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Background: Frailty is common in geriatric emergency surgery and associated with increased risk for poor postoperative outcomes. Frailty screening is challenging in emergency settings. The Edmonton Frail Scale (EFS) is a valid tool to screen for patients at high risk for poor postoperative outcomes. Recently, the EFS was modified to decrease dependence on staff to perform physical measures. This modification, the EFS-Acute Care (EFS-AC), has not been validated. We wish to assess the agreement between the EFS and the EFS-AC., Study Design: We performed a prospective cohort study from 10/2021 - 10/2022 screening 688 patients ≥ 65 years with both the EFS and EFS-AC preoperatively. We assessed the ability of the EFS-AC to discriminate frailty identified by the EFS and compared the association of both scales with loss of independence (LOI), hospital length of stay (LOS), ICU admissions, and ICU LOS. Receiver Operator Curves were used to estimate the discriminatory thresholds for LOI., Results: 688 patients with a median age 73 (IQR 68, 77) were enrolled. The EFS-AC was able to discriminate individuals' frailty status by the EFS with excellent agreement (AUC 0.971 [0.958, 0.983]). An EFS-AC threshold score of ≥ 6 points lead to 93.60% of individuals being correctly identified (77.87% sensitivity and 97.00% specificity). Both EFS and EFS-AC ≥ 6 were similarly associated with a higher risk for all clinical outcomes assessed and demonstrated similar ability to predict LOI., Conclusions: The EFS-AC is a valid preoperative frailty screen, and due to its self-reported nature, can be administered in the acute care setting, during virtual visits, or through digital health apps. Real-time screening can assist with better understanding patient needs and lead to interventions to prevent poor hospital outcomes., Competing Interests: Declaration of competing interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: April Ehrlich reports financial support was provided by National Institute on Aging. Eduardo Biala Jr. reports financial support was provided by National Institute on Aging. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflicts of Interest: None

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (-)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics.

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5-15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation. The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E. coli tRNA(Ala). The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase. Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site.

Thermal denaturation studies were carried out on a set of site-specific variants of a 22mer RNA hairpin comprising the aminoacyl acceptor stem sequence of E. coli tRNA(Ala). The pairing thermodynamics were calculated from the melting profiles.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.

The nonamer r(GCUUCGGC)dBrU, where dBrU is 5-bromo-2'-deoxyuridine, contains the tetraloop sequence UUCG. It crystallizes in the presence of Rh(NH3)6Cl3. In solution the oligomer is expected to form a hairpin loop but the x-ray structure analysis, to a resolution of 1.6 A, indicates an eight-base-pair A-RNA duplex containing a central block of two G.U and two C.U pairs. Self-pairs which approximate to Watson-Crick geometry are also formed in the extended crystal structure between symmetry-related BrU residues and are part of infinite double-helical stacks. The G.U pair is a wobble base pair analogous to the G.T pair found in DNA fragments. The C.U mismatch involves one hydrogen-bonded contact between the bases and a bridging water molecule which ensures a good fit of the base pair in the RNA helix. The BrU.BrU pair is held by two hydrogen bonds in an orientation which is compatible with duplex geometry. The structure observed within the crystal has some parallels with the structure of globular RNAs, and the presence of stable, noncanonical base pairs has implications for the prediction of RNA secondary structure.

Synthetic oligoribonucleotides having single uracil residues replaced by dU, dT, 2'-O-methylU or 5-bromodU have been prepared and used in the study of the interaction of HIV-1 tat protein with an RNA stem-loop. The preparation of phosphoramidites of 5-bromouridine and purine riboside suitable for use in solid-phase oligoribonucleotide synthesis is also described. The effect of adenine replacement by purine in a hammerhead ribozyme has also been determined.

An improved procedure for the transformation of 5'-O-monomethoxytrityl-2'-O-acetyl-3'-phosphates of uridine la, inosine ib and 6-N-benzoyladenosine lc into corresponding 3'/2,2,2-trichloroethyl, 2-cyanoethyl/-phosphates iiaic is reported. H NMR characterization of nucleoside 3'-phosphotriesters is presented. New conditions i.e. anhydrous triethylamine-pyridine treatment have been found for the selective removal of 2-cyanoethyl group from nucleoside 3'-phosphotriesters in the presence of neighbouring 2'-O-acetyl one.

Zinc/acetylacetone/pyridine treatment has been designed as a very efficient method for removal of 2,2,2,-trichloroethyl group from phosphoesters. Internucleotide and terminal 2,2,2-trichloroethylphosphotriesters were transformed to corresponding diesters quantitatively. Much less reactive 2,2,2-trichloroethylphosphodiesters produced monoesters with ca. 90% yield. 31P NMR spectroscopy has been proposed as a new tool for analysis of removal of internucleotide phosphate protecting groups-a crucial step in oligonucleotides synthesis via phosphotriester approach.