Recovering from the stress of the COVID-19 pandemic.

For our special issue on stress, we asked scientists about recovering from the stress of the pandemic, including some who shared insights with us in mid-2020. They discuss the importance of teamwork, reassessing priorities, and the added stresses of the cost-of-living crisis, funding cuts, and retaining scientists in academia.

TAK1 is an essential kinase for STING trafficking.

The translocation of stimulator of interferon genes (STING) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) enables its activation. However, the mechanism underlying the regulation of STING exit from the ER remains elusive. Here, we found that STING induces the activation of transforming growth factor beta-activated kinase 1 (TAK1) prior to STING trafficking in a TAK1 binding protein 1 (TAB1)-dependent manner. Intriguingly, activated TAK1 directly mediates STING phosphorylation on serine 355, which facilitates its interaction with STING ER exit protein (STEEP) and thereby promotes its oligomerization and translocation to the ERGIC for subsequent activation. Importantly, activation of TAK1 by monophosphoryl lipid A, a TLR4 agonist, boosts cGAMP-induced antitumor immunity dependent on STING phosphorylation in a mouse allograft tumor model. Taken together, TAK1 was identified as a checkpoint for STING activation by promoting its trafficking, providing a basis for combinatory tumor immunotherapy and intervention in STING-related diseases.

Cytoplasmic PARP1 links the genome instability to the inhibition of antiviral immunity through PARylating cGAS.

Virus infection modulates both host immunity and host genomic stability. Poly(ADP-ribose) polymerase 1 (PARP1) is a key nuclear sensor of DNA damage, which maintains genomic integrity, and the successful application of PARP1 inhibitors for clinical anti-cancer therapy has lasted for decades. However, precisely how PARP1 gains access to cytoplasm and regulates antiviral immunity remains unknown. Here, we report that DNA virus induces a reactive nitrogen species (RNS)-dependent DNA damage and activates DNA-dependent protein kinase (DNA-PK). Activated DNA-PK phosphorylates PARP1 on Thr594, thus facilitating the cytoplasmic translocation of PARP1 to inhibit the antiviral immunity both in vitro and in vivo. Mechanistically, cytoplasmic PARP1 interacts with and directly PARylates cyclic GMP-AMP synthase (cGAS) on Asp191 to inhibit its DNA-binding ability. Together, our findings uncover an essential role of PARP1 in linking virus-induced genome instability with inhibition of host immunity, which is of relevance to cancer, autoinflammation, and other diseases.

The kinase CK1ɛ controls the antiviral immune response by phosphorylating the signaling adaptor TRAF3.

The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1ɛ suppressed the production of type I interferon in response to viral infection. CK1ɛ interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1ɛ-deficient mice were more susceptible to viral infection. Our findings establish CK1ɛ as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1ɛ-mediated phosphorylation of TRAF3.

Host-mediated ubiquitination of a mycobacterial protein suppresses immunity.

Mycobacterium tuberculosis is an intracellular pathogen that uses several strategies to interfere with the signalling functions of host immune molecules. Many other bacterial pathogens exploit the host ubiquitination system to promote pathogenesis1,2, but whether this same system modulates the ubiquitination of M. tuberculosis proteins is unknown. Here we report that the host E3 ubiquitin ligase ANAPC2-a core subunit of the anaphase-promoting complex/cyclosome-interacts with the mycobacterial protein Rv0222 and promotes the attachment of lysine-11-linked ubiquitin chains to lysine 76 of Rv0222 in order to suppress the expression of proinflammatory cytokines. Inhibition of ANAPC2 by specific short hairpin RNA abolishes the inhibitory effect of Rv0222 on proinflammatory responses. Moreover, mutation of the ubiquitination site on Rv0222 impairs the inhibition of proinflammatory cytokines by Rv0222 and reduces virulence during infection in mice. Mechanistically, lysine-11-linked ubiquitination of Rv0222 by ANAPC2 facilitates the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6, preventing the lysine-63-linked ubiquitination and activation of TRAF6. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

A non-canonical role of endothelin converting enzyme 1 (ECE1) in promoting lung cancer development via directly targeting protein kinase B (AKT).

