Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Research on temperature detection method of liquor distilling pot feeding operation based on a compressed algorithm.

In the process of feeding the distilling bucket after vapor detection, the existing methods can only realize the lag detection after the steam overflow, and can not accurately detect the location of the steam, etc. At the same time, in order to effectively reduce the occupancy of the computational resources and improve the deployment performance, this study established infrared image dataset of fermented grains surface, and fused the YOLO v5n and the knowledge distillation and the model pruning algorithms, and an lightweight method YOLO v5ns-DP was proposed as as a model for detecting temperature changes in the surface layer of fermented grains during the process of feeding the distilling. The experimental results indicated that the improvement makes YOLOv5n improve its performance in all aspects. The number of parameters, GLOPs and model size of YOLO v5ns-DP have been reduced by 28.6%, 16.5%, and 26.4%, respectively, and the mAP has been improved by 0.6. Therefore, the algorithm is able to predict in advance and accurately detect the location of the liquor vapor, which effectively improves the precision and speed of the detection of the temperature of the surface fermented grains , and well completes the real-time detecting task.

Effects of nitrogen fertilization on the fate of high-risk antibiotic resistance genes in reclaimed water-irrigated soil and plants.

High-risk antibiotic resistance genes (ARGs) in reclaimed water-irrigated soil pose a potential threat to ecosystem and human health. Inorganic fertilization - including with nitrogen, a key ingredient in agricultural production - may affect the ARG profile in soil. However, little is known about nitrogen fertilization's influence on ARGs profiles in the soil-plant system. This study investigated the effects of different nitrogen fertilizer types (CO(NH2)2, NO3--N (NaNO3) and NH4+-N (NH4HCO3)) and different nitrogen fertilizer application rates (low, medium, high) on the distribution of high-risk ARGs in reclaimed water-irrigated soil and plants using quantitative PCR, high-throughput sequencing and metagenomic sequencing. Soil microcosms results revealed that nitrogen fertilization significantly affected the pattern of high-risk ARGs in soil, and also affected high-risk ARGs abundance and transfer capacity in plants. Compared with nitrogen fertilizer application rate, nitrogen fertilizer types significantly contributed to enhancing the soil resistome, with the order of CO(NH2)2 > NO3--N ≈ NH4+-N. The medium application of NO3--N and NH4+-N significantly reduced high-risk ARGs abundance in the leaf endophyte. Bacterial community mainly drove the variation of ARGs in nitrogen-fertilized soil-plant system, and class I integron and metal resistance genes (MRGs) also had direct effects on these high-risk ARGs. A similar high-risk ARGs pattern was also found in field plot experiments, and several dangerous pathogens were observed as the main high-risk ARGs potential hosts in nitrogen-fertilized soil. Based on an economic assessment, application of NH4+-N (NH4HCO3) could reduce costs by $1,312.83 ha-1 compared with NO3--N (NaNO3). These results showed that the more important role of nitrogen type might be an effective and economical way to control high-risk ARGs spread in soil-plant system under reclaimed water irrigation.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.

Advances in performance degradation mechanism and safety assessment of LiFePO4for energy storage.

In the context of 'energy shortage', developing a novel energy-based power system is essential for advancing the current power system towards low-carbon solutions. As the usage duration of lithium-ion batteries for energy storage increases, the nonlinear changes in their aging process pose challenges to accurately assess their performance. This paper focuses on the study LiFeO4(LFP), used for energy storage, and explores their performance degradation mechanisms. Furthermore, it introduces common battery models and data structures and algorithms, which used for predicting the correlation between electrode materials and physical parameters, applying to state of health assessment and thermal warning. This paper also discusses the establishment of digital management system. Compared to conventional battery networks, dynamically reconfigurable battery networks can realize real-time monitoring of lithium-ion batteries, and reduce the probability of fault occurrence to an acceptably low level.

The introduction of influent sulfamethoxazole loads induces changes in the removal pathways of sulfamethoxazole in vertical flow constructed wetlands featuring hematite substrate.

