Production of the anti-G-quadruplex antibody BG4 for efficient genome-wide analyses: From plasmid quality control to antibody validation.

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that can form at guanine-rich sequences of DNA and RNA in every kingdom of life. At the DNA level, G4s can form throughout genomes but they are prevalently found in promoter regions and at telomeres, and they have been attributed functions spanning from transcriptional regulation, to control of DNA replication, to maintenance of chromosome ends. Our understanding of the functions of G4s in cells has greatly improved with the development of specific anti-G4 antibodies, which allow the visualization of G4s by immunofluorescence but also the mapping of these secondary DNA structures genome wide. Whole genome identification of the location and abundance of G4s with techniques such as Chromatin Immunoprecipitation coupled with sequencing (ChIP-Seq) and Cleavage Under Target and Tagmentation (CUT&Tag) has allowed the profiling of G4 distribution across distinct cell types and deepen the understanding of G4 functions, particularly in the regulation of transcription. Crucial for these types of genome-wide studies is the availability of an anti-G4 antibody preparation with high affinity and specificity. Here, we describe a protocol for the expression and purification of the anti-DNA G4 structure antibody (BG4) first developed by the Balasubramanian group, which has been proven to selectively recognize G4 structures both in vitro and within cells, and which has great applicability in high-throughput techniques. We provide a detailed, step-by-step protocol to obtain active BG4 starting from a commercially available expression plasmid. We also describe three different approaches to validate the activity of the BG4 preparation.

Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern.

The high mutation rate of SARS-CoV-2 leads to the emergence of multiple variants, some of which are resistant to vaccines and drugs targeting viral elements. Targeting host dependency factors, e.g. cellular proteins required for viral replication, would help prevent resistance. However, it remains unclear whether different SARS-CoV-2 variants induce conserved cellular responses and exploit the same core host factors. To this end, we compared three variants of concern and found that the host transcriptional response was conserved, differing only in kinetics and magnitude. Through CRISPR screening, we identified host genes required for infection by each variant. Most of the genes were shared by multiple variants. We validated our hits with small molecules and repurposed Food and Drug Administration-approved drugs. All the drugs were highly active against all the variants tested, including new variants that emerged during the study (Delta and Omicron). Mechanistically, we identified reactive oxygen species production as a key step in early virus replication. Antioxidants such as N-acetyl cysteine (NAC) were effective against all the variants in both human lung cells and a humanised mouse model. Our study supports the use of available antioxidant drugs, such as NAC, as a general and effective anti-COVID-19 approach.

Naphthalene Diimide-Tetraazacycloalkane Conjugates Are G-Quadruplex-Based HIV-1 Inhibitors with a Dual Mode of Action.

Human immunodeficiency virus 1 (HIV-1) therapeutic regimens consist of three or more drugs targeting different steps of the viral life cycle to limit the emergence of viral resistance. In line with the multitargeting strategy, here we conjugated a naphthalene diimide (NDI) moiety with a tetraazacycloalkane to obtain novel naphthalene diimide (NDI)-tetraazacycloalkane conjugates. The NDI inhibits the HIV-1 promoter activity by binding to LTR G-quadruplexes, and the tetraazacycloalkane mimics AMD3100, which blocks HIV entry into cells by interfering with the CXCR4 coreceptor. We synthesized, purified, and tested the metal-free NDI-tetraazacycloalkane conjugate and the two derived metal-organic complexes (MOCs) that incorporate Cu2+ and Zn2+. The NDI-MOCs showed enhanced binding to LTR G4s as assessed by FRET and CD assays in vitro. They also showed enhanced activity in cells where they dose-dependently reduced LTR promoter activity and inhibited viral entry only of the HIV-1 strain that exploited the CXCR4 coreceptor. The time of addition assay confirmed the dual targeting at the different HIV-1 steps. Our results indicate that the NDI-MOC conjugates can simultaneously inhibit viral entry, by targeting the CXCR4 coreceptor, and LTR promoter activity, by stabilizing the LTR G-quadruplexes. The approach of combining multiple targets in a single compound may streamline treatment regimens and improve the overall patient outcomes.

