Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry

A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH^‡) and Eyring transition state entropies (ΔS^‡). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH_25°C = −41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH^‡ = −0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium (ΔH_25°C = −20.5 ± 2.1 kcal/mol, ΔH^‡ = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl₂). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.     

A high‐fat diet exacerbates the Alzheimer's disease pathology in the hippocampus of the App NL−F/NL−F knock‐in mouse model

Insulin resistance and diabetes mellitus are major risk factors for Alzheimer''s disease (AD), and studies with transgenic mouse models of AD have provided supportive evidence with some controversies. To overcome potential artifacts derived from transgenes, we used a knock‐in mouse model, App^{NL−F/NL−F} , which accumulates Aβ plaques from 6 months of age and shows mild cognitive impairment at 18 months of age, without the overproduction of APP. In the present study, 6‐month‐old male App^{NL−F/NL−F} and wild‐type mice were fed a regular or high‐fat diet (HFD) for 12 months. HFD treatment caused obesity and impaired glucose tolerance (i.e., T2DM conditions) in both wild‐type and App^{NL−F/NL−F} mice, but only the latter animals exhibited an impaired cognitive function accompanied by marked increases in both Aβ deposition and microgliosis as well as insulin resistance in the hippocampus. Furthermore, HFD‐fed App^{NL−F/NL−F} mice exhibited a significant decrease in volume of the granule cell layer in the dentate gyrus and an increased accumulation of 8‐oxoguanine, an oxidized guanine base, in the nuclei of granule cells. Gene expression profiling by microarrays revealed that the populations of the cell types in hippocampus were not significantly different between the two mouse lines, regardless of the diet. In addition, HFD treatment decreased the expression of the Aβ binding protein transthyretin (TTR) in App^{NL−F/NL−F} mice, suggesting that the depletion of TTR underlies the increased Aβ deposition in the hippocampus of HFD‐fed App^{NL−F/NL−F} mice.     

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.     

Fungal‐induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL‐22

Incorporation of microbiome data has recently become important for prevention, diagnosis, and treatment of colorectal cancer, and several species of bacteria were shown to be associated with carcinogenesis. However, the role of commensal fungi in colon cancer remains poorly understood. Here, we report that mice lacking the c‐type lectin Dectin‐3 (Dectin‐3 ^−/−) show increased tumorigenesis and Candida albicans burden upon chemical induction. Elevated C. albicans load triggered glycolysis in macrophages and interleukin‐7 (IL‐7) secretion. IL‐7 induced IL‐22 production in RORγt⁺ (group 3) innate lymphoid cells (ILC3s) via aryl hydrocarbon receptor and STAT3. Consistently, IL‐22 frequency in tumor tissues of colon cancer patients positively correlated with fungal burden, indicating the relevance of this regulatory axis in human disease. These results establish a C. albicans‐driven crosstalk between macrophages and innate lymphoid cells in the intestine and expand our understanding on how commensal mycobiota regulate host immunity and promote tumorigenesis.     

IFN‐γ+IL‐17+Th17 cells regulate fibrosis through secreting IL‐21 in systemic scleroderma

This study aimed to explore the function of IFN‐γ⁺IL‐17⁺Th17 cells on fibrosis in systemic scleroderma (SSc). Blood and skin samples were collected from 20 SSc cases and 10 healthy individuals. The percentage of IFN‐γ⁺IL‐17⁺Th17 cells was detected using flow cytometry. The in vitro induction of IFN‐γ⁺IL‐17⁺Th17 cells was performed adopting PHA and rIL‐12. Gene expression was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR), whereas western blot analysis was adopted for protein analysis. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with SSc stages (P = .031), disease duration (P = .016), activity (P = .025) and skin scores (P < .001). In vitro, IFN‐γ⁺IL‐17⁺Th17 cells could promote the expressions of α‐SMA and COL1A1, revealing increased fibroblasts’ proliferation and enhanced collagen‐secreting capacity. In addition, IL‐21 expression was significantly increased in co‐culture medium of IFN‐γ⁺IL‐17⁺Th17 cells and fibroblasts (P < .001). IL‐21 neutralizer treatment resulted in the down‐regulation of α‐SMA and COL1A1. IL‐21 was confirmed as an effector of IFN‐γ⁺IL‐17⁺Th17 cells in fibrosis process. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with disease activity. IFN‐γ⁺IL‐17⁺Th17 cells could promote fibroblast proliferation and enhance collagen‐secreting ability via producing IL‐21, thus contributing to fibrosis in SSc.     

