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In 2015, the Mw 7.8 Gorkha earthquake struck Nepal, triggering thousands of landslides across the central and eastern Himalayas. These landslides had many adverse effects, including causing widespread damage to low-grade transport routes (e.g. tracks, footpaths) in rural regions that depend on tourism for survival. Langtang Valley is a glacial–periglacial landscape located 60 km north of Kathmandu. It is one of the most popular trekking regions in Nepal and has been severely affected by Gorkha earthquake-triggered and monsoon-triggered landsliding. Here, qualitative and quantitative observations from fieldwork and remote sensing are used to describe the materials and geomorphology of the landslides across Langtang Valley, and to quantify the extent to which coseismic and monsoon-triggered landslides have affected Langtang's trekking infrastructure. The dominant bedrock materials involved within Langtang landslides are found to be a range of gneisses and intruded leucogranites. In total, 64 landslides are found to have intersected trekking paths across Langtang, with coseismic and monsoon-triggered landslides having an impact on c. 3 km and 0.8 km of path respectively. It is observed that the practice of reconstructing paths through unstable landslide deposits is leaving the trekking infrastructure across Langtang increasingly vulnerable to future failure. [ABSTRACT FROM AUTHOR]

River incision in upland areas is controlled by prevailing climatic and tectonic regimes, which are increasingly well described, and the nature of the bedrock lithology, which is still poorly constrained. Here, we calculated downstream variations in stream power and bedrock strength for six rivers crossing a normal fault in western Turkey, to derive new constraints on bedrock erodibility as function of rock type. These rivers were selected because they exhibit knick zones representing a transient response to an increase in throw rate, driven by fault linkage. Field measures of rock mass strength showed that the metamorphic units (gneisses and schists) in the catchments are ~2 times harder than the sedimentary lithologies. Stream power increases downstream in all rivers, reaching a maxima upstream of the fault within the metamorphic bedrock but declining markedly where softer sedimentary rocks are encountered. We demonstrate a positive correlation between throw rate and stream power in the metamorphic rocks, characteristic of rivers obeying a detachment-limited model of erosion. We estimated bedrock erodibility in the metamorphic rocks as kb = 2.2-6.3 x 10-14 m s² kg-1; in contrast, bedrock erodibility values were 5-30 times larger in the sedimentary units, with kb = 1.2-15 x 10-13 m s² kg-1. However, in the sedimentary units, stream power does not scale predictably with fault throw rate, and we evaluated the extent to which the friable nature of the outcropping clastic bedrock alters the long-term erosional dynamics of the rivers. This study places new constraints on bedrock erodibilities upstream of active faults and demonstrates that the strength and characteristics of underlying bedrock exert a fundamental influence on river behavior. [ABSTRACT FROM AUTHOR]

Optimization of oncolytic viruses for therapeutic applications requires the strategic removal or mutagenesis of virulence genes alongside the insertion of transgenes that enhance viral replication, spread and immunogenicity. However, the complexity of many viral genomes and the labour-intensive nature of methods for the generation and isolation of recombinant viruses have hindered the development of therapeutic oncolytic viruses. Here we report an iterative strategy that exploits the preferential susceptibility of viruses to certain antibiotics to accelerate the engineering of the genomes of oncolytic viruses for the insertion of immunomodulatory cytokine transgenes, and the identification of dispensable genes with regard to replication of the recombinant oncolytic viruses in tumour cells. We applied the strategy by leveraging insertional mutagenesis via the Sleeping Beauty transposon system, combined with long-read nanopore sequencing, to generate libraries of herpes simplex virus type 1 and vaccinia virus, identifying stable transgene insertion sites and gene deletions that enhance the safety and efficacy of the viruses., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Targeted antineoplastic immunotherapies have achieved remarkable clinical outcomes. However, resistance to these therapies due to target absence or antigen shedding limits their efficacy and excludes tumours from candidacy. To address this limitation, here we engineer an oncolytic rhabdovirus, vesicular stomatitis virus (VSVΔ51), to express a truncated targeted antigen, which allows for HER2-targeting with trastuzumab. The truncated HER2 (HER2T) lacks signaling capabilities and is efficiently expressed on infected cell surfaces. VSVΔ51-mediated HER2T expression simulates HER2-positive status in tumours, enabling effective treatment with the antibody-drug conjugate trastuzumab emtansine in vitro, ex vivo, and in vivo. Additionally, we combine VSVΔ51-HER2T with an oncolytic vaccinia virus expressing a HER2-targeted T-cell engager. This dual-virus therapeutic strategy demonstrates potent curative efficacy in vivo in female mice using CD3+ infiltrate for anti-tumour immunity. Our findings showcase the ability to tailor the tumour microenvironment using oncolytic viruses, thereby enhancing compatibility with "off-the-shelf" targeted therapies., (© 2024. The Author(s).)