High frequent detection of sulfamethoxazole (SMX) in wastewater cannot be effectively removed by constructed wetlands (CWs) with a traditional river sand substrate. The role of emerging substrate of hematite in promoting SMX removal and the effect of influent SMX loads remain unclear. The removal efficiency of SMX in hematite CWs was significantly higher than that in river sand CWs by 12.7-13.8% by improving substrate adsorption capacity, plant uptake and microbial degradation. With increasing influent SMX load, the removal efficiency of SMX in hematite CWs slightly increased, and the removal pathways varied significantly. The contribution of plant uptake was relatively small (< 0.1%) under different influent SMX loads. Substrate adsorption (37.8%) primarily contributed to SMX removal in hematite CWs treated with low-influent SMX. Higher influent SMX loads decreased the contribution of substrate adsorption, and microbial degradation (67.0%) became the main removal pathway. Metagenomic analyses revealed that the rising influent load increased the abundance of SMX-degrading relative bacteria and the activity of key enzymes. Moreover, the abundance of high-risk ARGs and sulfonamide resistance genes in hematite CWs did not increase with the increasing influent load. This study elucidates the potential improvements in CWs with hematite introduction under different influent SMX loads.

Genistin Represses the Proliferation and Angiogenesis While Accelerating the Apoptosis of Glioma Cells by Modulating the FOXC1-Mediated Wnt Signaling Pathway.

BACKGROUND: Glioma is a tumor originating from glial cells and is the most common primary brain tumor. At present, the main treatment methods for glioma include surgical resection and radiotherapy and chemotherapy, but the treatment effect is not very ideal. Genistin (GS) inhibits breast cancer cell growth while promoting apoptosis, but its effect and detailed molecular mechanism on glioma are yet to be defined. In addition, forkhead box C1 (FOXC1) has been found to be involved in the growth, invasion, and angiogenesis processes of glioma cells. METHODS: Human glioma cells in the Control, GS-6.25, GS-12.5, and GS25 (GS) groups were treated with 0, 6.25, 12.5, and 25 µM of Genistin, respectively, for 72 hours, and cells in the GS + NC (negative control) and GS + FOXC1 groups were transfected with negative control or forkhead box C1 (FOXC1) overexpression plasmids, respectively, prior to Genistin (25 µM) treatment for 72 hours. Next, the viability, proliferation, apoptosis, and angiogenesis of treated glioma cells were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'deoxyuridine (EdU) proliferation, flow cytometry, and tube formation assays. Meanwhile, the half-maximal inhibitory concentration (IC50) of Genistin in the treated glioma cells was calculated. Afterwards, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot quantified the levels of FOXC1, Wnt1, Wnt3a, glycogen synthase kinase-3ß (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß). RESULTS: Genistin inhibited viability, proliferation, and angiogenesis while promoting the apoptosis of glioma cells (p < 0.05, p < 0.001). Also, Genistin decreased the levels of FOXC1, Wnt1, and Wnt3a while increasing p-GSK3ß levels in glioma cells (p < 0.05, p < 0.01, p < 0.001). FOXC1 was up-regulated in glioma cells and tissues, and overexpressed FOXC1 overturned the effects of Genistin on the abovementioned factors in glioma cells (p < 0.05, p < 0.001). CONCLUSIONS: Genistin inhibits viability, proliferation, and angiogenesis while accelerating glioma cell apoptosis by modulating the FOXC1-mediated Wnt signaling pathway.

Structural and Functional Characterization of a Fish Type I Subgroup d IFN Reveals Its Binding to Receptors.

