Genome-wide mapping of i-motifs reveals their association with transcription regulation in live human cells.

i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate. We performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped iMs in two human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs. By simultaneously mapping G-quadruplexes (G4s), which form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.

Among the secondary structures alternative to the DNA double helix, i-Motifs (iMs) and G-quadruplexes (G4s) are four-stranded non-canonical nucleic acid structures that form in cytosine- and guanine-rich regions, respectively. Because iMs fold in vitro under acidic conditions, they were long thought to form only in vitro. We now show that iMs, like G4s, form in live human cells mainly at gene promoters in open chromatin. iMs that are unstable in vitro still form in cells. iMs and G4s are cell-type specific and associated with increased transcription; however, transcript levels are remarkably different: low for iMs and high for G4s, indicating their distinct activity as regulators of the cell transcriptome. The iM/G4 interplay may represent a novel therapeutic target in disease.

Targeting G-quadruplexes to achieve antiviral activity.

With the emergence of new viruses in the human population and the fast mutation rates of existing viruses, new antiviral targets and compounds are needed. Most existing antiviral drugs are active against proteins of a handful of viruses. Most of these proteins in the end affect viral nucleic acid processing, but direct nucleic acid targeting is less represented due to the difficulty of selectively acting at the nucleic acid of interest. Recently, nucleic acids have been shown to fold in structures alternative to the classic double helix and Watson and Crick base-pairing. Among these non-canonical structures, G-quadruplexes (G4s) have attracted interest because of their key biological roles that are being discovered. Molecules able to selectively target G4s have been developed and since G4s have been investigated as targets in several human pathologies, including viral infections. Here, after briefly introducing viruses, G4s and the G4-binding molecules with antiviral properties, we comment on the mechanisms at the base of the antiviral activity reported for G4-binding molecules. Understanding how G4-ligands act in infected cells will possibly help designing and developing next-generation antiviral drugs.

Biological relevance and therapeutic potential of G-quadruplex structures in the human noncoding transcriptome.

Noncoding RNAs are functional transcripts that are not translated into proteins. They represent the largest portion of the human transcriptome and have been shown to regulate gene expression networks in both physiological and pathological cell conditions. Research in this field has made remarkable progress in the comprehension of how aberrations in noncoding RNA drive relevant disease-associated phenotypes; however, the biological role and mechanism of action of several noncoding RNAs still need full understanding. Besides fulfilling its function through sequence-based mechanisms, RNA can form complex secondary and tertiary structures which allow non-canonical interactions with proteins and/or other nucleic acids. In this context, the presence of G-quadruplexes in microRNAs and long noncoding RNAs is increasingly being reported. This evidence suggests a role for RNA G-quadruplexes in controlling microRNA biogenesis and mediating noncoding RNA interaction with biological partners, thus ultimately regulating gene expression. Here, we review the state of the art of G-quadruplexes in the noncoding transcriptome, with their structural and functional characterization. In light of the existence and further possible development of G-quadruplex binders that modulate G-quadruplex conformation and protein interactions, we also discuss the therapeutic potential of G-quadruplexes as targets to interfere with disease-associated noncoding RNAs.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17-26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections.

Selective targeting of mutually exclusive DNA G-quadruplexes: HIV-1 LTR as paradigmatic model.

Targeting of G-quadruplexes, non-canonical conformations that form in G-rich regions of nucleic acids, has been proposed as a novel therapeutic strategy toward several diseases, including cancer and infections. The unavailability of highly selective molecules targeting a G-quadruplex of choice has hampered relevant applications. Herein, we describe a novel approach, based on naphthalene diimide (NDI)-peptide nucleic acid (PNA) conjugates, taking advantage of the cooperative interaction of the NDI with the G-quadruplex structure and hybridization of the PNA with the flanking region upstream or downstream the targeted G-quadruplex. By biophysical and biomolecular assays, we show that the NDI-PNA conjugates are able to specifically recognize the G-quadruplex of choice within the HIV-1 LTR region, consisting of overlapping and therefore mutually exclusive G-quadruplexes. Additionally, the conjugates can induce and stabilize the least populated G-quadruplex at the expenses of the more stable ones. The general and straightforward design and synthesis, which readily apply to any G4 target of choice, together with both the red-fluorescent emission and the possibility to introduce cellular localization signals, make the novel conjugates available to selectively control G-quadruplex folding over a wide range of applications.