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S) and nucleocapsid (N) proteins as well as a unique poly-epitope antigen that contains immunodominant T cell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates T cells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust T cell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines., Competing Interests: All authors have declared that they have no conflicts of interest with this study., (© 2023 The Author(s).)

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks., Competing Interests: Conflicts of interest R. S. is currently employed by the Public Health Agency of Canada (PHAC). His contribution to the study described herein was conducted prior to his employment at PHAC. The work was not undertaken under the auspices of PHAC as part of their employment responsibilities and does not represent the views of PHAC. All other authors have declared that they have no conflicts of interest with this study., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design., (© 2023. The Author(s).)

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies., Competing Interests: JB and BL are scientific co-founders of Turnstone Biologics, which develops oncolytic vaccinia viruses. JD, MH, MT, and AP have worked for Turnstone Biologics. JB and BL are shareholders in Turnstone Biologics. JB, CI, RS, FW and BL are co-inventors on a patent regarding the expression of bacterial cyclases and their therapeutic utility in mammals. RS is currently employed by the Public Health Agency of Canada (PHAC). His contribution to the study described herein was conducted prior to his employment at PHAC. The work was not undertaken under the auspices of PHAC and does not represent the views of PHAC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Whelan, Singaravelu, Wang, Pelin, Tamming, Pugliese, Martin, Crupi, Petryk, Austin, He, Marius, Duong, Jones, Fekete, Alluqmani, Chen, Boulton, Huh, Tang, Taha, Scut, Diallo, Azad, Lichty, Ilkow and Bell.)

Viruses share many attributes in common with extracellular vesicles (EVs). The cellular machinery that is used for EV production, packaging of substrates and secretion is also commonly manipulated by viruses for replication, assembly and egress. Viruses can increase EV production or manipulate EVs to spread their own genetic material or proteins, while EVs can play a key role in regulating viral infections by transporting immunomodulatory molecules and viral antigens to initiate antiviral immune responses. Ultimately, the interactions between EVs and viruses are highly interconnected, which has led to interesting discoveries in their associated roles in the progression of different diseases, as well as the new promise of combinational therapeutics. In this review, we summarize the relationships between viruses and EVs and discuss major developments from the past five years in the engineering of virus-EV therapies.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive T reg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours., Competing Interests: We declare that JB has an interest in Turnstone Biologics, which develops the oncolytic vaccinia virus as an OV platform. LP, MH, JD, AP, CB, DS and MB have worked for Turnstone Biologics. JB, CB, DS and MB are shareholders in Turnstone Biologics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Crupi, Taha, Janssen, Petryk, Boulton, Alluqmani, Jirovec, Kassas, Khan, Vallati, Lee, Huang, Huh, Pikor, He, Marius, Austin, Duong, Pelin, Neault, Azad, Breitbach, Stojdl, Burgess, McComb, Auer, Diallo, Ilkow and Bell.)

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Protein kinase G (PKG) is a major receptor of cGMP, and controls signaling pathways distinct from those regulated by cAMP. However, the contributions of the two substituents that differentiate cGMP from cAMP (i.e. 6-oxo and 2-NH 2 ) to the cGMP-versus-cAMP selectivity of PKG remain unclear. Here, using NMR to map how binding affinity and dynamics of the protein and ligand vary along a ligand double-substitution cycle, we show that the contributions of the two substituents to binding affinity are surprisingly non-additive. Such non-additivity stems primarily from mutual protein-ligand conformational selection, whereby not only does the ligand select for a preferred protein conformation upon binding, but also, the protein selects for a preferred ligand conformation. The 6-oxo substituent mainly controls the conformational equilibrium of the bound protein, while the 2-NH 2 substituent primarily controls the conformational equilibrium of the unbound ligand (i.e. syn versus anti). Therefore, understanding the conformational dynamics of both the protein and ligand is essential to explain the cGMP-versus-cAMP selectivity of PKG., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function., Competing Interests: The authors declare no competing interest.