Teleost fish type I IFNs and the associated receptors from the cytokine receptor family B (CRFB) are characterized by remarkable diversity and complexity. How the fish type I IFNs bind to their receptors is still not fully understood. In this study, we demonstrate that CRFB1 and CRFB5 constitute the receptor pair through which type I subgroup d IFN (IFNd) from large yellow croaker, Larimichthys crocea, activates the conserved JAK-STAT signaling pathway as a part of the antiviral response. Our data suggest that L. crocea IFNd (LcIFNd) has a higher binding affinity with L. crocea CRFB5 (LcCRFB5) than with LcCRFB1. Furthermore, we report the crystal structure of LcIFNd at a 1.49-Å resolution and construct structural models of LcIFNd in binary complexes with predicted structures of extracellular regions of LcCRFB1 and LcCRFB5, respectively. Despite striking similarities in overall architectures of LcIFNd and its ortholog human IFN-ω, the receptor binding patterns between LcIFNd and its receptors show that teleost and mammalian type I IFNs may have differentially selected helices that bind to their homologous receptors. Correspondingly, key residues mediating binding of LcIFNd to LcCRFB1 and LcCRFB5 are largely distinct from the receptor-interacting residues in other fish and mammalian type I IFNs. Our findings reveal a ligand/receptor complex binding mechanism of IFNd in teleost fish, thus providing new insights into the function and evolution of type I IFNs.

Effect of acid-modified biochar coupled with alternate wetting and drying on P leaching, soil P retention and plant P uptake in paddy fields.

H2SO4-modified biochar has been recognized as a means to achieve the advantages of carbon sequestration, and nitrogen loss reduction. However, little information is available on its effect on phosphorus (P) uptake, soil available P, and P leaching under alternate wetting and drying irrigation (IAWD). A split-plot experimental layout was carried out with two irrigation regimes (conventional continuous flooding, ICF, and alternate wetting and drying, IAWD) as main plots and three biochar additions (biochar-free control, B0, non-acidified biochar, B20, and acid-modified biochar, B20A) as subplots. Results indicated that IAWD decreased water percolation by 9.26%-14.74% and P leaching by 50.14%-106.64% and increased surface soil available P by 10.88-29.08%, resulting in 14.21-35.03% apparent phosphorus balance (APB) over the three years as compared with ICF. B20 produced a 6.23% lower grain yield in the 1st year and 5.06% and 11.02% higher yields in the 2nd and 3rd years, while B20A increased or tended to increase it throughout the three years. Both B20 and B20A significantly decreased total water percolation (9.68-28.37%), P leaching (18.26-152.00%), and increased soil available P (9.90-46.24%), dissolved P in surface soil (10.00-62.50%), and P uptake (4.31-49.71%), and thereafter enhanced apparent phosphorus balance (11.06-40.78%). Compared with B20, B20A achieved a better APB due to a 113% lower P leaching and 52.9% lower dissolved P at 60 cm soil profiles. IAWDB20A-M produced the highest APB, surface soil available and dissolved P, and the lowest P leaching, which increased grain yield, APB, surface soil available P, and dissolved P by 9.54%, 129.61%, and 53.19%, and decreased P leaching by 257% over ICFB0, respectively. Therefore, the use of H2SO4-modified biochar could produce higher grain yield with lower P leaching and higher APB for IAWD paddy systems, which is beneficial to enhancing plant P uptake, mitigating P leaching, and ensuring sustainable agricultural production.

Effects of five-year field aged zeolite on grain yield and reactive gaseous N losses in alternate wetting and drying paddy system.

