DNA Damage Accelerates G-Quadruplex Folding in a Duplex-G-Quadruplex-Duplex Context.

Molecular details for the impact of DNA damage on folding of potential G-quadruplex sequences (PQSs) to noncanonical DNA structures involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the VEGF promoter sequence embedded between two DNA duplex anchors, termed a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg2+ ions and G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D 1H NMR and CD analyses confirmed that isothermal folding of the VEGF-DGD constructs yielded spectral signatures that suggest the formation of G4 motifs and demonstrated a folding dependency on the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames.

DNA Damage Accelerates G-Quadruplex Folding in a Duplex-G-Quadruplex-Duplex Context.

Molecular details for DNA damage impact on the folding of potential G-quadruplex sequences (PQS) to non-canonical DNA structures that are involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the VEGF promoter sequence embedded between two DNA duplex anchors, referred to as a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg2+ ions and the G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D 1H-NMR and CD analyses confirmed that isothermal folding of the VEGF-DGD constructs yielded spectral signatures that suggest formation of G4 motifs, and demonstrated a folding dependency with the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames.

Abduction/Adduction Assistance From Powered Hip Exoskeleton Enables Modulation of User Step Width During Walking.

Using wearable robotics to modulate step width in normal walking for enhanced mediolateral balance has not been demonstrated in the field. We designed a bilateral hip exoskeleton with admittance control to power hip abduction and adduction to modulate step width. OBJECTIVE: As the first step to show its potential, the objective of this study was to investigate how human's step width reacted to hip exoskeleton's admittance control parameter changes during walking. METHODS: Ten non-disabled individuals walked on a treadmill at a self-selected speed, while wearing our bilateral robotic hip exoskeleton. We used two equilibrium positions to define the direction of assistance. We studied the influence of multiple stiffness values in the admittance control on the participants' step width, step length, and electromyographic (EMG) activity of the gluteus medius. RESULTS: Step width were significantly modulated by the change of stiffness in exoskeleton control, while step length did not show significant changes. When the stiffness changed from zero to our studied stiffness values, the participants' step width started to modulate immediately. Within 4 consecutive heel strikes right after a stiffness change, the step width showed a significant change. Interestingly, EMG activity of the gluteus medius did not change significantly regardless the applied stiffness and powered direction. CONCLUSION: Tuning of stiffness in admittance control of a hip exoskeleton, acting in mediolateral direction, can be a viable way for controlling step width in normal walking. Unvaried gluteus medius activity indicates that the increase in step width were mainly caused by the assistive torque applied by the exoskeleton. SIGNIFICANCE: Our study results pave a new way for future design and control of wearable robotics in enhancing mediolateral walking balance for various rehabilitation applications.

Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications.

Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (Ψ) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose adducts at C1' of Ψ. The adducted sites produced greater base call errors in the form of deletion signatures compared to Ψ. Sequencing studies on E. coli rRNA and tmRNA before and after the pH 7 bisulfite reaction demonstrated that using chemically-assisted nanopore sequencing has distinct advantages for minimization of false positives and false negatives in the data. The rRNA from E. coli has 19 known U/C sequence variations that give similar base call signatures as Ψ, and therefore, are false positives when inspecting base call data; however, these sites are refractory to reacting with bisulfite as is easily observed in nanopore data. The E. coli tmRNA has a low occupancy Ψ in a pyrimidine-rich sequence context that is called a U representing a false negative; partial occupancy by Ψ is revealed after the bisulfite reaction. In a final study, 5-methylcytidine (m5C) in RNA can readily be observed after the pH 5 bisulfite reaction in which the parent C deaminates to U and the modified site does not react. This locates m5C when using bisulfite-assisted nanopore direct RNA sequencing, which is otherwise challenging to observe. The advantages and challenges of the overall approach are discussed.

Neural prosthesis control restores near-normative neuromechanics in standing postural control.