Motivation: Correlated Nuclear Magnetic Resonance (NMR) chemical shift changes identified through the CHEmical Shift Projection Analysis (CHESPA) and CHEmical Shift Covariance Analysis (CHESCA) reveal pathways of allosteric transitions in biological macromolecules. To address the need for an automated platform that implements CHESPA and CHESCA and integrates them with other NMR analysis software packages, we introduce here integrated plugins for NMRFAM-SPARKY that implement the seamless detection and visualization of allosteric networks., Availability and Implementation: CHESCA-SPARKY and CHESPA-SPARKY are available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). The assigned spectra involved in this study and tutorial videos using this dataset are available at https://sites.google.com/view/chescachespa-sparky., Supplementary Information: Supplementary data are available at Bioinformatics Online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As the COVID-19 pandemic continues, there is an imminent need for rapid diagnostic tools and effective antivirals targeting SARS-CoV-2. We have developed a novel bioluminescence-based biosensor to probe a key host-virus interaction during viral entry: the binding of SARS-CoV-2 viral spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2). Derived from Nanoluciferase binary technology (NanoBiT), the biosensor is composed of Nanoluciferase split into two complementary subunits, Large BiT and Small BiT, fused to the Spike S1 domain of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. The ACE2-S1 interaction results in reassembly of functional Nanoluciferase, which catalyzes a bioluminescent reaction that can be assayed in a highly sensitive and specific manner. We demonstrate the biosensor's large dynamic range, enhanced thermostability and pH tolerance. In addition, we show the biosensor's versatility towards the high-throughput screening of drugs which disrupt the ACE2-S1 interaction, as well as its ability to act as a surrogate virus neutralization assay. Results obtained with our biosensor correlate well with those obtained with a Spike-pseudotyped lentivirus assay. This rapid in vitro tool does not require infectious virus and should enable the timely development of antiviral modalities targeting SARS-CoV-2 entry., (Copyright © 2021. Published by Elsevier B.V.)

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis., Competing Interests: The authors have declared that no competing interests exist.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

Quantifying chemical substituent contributions to ligand-binding free energies is challenging due to nonadditive effects. Protein allostery is a frequent cause of nonadditivity, but the underlying allosteric mechanisms often remain elusive. Here, we propose a general NMR-based approach to elucidate such mechanisms and we apply it to the HCN4 ion channel, whose cAMP-binding domain is an archetypal conformational switch. Using NMR, we show that nonadditivity arises not only from concerted conformational transitions, but also from conformer-specific effects, such as steric frustration. Our results explain how affinity-reducing functional groups may lead to affinity gains if combined. Surprisingly, our approach also reveals that nonadditivity depends markedly on the receptor conformation. It is negligible for the inhibited state but highly significant for the active state, opening new opportunities to tune potency and agonism of allosteric effectors., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Introduction: Immunotherapy is a rapidly evolving area of cancer therapeutics aimed at driving a systemic immune response to fight cancer. Oncolytic viruses (OVs) are at the cutting-edge of innovation in the immunotherapy field. Successful OV platforms must be effective in reshaping the tumor microenvironment and controlling tumor burden, but also be highly specific to avoid off-target side effects. Large DNA viruses, like vaccinia virus (VACV), have a large coding capacity, enabling the encoding of multiple immunostimulatory transgenes to reshape the tumor immune microenvironment. VACV-based OVs have shown promising results in both pre-clinical and clinical studies, including safe and efficient intravenous delivery to metastatic tumors., Area Covered: This review summarizes attenuation strategies to generate a recombinant VACV with optimal tumor selectivity and immunogenicity. In addition, we discuss immunomodulatory transgenes that have been introduced into VACV and summarize their effectiveness in controlling tumor burden., Expert Opinion: VACV encodes several immunomodulatory genes which aid the virus in overcoming innate and adaptive immune responses. Strategic deletion of these virulence factors will enable an optimal balance between viral persistence and immunogenicity, robust tumor-specific expression of payloads and promotion of a systemic anti-cancer immune response. Rational selection of therapeutic transgenes will maximize the efficacy of OVs and their synergy in combinatorial immunotherapy schemes.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presents an urgent need for an effective vaccine. Molecular characterization of SARS-CoV-2 is critical to the development of effective vaccine and therapeutic strategies. In the present study, we show that the fusion of the SARS-CoV-2 spike protein receptor-binding domain to its transmembrane domain is sufficient to mediate trimerization. Our findings may have implications for vaccine development and therapeutic drug design strategies targeting spike trimerization. As global efforts for developing SARS-CoV-2 vaccines are rapidly underway, we believe this observation is an important consideration for identifying crucial epitopes of SARS-CoV-2.

