PCIF1-mediated deposition of 5'-cap N6,2'-O-dimethyladenosine in ACE2 and TMPRSS2 mRNA regulates susceptibility to SARS-CoV-2 infection.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major health problem worldwide. Due to the fast emergence of SARS-CoV-2 variants, understanding the molecular mechanisms of viral pathogenesis and developing novel inhibitors are essential and urgent. Here, we investigated the potential roles of N6,2'-O-dimethyladenosine (m6Am), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells. Using genome-wide m6Am-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N6-methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses. We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m6Am-dependent mRNA stabilization. In PCIF1-depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1. These findings suggest a role for PCIF1 and cap m6Am in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection.

Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern.

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.

Serum C-reactive protein is associated with earlier mortality across different interstitial lung diseases.

BACKGROUND AND OBJECTIVE: The acute-phase protein C-reactive protein (CRP) is known to be associated with poor outcomes in cancer and cardiovascular disease, but there is limited evidence of its prognostic implications in interstitial lung diseases (ILDs). We therefore set out to test whether baseline serum CRP levels are associated with mortality in four different ILDs. METHODS: In this retrospective study, clinically measured CRP levels, as well as baseline demographics and lung function measures, were collected for ILD patients first presenting to the Royal Brompton Hospital between January 2010 and December 2019. Cox regression analysis was used to determine the relationship with 5-year mortality. RESULTS: Patients included in the study were: idiopathic pulmonary fibrosis (IPF) n = 422, fibrotic hypersensitivity pneumonitis (fHP) n = 233, rheumatoid arthritis associated ILD (RA-ILD) n = 111 and Systemic Sclerosis associated ILD (SSc-ILD) n = 86. Patients with a recent history of infection were excluded. Higher CRP levels were associated with shorter 5-year survival in all four disease groups on both univariable analyses, and after adjusting for age, gender, smoking history, immunosuppressive therapy and baseline disease severity (IPF: HR (95% CI): 1.3 (1.1-1.5), p = 0.003, fHP: 1.5 (1.2-1.9), p = 0.001, RA-ILD: 1.4 (1.1-1.84), p = 0.01 and SSc-ILD: 2.7 (1.6-4.5), p < 0.001). CONCLUSION: Higher CRP levels are independently associated with reduced 5-year survival in IPF, fHP, RA-ILD and SSc-ILD.

Virologic and Immunologic Characterization of Coronavirus Disease 2019 Recrudescence After Nirmatrelvir/Ritonavir Treatment.

We isolated a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2 variant from a person with coronavirus disease 2019 recrudescence after nirmatrelvir/ritonavir treatment. Antiviral sensitivity and neutralizing antibody testing were performed with both parental SARS-CoV-2 and multiple variants of concern. We found that neither nirmatrelvir resistance nor absence of neutralizing immunity was a likely cause of the recrudescence.

Aging and Intermittent Fasting Impact on Transcriptional Regulation and Physiological Responses of Adult Drosophila Neuronal and Muscle Tissues.

The progressive decline of the nervous system, including protein aggregate formation, reflects the subtle dysregulation of multiple functional pathways. Our previous work has shown intermittent fasting (IF) enhances longevity, maintains adult behaviors and reduces aggregates, in part, by promoting autophagic function in the aging Drosophila brain. To clarify the impact that IF-treatment has upon aging, we used high throughput RNA-sequencing technology to examine the changing transcriptome in adult Drosophila tissues. Principle component analysis (PCA) and other analyses showed ~1200 age-related transcriptional differences in head and muscle tissues, with few genes having matching expression patterns. Pathway components showing age-dependent expression differences were involved with stress response, metabolic, neural and chromatin remodeling functions. Middle-aged tissues also showed a significant increase in transcriptional drift-variance (TD), which in the CNS included multiple proteolytic pathway components. Overall, IF-treatment had a demonstrably positive impact on aged transcriptomes, partly ameliorating both fold and variance changes. Consistent with these findings, aged IF-treated flies displayed more youthful metabolic, behavioral and basal proteolytic profiles that closely correlated with transcriptional alterations to key components. These results indicate that even modest dietary changes can have therapeutic consequences, slowing the progressive decline of multiple cellular systems, including proteostasis in the aging nervous system.

Interferon-Inducible Guanylate-Binding Protein 5 Inhibits Replication of Multiple Viruses by Binding to the Oligosaccharyltransferase Complex and Inhibiting Glycoprotein Maturation.