Dynamics of NK,CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8⁺ T, CD4⁺ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8⁺ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3⁺ Tfh‐like cell and CD226⁺ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS⁺ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8⁺ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4⁺ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.     

Cyclic di‐GMP modulates sessile‐motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice‐growing countries. We showed recently that the universal bacterial second messenger c‐di‐GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c‐di‐GMP receptor domain, known as the PilZ‐domain. By deleting all the genes encoding c‐di‐GMP‐degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c‐di‐GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild‐type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c‐di‐GMP, which together play a critical role in regulating the c‐di‐GMP‐associated functionality. The findings from this study fill a gap in our understanding of how c‐di‐GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.     

LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS ^−/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS ^−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.     

Short and dysfunctional telomeres protect from allergen‐induced airway inflammation

Asthma is a chronic inflammatory disease affecting 300 million people worldwide. As telomere shortening is a well‐established hallmark of aging and that asthma incidence decreases with age, here we aimed to study the role of short telomeres in asthma pathobiology. To this end, wild‐type and telomerase‐deficient mice with short telomeres (third‐generation (G3 Tert ^−/− mice)) were challenged with intranasal house dust mite (HDM) extract. We also challenged with HDM wild‐type mice in which we induced a telomere dysfunction by the administration of 6‐thio‐2´‐deoxyguanosine (6‐thio‐dG). Following HDM exposure, G3 Tert ^−/− and 6‐thio‐dG treated mice exhibited attenuated eosinophil counts and presence of hematopoietic stem cells in the bone marrow, as well as lower levels of IgE and circulating eosinophils. Accordingly, both G3 Tert ^−/− and 6‐thio‐dG treated wild‐type mice displayed reduced airway hyperresponsiveness (AHR), as indicated by decreased airway remodeling and allergic airway inflammation markers in the lung. Furthermore, G3 Tert ^−/− and 6‐thio‐dG treated mice showed lower differentiation of Club cells, attenuating goblet cell hyperplasia. Club cells of G3 Tert ^−/− and 6‐thio‐dG treated mice displayed increased DNA damage and senescence and reduced proliferation. Thus, short/dysfunctional telomeres play a protective role in murine asthma by impeding both AHR and mucus secretion after HDM exposure. Therefore, our findings imply that telomeres play a relevant role in allergen‐induced airway inflammation.     

Toll‐7 promotes tumour growth and invasion in Drosophila

Objectives Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well‐established Drosophila tumour models, we identified Toll‐7 as a novel regulator of tumour growth and invasion.Materials and methodsTransgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT‐qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0.ResultsLoss of Toll‐7 suppresses Ras^V12/lgl ^−/−‐induced tumour growth and invasion, as well as cell polarity disruption‐induced invasive cell migration, whereas expression of a constitutively active allele of Toll‐7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr‐JNK signalling is necessary and sufficient for Toll‐7‐induced invasive cell migration. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll‐7 activates the EGFR‐Ras signalling, which cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Finally, Toll‐7 is necessary and sufficient for the proper maintenance of EGFR protein level.ConclusionsOur findings characterized Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion in Drosophila, which shed light on the pro‐tumour function of mammalian Toll‐like receptors (TLRs).     

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C

Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu²⁺ ≥ Zn²⁺ > Ni²⁺ > Co²⁺) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k _cat rather than K _M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn²⁺ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.     