Evaluation of Student Peer- and Self-Grading in an Integrated Pharmacotherapy Course.

OBJECTIVE: To determine if student peer- and self-grades correlate with faculty grades on case vignettes. METHODS: This study involved first professional-year students enrolled in an Integrated Pharmacotherapy course. The course included three modified team-based learning (TBL) activities (each consisting of individual and team readiness assurance tests, followed by three open-note case vignettes completed in teams). Each student uploaded completed case vignettes to the learning management system and was assigned to complete a self- and a random, anonymous peer-grade using a provided key. Peer- and self-grades were compared to faculty grades using a null multilevel model to determine the intraclass correlation coefficient (ICC). Faculty time spent grading was captured, and students were surveyed to determine the perceived value of peer- and self-grading. RESULTS: Faculty- and peer-grades had a slightly higher correlation than faculty- and self-grades (ICC = 0.75 vs 0.73, respectively). The ICC between all three grader groups was 0.74. Faculty spent an average of 2.5 h grading the cases after each TBL session. Students reported spending a median of 36 min on the peer- and self-grades for each TBL session. Overall, students agreed that both the self- and peer-grading activities helped identify gaps in knowledge (90% and 56%, respectively). A total of 78% of students agreed that self-grading was beneficial for their learning. CONCLUSION: There was a moderate-to-good correlation between peer-, self-, and faculty- grades for case vignettes. Faculty time may be saved through student self- or peer-grading.

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

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.

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.

TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability, and telomere dysfunction is an important cause of acquired genomic alterations. Telomeric repeat-containing RNA (TERRA) transcripts are long non-coding RNAs involved in telomere stability through the interaction with shelterin complex. Dysregulation of TERRAs has been reported across several cancer types. We recently identified a small molecule, hit 17, which stabilizes the secondary structure of TERRA. In this study, we investigated in vitro and in vivo anti-MM activities of hit 17. METHODS: Anti-proliferative activity of hit 17 was evaluated in different MM cell lines by cell proliferation assay, and the apoptotic process was analyzed by flow cytometry. Gene and protein expressions were detected by RT-qPCR and western blotting, respectively. Microarray analysis was used to analyze the transcriptome profile. The effect of hit 17 on telomeric structure was evaluated by chromatin immunoprecipitation. Further evaluation in vivo was proceeded upon NCI-H929 and AMO-1 xenograft models. RESULTS: TERRA G4 stabilization induced in vitro dissociation of telomeric repeat-binding factor 2 (TRF2) from telomeres leading to the activation of ATM-dependent DNA damage response, cell cycle arrest, proliferation block, and apoptotic death in MM cell lines. In addition, up-regulation of TERRA transcription was observed upon DNA damage and TRF2 loss. Transcriptome analysis followed by gene set enrichment analysis (GSEA) confirmed the involvement of the above-mentioned processes and other pathways such as E2F, MYC, oxidative phosphorylation, and DNA repair genes as early events following hit 17-induced TERRA stabilization. Moreover, hit 17 exerted anti-tumor activity against MM xenograft models. CONCLUSION: Our findings provide evidence that targeting TERRA by hit 17 could represent a promising strategy for a novel therapeutic approach to MM.

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.

Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1.

G-quadruplexes (G4s) are non-canonical nucleic acid structures that regulate key biological processes, from transcription to genome replication both in humans and viruses. The herpes simplex virus-1 (HSV-1) genome is prone to form G4s that, along with proteins, regulate its viral cycle. General G4 ligands have been shown to hamper the viral cycle, pointing to viral G4s as original antiviral targets. Because cellular G4s are also normally present in infected cells, the quest for improved anti-HSV-1 G4 ligands is still open. Here, we evaluated a series of new quindoline-derivatives which showed high binding to and stabilization of the viral G4s. They displayed nanomolar-range anti-HSV-1 activity paralleled by negligible cytotoxicity in human cells, thus proving remarkable selectivity. The best-in-class compound inhibited the viral life cycle at the early times post infection up to the step of viral genome replication. In infected human cells, it reduced expression of ICP4, the main viral transcription factor, by stabilizing the G4s embedded in ICP4 promoter. Quindoline-derivatives thus emerge as a new class of G4 ligands with potent dual anti HSV-1 activity.

