Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

TMPRSS2 expression dictates the entry route used by SARS‐CoV‐2 to infect host cells

SARS‐CoV‐2 is a newly emerged coronavirus that caused the global COVID‐19 outbreak in early 2020. COVID‐19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS‐CoV‐2–host cell interactions and entry mechanisms remain poorly understood. Investigating SARS‐CoV‐2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS‐CoV‐2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH‐independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS‐CoV‐2 entered the cytosol via acid‐activated cathepsin L protease 40–60 min post‐infection. Overexpression of TMPRSS2 in non‐TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS‐CoV‐2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS‐CoV‐2 sorting into either pathway.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

CTD of SARS‐CoV‐2 N protein is a cryptic domain for binding ATP and nucleic acid that interplay in modulating phase separation

SARS‐CoV‐2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N‐/C‐terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid–liquid phase separation (LLPS) depend on the capacity in binding various viral/host‐cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high‐order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS‐CoV‐2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2–16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP‐binding residues of NTD/CTD are identical in the SARS‐CoV‐2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS‐CoV‐2 to complete its life cycle in the host cell. Most importantly, the unique ATP‐binding pockets on NTD/CTD may offer promising targets for design of specific anti‐SARS‐CoV‐2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP.     

High diversity in Delta variant across countries revealed by genome‐wide analysis of SARS‐CoV‐2 beyond the Spike protein

The highly contagious Delta variant of SARS‐CoV‐2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant’s Spike protein, the mutational landscape of the rest of the SARS‐CoV‐2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS‐CoV‐2 proteins from nearly 2 million SARS‐CoV‐2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle‐associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant’s proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.     

A new l‐arginine oxidase engineered from l‐glutamate oxidase

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l‐Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6 catalyzes the oxidative deamination of l‐glutamate to produce 2‐ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l‐glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l‐glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l‐arginine. The oxidative deamination activity of LGOX to l‐arginine is higher than that of l‐arginine oxidase form from Pseudomonas sp. TPU 7192. X‐ray crystal structure analysis revealed that the guanidino group of l‐arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild‐type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l‐arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l‐arginine by LGOX R305E, is proportional to the concentration of l‐arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l‐arginine. Thus, LGOX R305E is suitable for the determination of l‐arginine.     

Sildenafil improves radiation‐induced oral mucositis by attenuating oxidative stress,NF‐κB,ERK and JNK signalling pathways

Radiation‐induced oral mucositis is a common and dose‐limiting complication of head and neck radiotherapy with no effective treatment. Previous studies revealed that sildenafil, a phosphodiesterase 5 inhibitor, has anti‐inflammatory and anti‐cancer effects. In this study, we investigated the effect of sildenafil on radiation‐induced mucositis in rats. Two doses of radiation (8 and 26 Gy X‐ray) were used to induce low‐grade and high‐grade oral mucositis, separately. A control group and three groups of sildenafil citrate‐treated rats (5, 10, and 40 mg/kg/day) were used for each dose of radiation. Radiation increased MDA and activated NF‐κB, ERK and JNK signalling pathways. Sildenafil significantly decreased MDA level, nitric oxide (NO) level, IL1β, IL6 and TNF‐α. The most effective dose of sildenafil was 40 mg/kg/day in this study. Sildenafil also significantly inhibited NF‐κB, ERK and JNK signalling pathways and increased bcl2/bax ratio. In addition, high‐dose radiation severely destructed the mucosal layer in histopathology and led to mucosal cell apoptosis in the TUNEL assay. Sildenafil significantly improved mucosal structure and decreased inflammatory cell infiltration after exposure to high‐dose radiation and reduced apoptosis in the TUNEL assay. These findings show that sildenafil can improve radiation‐induced oral mucositis and decrease the apoptosis of mucosal cells via attenuation of inflammation and oxidative stress.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

Sex‐ and strain‐specific effects of mitochondrial uncoupling on age‐related metabolic diseases in high‐fat diet‐fed mice

