James F. Conway

Postoperative surgical draining fluid (SDF) in oral squamous cell carcinoma (OSCC) was reported to contain circulating free DNA and extracellular vesicles (EVs). The potential role of SDF DNA as a cancer biomarker in OSCC patients is investigated. To determine whether EVs contribute to the DNA content of SDF, small EVs (sEVs) were isolated by size exclusion chromatography and compared with those in paired plasma. sEV obtained from a cohort of HPV(+) and HPV(−) patients were evaluated for the endocytic origin, size, vesicular morphology, and the DNA content. The sEV from HPV(+) and HPV(−) SDF were similar. The sEVs from SDF were larger and contained more DNA than the sEV from plasma. Treatments of sEV with the DNase/RNase cocktail established the presence of DNA on the surface and in the sEV lumen. Furthermore, 20 to 30% of the total SDF DNA was associated with sEV. Fragmentomic analyses identified the largest DNA (>10,000 bp) on the vesicle surface. The DNA in the vesicle lumen consisted of smaller (~5000 bp) DNA fragments. Electron microscopy of the enzyme-treated vesicles indicated that surface DNA/RNA may be involved in maintaining vesicular integrity and modulating the vesicle entry into cells. The sEV-carrying surface and luminal DNA emerge as significant contributors to the total SDF DNA and, following validation, might serve as useful biomarkers of disease presence/progression in OSCC.

Background: Tumor-derived small extracellular vesicles (sEV), which we call TEX, carry a cargo of molecules that resembles the producer tumor cells. Circulating freely in body fluids, TEX potentially serve as a liquid tumor biopsy. TEX horizontally transfer their cargo to various recipient cells, imparting to them pro-tumor activity. Mechanisms of TEX-driven reprogramming might involve nucleic acids, especially double-stranded (ds)DNA. Methods: TEX isolated from supernatants of human tumor cells were identified as sEV, based on their size, endocytic origin and morphology. TEX treated with DNase/RNase cocktail were examined by transmission and cryo-electron microscopy and tested for biologic activity. DNA was extracted from enzyme-treated TEX, quantified by Qubit and analyzed for fragment sizes. The presence of genomic DNA in TEX was confirmed by PCR, and sequencing of the TP53 gene fragment for a mutational signature was performed. Results: Enzymatic and microscopic studies of TEX showed that nucleic acids are present in the biocorona on the outer surface. Their removal interfered with the biocorona integrity. A short TEX exposure to DNase/RNase altered their morphology without impairing vesicle functions; longer treatments induced TEX re-organization into smaller membrane-bound vesicles. The TEX lumen contained long fragments of protected genomic DNA with a mutational signature reflecting that of the tumor. Conclusions: Nucleic acids present on the TEX surface support the vesicular integrity. The TEX lumen contains membrane-protected large (ds)DNA fragments with the mutational signature of the parent tumor. The presence of surface and luminal nucleic acids in TEX, and especially their mutational signature, suggests that TEX may serve as highly promising cancer-specific biomarkers.

Tailed bacteriophages comprise the largest structural family of viruses with close relatives in archaea and the eukaryotic herpesviruses. The common assembly pathway produces an icosahedrally symmetric protein shell, called capsid, into which the double-stranded DNA genome is packaged. While capsid sizes and amino acid sequences vary considerably, the major capsid protein (MCP) folds are remarkably similar throughout the family. To investigate the mechanisms governing capsid size, we characterize the procapsid and mature capsid of phage D3, which expresses an icosahedral lattice with Triangulation number T = 9. We find that the MCP scaffold domain binds to the interior capsid surface, acting as a clamp to constrain subunit interactions. Following scaffold digestion, the MCP capsid domains form strong interactions that maintain capsid structure throughout maturation. The scaffold constraints appear critical for capsid size determination and provide important understanding of the factors governing capsid assembly in general and expands our understanding of these ecologically and biomedically important viruses.

Biofilm-protected Pseudomonas aeruginosa causes chronic infections that are difficult to treat. FapC, the major biofilm-forming functional amyloid in Pseudomonas , is essential for biofilm integrity, yet its structural details remain unresolved. Using an integrative structural biology approach, we combine a solution nuclear magnetic resonance–based structural ensemble of unfolded monomeric FapC, a ~3.3-angstrom-resolution cryo–electron microscopy (cryo-EM) density map of FapC fibril, and all-atom molecular dynamics (MD) simulations to capture the transition from the unfolded to folded monomer to the fibrillar fold, providing a complete structural view of FapC biogenesis. Cryo-EM reveals a unique irregular triple-layer β solenoid cross-β fibril composed of a single protofilament. MD simulations initiated from monomeric and fibrillar FapC mapped structural transitions, offering mechanistic insights into amyloid assembly and disassembly. Understanding FapC reveals how Pseudomonas exploits functional amyloids for biofilm formation, and establishes a structural and mechanistic foundation for developing therapeutics targeting biofilm-related infection and antimicrobial resistance.