Angiotensin II Promotes SARS-CoV-2 Infection via Upregulation of ACE2 in Human Bronchial Cells.

Blockers of the renin-angiotensin system (RAS) have been reported to increase the angiotensin converting enzyme (ACE)2, the cellular receptor of SARS-CoV-2, and thus the risk and course of COVID-19. Therefore, we investigated if angiotensin (Ang) II and RAS blockers affected ACE2 expression and SARS-CoV-2 infectivity in human epithelial bronchial Calu-3 cells. By infectivity and spike-mediated cell-cell fusion assays, we showed that Ang II acting on the angiotensin type 1 receptor markedly increased ACE2 at mRNA and protein levels, resulting in enhanced SARS-CoV-2 cell entry. These effects were abolished by irbesartan and not affected by the blockade of ACE-1-mediated Ang II formation with ramipril, and of ACE2- mediated Ang II conversion into Ang 1-7 with MLN-4760. Thus, enhanced Ang II production in patients with an activated RAS might expose to a greater spread of COVID-19 infection in lung cells. The protective action of Angiotensin type 1 receptor antagonists (ARBs) documented in these studies provides a mechanistic explanation for the lack of worse outcomes in high-risk COVID-19 patients on RAS blockers.

Selective Recognition of a Single HIV-1 G-Quadruplex by Ultrafast Small-Molecule Screening.

G-quadruplexes (G4s) are implicated in pathological processes such as cancer and infective diseases. Their targeting with G4-ligands has shown therapeutic capacity. Most of the current G4-ligands are planar molecules, do not discriminate among G4s, and have poor druglike properties. The available methods to identify compounds selective for one single G4 are often time-consuming. Here, we describe the development, validation, and application of an affinity-selection mass spectrometry method that employs unlabeled G4 oligonucleotides as targets and allows testing of up to 320 unmodified small molecules in a single tube. As a proof of concept, this method was applied to screen a library of 40 000 druglike molecules against two G4s, transcriptional regulators of the HIV-1 LTR promoter. We identified nonplanar pyrazolopyrimidines that selectively recognize and stabilize the major HIV-1 LTR G4 possibly by fitting and binding through H-bonding in its unique binding pocket. The compounds inhibit LTR promoter activity and HIV-1 replication. We propose this method to prompt the fast development of new G4-based therapeutics.

Voluntary and magnetically evoked muscle contraction protocol in males with Duchenne muscular dystrophy: Safety, feasibility, reliability, and validity.

INTRODUCTION/AIMS: Clinical trials addressing treatments for Duchenne muscular dystrophy (DMD) require reliable and valid measurement of muscle contractile function across all disease severity levels. In this work we aimed to evaluate a protocol combining voluntary and evoked contractions to measure strength and excitability of wrist extensor muscles for safety, feasibility, reliability, and discriminant validity between males with DMD and controls. METHODS: Wrist extensor muscle strength and excitability were assessed in males with DMD (N = 10; mean ± standard deviation: 15.4 ± 5.9 years of age), using the Brooke Upper Extremity Rating Scale (scored 1-6), and age-matched healthy male controls (N = 15; 15.5 ± 5.0 years of age). Torque and electromyographic (EMG) measurements were analyzed under maximum voluntary and stimulated conditions at two visits. RESULTS: A protocol of multiple maximal voluntary contractions (MVCs) and evoked twitch contractions was feasible and safe, with 96% of the participants completing the protocol and having a less than 7% strength decrement on either measure for both DMD patients and controls (P ≥ .074). Reliability was excellent for voluntary and evoked measurements of torque and EMG (intraclass correlation coefficient [ICC] over 0.90 and over 0.85 within and between visits, respectively). Torque, EMG, and timing of twitch-onset measurements discriminated between DMD and controls (P < .001). Twitch contraction time did not differ significantly between groups (P = .10). DISCUSSION: Findings from this study show that the protocol is a safe, feasible, reliable, and a valid method to measure strength and excitability of wrist extensors in males with DMD.