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti‐aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled‐release mitochondrial protonophore (CRMP) that is functionally liver‐directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver‐directed fashion could reduce oxidative damage and improve age‐related metabolic disease and lifespan in diet‐induced obese mice. Oral administration of CRMP (20 mg/[kg‐day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74‐week‐old) high‐fat diet (HFD)‐fed C57BL/6J male mice, independently of changes in body weight, whole‐body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long‐term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94–104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex‐specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof‐of‐concept data to support further studies investigating the use of liver‐directed mitochondrial uncouplers to promote healthy aging in humans.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

Dissecting molecular details and functional effects of the high‐affinity copper binding site in plasminogen activator Inhibitor‐1

Plasminogen activator inhibitor‐1 (PAI‐1) is the primary inhibitor for plasminogen activators, tissue‐type plasminogen activator (tPA) and urokinase‐type plasminogen activator (uPA). As a unique member in the serine protease inhibitor (serpin) family, PAI‐1 is metastable and converts to an inactive, latent structure with a half‐life of 1–2 hr under physiological conditions. Unusual effects of metals on the rate of the latency conversion are incompletely understood. Previous work has identified two residues near the N‐terminus, H2 and H3, which reside in a high‐affinity copper‐binding site in PAI‐1 [Bucci JC, McClintock CS, Chu Y, Ware GL, McConnell KD, Emerson JP, Peterson CB (2017) J Biol Inorg Chem 22:1123–1,135]. In this study, neighboring residues, H10, E81, and H364, were tested as possible sites that participate in Cu(II) coordination at the high‐affinity site. Kinetic methods, gel sensitivity assays, and isothermal titration calorimetry (ITC) revealed that E81 and H364 have different roles in coordinating metal and mediating the stability of PAI‐1. H364 provides a third histidine in the metal‐coordination sphere with H2 and H3. In contrast, E81 does not appear to be required for metal ligation along with histidines; contacts made by the side‐chain carboxylate upon metal binding are perturbed and, in turn, influence dynamic fluctuations within the region encompassing helices D, E, and F and the W86 loop that are important in the pathway for the PAI‐1 latency conversion. This investigation underscores a prominent role of protein dynamics, noncovalent bonding networks and ligand binding in controlling the stability of the active form of PAI‐1.     

Aptamer‐mediated synthesis of multifunctional nano‐hydroxyapatite for active tumour bioimaging and treatment

ObjectivesThe nano‐hydroxyapatite (nHAp) is widely used to develop imaging probes and drug carriers due to its excellent bioactivity and biocompatibility. However, traditional methods usually need cumbersome and stringent conditions such as high temperature and post‐modification to prepare the functionalized nHAp, which do not benefit the particles to enter cells due to the increased particle size. Herein, a biomimetic synthesis strategy was explored to achieve the AS1411‐targeted tumour dual‐model bioimaging using DNA aptamer AS1411 as a template. Then, the imaging properties and the biocompatibility of the synthesized AS‐nFAp:Gd/Tb were further investigated.Materials and methodsThe AS‐nFAp:Gd/Tb was prepared under mild conditions through a one‐pot procedure with AS1411 as a template. Besides, the anticancer drug DOX was loaded to AS‐nFAp:Gd/Tb so as to achieve the establishment of a multifunctional nano‐probe that integrated the tumour diagnosis and treatment. The AS‐nFAp:Gd/Tb was characterized by transmission electron microscopy (TEM), energy disperse X‐ray Spectroscopy (EDS) mapping, X‐ray photoelectron spectroscopy (XPS) spectrum, X‐ray diffraction (XRD), fourier‐transformed infrared (FTIR) spectroscopy, capillary electrophoresis analyses, zeta potential and particle sizes. The in vitro magnetic resonance imaging (MRI) and fluorescence imaging were performed on an MRI system and a confocal laser scanning microscope, respectively. The potential of the prepared multifunctional nHAp for a targeted tumour therapy was investigated by a CCK‐8 kit. And the animal experiments were conducted on the basis of the guidelines approved by the Animal Care and Use Committee of Sichuan University, China.ResultsIn the presence of AS1411, the as‐prepared AS‐nFAp:Gd/Tb presented a needle‐like morphology with good monodispersity and improved imaging performance. Furthermore, due to the specific binding between AS1411 and nucleolin up‐expressed in cancer cells, the AS‐nFAp:Gd/Tb possessed excellent AS1411‐targeted fluorescence and MRI imaging properties. Moreover, after loading chemotherapy drug DOX, in vitro and in vivo studies showed that DOX@AS‐nFAp:Gd/Tb could effectively deliver DOX to tumour tissues and exert a highly effective tumour inhibition without systemic toxicity compared with pure DOX.ConclusionsThe results indicated that the prepared multifunctional nHAp synthesized by a novel biomimetic strategy had outstanding capabilities of recognition and treatment for the tumour and had good biocompatibility; hence, it might have a potential clinical application in the future.     