Viral infection induces production of type I interferons and expression of interferon-stimulated genes (ISGs) that play key roles in inhibiting viral infection. Here, we show that the ISG guanylate-binding protein 5 (GBP5) inhibits N-linked glycosylation of key proteins in multiple viruses, including SARS-CoV-2 spike protein. GBP5 binds to accessory subunits of the host oligosaccharyltransferase (OST) complex and blocks its interaction with the spike protein, which results in misfolding and retention of spike protein in the endoplasmic reticulum likely due to decreased N-glycan transfer, and reduces the assembly and release of infectious virions. Consistent with these observations, pharmacological inhibition of the OST complex with NGI-1 potently inhibits glycosylation of other viral proteins, including MERS-CoV spike protein, HIV-1 gp160, and IAV hemagglutinin, and prevents the production of infectious virions. Our results identify a novel strategy by which ISGs restrict virus infection and provide a rationale for targeting glycosylation as a broad antiviral therapeutic strategy.

Implementing Smartphone Tutorials for Patients With Severe Glaucoma.

Purpose The purpose of the study is to determine if instructional videos detailing the use of smartphone accessibility features may be used to improve quality of life and comfort with phone usage among patients with severe glaucoma. Design The design of the present study is an interventional case series. Methods The patients with vision loss due to severe glaucoma were recruited from one institution. Two surveys were completed to provide baseline data: one detailed their current use of smartphone accessibility features, and the other provided survey was the EuroQol 5 Dimension 5 Level (EQ-5D-5L) (EuroQol Group, Rotterdam, Netherlands), which is used to assess the quality of life. Then, the patients were shown a brief video with instructions on configuring the use of voice-over, magnification, and zoom functions, along with other features. To conclude, the patients completed the same surveys either at follow-up visits or by phone calls. Results Fifteen patients were recruited to participate in the study. At baseline, the participants used a median of one accessibility feature, with the most common feature being "text sizing/bolding." At follow-up, the participants averaged the gain of use of one accessibility feature and reported a decrease in text messaging visual limitation, although these findings did not reach statistical significance. Overall, the quality of life, as measured by the EQ-5D-5L, demonstrated a non-statistically significant increase of six points. Conclusions Despite the lack of statistical significance, our results indicate that providing instructional videos may benefit the patients' ability to navigate on their smartphones. Incorporating links or Quick Response (QR) codes to these instructional videos provides an opportunity to improve the quality of life at no additional risk to the patient. Further studies are needed with an increased population to investigate for any significance of our findings.

Neutralizing Antibody Responses After Severe Acute Respiratory Syndrome Coronavirus 2 BA.2 and BA.2.12.1 Infection Do Not Neutralize BA.4 and BA.5 and Can Be Blunted by Nirmatrelvir/Ritonavir Treatment.

The factors contributing to the rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.4 and BA.5 subvariants in populations that experienced recent surges of BA.2 and BA.2.12.1 infections are not understood. Neutralizing antibodies (NAbs) are likely to protect against severe disease if present in sufficient quantity. We found that after BA.2 or BA.2.12.1 infection, NAb responses were largely cross-neutralizing but were much less effective against BA.5. In addition, individuals who were infected and treated early with nirmatrelvir/ritonavir (Paxlovid) had lower NAb levels than untreated individuals.

1-O-Octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-GS-441524 (V2043). Evaluation of Oral V2043 in a Mouse Model of SARS-CoV-2 Infection and Synthesis and Antiviral Evaluation of Additional Phospholipid Esters with Enhanced Anti-SARS-CoV-2 Activity.

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.

Virologic and Immunologic Characterization of COVID-19 Recrudescence after Nirmatrelvir/Ritonavir Treatment.

We isolated a SARS-CoV-2 BA.2 variant from a person with COVID-19 recrudescence after nirmatrelvir/ritonavir treatment. Antiviral sensitivity and neutralizing antibody testing was performed and compared with parental SARS-CoV-2 and multiple variants of concern. We found that neither NM resistance nor absence of neutralizing immunity were likely causes of the recrudescence.

Rational Design and Optimization of m6A-RNA Demethylase FTO Inhibitors as Anticancer Agents.