A randomized trial of medical cannabis in patients with stage IV cancers to assess feasibility, dose requirements, impact on pain and opioid use, safety, and overall patient satisfaction.

PURPOSE: The prevalence of medical cannabis (MC) use in patients with cancer is growing, but questions about safety, efficacy, and dosing remain. Conducting randomized, controlled trials (RCTs) using state-sponsored MC programs is novel and could provide data needed to guide patients and providers. METHODS: A pilot RCT of patients with stage IV cancer requiring opioids was conducted. Thirty patients were randomized 1:1 to early cannabis (EC, n = 15) versus delayed start cannabis (DC, n = 15). The EC group obtained 3 months (3 M) of MC through a state program at no charge, while the DC group received standard oncology care without MC for the first 3 M. Patients met with licensed pharmacists at one of two MC dispensaries to determine a suggested MC dosing, formulation, and route. Patients completed surveys on pain levels, opioid/MC use, side effects, and overall satisfaction with the study. RESULTS: Interest in the study was high as 36% of patients who met eligibility criteria ultimately enrolled. The estimated mean daily THC and CBD allotments at 3 M were 34 mg and 17 mg, respectively. A higher proportion of EC patients achieved a reduction in opioid use and improved pain control. No serious safety issues were reported, and patients reported high satisfaction. CONCLUSION: Conducting RCTs using a state cannabis program is feasible. The addition of MC to standard oncology care was well-tolerated and may lead to improved pain control and lower opioid requirements. Conducting larger RCTs with MC in state-sponsored programs may guide oncology providers on how to safely and effectively incorporate MC for interested patients.

Defining adult e-cigarette prevalence: comparing a categorical definition with days of use.

INTRODUCTION: Electronic cigarette use has grown substantially and the health effects are being closely monitored. Tracking the evolving market place and the profile of adult users is important for tobacco control efforts; however, several different ways of measuring current use have been reported. This paper examines how well a categorical definition aligns with days of use. METHODS: Data from the 2018 Minnesota Adult Tobacco Survey assessed e-cigarette use based on days of use in the past month and currently using 'every day, some days, or not at all'. Prevalence of current use and agreement of >1, >5 and >20 days of use with every day or some days were calculated. RESULTS: The prevalence of e-cigarette use varied by category of use from 2.4% (≥20 days/30) to 6.0% (≥1 day/30). The highest prevalence was found among young adults reporting any use in the past 30 days (21.9%). Never smokers had low prevalence overall; however, 4.4% reported using in the past 30 days. Using at least 1 day in the past 30 days included a higher proportion of young adults (p<0.001) and never smokers (p<0.001) compared with other current use categories. Compared with every day or some days, the per cent agreement with days of use categories ranged from 89.7% to 94.4% and kappa ranged from 0.60 to 0.81. CONCLUSIONS: Prevalence and sociodemographics varied by definition of use. Asking 'every day, some days or not at all' in population-based studies has the advantage of aligning with cigarette smoking current use definition.

A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription.

I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif.

G-Quadruplex Targeting in the Fight against Viruses: An Update.

G-quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of key cellular processes, such as transcription and replication. Since their discovery, G4s have been mainly investigated for their role in cancer and as targets in anticancer therapy. More recently, exploration of the presence and role of G4s in viral genomes has led to the discovery of G4-regulated key viral pathways. In this context, employment of selective G4 ligands has helped to understand the complexity of G4-mediated mechanisms in the viral life cycle, and highlighted the possibility to target viral G4s as an emerging antiviral approach. Research in this field is growing at a fast pace, providing increasing evidence of the antiviral activity of old and new G4 ligands. This review aims to provide a punctual update on the literature on G4 ligands exploited in virology. Different classes of G4 binders are described, with emphasis on possible antiviral applications in emerging diseases, such as the current COVID-19 pandemic. Strengths and weaknesses of G4 targeting in viruses are discussed.