The Hippo pathway plays a critical role in tissue and organ growth under normal physiological conditions, and its dysregulation in malignant growth has made it an attractive target for therapeutic intervention in the fight against cancer. To date, its complex signaling mechanisms have made it difficult to identify strong therapeutic candidates. Hippo signaling is largely carried out by two main activated signaling pathways involving receptor tyrosine kinases (RTKs)-the RTK/RAS/PI3K and the RTK-RAS-MAPK pathways. However, several RTKs have also been shown to regulate this pathway to engage downstream Hippo effectors and ultimately influence cell proliferation. In this text, we attempt to review the diverse RTK signaling pathways that influence Hippo signaling in the context of oncogenesis.

The exchange protein activated by cAMP (EPAC) is a promising drug target for a wide disease range, from neurodegeneration and infections to cancer and cardiovascular conditions. A novel partial agonist of the EPAC isoform 1 (EPAC1), I942, was recently discovered, but its mechanism of action remains poorly understood. Here, we utilize NMR spectroscopy to map the I942-EPAC1 interactions at atomic resolution and propose a mechanism for I942 partial agonism. We found that I942 interacts with the phosphate binding cassette (PBC) and base binding region (BBR) of EPAC1, similar to cyclic adenosine monophosphate (cAMP). These results not only reveal the molecular basis for the I942 vs cAMP mimicry and competition, but also suggest that the partial agonism of I942 arises from its ability to stabilize an inhibition-incompetent activation intermediate distinct from both active and inactive EPAC1 states. The mechanism of action of I942 may facilitate drug design for EPAC-related diseases.

Polo-like kinases (Plks) are key cell cycle regulators. They contain a kinase domain followed by a polo-box domain that recognizes phosphorylated substrates and enhances their phosphorylation. The regulatory subunit of the Dbf4-dependent kinase complex interacts with the polo-box domain of Cdc5 (the sole Plk in Saccharomyces cerevisiae) in a phosphorylation-independent manner. We have solved the crystal structures of the polo-box domain of Cdc5 on its own and in the presence of peptides derived from Dbf4 and a canonical phosphorylated substrate. The structure bound to the Dbf4-peptide reveals an additional density on the surface opposite to the phospho-peptide binding site that allowed us to propose a model for the interaction. We found that the two peptides can bind simultaneously and non-competitively to the polo-box domain in solution. Furthermore, point mutations on the surface opposite to the phosphopeptide binding site of the polo-box domain disrupt the interaction with the Dbf4 peptide in solution and cause an early anaphase arrest phenotype distinct from the mitotic exit defect typically observed in cdc5 mutants. Collectively, our data illustrates the importance of non-canonical interactions mediated by the polo-box domain and provide key mechanistic insights into the combinatorial recognition of substrates by Polo-like kinases.

The universal second messenger cAMP regulates diverse intracellular processes by interacting with ubiquitously expressed proteins, such as Protein Kinase A (PKA) and the Exchange Protein directly Activated by cAMP (EPAC). EPAC is implicated in multiple pathologies, thus several EPAC-specific inhibitors have been identified in recent years. However, the mechanisms and molecular interactions underlying the EPAC inhibition elicited by such compounds are still poorly understood. Additionally, being hydrophobic low molecular weight species, EPAC-specific inhibitors are prone to forming colloidal aggregates, which result in non-specific aggregation-based inhibition (ABI) in aqueous systems. Here, we review from a biophysical perspective the molecular basis of the specific and non-specific interactions of two EPAC antagonists-CE3F4R, a non-competitive inhibitor, and ESI-09, a competitive inhibitor of EPAC. Additionally, we discuss the value of common ABI attenuators (e.g., TX and HSA) to reduce false positives at the expense of introducing false negatives when screening aggregation-prone compounds. We hope this review provides the EPAC community effective criteria to evaluate similar compounds, aiding in the optimization of existing drug leads, and informing the development of the next generation of EPAC-specific inhibitors.