Aberrant regulation of N6-methyladenosine (m6A) RNA modification has been implicated in the progression of multiple diseases, including cancer. Previously, we identified a small molecule inhibitor of the m6A demethylase fat mass- and obesity-associated protein (FTO), which removes both m6A and N6,2'-O-dimethyladenosine (m6Am) RNA modifications. In this work, we describe the rational design and optimization of a new class of FTO inhibitors derived from our previous lead FTO-04 with nanomolar potency and high selectivity against the homologous m6A RNA demethylase ALKBH5. The oxetanyl class of compounds comprise competitive inhibitors of FTO with potent antiproliferative effects in glioblastoma, acute myeloid leukemia, and gastric cancer models where lead FTO-43 demonstrated potency comparable to clinical chemotherapeutic 5-fluorouracil. Furthermore, FTO-43 increased m6A and m6Am levels in a manner comparable to FTO knockdown in gastric cancer cells and regulated Wnt/PI3K-Akt signaling pathways. The oxetanyl class contains significantly improved anticancer agents with a variety of applications beyond glioblastoma.

Discovery and Mechanism of SARS-CoV-2 Main Protease Inhibitors.

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.

Finite element derived femoral strength is a better predictor of hip fracture risk than aBMD in the AGES Reykjavik study cohort.

Hip fractures associated with a high economic burden, loss of independence, and a high rate of post-fracture mortality, are a major health concern for modern societies. Areal bone mineral density is the current clinical metric of choice when assessing an individual's future risk of fracture. However, this metric has been shown to lack sensitivity and specificity in the targeted selection of individuals for preventive interventions. Although femoral strength derived from computed tomography based finite element models has been proposed as an alternative based on its superior femoral strength prediction ex vivo, such predictions have only shown marginal or no improvement for assessing hip fracture risk. This study compares finite element derived femoral strength to aBMD as a metric for hip fracture risk assessment in subjects (N = 601) from the AGES Reykjavik Study cohort and analyses the dependence of femoral strength predictions and classification accuracy on the material model and femoral loading alignment. We found hip fracture classification based on finite element derived femoral strength to be significantly improved compared to aBMD. Finite element models with non-linear material models performed better at classifying hip fractures compared to finite element models with linear material models and loading alignments with low internal rotation and adduction, which do not correspond to weak femur alignments, were found to be most suitable for hip fracture classification.

Strain rate dependency of bovine trabecular bone under impact loading at sideways fall velocity.

There is currently a knowledge gap in scientific literature concerning the strain rate dependent properties of trabecular bone at intermediate strain rates. Meanwhile, strain rates between 10 and 200/s have been observed in previous dynamic finite element models of the proximal femur loaded at realistic sideways fall speeds. This study aimed to quantify the effect of strain rate (ε̇) on modulus of elasticity (E), ultimate stress (σu), failure energy (Uf), and minimum stress (σm) of trabecular bone in order to improve the biofidelity of material properties used in dynamic simulations of sideways fall loading on the hip. Cylindrical cores of trabecular bone (D = 8 mm, Lgauge = 16 mm, n = 34) from bovine proximal tibiae and distal femurs were scanned in µCT (10 µm), quantifying apparent density (ρapp) and degree of anisotropy (DA), and subsequently impacted within a miniature drop tower. Force of impact was measured using a piezoelectric load cell (400 kHz), while displacement during compression was measured from high speed video (50,000 frames/s). Four groups, with similar density distributions, were loaded at different impact velocities (0.84, 1.33, 1.75, and 2.16 m/s) with constant kinetic energy (0.4 J) by adjusting the impact mass. The mean strain rates of each group were significantly different (p < 0.05) except for the two fastest impact speeds (p = 0.09). Non-linear regression models correlated strain rate, DA, and ρapp with ultimate stress (R2 = 0.76), elastic modulus (R2 = 0.63), failure energy (R2 = 0.38), and minimum stress (R2 = 0.57). These results indicate that previous estimates of σu could be under predicting the mechanical properties at strain rates above 10/s.

Correction: On the internal reaction forces, energy absorption, and fracture in the hip during simulated sideways fall impact.

[This corrects the article DOI: 10.1371/journal.pone.0200952.].

On the internal reaction forces, energy absorption, and fracture in the hip during simulated sideways fall impact.

The majority of hip fractures have been reported to occur as a result of a fall with impact to the greater trochanter of the femur. Recently, we developed a novel cadaveric pendulum-based hip impact model and tested two cadaveric femur-pelvis constructs, embedded in a soft tissue surrogate. The outcome was a femoral neck fracture in a male specimen while a female specimen had no fracture. The aim of the present study was, first, to develop a methodology for constructing and assessing the accuracy of explicit Finite Element Models (FEMs) for simulation of sideways falls to the hip based on the experimental model. Second, to use the FEMs for quantifying the internal reaction forces and energy absorption in the hip during impact. Third, to assess the potential of the FEMs in terms of separating a femoral fracture endpoint from a non-fracture endpoint. Using a non-linear, strain rate dependent, and heterogeneous material mapping strategy for bone tissue in these models, we found the FEM-derived results to closely match the experimental test results in terms of impact forces and displacements of pelvic video markers up to the time of peak impact force with errors below 10%. We found the internal reaction forces in the femoral neck on the impact side to be approximately 35% lower than the impact force measured between soft tissue and ground for both specimens. In addition, we found the soft tissue to be the component that absorbed the largest part of the energy of the tissue types in the hip region. Finally, we found surface strain patterns derived from FEM results to match the fracture location and extent based on post testing x-rays of the specimens. This is the first study with quantitative data on the energy absorption in the pelvic region during a sideways fall.