BACKGROUND: Lung cancer is the second most common malignancy in the world, and lung adenocarcinoma (LUAD) in particular is the leading cause of cancer death worldwide. Endothelin converting enzyme 1 (ECE1) is a membrane-bound metalloprotease involved in endothelin-1 (ET-1) processing and regulates vasoconstriction. However, very few studies have reported the involvement of ECE1 in regulating tumor cell proliferation, and the mechanism remains poorly understood. Therefore, we aimed to determine the role of ECE1 in lung cancer development. METHODS: The Cancer Genome Atlas database and Kaplan-Meier plotter were used to assess the association between ECE1 and lung cancer. The expression of ECE1 was detected using immunohistochemistry staining and western blotting. A variety of in vitro assays were performed to evaluate the effects of ECE1 on the colony formation, proliferation, migration and invasion using ECE1 knockdown lung cancer cells. The gene expression profiles regulated by ECE1 were investigated by RNA sequencing. An immunoprecipitation assay and immunofluorescence assay were used to evaluate the mechanism underlying the regulatory effect of ECE1 on protein kinase B (AKT). The effect of ECE1 on tumor development was assessed by xenografted lung cancer cells in either C57BL/6 mice or nude mice. RESULTS: ECE1 was upregulated in LUAD and correlated with the poor prognosis of patients with LUAD. Functional studies showed that knockdown of ECE1 retarded the progression of tumors formed by lung cancer cells at least partly by inhibiting tumor cell proliferation. Moreover, ECE1 accelerated tumor cell proliferation through promoting AKT activation dispensable of its canonical target ET-1. Mechanically, ECE1 interacted with the pleckstrin homology (PH) domain of AKT and facilitated its translocation to the plasma membrane for activation. Furthermore, the inhibition of AKT activity counteracted the lung cancer cell growth inhibition observed both in vitro and in xenografts caused by ECE1 suppression. CONCLUSIONS: The present study reveals a non-canonical function of ECE1 in regulating AKT activation and cell proliferation, which provides the basis for the development of a novel strategy for the intervention of cancer including LUAD by abrogating ECE1-AKT signaling.

Alpha-Emitter Radium-223 Induces STING-Dependent Pyroptosis to Trigger Robust Antitumor Immunity.

Radium-223 (223 Ra) is the first-in-class alpha-emitter to mediate tumor eradication, which is commonly thought to kill tumor cells by directly cleaving double-strand DNA. However, the immunogenic characteristics and cell death modalities triggered by 223 Ra remain unclear. Here, it is reported that the 223 Ra irradiation induces the pro-inflammatory damage-associated molecular patterns including calreticulin, HMGB1, and HSP70, hallmarks of tumor immunogenicity. Moreover, therapeutic 223 Ra retards tumor progression by triggering pyroptosis, an immunogenic cell death. Mechanically, 223 Ra-induced DNA damage leads to the activation of stimulator of interferon genes (STING)-mediated DNA sensing pathway, which is critical for NLRP3 inflammasome-dependent pyroptosis and subsequent DCs maturation as well as T cell activation. These findings establish an essential role of STING in mediating alpha-emitter 223 Ra-induced antitumor immunity, which provides the basis for the development of novel cancer therapeutic strategies and combinatory therapy.

cGAS exacerbates Schistosoma japonicum infection in a STING-type I IFN-dependent and independent manner.