Background and Purpose: Little is known about complementary and integrative health intervention usage in the primary brain tumor population. We aimed to identify the percentage of patients using these practices and explore the impact on quality of life., Materials and Methods: Clinical records from patients seen in clinic between December 16, 2013 and February 28, 2014 were reviewed retrospectively. The questionnaires used were a modified version of the International Complementary and Alternative Medicine Questionnaire, the Functional Assessment of Cancer Therapy- Brain Cancer and the Functional Assessment of Chronic Illness Therapy- Fatigue., Results: 76% of patients utilized a complementary and integrative health modality. The most frequently reported modalities used were vitamins, massage, and spiritual healing, prayer, diet and meditation., Conclusion: These results confirm the usage of complementary and integrative health practices within the primary brain tumor population; however, there was no evidence of association between use and quality of life., (Copyright © 2019. Published by Elsevier Ltd.)

A common source of false positives in drug discovery is ligand self-association into large colloidal assemblies that nonspecifically inhibit target proteins. However, the mechanisms of aggregation-based inhibition (ABI) and ABI-attenuation by additives, such as Triton X-100 (TX) and human serum albumin (HSA), are not fully understood. Here, we investigate the molecular basis of ABI and ABI-attenuation through the lens of NMR and coupled thermodynamic cycles. We unexpectedly discover a new class of aggregating ligands that exhibit negligible interactions with proteins but act as competitive sinks for the free inhibitor, resulting in bell-shaped dose-response curves. TX attenuates ABI by converting inhibitory, protein-binding aggregates into nonbinding coaggregates, whereas HSA minimizes nonspecific ligand interactions by functioning as a reservoir for free inhibitor and preventing self-association. Hence, both TX and HSA are useful tools to minimize false positives arising from nonspecific binding but at the cost of potentially introducing false negatives due to suppression of specific interactions.

Soluble amyloid beta assemblies (Aβ n ) are neurotoxic and play a central role in the early phases of the pathogenesis cascade leading to Alzheimer's disease. However, the current knowledge about the molecular determinants of Aβ n toxicity is at best scant. Here, we comparatively analyze Aβ n prepared in the absence or presence of a catechin library that modulates cellular toxicity. By combining solution NMR with dynamic light scattering, fluorescence spectroscopy, electron microscopy, wide-angle X-ray diffraction and cell viability assays, we identify a cluster of unique molecular signatures that distinguish toxic vs. nontoxic Aβ assemblies. These include the exposure of a hydrophobic surface spanning residues 17-28 and the concurrent shielding of the highly charged N-terminus. We show that the combination of these two dichotomous structural transitions promotes the colocalization and insertion of β-sheet rich Aβ n into the membrane, compromising membrane integrity. These previously elusive toxic surfaces mapped here provide an unprecedented foundation to establish structure-toxicity relationships of Aβ assemblies.

Nucleic acids are potential pathogen-associated or danger-associated molecular patterns that modulate immune responses and the development of autoimmune disorders. Class A scavenger receptors (SR-As) are a diverse group of pattern recognition receptors that recognize a variety of polyanionic ligands including nucleic acids. While SR-As are important for the recognition and internalization of extracellular dsRNA, little is known about extracellular DNA, despite its association with chronic infections and autoimmune disorders. In this study, we investigated the specificity of and requirement for SR-As in binding and internalizing different species, sequences and lengths of nucleic acids. We purified recombinant coiled-coil/collagenous and scavenger receptor cysteine-rich (SRCR) domains that have been implicated as potential ligand-binding domains. We detected a direct interaction of RNA and DNA species with the coiled-coil/collagenous domain, but not the SRCR domain. Despite the presence of additional surface receptors that bind nucleic acids, SR-As were found to be sufficient for nucleic acid binding and uptake in A549 human lung epithelial cells. Moreover, these findings suggest that the coiled-coil/collagenous domain of SR-As is sufficient to bind nucleic acids independent of species, sequence or length., (© 2018 Australasian Society for Immunology Inc.)