Interactive graph-cut segmentation for fast creation of finite element models from clinical ct data for hip fracture prediction.

In this study, we propose interactive graph cut image segmentation for fast creation of femur finite element (FE) models from clinical computed tomography scans for hip fracture prediction. Using a sample of N = 48 bone scans representing normal, osteopenic and osteoporotic subjects, the proximal femur was segmented using manual (gold standard) and graph cut segmentation. Segmentations were subsequently used to generate FE models to calculate overall stiffness and peak force in a sideways fall simulations. Results show that, comparable FE results can be obtained with the graph cut method, with a reduction from 20 to 2-5 min interaction time. Average differences between segmentation methods of 0.22 mm were not significantly correlated with differences in FE derived stiffness (R2 = 0.08, p = 0.05) and weakly correlated to differences in FE derived peak force (R2 = 0.16, p = 0.01). We further found that changes in automatically assigned boundary conditions as a consequence of small segmentation differences were significantly correlated with FE derived results. The proposed interactive graph cut segmentation software MITK-GEM is freely available online at https://simtk.org/home/mitk-gem .

Mapping anisotropy of the proximal femur for enhanced image based finite element analysis.

Finite element (FE) models of bone derived from quantitative computed tomography (QCT) rely on realistic material properties to accurately predict bone strength. QCT cannot resolve bone microarchitecture, therefore QCT-based FE models lack the anisotropy apparent within the underlying bone tissue. This study proposes a method for mapping femoral anisotropy using high-resolution peripheral quantitative computed tomography (HR-pQCT) scans of human cadaver specimens. Femur HR-pQCT images were sub-divided into numerous overlapping cubic sub-volumes and the local anisotropy was quantified using a 'direct-mechanics' method. The resulting directionality reflected all the major stress lines visible within the trabecular lattice, and provided a realistic estimate of the alignment of Harvesian systems within the cortical compartment. QCT-based FE models of the proximal femur were constructed with isotropic and anisotropic material properties, with directionality interpolated from the map of anisotropy. Models were loaded in a sideways fall configuration and the resulting whole bone stiffness was compared to experimental stiffness and ultimate strength. Anisotropic models were consistently less stiff, but no statistically significant differences in correlation were observed between material models against experimental data. The mean difference in whole bone stiffness between model types was approximately 26%, suggesting that anisotropy can still effect considerable change in the mechanics of proximal femur models. The under prediction of whole bone stiffness in anisotropic models suggests that the orthotropic elastic constants require further investigation. The ability to map mechanical anisotropy from high-resolution images and interpolate information into clinical-resolution models will allow testing of new anisotropic material mapping strategies.

Multi-unit sustained vibration loading platform for biological tissues: design, validation and experimentation.

The relationships between mechanical inputs and resulting biological tissue structure, composition, and metabolism are critical to detailing the nuances of tissue mechanobiology in both healthy and injured tissues. Developing a model system to test the mechanobiology of tissues ex-vivo is a complex task, as controlling chemical and mechanical boundary layers in-vitro are difficult to replicate. A novel multi-unit vibration loading platform for intervertebral discs was designed and validated with both independent electronic data and experimental loading of 6 bovine intervertebral discs (IVDs) and an equal number of unloaded controls. Sustained vibration was applied using closed-loop positional control of pushrods within four independent bioreactors with circulating phosphate buffered saline. The bioreactors were designed to be modular with removable components allowing for easy cleaning and replacement. The loading regime was chosen to maximize target mRNA expression as reported in previous research. Aggrecan, decorin, and versican mRNA all reported statistically significant increases above control levels. Biglycan, collagen type I and II showed no significant difference from the control group. Further study is required to determine the resulting effect of increased mRNA expressions on long-term disc health. However these results indicate that this research is past the proof of concept stage, supporting future studies of mechanobiology utilizing this new device. The next stage in developing this novel loading platform should consider modifying the tissue grips to explore the effects of different directional loading on different gene expression, and also loading different types of tissues.