounds 1 and 2 were inactive or less active, respectively.