The Multiple Effects of Vitamin D against Chronic Diseases: From Reduction of Lipid Peroxidation to Updated Evidence from Clinical Studies.

BACKGROUND: Vitamin D exerts multiple beneficial effects in humans, including neuronal, immune, and bone homeostasis and the regulation of cardiovascular functions. Recent studies correlate vitamin D with cancer cell growth and survival, but meta-analyses on this topic are often not consistent. METHODS: A systematic search of the PubMed database and the Clinical Trial Register was performed to identify all potentially relevant English-language scientific papers containing original research articles on the effects of vitamin D on human health. RESULTS: In this review, we analyzed the antioxidant and anti-inflammatory effects of vitamin D against acute and chronic diseases, focusing particularly on cancer, immune-related diseases, cardiomyophaties (including heart failure, cardiac arrhythmias, and atherosclerosis) and infectious diseases. CONCLUSIONS: Vitamin D significantly reduces the pro-oxidant systemic and tissue biomarkers involved in the development, progression, and recurrence of chronic cardiometabolic disease and cancer. The overall picture of this review provides the basis for new randomized controlled trials of oral vitamin D supplementation in patients with cancer and infectious, neurodegenerative, and cardiovascular diseases aimed at reducing risk factors for disease recurrence and improving quality of life.

Human Virus Genomes Are Enriched in Conserved Adenine/Thymine/Uracil Multiple Tracts That Pause Polymerase Progression.

The DNA secondary structures that deviate from the classic Watson and Crick base pairing are increasingly being reported to form transiently in the cell and regulate specific cellular mechanisms. Human viruses are cell parasites that have evolved mechanisms shared with the host cell to support their own replication and spreading. Contrary to human host cells, viruses display a diverse array of nucleic acid types, which include DNA or RNA in single-stranded or double-stranded conformations. This heterogeneity improves the possible occurrence of non-canonical nucleic acid structures. We have previously shown that human virus genomes are enriched in G-rich sequences that fold in four-stranded nucleic acid secondary structures, the G-quadruplexes.Here, by extensive bioinformatics analysis on all available genomes, we showed that human viruses are enriched in highly conserved multiple A (and T or U) tracts, with such an array that they could in principle form quadruplex structures. By circular dichroism, NMR, and Taq polymerase stop assays, we proved that, while A/T/U-quadruplexes do not form, these tracts still display biological significance, as they invariably trigger polymerase pausing within two bases from the A/T/U tract. "A" bases display the strongest effect. Most of the identified A-tracts are in the coding strand, both at the DNA and RNA levels, suggesting their possible relevance during viral translation. This study expands on the presence and mechanism of nucleic acid secondary structures in human viruses and provides a new direction for antiviral research.

Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes.

HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses.

Chromene Derivatives as Selective TERRA G-Quadruplex RNA Binders with Antiproliferative Properties.

In mammalian cells, telomerase transcribes telomeres in large G-rich non-coding RNA, known as telomeric repeat-containing RNA (TERRA), which folds into noncanonical nucleic acid secondary structures called G-quadruplexes (G4s). Since TERRA G4 has been shown to be involved in telomere length and translation regulation, it could provide valuable insight into fundamental biological processes, such as cancer growth, and TERRA G4 binders could represent an innovative strategy for cancer treatment. In this work, the three best candidates identified in our previous virtual screening campaign on bimolecular DNA/RNA G4s were investigated on the monomolecular Tel DNA and TERRA G4s by means of molecular modelling simulations and in vitro and in cell analysis. The results obtained in this work highlighted the stabilizing power of all the three candidates on TERRA G4. In particular, the two compounds characterized by a chromene scaffold were selective TERRA G4 binders, while the compound with a naphthyridine core acted as a dual Tel/TERRA G4-binder. A biophysical investigation by circular dichroism confirmed the relative stabilization efficiency of the compounds towards TERRA and Tel G4s. The TERRA G4 stabilizing hits showed good antiproliferative activity against colorectal and lung adenocarcinoma cell lines. Lead optimization to increase TERRA G4 stabilization may provide new powerful tools against cancer.