Structural characterization of the self‐association domain of swallow

Swallow, a 62 kDa multidomain protein, is required for the proper localization of several mRNAs involved in the development of Drosophila oocytes. The dimerization of Swallow depends on a 71‐residue self‐association domain in the center of the protein sequence, and is significantly stabilized by a binding interaction with dynein light chain (LC8). Here, we detail the use of solution‐state nuclear magnetic resonance spectroscopy to characterize the structure of this self‐association domain, thereby establishing that this domain forms a parallel coiled‐coil and providing insight into how the stability of the dimerization interaction is regulated.     

Association between IGF‐1 levels ranges and all‐cause mortality: A meta‐analysis

The association between IGF‐1 levels and mortality in humans is complex with low levels being associated with both low and high mortality. The present meta‐analysis investigates this complex relationship between IGF‐1 and all‐cause mortality in prospective cohort studies. A systematic literature search was conducted in PubMed/MEDLINE, Scopus, and Cochrane Library up to September 2019. Published studies were eligible for the meta‐analysis if they had a prospective cohort design, a hazard ratio (HR) and 95% confidence interval (CI) for two or more categories of IGF‐1 and were conducted among adults. A random‐effects model with a restricted maximum likelihood heterogeneity variance estimator was used to find combined HRs for all‐cause mortality. Nineteen studies involving 30,876 participants were included. Meta‐analysis of the 19 eligible studies showed that with respect to the low IGF‐1 category, higher IGF‐1 was not associated with increased risk of all‐cause mortality (HR = 0.84, 95% CI = 0.68–1.05). Dose–response analysis revealed a U‐shaped relation between IGF‐1 and mortality HR. Pooled results comparing low vs. middle IGF‐1 showed a significant increase of all‐cause mortality (HR = 1.33, 95% CI = 1.14–1.57), as well as comparing high vs. middle IGF‐1 categories (HR = 1.23, 95% CI = 1.06–1.44). Finally, we provide data on the association between IGF‐1 levels and the intake of proteins, carbohydrates, certain vitamins/minerals, and specific foods. Both high and low levels of IGF‐1 increase mortality risk, with a specific 120–160 ng/ml range being associated with the lowest mortality. These findings can explain the apparent controversy related to the association between IGF‐1 levels and mortality.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

Pirfenidone modulates macrophage polarization and ameliorates radiation‐induced lung fibrosis by inhibiting the TGF‐β1/Smad3 pathway

Radiation‐induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation‐induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)‐approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X‐ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor‐β1 (TGF‐β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation‐induced expression of TGF‐β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL‐4–induced M2 macrophage polarization and IL‐13–induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase‐1 (ARG‐1), chitinase 3‐like 3 (YM‐1) and TGF‐β1. Notably, the PFD‐induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa‐B (NF‐κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation‐induced chemokine secretion in MLE‐12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF‐β1 from M2 macrophages by attenuating the activation of TGF‐β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF‐β1/Smad3.