Tailed bacteriophages comprise the largest structural family of viruses with close relatives in archaea and the eukaryotic herpesviruses. The common assembly pathway produces an icosahedrally symmetric protein shell, called capsid, into which the double-stranded DNA genome is packaged. While capsid sizes and amino acid sequences vary considerably, the major capsid protein (MCP) folds are remarkably similar throughout the family. To investigate the mechanisms governing capsid size, we characterized the procapsid and mature capsid of phage D3, which expresses an icosahedral lattice with Triangulation number T=9. We found that the MCP scaffold domain binds to the interior capsid surface, acting as a clamp to constrain subunit interactions. Following scaffold digestion, the MCP capsid domains form strong interactions that maintain capsid structure throughout maturation. The scaffold constraints appear critical for capsid size determination and provide important understanding of the factors governing capsid assembly in general and expands our understanding of these ecologically and biomedically important viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a highly transmissible acute respiratory infection that can result in severe pneumonia and death. Many details of SARS-CoV-2 infection are not fully understood, including the cell biology and host-virus interactions involved in coronavirus assembly and release, in which the Envelope (E) structural protein is instrumental. Deletion of E in other coronaviruses has been shown previously to either attenuate or abrogate infection. To determine the role of E on SARS-CoV-2 virus production and infectivity, we produced reporter SARS-CoV-2 with or without the E gene deleted using a bacterial artificial chromosome. Replication of ΔE SARS-CoV-2 was attenuated in Vero E6 cells expressing human ACE2 and TMPRSS2 and in human epithelial cell lines. Electron and immunofluorescence microscopy and virology assays showed that ΔE SARS-CoV-2 increased cell surface expression of Spike (S) glycoprotein, leading to reduced S incorporation into ΔE SARS-CoV-2 particles and promotion of increased cell-to-cell transmission that evades neutralizing antibody inhibition. Trans-complementation of E partially rescued ΔE SARS-CoV-2 S incorporation and restored cell-free transmission. In addition to validating the role of E in retention of S in the ER-Golgi intermediate complex (ERGIC), our results showed that a lack of E led to reorganization of the ERGIC during SARS-CoV-2 infection. Improved understanding of E in SARS-CoV-2 replication and host pathogenesis may help development of novel therapeutics. Importance Non-S coronavirus structural proteins, including E, are conserved, making them potential pan-coronavirus therapeutic targets. Many details about these proteins and their roles in viral replication and host pathogenesis are unknown. In this study, we showed that SARS-CoV-2 replicates without E but is attenuated and impaired for virus particle formation, with less S incorporated into virions and more S expressed on the cell surface compared to wild-type virus. SARS-CoV-2 lacking E spread primarily via cell fusion and evaded neutralizing antibodies. In addition, the absence of E resulted in the reorganization of the ERGIC cell secretory compartment during SARS-CoV-2 infection. A better understanding of how E influences SARS-CoV-2 replication could guide directed design of novel therapeutics for treatment of COVID-19 patients, as well as the potential for pan-coronavirus protection against future coronavirus outbreaks.

SERPINB9, an endogenous inhibitor of granzyme B (GzmB), has emerged as a critical factor in the resistance to immunotherapy by protecting cancer cells from GzmB-induced cytotoxicity. However, its role in chemosensitivity remains unknown. In this study, we show that gemcitabine (GEM) treatment upregulates SERPINB9 through transcription factor ATF-3. Interestingly, GEM also induces the expression of GzmB and knockout or knockdown of SERPINB9 results in enhanced response of tumor cells to GEM, suggesting a role of GzmB/SERPINB9 axis in regulating chemosensitivity. To facilitate the therapeutic translation of these findings, we engineer POEM nanocarrier (consisting of lipid-derivatized polylysine (PEG-PLL-Oleic acid, PPO), and GEM-conjugated polylysine (PEG-PLL-OA-GEM, PPOGEM), PPO/PPOGEM (POEM)) that is highly effective in codelivery of built-in GEM and loaded SERPINB9 short interfering RNA (siSPB9). GEM conjugation introduces an additional mechanism of carrier/siRNA interaction in addition to charge-mediated interaction and enables efficient i.v. delivery at lower N/P ratios. Here, we show that co-delivery of GEM and siSPB9 significantly improves antitumor efficacy and remodels the tumor immune microenvironment in pancreatic cancer models, supporting a promising therapeutic strategy.

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria and, in doing so, may promote the spread of drug resistance genes in the population. IMPORTANCE: This work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here, we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface and that this DNA can be imported by the transformation machinery, facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg²⁺ (site B) inducing formation of a negatively charged pocket where a second floating Mg²⁺ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg²⁺ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.

Biofilm-protected pathogenic Staphylococcus aureus causes chronic infections that are difficult to treat. An essential building block of these biofilms are functional amyloid fibrils that assemble from phenol-soluble modulins (PSMs). PSMα1 cross-seeds other PSMs into cross-β amyloid folds and is therefore a key element in initiating biofilm formation. However, the paucity of high-resolution structures hinders efforts to prevent amyloid assembly and biofilm formation. Here, we present a 3.5 Å resolution density map of the major PSMα1 fibril form revealing a left-handed cross-β fibril composed of two C ₂ -symmetric U-shaped protofilaments whose subunits are unusually tilted out-of-plane. Monomeric α-helical PSMα1 is extremely cytotoxic to cells, despite the moderate toxicity of the cross-β fibril. We suggest mechanistic insights into the PSM functional amyloid formation and conformation transformation on the path from monomer-to-fibril formation. Details of PSMα1 assembly and fibril polymorphism suggest how S. aureus utilizes functional amyloids to form biofilms and establish a framework for developing therapeutics against infection and antimicrobial resistance.