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.

Multimeric G-quadruplexes: A review on their biological roles and targeting.

In human cells, nucleic acids adopt several non-canonical structures that regulate key cellular processes. Among them, G-quadruplexes (G4s) are stable structures that form in guanine-rich regions in vitro and in cells. G4 folded/unfolded state shapes numerous cellular processes, including genome replication, transcription, and translation. Moreover, G4 folding is involved in genomic instability. G4s have been described to multimerize, forming high-order structures in both DNA and/or RNA strands. Multimeric G4s can be formed by adjacent intramolecular G4s joined by stacking interactions or connected by short loops. Multimeric G4s can also originate from the assembly of guanines embedded on independent DNA or RNA strands. Notably, crucial regions of the human genome, such as the 3'-terminal overhang of the telomeric DNA as well as the open reading frame of genes involved in the preservation of neuron viability in the human central and peripheral nervous system are prone to form multimeric G4s. The biological importance of such structures has been recently described, with multimeric G4s playing potentially protective or deleterious effects in the pathogenic cascade of various diseases. Here, we portray the multifaceted scenario of multimeric G4s, in terms of structural properties, biological roles, and targeting strategies.

Comparing Patch vs Pen Bolus Insulin Delivery in Type 2 Diabetes Using Continuous Glucose Monitoring Metrics and Profiles.

OBJECTIVE: CeQur Simplicity™ (CeQur, Marlborough, MA) is a 3-day insulin delivery patch designed to meet mealtime insulin requirements. A recently reported 48-week, randomized, multicenter, interventional trial compared efficacy, safety and self-reported outcomes in 278 adults with type 2 diabetes (T2D) on basal insulin therapy who initiated and managed mealtime insulin therapy with a patch pump versus insulin pen. We assessed changes in key glycemic metrics among a subset of patients who wore a continuous glucose monitoring (CGM) device. METHODS: Study participants (patch, n = 49; pen, n = 48) wore a CGM device in masked setting during the baseline period and prior to week 24. Glycemic control was assessed using international consensus guidelines for percentage of Time In Range (%TIR: >70% at 70-180 mg/dL), Time Below Range (%TBR: <4% at <70 mg/dL; <1% at <54 mg/dL), and Time Above Range (%TAR: <25% at >180 mg/dL; <5% at >250 mg/dL). RESULTS: Both the patch and pen groups achieved recommended targets in %TIR (74.1% ± 18.7%, 75.2 ± 16.1%, respectively) and marked reductions in %TAR >180 mg/dL (21.1% ± 19.9%, 19.7% ± 17.5%, respectively) but with increased %TBR <70 mg/dL (4.7% ± 5.2%, 5.1 ± 5.8, respectively), all P < .0001. No significant between-group differences in glycemic improvements or adverse events were observed. CONCLUSIONS: CGM confirmed that the patch or pen can be used to safely initiate and optimize basal-bolus therapy using a simple insulin adjustment algorithm with SMBG. Preference data suggest that use of the patch vs pen may enhance treatment adherence.

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.

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.

Electronic Health Records in Pharmacy Skills-based Curricula.

Electronic health records (EHRs) are integral to contemporary pharmacy practice. The use of EHRs and associated skill development in curricula across pharmacy education is variable. Skills-based courses in the Doctor of Pharmacy curriculum are ideal areas to develop these competencies' and integrate EHR use and skills with the Pharmacists' Patient Care Process. Consideration should be given by each school and college of pharmacy for having an EHR curriculum embedded within skills-based courses to prepare students for advanced pharmacy practice experiences as well as professional practice after graduation. A consensus on what skills or competencies should be consistently included in pharmacy curricula should be developed across pharmacy education to increase consistency in the delivery of EHR skills education and assessment. Emphasis on EHR skills and incorporation of them into national pharmacy education standards would help further guide development and assessment, as well as ensure new pharmacists are on the cutting edge of patient care and technology.