Classical uncompetitive inhibitors are potent pharmacological modulators of enzyme function. Since they selectively target enzyme-substrate complexes (E:S), their inhibitory potency is amplified by increasing substrate concentrations. Recently, an unconventional uncompetitive inhibitor, called CE3F4R, was discovered for the exchange protein activated by cAMP isoform 1 (EPAC1). Unlike conventional uncompetitive inhibitors, CE3F4R is uncompetitive with respect to an allosteric effector, cAMP, as opposed to the substrate (i.e., CE3F4R targets the E:cAMP rather than the E:S complex). However, the mechanism of CE3F4R as an uncompetitive inhibitor is currently unknown. Here, we elucidate the mechanism of CE3F4R's action using NMR spectroscopy. Due to limited solubility and line broadening, which pose major challenges for traditional structural determination approaches, we resorted to a combination of protein- and ligand-based NMR experiments to comparatively analyze EPAC mutations, inhibitor analogs, and cyclic nucleotide derivatives that trap EPAC at different stages of activation. We discovered that CE3F4R binds within the EPAC cAMP-binding domain (CBD) at a subdomain interface distinct from the cAMP binding site, acting as a wedge that stabilizes a cAMP-bound mixed-intermediate. The mixed-intermediate includes attributes of both the apo/inactive and cAMP-bound/active states. In particular, the intermediate targeted by CE3F4R traps a CBD's hinge helix in its inactive conformation, locking EPAC into a closed domain topology that restricts substrate access to the catalytic domain. The proposed mechanism of action also explains the isoform selectivity of CE3F4R in terms of a single EPAC1 versus EPAC2 amino acid difference that destabilizes the active conformation of the hinge helix.

Type IVa pili (T4aP) are long, thin surface filaments involved in attachment, motility, biofilm formation, and DNA uptake. They are important virulence factors for many bacteria, including Pseudomonas aeruginosa, an opportunistic pathogen and common cause of hospital-acquired infections. Each helical filament contains thousands of monomers of the major pilin subunit, PilA. Each P. aeruginosa strain expresses one of five phylogenetically distinct major pilins, which vary in sequence and the nature of their associated accessory protein(s). Here, we present the backbone resonance assignment of the C-terminal domain of the group III PilA from strain PA14, a highly virulent, globally distributed clone. Secondary structure probabilities calculated from chemical shifts were in excellent agreement with previous homology modeling using a group V pilin structural template. The analysis revealed that the distal segment of the αβ loop had high microsecond-millisecond dynamics compared with other loop regions. Shortening of this segment by internal deletion abrogated pilus assembly in a dominant negative manner, suggesting a potential role in pilin polymerization. Pilin conformations that support optimal interactions of both the conserved hydrophobic N-termini in the pilus core and hydrophilic loops creating the filament surface may be necessary to produce stable filaments.

Mapping allosteric sites is emerging as one of the central challenges in physiology, pathology, and pharmacology. Nuclear Magnetic Resonance (NMR) spectroscopy is ideally suited to map allosteric sites, given its ability to sense at atomic resolution the dynamics underlying allostery. Here, we focus specifically on the NMR CHEmical Shift Covariance Analysis (CHESCA), in which allosteric systems are interrogated through a targeted library of perturbations (e.g., mutations and/or analogs of the allosteric effector ligand). The atomic resolution readout for the response to such perturbation library is provided by NMR chemical shifts. These are then subject to statistical correlation and covariance analyses resulting in clusters of allosterically coupled residues that exhibit concerted responses to the common set of perturbations. This chapter provides a description of how each step in the CHESCA is implemented, starting from the selection of the perturbation library and ending with an overview of different clustering options.