Schistosomiasis, which is caused by infection with Schistosoma spp., is characterized by granuloma and fibrosis in response to egg deposition. Pattern recognition receptors are important to sense invading Schistosoma, triggering an innate immune response, and subsequently shaping adaptive immunity. Cyclic GMP-AMP synthase (cGAS) was identified as a major cytosolic DNA sensor, which catalyzes the formation of cyclic GMP-AMP (cGAMP), a critical second messenger for the activation of the adaptor protein stimulator of interferon genes (STING). The engagement of STING by cGAMP leads to the activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and the subsequent type I interferon (IFN) response. cGAS is suggested to regulate infectious diseases, autoimmune diseases, and cancer. However, the function of cGAS in helminth infection is unclear. In this study, we found that Cgas deficiency enhanced the survival of mice infected with S. japonicum markedly, without affecting the egg load in the liver. Consistently, Cgas deletion alleviated liver pathological impairment, reduced egg granuloma formation, and decreased fibrosis severity. In contrast, Sting deletion reduced the formation of egg granulomas markedly, but not liver fibrosis. Notably, Cgas or Sting deficiency reduced the production of IFNß drastically in mice infected with S. japonicum. Intriguingly, intravenous administration of recombinant IFNß exacerbated liver damage and promoted egg granuloma formation, without affecting liver fibrosis. Clodronate liposome-mediated depletion of macrophages indicated that macrophages are the major type of cells contributing to the induction of the type I IFN response during schistosome infection. Moreover, cGAS is important for type I IFN production and phosphorylation of TBK1 and IRF3 in response to stimulation with S. japonicum egg- or adult worm-derived DNA in macrophages. Our results clarified the immunomodulatory effect of cGAS in the regulation of liver granuloma formation during S. japonicum infection, involving sensing schistosome-derived DNA and producing type I IFN. Additionally, we showed that cGAS regulates liver fibrosis in a STING-type I-IFN-independent manner.

CDK inhibitor Palbociclib targets STING to alleviate autoinflammation.

Aberrant activation of stimulator of interferon genes (STING) is tightly associated with multiple types of disease, including cancer, infection, and autoimmune diseases. However, the development of STING modulators for the therapy of STING-related diseases is still an unmet clinical need. We employed a high-throughput screening approach based on the interaction of small-molecule chemical compounds with recombinant STING protein to identify functional STING modulators. Intriguingly, the cyclin-dependent protein kinase (CDK) inhibitor Palbociclib was found to directly bind STING and inhibit its activation in both mouse and human cells. Mechanistically, Palbociclib targets Y167 of STING to block its dimerization, its binding with cyclic dinucleotides, and its trafficking. Importantly, Palbociclib alleviates autoimmune disease features induced by dextran sulphate sodium or genetic ablation of three prime repair exonuclease 1 (Trex1) in mice in a STING-dependent manner. Our work identifies Palbociclib as a novel pharmacological inhibitor of STING that abrogates its homodimerization and provides a basis for the fast repurposing of this Food and Drug Administration-approved drug for the therapy of autoinflammatory diseases.

Comparative effectiveness of sodium-glucose cotransporter-2 inhibitors for new-onset gastric cancer and gastric diseases in patients with type 2 diabetes mellitus: a population-based cohort study.

OBJECTIVE: To compare the risks of gastric cancer and other gastric diseases in patients with type-2 diabetes mellitus (T2DM) exposed to sodium-glucose cotransporter 2 inhibitors (SGLT2I), dipeptidyl peptidase-4 inhibitors (DPP4I) or glucagon-like peptide-1 receptor agonists (GLP1a). DESIGN: This was a population-based cohort study of prospectively collected data on patients with T2DM prescribed SGLT2I, DPP4I or GLP1a between January 1st 2015 and December 31st 2020 from Hong Kong. The outcomes were new-onset gastric cancer, peptic ulcer (PU), acute gastritis, non-acute gastritis, and gastroesophageal reflux disease (GERD). Propensity score matching (1:1) using the nearest neighbour search was performed, and multivariable Cox regression was applied. A three-arm comparison between SGLT2I, DPP4I and GLP1a was conducted using propensity scores with inverse probability of treatment weighting. RESULTS: A total of 62,858 patients (median age: 62.2 years old [SD: 12.8]; 55.93% males; SGLT2I: n = 23,442; DPP4I: n = 39,416) were included. In the matched cohort, the incidence of gastric cancer was lower in SGLT2I (Incidence rate per 1000 person-year, IR: 0.32; 95% confidence interval, CI 0.23-0.43) than in DPP4I (IR per 1000 person-year: 1.22; CI 1.03-1.42) users. Multivariable Cox regression found that SGLT2I use was associated with lower risks of gastric cancer (HR 0.30; 95% CI 0.19-0.48), PU, acute gastritis, non-acute gastritis, and GERD (p < 0.05) compared to DPP4I use. In the three-arm analysis, GLP1a use was associated with higher risks of gastric cancer and GERD compared to SGLT2I use. CONCLUSIONS: The use of SGLT2I was associated with lower risks of new-onset gastric cancer, PU, acute gastritis, non-acute gastritis, and GERD after matching and adjustments compared to DPP4I use. SGLT2I use was associated with lower risks of GERD and gastric cancer compared to GLP1a use.

Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

Evaluating the consistency in different methods for measuring left atrium diameters.

BACKGROUND: The morphological information of the pulmonary vein (PV) and left atrium (LA) is of immense clinical importance for effective atrial fibrillation ablation. The aim of this study is to examine the consistency in different LA diameter measurement techniques. METHODS: Retrospective imaging data from 87 patients diagnosed with PV computed tomography angiography were included. The patients consisted of 50 males and 37 females, with an average age of (60.74 ± 8.70) years. Two physicians independently measured the anteroposterior diameter, long diameter, and transverse diameter of the LA using six different methods. Additionally, we recorded the post-processing time of the images. Physician 1 conducted measurements twice with a one-month interval between the measurements to assess intra-rater reliability. Using the intraclass correlation coefficient (ICC), the consistency of each LA diameter measurement by the two physicians was evaluated. We compared the differences in the LA diameter and the time consumed for measurements using different methods. This was done by employing the rank sum test of a randomized block design (Friedman M test) and the q test for pairwise comparisons among multiple relevant samples. RESULTS: (1) The consistency of the measured LA diameter by the two physicians was strong or very strong. (2) There were statistical differences in the anteroposterior diameter, long diameter, and transverse diameter of LA assessed using different methods (χ2 = 222.28, 32.74, 293.83, P < 0.001). (3) Different methods for measuring the diameters of LA required different amounts of time (χ2 = 333.10, P < 0.001). CONCLUSION: The results of left atrium (LA) diameter measurements conducted by different physicians were found to be reliable. However, the LA diameters obtained through various techniques exhibited variations. It was observed that measuring LA long diameters using only the VR (volume rendering) picture was the most clinically applicable method.

Phantom study of a self-shielded X-ray bone age assessment instrument against scattered radiation in children.

Hand-wrist radiography is the most common and accurate method for evaluating children's bone age. To reduce the scattered radiation of radiosensitive organs in bone age assessment, we designed a small X-ray instrument with radioprotection function by adding metal enclosure for X-ray shielding. We used a phantom operator to compare the scattered radiation doses received by sensitive organs under three different protection scenarios (proposed instrument, radiation personal protective equipment, no protection). The proposed instrument showed greater reduction in the mean dose of a single exposure compared with radiation personal protective equipment especially on the left side which was proximal to the X-ray machine (≥80.0% in eye and thyroid, ≥99.9% in breast and gonad). The proposed instrument provides a new pathway towards more convenient and efficient radioprotection.

A Wide-Band Low-Profile Antenna for a High-Integration Phased Array System.

In this paper, a wide-band, low-profile antenna is presented for a high-integration phased array system. The proposed antenna, implemented using a tightly coupled array, operates over roughly the X-K frequency band and is performant at 8 GHz-18.5 GHz. The antenna can scan to ±60 degrees in both the E- and H-planes. Compared to previous tightly coupled antennas with smaller element spacing, the antenna in this paper reaches 9.4 mm, which corresponds to 0.58 λ of high frequency, suitable for engineering application conditions in production. The antenna can be soldered to BGA T/R chips in this space. Additionally, to facilitate flexible assembly for large arrays, the antenna is manufactured modularly using four elements and its parasitic radiation is analyzed. Then, a method for repressing parasitic radiation is presented. Finally, the antenna is fabricated and measured in a microwave chamber, exhibiting an excellent pattern and scanning radiation. The measured performance agrees with the full-wave finite array simulations.

Protein Tyrosine Phosphatase 1B Inhibitors of Pueraria lobata Based on the Spectrum-Effect Relationship by Q-Marker Selection.