Current lower-limb prostheses do not provide active assistance in postural control tasks to maintain the user's balance, particularly in situations of perturbation. In this study, we aimed to address this missing function by enabling neural control of robotic lower-limb prostheses. Specifically, electromyographic (EMG) signals (amplified neural control signals) recorded from antagonistic residual ankle muscles were used to drive a robotic prosthetic ankle directly and continuously. Participants with transtibial amputation were recruited and trained in using the EMG-driven robotic ankle. We studied how using the EMG-controlled ankle affected the participants' anticipatory and compensatory postural control strategies and stability under expected perturbations compared with using their daily passive devices. We investigated the similarity of neuromuscular coordination (by analyzing motor modules) of the participants, using either device in a postural sway task, to that of able-bodied controls. Results showed that, compared with their passive prosthesis, the EMG-controlled prosthesis enabled participants to use near-normative postural control strategies, as evidenced by improved between-limb symmetry in intact-prosthetic center-of-pressure and joint angle excursions. Participants substantially improved postural stability, as evidenced by a reduction in steps or falls using the EMG-controlled prosthetic ankle. Furthermore, after relearning to use residual ankle muscles to drive the robotic ankle in postural control, nearly all participants' motor module structure shifted toward that observed in individuals without limb amputations. Here, we have demonstrated the potential benefit of direct EMG control of robotic lower limb prostheses to restore normative postural control strategies (both neural and biomechanical) toward enhancing standing postural stability in amputee users.

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA.

Nucleophilic addition of bisulfite to pyrimidine bases has been known for a half century, and the reaction has been in use for at least a quarter of a century for identifying 5-methylcytidine in DNA. This account focuses on the chemistry of bisulfite with pseudouridine, an isomer of the RNA nucleoside uridine in which the uracil base is connected to C1' of ribose via C5 instead of N1. Pseudouridine, Ψ, is the most common nucleotide modification found in cellular RNA overall, in part due to its abundance in rRNAs and tRNAs. It has a stabilizing influence on RNA structure because N1 is now available for additional hydrogen bonding and because the heterocycle is slightly better at π stacking. The isomerization of U to Ψ in RNA strands is catalyzed by 13 different enzymes in humans and 11 in E. coli; some of these enzymes are implicated in disease states which is testament to the biological importance of pseudouridine in cells. Recently, pseudouridine came into the limelight as the key modification that, after N1 methylation, enables mRNA vaccines to be delivered efficiently into human tissue with minimal generation of a deleterious immunogenic response. Here we describe the bisulfite reaction with pseudouridine which gives rise to a chemical sequencing method to map the modified base in the epitranscriptome. Unlike the reaction with cytidine, the addition of bisulfite to Ψ leads irreversibly to form an adduct that is bypassed during cDNA synthesis by reverse transcriptases yielding a characteristic deletion signature. Although there were hints to the structure of the bisulfite adduct(s) 30 to 50 years ago, it took modern spectroscopic and computational methods to solve the mystery. Raman spectroscopy along with extensive NMR, ECD, and computational work led to the assignment of the major product as the (R) diastereomer of an oxygen adduct at C1' of a ring-opened pseudouridine. Mechanistically, this arose from a succession of conjugate addition, E2 elimination, and a [2,3] sigmatropic rearrangement, all of which are stereodefined reactions. A minor reaction with excess bisulfite led to the (S) isomer of a S-adducted SO3- group. Understanding structure and mechanism aided the design of a Ψ-specific sequencing reaction and guided attempts to improve the utility and specificity of the method. Separately, we have been investigating the use of nanopore direct RNA sequencing, a single-molecule method that directly analyzes RNA strands isolated from cells after end-ligation of adaptor sequences. By combining the electrical current and base-calling data from the nanopore with dwell-time analysis from the helicase employed to deliver RNA to the nanopore, we were able to map Ψ sites in nearly all sequence contexts. This analysis was employed to find Ψ residues in the SARS-CoV-2 vRNA, to analyze the sequence context effects of mRNA vaccine synthesis via in vitro transcription, and to evaluate the impact of stress on chemical modifications in the E. coli ribosome. Most recently, we found that bisulfite treatment of RNA leading to Ψ adducts could modulate the nanopore signal to help in mapping modifications of low occupancy.

Sequencing for oxidative DNA damage at single-nucleotide resolution with click-code-seq v2.0.

Oxidative damage to DNA nucleotides has many cellular outcomes that could be aided by the development of sequencing methods. Herein, the previously reported click-code-seq method for sequencing a single damage type is redeveloped to enable the sequencing of many damage types by making simple changes to the protocol (i.e., click-code-seq v2.0).