Use of Nonselective ß-Blockers in Patients With End-Stage Liver Disease and Select Complications.

Objective: To review the literature and recommendations for nonselective ß-blockers (NSBBs) in the setting of variceal bleeding prophylaxis and decompensated liver disease. Data Sources: Literature search of MEDLINE was performed (1988 to October 2019) using the following search terms: cirrhosis, advanced cirrhosis, ß-blocker, decompensation, prophylaxis. Abstracts, peer-reviewed publications, clinical practice guidelines, and product monographs were reviewed. Study Selection and Data Extraction: Relevant English language studies and those conducted in humans were considered for analysis and inclusion. Data Synthesis: Evidence that suggests that NSBBs are harmful in advanced cirrhosis is overshadowed by confounding variables and small patient populations. The majority of the available evidence suggests neutral or beneficial effects on mortality with continuation of NSBBs despite liver disease progression. Based on the available literature, guidelines, and expert consensuses, NSBBs can be considered within this patient population and may have a positive impact on the majority of these patients. Relevance to Patient Care and Clinical Practice: This review summarizes current place in therapy for NSBBs in the setting of cirrhosis and variceal bleeding prophylaxis. It also includes a discussion of the literature for use of NSBBs within the setting of different acute decompensations in which the data and recommendations for use are less clear. Conclusions: Recent evidence shows neutral or positive results for NSBB use in particular decompensation subgroups, which suggests that NSBBs can be used cautiously with close monitoring in patients with advanced cirrhosis. Questions still remain regarding optimal agent and dose and whether agents can be safely restarted after an acute decompensation episode.

G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy.

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. G4s have been extensively described in the human genome, especially in telomeres and oncogene promoters; in recent years the presence of G4s in viruses has attracted increasing interest. Indeed, G4s have been reported in several viruses, including those involved in recent epidemics, such as the Zika and Ebola viruses. Viral G4s are usually located in regulatory regions of the genome and implicated in the control of key viral processes; in some cases, they have been involved also in viral latency. In this context, G4 ligands have been developed and tested both as tools to study the complexity of G4-mediated mechanisms in the viral life cycle, and as therapeutic agents. In general, G4 ligands showed promising antiviral activity, with G4-mediated mechanisms of action both at the genome and transcript level. This review aims to provide an updated close-up of the literature on G4s in viruses. The current state of the art of G4 ligands in antiviral research is also reported, with particular focus on the structural and physicochemical requirements for optimal biological activity. The achievements and the to-dos in the field are discussed.

Dyads of G-Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration.

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C7 linker exhibit extremely low IC50 values, below 10 nm, on different cancer cell lines. Contrary, the dyads with the shorter C4 linker were much less effective, with IC values increasing up to 1 µm. Among the three dyads with the longest linker, small differences in the IC50 values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents.

G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide.

G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in organisms. G-quadruplexes are present in eukaryotes, prokaryotes, and viruses. In the latter, mounting evidence indicates their key biological activity. Since data on viruses are scattered, we here present a comprehensive analysis of potential quadruplex-forming sequences (PQS) in the genome of all known viruses that can infect humans. We show that occurrence and location of PQSs are features characteristic of each virus class and family. Our statistical analysis proves that their presence within the viral genome is orderly arranged, as indicated by the possibility to correctly assign up to two-thirds of viruses to their exact class based on the PQS classification. For each virus we provide: i) the list of all PQS present in the genome (positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation among strains of each PQS in its genome context, iv) the statistical significance of PQS abundance. This information is accessible from a database to allow the easy navigation of the results: http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=g4virus. The availability of these data will greatly expedite research on G-quadruplex in viruses, with the possibility to accelerate finding therapeutic opportunities to numerous and some fearsome human diseases.

Predictors of Treatment Failure Following De-escalation to a Fluoroquinolone in Culture-Negative Nosocomial Pneumonia.