Caveolin‐1 negatively regulates inflammation and fibrosis in silicosis

Inhalation of crystalline silica causes silicosis, the most common and serious occupational disease, which is characterized by progressive lung inflammation and fibrosis. Recent studies revealed the anti‐inflammatory and anti‐fibrosis role of Caveolin‐1 (Cav‐1) in lung, but this role in silicosis has not been investigated. Thus, this study evaluated Cav‐1 regulatory effects in silicosis. It was found that Cav‐1 levels were significantly reduced in the lung from silicosis patients and silicotic mice. The silicosis models were established in C57BL/6 (wild‐type) and Cav‐1 deficiency (Cav‐1 ^−/−) mice, and Cav‐1 ^−/− mice displayed wider alveolar septa, increased collagen deposition and more silicotic nodules. The mice peritoneal‐derived macrophages were used to explore the role of Cav‐1 in silica‐induced inflammation, which plays a central role in mechanism of silicosis. Cav‐1 inhibited silica‐induced infiltration of inflammatory cells and secretion of inflammatory factors in vitro and in vivo, partly by downregulating NF‐κB pathway. Additionally, silica uptake and expression of 4‐hydroxynonenal in silicotic mice were observed, and it was found that Cav‐1 absence triggered excessive silica deposition, causing a stronger oxidative stress response. These findings demonstrate the protective effects of Cav‐1 in silica‐induced lung injury, suggesting its potential therapeutic value in silicosis.     

Thermodynamic analysis of F1‐ATPase rotary catalysis using high‐speed imaging

F₁-ATPase (F₁) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F₁ rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F₁ catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F₁ rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (P_i). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, P_i release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG^‡ = 53–56 kJ mol⁻¹, the contributions of the enthalpy (ΔH^‡), and entropy (ΔS^‡) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH^‡ and ΔS^‡. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.     

Genotyping validates the efficacy of photographic identification in a capture‐mark‐recapture study based on the head scale patterns of the prairie lizard (Sceloporus consobrinus)

Population studies often incorporate capture‐mark‐recapture (CMR) techniques to gather information on long‐term biological and demographic characteristics. A fundamental requirement for CMR studies is that an individual must be uniquely and permanently marked to ensure reliable reidentification throughout its lifespan. Photographic identification involving automated photographic identification software has become a popular and efficient noninvasive method for identifying individuals based on natural markings. However, few studies have (a) robustly assessed the performance of automated programs by using a double‐marking system or (b) determined their efficacy for long‐term studies by incorporating multi‐year data. Here, we evaluated the performance of the program Interactive Individual Identification System (I³S) by cross‐validating photographic identifications based on the head scale pattern of the prairie lizard (Sceloporus consobrinus) with individual microsatellite genotyping (N = 863). Further, we assessed the efficacy of the program to identify individuals over time by comparing error rates between within‐year and between‐year recaptures. Recaptured lizards were correctly identified by I³S in 94.1% of cases. We estimated a false rejection rate (FRR) of 5.9% and a false acceptance rate (FAR) of 0%. By using I³S, we correctly identified 97.8% of within‐year recaptures (FRR = 2.2%; FAR = 0%) and 91.1% of between‐year recaptures (FRR = 8.9%; FAR = 0%). Misidentifications were primarily due to poor photograph quality (N = 4). However, two misidentifications were caused by indistinct scale configuration due to scale damage (N = 1) and ontogenetic changes in head scalation between capture events (N = 1). We conclude that automated photographic identification based on head scale patterns is a reliable and accurate method for identifying individuals over time. Because many lizard or reptilian species possess variable head squamation, this method has potential for successful application in many species.     

Inherited genetic variants associated with glucocorticoid sensitivity in leukaemia cells