NEIL3 promoter G-quadruplex with oxidatively modified bases shows magnesium-dependent folding that stalls polymerase bypass.

Oxidative stress unleashes reactive species capable of oxidizing 2'-deoxyguanosine (G) nucleotides in G-rich sequences of the genome, such as the potential G-quadruplex forming sequencing (PQS) in the NEIL3 gene promoter. Oxidative modification of G yields 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) that can be further oxidized to hydantoin products. Herein, OG was synthesized into the NEIL3 PQS that was allowed to fold to a G-quadruplex (G4) in K+ ion solutions with varying amounts of Mg2+ in the physiological range. The Mg2+ dependency in the oxidatively modified NEIL3 G4 to stall a replicative DNA polymerase was evaluated. The polymerase was found to stall at the G4 or OG, as well as continue to full-length extension with dependency on the location of the modification and the concentration of Mg2+. To provide some clarity on these findings, OG or the hydantoins were synthesized in model NEIL3 G4 folding sequences at the positions of the polymerase study. The model G4 sequences were allowed to fold in K+ ion solutions with varying levels of Mg2+ to identify how the presence of the divalent metal impacted G4 folding depending on the location of the modification. The presence of Mg2+ either caused the transition of the NEIL3 G4 folds from an antiparallel to parallel orientation of the strands or had no impact. Structural models are proposed to understand the findings using the literature as a guide. The biological significance of the results is discussed.

Direct Nanopore Sequencing for the 17 RNA Modification Types in 36 Locations in the E. coli Ribosome Enables Monitoring of Stress-Dependent Changes.

The bacterium Escherichia coli possesses 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Nanopore direct RNA sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to unmodified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification's signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. The double modification m4Cm1402 resides in 16S rRNA, and it decreased with both stressors. Using the helicase dwell time, it was determined that the N4 methyl group is lost during both stressors, and the 2'-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

Promoters vs. telomeres: AP-endonuclease 1 interactions with abasic sites in G-quadruplex folds depend on topology.

The DNA repair endonuclease APE1 is responsible for the cleavage of abasic sites (AP) in DNA as well as binding AP in promoter G-quadruplex (G4) folds in some genes to regulate transcription. The present studies focused on the topological properties of AP-bearing G4 folds and how they impact APE1 interaction. The human telomere sequence with a tetrahydrofuran model (F) of an AP was folded in K+- or Na+-containing buffers to adopt hybrid- or basket-folds, respectively. Endonuclease and binding assays were performed with APE1 and the G4 substrates, and the data were compared to prior work with parallel-stranded VEGF and NEIL3 promoter G4s to identify topological differences. The APE1-catalyzed endonuclease assays led to the conclusion that telomere G4 folds were slightly better substrates than the promoter G4s, but the yields were all low compared to duplex DNA. In the binding assays, G4 topological differences were observed in which APE1 bound telomere G4s with dissociation constants similar to single-stranded DNA, and promoter G4s were bound with nearly ten-fold lower values similar to duplex DNA. An in-cellulo assay with the telomere G4 in a model promoter bearing a lesion failed to regulate transcription. These data support a hypothesis that G4 topology in gene promoters is a critical feature that APE1 recognizes for gene regulation.

Nanopore sequencing for N1-methylpseudouridine in RNA reveals sequence-dependent discrimination of the modified nucleotide triphosphate during transcription.

Direct RNA sequencing with a commercial nanopore platform was used to sequence RNA containing uridine (U), pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ) in >100 different 5-nucleotide contexts. The base calling data for Ψ or m1Ψ were similar but different from U allowing their detection. Understanding the nanopore signatures for Ψ and m1Ψ enabled a running start T7 RNA polymerase assay to study the selection of UTP versus ΨTP or m1ΨTP competing mixtures in all possible adjacent sequence contexts. A significant sequence context dependency was observed for T7 RNA polymerase with insertion yields for ΨTP versus UTP spanning a range of 20-65%, and m1ΨTP versus UTP producing variable yields that differ by 15-70%. Experiments with SP6 RNA polymerase, as well as chemically-modified triphosphates and DNA templates provide insight to explain the observations. The SP6 polymerase introduced m1ΨTP when competed with UTP with a smaller window of yields (15-30%) across all sequence contexts studied. These results may aid in future efforts that employ RNA polymerases to make therapeutic mRNAs with sub-stoichiometric amounts of m1Ψ.