Primary brain tumor patients experience high levels of distress. The purpose of this cross-sectional, retrospective study is to evaluate the level and different sources of psychosocial distress and how these pertain to health-related quality of life (HRQoL). The Primary and Recurrent Glioma registry at Duke's The Preston Robert Tisch Brain Tumor Center was queried retrospectively for demographic and clinical information on patients seen between December 2013 and February 2014. Data also included the National Comprehensive Cancer Network's Distress Thermometer (NCCN-DT), Functional Assessment of Cancer Therapy-Brain Cancer (FACT-Br), and Functional Assessment of Chronic Illness Therapy- Fatigue (FACIT-F). 829 subjects completed questionnaires. 54% were male; 96% completed the NCCN-DT; 33.3% had a DT score ≥4 (moderate/severe distress). Women reported DT ≥ 4 more often than men (38.6 vs 29.0%; p = 0.005). Patients within 1 year of diagnosis reported DT ≥ 4 more often than those 1+ years after diagnosis (38.8 vs 30.9%; p = 0.034). 73.0% reported physical problems; the most frequent being fatigue (43.2%) and memory/concentration (40.9%). 42.0% complained of emotional problems with worry (29.4%) and nervousness (22.4%) being the most common. Patients who reported at least one practical, family, emotional or physical problem had significantly lower HRQoL scores (p < 0.001). Primary brain tumor patients experience memory dysfunction, fatigue, nervousness, worry, and financial concerns, which have a negative effect on the patient's HRQoL. By identifying and addressing these stressors, it may be possible to improve patient HRQoL.

The hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo- and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Drug-induced photosensitivity occurs when a drug is capable of absorbing radiation from the sun (usually ultraviolet A) leading to chemical reactions that cause cellular damage (phototoxicity) or, more rarely, form photoallergens (photoallergy). The manifestation varies considerably in presentation and severity from mild pain to severe blistering. Despite screening strategies and guidelines in place to predict photoreactive drugs during development there are still new drugs coming onto the market that cause photosensitivity. Thus, there is a continuing need for dermatologists to be aware of the different forms of presentation and the culprit drugs. Management usually involves photoprotection and cessation of drug treatment. However, there are always cases where the culprit drug is indispensable. The reason why some patients are susceptible while others remain asymptomatic is not known. A potential mechanism for the phototoxic reactions is the generation of reactive oxygen species (ROS), and there are a number of reasons why some patients might be less able to cope with enhanced levels of ROS., (© 2016 British Association of Dermatologists.)

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an 'AND' logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

The last five years have witnessed major developments in the understanding of the allosteric phenomenon, broadly defined as coupling between remote molecular sites. Such advances have been driven not only by new theoretical models and pharmacological applications of allostery, but also by progress in the experimental approaches designed to map allosteric sites and transitions. Among these techniques, NMR spectroscopy has played a major role given its unique near-atomic resolution and sensitivity to the dynamics that underlie allosteric couplings. Here, we highlight recent progress in the NMR methods tailored to investigate allostery with the goal of offering an overview of which NMR approaches are best suited for which allosterically relevant questions. The picture of the allosteric "NMR toolbox" is provided starting from one of the simplest models of allostery (i.e., the four-state thermodynamic cycle) and continuing to more complex multistate mechanisms. We also review how such an "NMR toolbox" has assisted the elucidation of the allosteric molecular basis for disease-related mutations and the discovery of novel leads for allosteric drugs. From this overview, it is clear that NMR plays a central role not only in experimentally validating transformative theories of allostery, but also in tapping the full translational potential of allosteric systems.

Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off., (© 2015 FEBS.)

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce "protein denaturation". Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Allostery is an essential regulatory mechanism of biological function. Allosteric sites are also pharmacologically relevant as they are often targeted with higher selectivity than orthosteric sites. However, a comprehensive map of allosteric sites poses experimental challenges because allostery is driven not only by structural changes, but also by modulations in dynamics that typically remain elusive to classical structure determination methods. An avenue to overcome these challenges is provided by the NMR chemical shift covariance analysis (CHESCA), as chemical shifts are exquisitely sensitive to redistributions in dynamic conformational ensembles. Here, we propose a set of complementary CHESCA algorithms designed to reliably detect allosteric networks with minimal occurrences of false positives or negatives. The proposed CHESCA toolset was tested for two allosteric proteins (PKA and EPAC) and is expected to complement traditional comparative structural analyses in the comprehensive identification of functionally relevant allosteric sites, including those in otherwise elusive partially unstructured regions.