Identification of genetic variants associated with glucocorticoids (GC) sensitivity of leukaemia cells may provide insight into potential drug targets and tailored therapy. In the present study, within 72 leukaemic cell lines derived from Japanese patients with B‐cell precursor acute lymphoblastic leukaemia (ALL), we conducted genome‐wide genotyping of single nucleotide polymorphisms (SNP) and attempted to identify genetic variants associated with GC sensitivity and NR3C1 (GC receptor) gene expression. IC50 measures for prednisolone (Pred) and dexamethasone (Dex) were available using an alamarBlue cell viability assay. IC50 values of Pred showed the strongest association with rs904419 (P = 4.34 × 10⁻⁸), located between the FRMD4B and MITF genes. The median IC50 values of prednisolone for cell lines with rs904419 AA (n = 13), AG (n = 31) and GG (n = 28) genotypes were 0.089, 0.139 and 297 µmol/L, respectively. For dexamethasone sensitivity, suggestive association was observed for SNP rs2306888 (P = 1.43 × 10⁻⁶), a synonymous SNP of the TGFBR3 gene. For NR3C1 gene expression, suggestive association was observed for SNP rs11982167 (P = 6.44 × 10⁻⁸), located in the PLEKHA8 gene. These genetic variants may affect GC sensitivity of ALL cells and may give rise to opportunities in personalized medicine for effective and safe chemotherapy in ALL patients.     

Removal of gut symbiotic bacteria negatively affects life history traits of the shield bug,Graphosoma lineatum

The shield bug, Graphosoma lineatum (Heteroptera, Pentatomidae), harbors extracellular Pantoea‐like symbiont in the enclosed crypts of the midgut. The symbiotic bacteria are essential for normal longevity and fecundity of this insect. In this study, life table analysis was used to assess the biological importance of the gut symbiont in G. lineatum. Considering vertical transmission of the bacterial symbiont through the egg surface contamination, we used surface sterilization of the eggs to remove the symbiont. The symbiont population was decreased in the newborn nymphs hatched from the surface‐sterilized eggs (the aposymbiotic insects), and this reduction imposed strongly negative effects on the insect host. We found significant differences in most life table parameters between the symbiotic insects and the aposymbiotics. The intrinsic rate of increase in the control insects (0.080 ± 0.003 day⁻¹) was higher than the aposymbiotic insects (0.045 ± 0.007 day⁻¹). Also, the net reproductive and gross reproductive rates were decreased in the aposymbiotic insects (i.e., 20.770 ± 8.992 and 65.649 ± 27.654 offspring/individual, respectively), compared with the symbiotic insects (i.e., 115.878 ± 21.624 and 165.692 ± 29.058 offspring/individual, respectively). These results clearly show biological importance of the symbiont in G. lineatum.     

Pressure-accelerated dissociation of amyloid fibrils in wild-type hen lysozyme

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml⁻¹ fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (k_obs = 0.00193 min⁻¹ at 0.1 MPa, 0.0348 min⁻¹ at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and the activation compressibility Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹ bar⁻¹ for the dissociation reaction. The negative ΔV^0‡ and Δκ^‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.     

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2^LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2^LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2^LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.     

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.     

Trade‐off between flight capability and reproduction in Acridoidea (Insecta: Orthoptera)

In many insect taxa, there is a well‐established trade‐off between flight capability and reproduction. The wing types of Acridoidea exhibit extremely variability from full length to complete loss in many groups, thus, provide a good model for studying the trade‐off between flight and reproduction. In this study, we completed the sampling of 63 Acridoidea species, measured the body length, wing length, body weight, flight muscle weight, testis and ovary weight, and the relative wing length (RWL), relative flight muscle weight (RFW), and gonadosomatic index (GSI) of different species were statistically analyzed. The results showed that there were significant differences in RWL, RFW, and GSI among Acridoidea species with different wing types. RFW of long‐winged species was significantly higher than that of short‐winged and wingless species (p < .01), while GSI of wingless species was higher than that of long‐winged and short‐winged species. The RWL and RFW had a strong positive correlation in species with different wing types (correlation coefficient r = .8344 for male and .7269 for female, and p < .05), while RFW was strong negatively correlated with GSI (r = −.2649 for male and −.5024 for female, and p < .05). For Acridoidea species with wing dimorphism, males with relatively long wings had higher RFW than that of females with relatively short wings, while females had higher GSI. Phylogenetic comparative analysis showed that RWL, RFW, and GSI all had phylogenetic signals and phylogenetic dependence. These results revealed that long‐winged individuals are flight capable at the expense of reproduction, while short‐winged and wingless individuals cannot fly, but has greater reproductive output. The results support the trade‐off between flight and reproduction in Acridoidea.