The Impact of COVID-19 on Emergency Medicine Rotations.

Introduction The impact of modifications in curriculum and clinical rotations made secondary to the COVID-19 pandemic on medical education has yet to be fully investigated. We observed differences in the types of patients seen by medical students that may have resulted from clinical disruptions due to the COVID-19 pandemic. We then evaluated what impact these disruptions had on the students' clinical competency. Methods We obtained patient logbooks of third-year medical students (M3) and fourth-year sub-interns (M4) from the first three emergency medicine (EM) rotation blocks of the 2019-2020 (Y19) and 2020-2021 (Y20) academic years. We then reviewed and categorized the chief complaints seen and procedures in which they participated. A robust t-test was used to detect differences in chief complaints and procedures. Finally, we looked for objective differences using the chi-square test in clinical performance between the class of 2021 (Class21) and the class of 2022 (Class22), as assessed by performance on our institution's clinical competency examination. Results Overall, students saw a 25.3% decrease in average number of patient encounters. Statistically significant decreased average numbers of infectious (-28.3%, p=0.013); musculoskeletal (-22.2%, p=0.018); gastrointestinal (GI) (-24.6%, p<0.01); genitourinary (GU) (-33.2%, p<0.01); head, eyes, ears, nose, throat (HEENT) (-31.1%, p<0.01); trauma (-33.0%, p<0.01); and respiratory (-45.4%, p<0.001) complaints were observed. Both M3s and M4s encountered significantly less GU (-25.6%, p=0.048; -41.7%, p=0.016) and trauma (-29.1%, p=0.023; -33.2%, p=0.032) complaints in Y20. M4s saw significantly less GI complaints (-42.6%, p<0.001) in Y20, whereas M3s encountered significantly less psychiatric and HEENT complaints (-30.3%, p=0.046; -34.6%, p=0.013). Both classes saw significantly less respiratory complaints in Y20 but more so for M4s (-65.3%, p<0.001) than for M3s (-27.9%, p=0.033). There were no significant differences in average number of procedures between years. We did not observe any differences in overall clinical performance between the two selected classes. While class of 2021 scored a significantly higher average on a case of fatigue (p=0.0004) and class of 2022 on a case of abdominal pain (p<0.0001), there were no significant differences in the primary chief complaints that would be attributed to COVID-19, such as dyspnea. Conclusion Modifications made to curricula and clinical rotations due to the COVID-19 pandemic led to students encountering less patients overall, with significant decreases in multiple chief complaint types compared to Y19 but no significant change in procedure numbers. Notably, there was no major impact seen on clinical competency providing a positive argument for considering innovative teaching and learning methods.

Fluorophore-mediated Photooxidation of the Guanine Heterocycle.

Fluorescent dyes are routinely used to visualize DNA or RNA in various experiments, and some dyes also act as photosensitizers capable of catalyzing oxidation reactions. The present studies explored whether the common labeling dyes fluorescein, rhodamine, BODIPY, or cyanine3 (Cy3) can function as photosensitizers to oxidize nucleic acid polymers. Photoirradiation of each dye in the presence of the guanine (G) heterocycle, which is the most sensitive toward oxidation, identified slow rates of nucleobase oxidation in the nucleoside and DNA contexts. For all four fluorophores studied, the only product detected was spiroiminodihydantoin (Sp) suggesting the dyes functioned as Type II photosensitizers and generate singlet oxygen (1O2). The nucleoside reactions were then conducted in D2O solutions, known to increase the lifetime of 1O2, which resulted in a ~6-fold increase in the Sp yield, further supporting the classification of these dyes as Type II photosensitizers. Lastly, we inspected the pattern of G reactivity with the dyes upon photoirradiation in the context of a parallel-stranded G-quadruplex. The G nucleotides in the two exterior G-tetrads were found to be oxidation prone, providing the third line of evidence that the dyes are Type II photooxidants. The present work found that the common dyes fluorescein, rhodamine, BODIPY, or Cy3 can drive G oxidation but with a slow rate and low overall yield. This will likely not impact many experiments using dyes to study nucleic acids except for those that have long exposures with high-intensity lights, such as sequencing-by-synthesis experiments using fluorescence as the readout.

Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA.

The binding interaction between the DNA repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the VEGF promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the VEGF promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of ∼100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the VEGF G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.

Pseudouridine and N1-Methylpseudouridine Display pH-Independent Reaction Rates with Bisulfite Yielding Ribose Adducts.

In RNA, pseudouridine (Ψ) and 5-methylcytidine (m5C) are located by their differential reactions with NaHSO3 at pH 5. The pyrimidines were allowed to react with NaHSO3, NaN3, NaCN, or NaSCN at pH 5 to find that NaHSO3 was unique in achieving quantitative yields. Pseudouridine reaction selectivity with NaHSO3 was found at pH 7 supported by the reaction rate constants. The Ψ derivative N1-methylpseudouridine found in mRNA vaccines reacts similarly with bisulfite to yield ribose adducts.

Insights into the 5-Carboxamido-5-Formamido-2-Iminohydantoin Structural Isomerization Equilibria.

Exposure of DNA to oxidants results in modification of the electron-rich guanine heterocycle including formation of the mutagenic 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) lesion. Previously thought to exist solely as a pair of diastereomers, we found under biologically relevant conditions that 2Ih reversibly closes to a formerly hypothetical intermediate and opens into a newly discovered regioisomer. In a nucleoside model, only ∼80% of 2Ih existed as the structure studied over the last 20 years with significant isomeric products persisting in buffered aqueous solution.

Cysteine Oxidation to Sulfenic Acid in APE1 Aids G-Quadruplex Binding While Compromising DNA Repair.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a base excision repair (BER) enzyme that is also engaged in transcriptional regulation. Previous work demonstrated that the enzymatic stalling of APE1 on a promoter G-quadruplex (G4) recruits transcription factors during oxidative stress for gene regulation. Also, during oxidative stress, cysteine (Cys) oxidation is a post-translational modification (PTM) that can change a protein's function. The current study provides a quantitative survey of cysteine oxidation to sulfenic acid in APE1 and how this PTM at specific cysteine residues affects the function of APE1 toward the NEIL3 gene promoter G4 bearing an abasic site. Of the seven cysteine residues in APE1, five (C65, C93, C208, C296, and C310) were prone to carbonate radical anion oxidation to yield sulfenic acids that were identified and quantified by mass spectrometry. Accordingly, five Cys-to-serine (Ser) mutants of APE1 were prepared and found to have attenuated levels of endonuclease activity, depending on the position, while KD values generally decreased for G4 binding, indicating greater affinity. These data support the concept that cysteine oxidation to sulfenic acid can result in modified APE1 that enhances G4 binding at the expense of endonuclease activity during oxidative stress. Cysteine oxidation to sulfenic acid residues should be considered as one of the factors that may trigger a switch from base excision repair activity to transcriptional modulation by APE1.

Group 3 innate lymphocytes make a distinct contribution to type 17 immunity in bladder defence.

Bladder infection affects a hundred million people annually, but our understanding of bladder immunity is incomplete. We found type 17 immune response genes among the most up-regulated networks in mouse bladder following uropathogenic Escherichia coli (UPEC) challenge. Intravital imaging revealed submucosal Rorc+ cells responsive to UPEC challenge, and we found increased Il17 and IL22 transcripts in wild-type and Rag2 -/- mice, implicating group 3 innate lymphoid cells (ILC3s) as a source of these cytokines. NCR-positive and negative ILC3 subsets were identified in murine and human bladders, with local proliferation increasing IL17-producing ILC3s post infection. ILC3s made a more limited contribution to bladder IL22, with prominent early induction of IL22 evident in Th17 cells. Single-cell RNA sequencing revealed bladder NCR-negative ILC3s as the source of IL17 and identified putative ILC3-myeloid cell interactions, including via lymphotoxin-ß-LTBR. Altogether, our data provide important insights into the orchestration and execution of type 17 immunity in bladder defense.