Pueraria lobata (P. lobata), a traditional anti-diabetic medicine mainly composed of flavonoids and isoflavones, has a long history in diabetes treatment in China. However, the anti-diabetic active component is still unclear. Recently, protein tyrosine phosphatase 1B (PTP1B) has been a hot therapeutic target by negatively regulating insulin signaling pathways. In this study, the spectrum-effect relationship analysis method was first used to identify the active components of P. lobata that inhibit PTP1B. The fingerprints of 12 batches of samples were established using high-performance liquid chromatography (HPLC), and sixty common peaks were identified. Meanwhile, twelve components were identified by a comparison with the standards. The inhibition of PTP1B activity was studied in vitro by using the p-nitrophenol method, and the partial least squares discriminant analysis, grey relational analysis, bivariate correlation analysis, and cluster analysis were used to analyze the bioactive compounds in P. lobata. Peaks 6, 9 (glycitin), 11 (genistin), 12 (4'-methoxypuerarin), 25, 34, 35, 36, 53, and 59 were considered as potentially active substances that inhibit PTP1B. The in vitro PTP1B inhibitory activity was confirmed by glycitin, genistin, and 4'-methoxypuerarin. The IC50s of the three compounds were 10.56 ± 0.42 µg/mL, 16.46 ± 0.29 µg/mL, and 9.336 ± 0.56 µg/mL, respectively, indicating the obvious PTP1B inhibitory activity. In brief, we established an effective method to identify PTP1B enzyme inhibitors in P. lobata, which is helpful in clarifying the material basis of P. lobata on diabetes. Additionally, it is evident that the spectrum-effect relationship method serves as an efficient approach for identifying active compounds, and this study can also serve as a reference for screening bioactive constituents in traditional Chinese medicine.

The Medial Canthus Fibrous Band's Impact on Epicanthal Fold Severity and Classification in Asians: Implications for Epicanthoplasty.

BACKGROUND: The epicanthal fold (EF) is a semilunar skin fold located in the medial canthus in most Asians. The medial canthus fibrous band (MCFB) reportedly plays a critical role in EF formation. Variations in MCFB shape and size affect the severity and type of EF. OBJECTIVES: We aimed to analyze MCFB variations in different types and severities of EF and explore the effect of the MCFB resection epicanthoplasty technique (MCFB epicanthoplasty). METHODS: Surgical videos of 40 patients undergoing MCFB epicanthoplasty in our department were reviewed. The MCFB (area), transverse dimension, vertical dimension, upper eyelid direction length (UEDL), and lower eyelid direction length (LEDL) were measured. For aesthetic assessment, 37 patients were followed up for 6 months; intercanthal distance (ICD) and horizontal lid fissure length (HLFL) were measured. Preoperative and postoperative ICD/HLFL ratios were compared. Postoperative scar recovery was evaluated with the Patient and Observer Scar Assessment Scale. Statistical significance was set at P < .05. RESULTS: The MCFB diameter and area were larger for severe EF than for moderate EF (P < .01). Patients with severe EF had larger LEDL than UEDL (P < .01). The tarsalis type had a larger LEDL than the palpebralis type with the same severity (P < .01). MCFB epicanthoplasty yielded favorable postoperative cosmetic effects and scar recovery. Postoperative ICD decreased, while HLFL increased compared to preoperative values (P < .001). The ICD/HLFL ratio was significantly lower postoperatively than preoperatively (P < .001). Postoperative ICD/HLFL ratio was 1.2:1. CONCLUSIONS: The MCFB affects the severity and type of EF. MCFB epicanthoplasty effectively corrected moderate to severe EF.

Invasion and Propagation of White Spot Syndrome Virus: Hijacking of the Cytoskeleton, Intracellular Transport Machinery, and Nuclear Import Transporters.