Background: Little evidence exists for de-escalation of nosocomial pneumonia therapy without positive cultures. Objective: The purpose of this study was to identify potential predictors of treatment failure following de-escalation to a fluoroquinolone in culture-negative nosocomial pneumonia. Methods: The study involved a single-center, retrospective cohort of patients admitted with diagnosis of nosocomial pneumonia and positive chest radiography who received at least 24 hours of fluoroquinolone monotherapy following at least 24 hours of appropriate empirical antibiotics. Treatment failure was defined using a composite of all-cause death within 30 days of discharge, treatment re-escalation, or readmission for pneumonia within 30 days of discharge. The Cox proportional hazards model was used to analyze predictors of treatment failure. Duration of empirical antibiotics and significant univariable exploratory predictors were included in multivariable analysis. Results: Of 164 patients, 23 (14%) failed de-escalation. Duration of empirical antibiotics (68.5 ± 32.1 vs 65.8 ± 35 hours) was not associated with treatment failure in univariable (Hazard Ratio [HR] = 1.002 [95% CI = 0.991-1.013]) or multivariable analyses (HR = 1.003 [95% CI = 0.991-1.015]). Significant exploratory predictors on univariable analysis included active cancer, intensive care unit (ICU) admission at empirical initiation, APACHE II score, and steroid use ≥20-mg prednisone equivalent. ICU admission at empirical initiation (HR = 2.439 [95% CI = 1.048-5.676]) and steroid use ≥20-mg prednisone equivalent (HR = 2.946 [95% CI = 1.281-6.772]) were associated with treatment failure on multivariable analysis. Conclusion and Relevance: Duration of empirical antibiotics does not appear to influence failure of de-escalation to fluoroquinolone monotherapy in culture-negative nosocomial pneumonia.

EVALUATION OF INSULIN GLARGINE AND EXENATIDE ALONE AND IN COMBINATION: A RANDOMIZED CLINICAL TRIAL WITH CONTINUOUS GLUCOSE MONITORING AND AMBULATORY GLUCOSE PROFILE ANALYSIS.

Objective: Characterize the effectiveness of insulin glargine alone, exenatide alone, or combined in subjects taking stable doses of metformin and evaluate their impact on hemoglobin A1C, hypoglycemia, weight, and glucose variability. Methods: Open-label, randomized, parallel-arm study of adults with type 2 diabetes naïve to both insulin and glucagon-like peptide 1 (GLP-1) agonist who were not at A1C goal despite treatment with metformin. This prospective interventional study employed blinded continuous glucose monitoring ambulatory glucose profile (AGP) reports over 32 weeks. Subjects were randomized to treatment with glargine (Iglar), exenatide (GLP-1), or combination of glargine and exenatide (Iglar + GLP-1). At midpoint, those not at A1C target had the second medication added; those on Iglar + GLP-1 continued therapy optimization. Results: Decreases in A1C were: 7.6 to 6.2% for Iglar + GLP-1, 7.5 to 6.6% for Iglar, and 7.5 to 6.4% for GLP-1. Iglar + GLP-1 achieved A1C targets faster (14 to 16 weeks) but had more hypoglycemia. Hypoglycemia rates increased slightly for all arms. Weight loss was achieved in all regimens including GLP-1. Glucose variability was not reduced to the same extent in the Iglar arm as the GLP-1 arm. Conclusion: Addition of Iglar and/or GLP-1 to metformin for patients not at treatment goal was safe and effective. The order of medication addition needs to consider individualized AGP patterns and goals. Iglar + GLP-1 resulted in rapid A1C lowering, whereas GLP-1 was noted to have less hypoglycemia. Weight loss was most pronounced in GLP-1 monotherapy, suggesting that GLP-1 may mitigate the weight gain of Iglar. Any treatment with GLP-1 showed significant decreases in glucose variability. Abbreviations: A1C = hemoglobin A1c; AGP = ambulatory glucose profile; CGM = continuous glucose monitoring; GLM = general linear model; GLP-1 = glucagon-like peptide 1 (exenatide); Iglar = insulin glargine; SGLT2 = sodium-glucose cotransporter 2; SMBG = self-monitoring blood glucose; SU = sulfonylurea; T2D = type 2 diabetes mellitus.

Conserved G-Quadruplexes Regulate the Immediate Early Promoters of Human Alphaherpesviruses.

Human Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.