The pathogenesis of white spot syndrome virus (WSSV) is largely unclear. In this study, we found that actin nucleation and clathrin-mediated endocytosis were recruited for internalization of WSSV into crayfish hematopoietic tissue (Hpt) cells. This internalization was followed by intracellular transport of the invading virions via endocytic vesicles and endosomes. After envelope fusion within endosomes, the penetrated nucleocapsids were transported along microtubules toward the periphery of the nuclear pores. Furthermore, the nuclear transporter CqImportin α1/ß1, via binding of ARM repeat domain within CqImportin α1 to the nuclear localization sequences (NLSs) of viral cargoes and binding of CqImportin ß1 to the nucleoporins CqNup35/62 with the action of CqRan for docking to nuclear pores, was hijacked for both targeting of the incoming nucleocapsids toward the nuclear pores and import of the expressed viral structural proteins containing NLS into the cell nucleus. Intriguingly, dysfunction of CqImportin α1/ß1 resulted in significant accumulation of incoming nucleocapsids on the periphery of the Hpt cell nucleus, leading to substantially decreased introduction of the viral genome into the nucleus and remarkably reduced nuclear import of expressed viral structural proteins with NLS; both of these effects were accompanied by significantly inhibited viral propagation. Accordingly, the survival rate of crayfish post-WSSV challenge was significantly increased after dysfunction of CqImportin α1/ß1, also showing significantly reduced viral propagation, and was induced either by gene silencing or by pharmacological blockade via dietary administration of ivermectin per os. Collectively, our findings improve our understanding of WSSV pathogenesis and support future antiviral designing against WSSV. IMPORTANCE As one of the largest animal DNA viruses, white spot syndrome virus (WSSV) has been causing severe economical loss in aquaculture due to the limited knowledge on WSSV pathogenesis for an antiviral strategy. We demonstrate that the actin cytoskeleton, endocytic vesicles, endosomes, and microtubules are hijacked for WSSV invasion; importantly, the nuclear transporter CqImportin α1/ß1 together with CqRan were recruited, via binding of CqImportin ß1 to the nucleoporins CqNup35/62, for both the nuclear pore targeting of the incoming nucleocapsids and the nuclear import of expressed viral structural proteins containing the nuclear localization sequences (NLSs). This is the first report that NLSs from both viral structure proteins and host factor are elaborately recruited together to facilitate WSSV infection. Our findings provide a novel explanation for WSSV pathogenesis involving systemic hijacking of host factors, which can be used for antiviral targeting against WSSV disease, such as the blockade of CqImportin α1/ß1 with ivermectin.

Sensing of mycobacterial arabinogalactan by galectin-9 exacerbates mycobacterial infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor ß-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

Enhancing Antigen Presentation and Inducing Antigen-Specific Immune Tolerance with Amphiphilic Peptides.

Ag-specific immunotherapy to restore immune tolerance to self-antigens, without global immune suppression, is a long-standing goal in the treatment of autoimmune disorders such as type 1 diabetes (T1D). However, vaccination with autoantigens such as insulin or glutamic acid decarboxylase have largely failed in human T1D trials. Induction and maintenance of peripheral tolerance by vaccination requires efficient autoantigen presentation by APCs. In this study, we show that a lipophilic modification at the N-terminal end of CD4+ epitopes (lipo-peptides) dramatically improves peptide Ag presentation. We designed amphiphilic lipo-peptides to efficiently target APCs in the lymph nodes by binding and trafficking with endogenous albumin. Additionally, we show that lipophilic modification anchors the peptide into the membranes of APCs, enabling a bivalent cell-surface Ag presentation. The s.c. injected lipo-peptide accumulates in the APCs in the lymph node, enhances the potency and duration of peptide Ag presentation by APCs, and induces Ag-specific immune tolerance that controls both T cell- and B cell-mediated immunity. Immunization with an amphiphilic insulin B chain 9-23 peptide, an immunodominant CD4+ T cell epitope in NOD mice, significantly suppresses the activation of T cells, increases inhibitory cytokine production, induces regulatory T cells, and delays the onset and lowers the incidence of T1D. Importantly, treatment with a lipophilic ß-cell peptide mixture delays progression to end-stage diabetes in acutely diabetic NOD mice, whereas the same doses of standard soluble peptides were not effective. Amphiphilic modification effectively enhances Ag presentation for peptide-based immune regulation of autoimmune diseases.