Clinoptilolite zeolite has been widely used in agricultural production systems for enhancing water and fertilizer savings, mitigating greenhouse gas emissions, and increasing yield. However, there is little information on field-aged effects of zeolite on reactive gaseous N losses under alternate wetting and drying irrigation (AWD). We conducted a five-year field experiment to investigate field-aged effect of natural zeolite addition at 0 (Z0), 5 (Z5), and 10 (Z10) t ha-1 on reactive gaseous N losses (NH3, N2O), N-related global warming potential (GWPN), soil properties and grain yield under two irrigation regimes (CF: continuous flooding irrigation; AWD) in the 4th (2020) and 5th (2021) years since its initial application in 2017. As compared with CF, AWD did not significantly affect grain yield and NH3 volatilization but increased seasonal N2O emissions by 46 %-71 % over two years. Zeolite increased rice yield for five consecutive years. Z10 reduced averaged cumulative NH3 volatilization and GWPN by 23 % and 26 %, compared to zeolite-free treatment, respectively, in the 4th and 5th years. Soil NH4+-N was increased with the increased rate of Z application under both CF and AWD. Z10 increased soil NH4+-N by 27 %-38 % and NO3--N by 14 %-22 % in five years, compared to Z0, respectively. Compared to AWD without zeolite, the addition of 10 t ha-1 zeolite under AWD lowered NH3 volatilization, cumulative N2O emissions, and GWPN by an average of 28 %, 29 %, and 30 % in two years, respectively. IAWDZ10 did not differ from ICFZ0 on reactive gaseous N losses but significantly lowered reactive gaseous losses relative to IAWDZ0. Therefore, zeolite addition could mitigate the reactive gaseous N losses and GWPN for at least five years after initial application, which is beneficial to promoting zeolite application and ensuring sustainable agriculture.

Effects of acid modified biochar on potassium uptake, leaching and balance in an alternate wetting and drying paddy ecosystem.

Straw biochar amended soils reduce fertilizer losses and alleviate soil K-exhaustion, while decrease grain yield due to its high pH. H2SO4-modified biochar has been studied as a means to enhance the advantages of biochar and address yield decrease. However, little information is available on its effects on aboveground K uptake, soil K fixation, K leaching, and utilization in paddy rice systems, especially under water stress. A 3-year field experiment was conducted with two irrigation regimes (continuously flooded irrigation, ICF and alternate wetting and drying irrigation, IAWD) as main plots and 0 (control), 20 t ha-1 biochar (B20), and 20 t ha-1 acid-modified biochar (B20A-M) as subplots. The results showed that IAWD significantly decreased water percolation by 9.26 %-14.74 % but increased K leaching by 10.84 %-15.66 %. Compared to B0, B20 and B20A-M significantly increased K leaching by 32.40 % and 30.42 % in 2019, while decreased it by 11.60 %-14.01 % in 2020 and 2021. Both B20 and B20A-M significantly improved aboveground K uptake by 3.45 %-6.71 % throughout the three years. B20 reduced grain yield in 2019 and increased it in 2020 and 2021, while B20A-M increased grain yield throughout the three years. Apparent K balance (AKB) from pre-transplanting to post-harvest over the three years suggested that IAWD significantly increased the risk of soil K depletion but B20 and B20A-M significantly increased AKB, thereby addressing the depletion of it. IAWDB20A-M have a comparable AKB with ICFB20A-M, but had up to 18.3 % and 21.61 % higher AKB than IAWDB20 and ICFB20. Therefore, the use of H2SO4 modified biochar could produce higher grain yield with lower K leaching for addition in IAWD paddy systems, which is beneficial to mitigate soil K depletion and ensure a sustainable agricultural production.

[Integrated management during the perinatal period for total anomalous pulmonary venous connection]. / å®Œå ¨æ€§è‚ºé™è„‰å¼‚ä½è¿žæŽ¥çš„å›´ç”ŸæœŸä¸€ä½“åŒ–ç®¡ç†.

OBJECTIVES: To evaluate the clinical effectiveness of integrated management during the perinatal period for fetuses diagnosed with total anomalous pulmonary venous connection (TAPVC) by prenatal echocardiography. METHODS: Clinical data of 64 cases of TAPVC fetuses diagnosed by prenatal echocardiography and managed with integrated perinatal care in Qingdao Women and Children's Hospital from January 2017 to December 2021 were retrospectively analyzed. Integrated perinatal care included multidisciplinary collaboration among obstetrics, fetal medicine, ultrasound, pediatric cardiology, pediatric anesthesia, and neonatology. RESULTS: Among the 64 TAPVC fetuses, there were 29 cases of supracardiac type, 27 cases of intracardiac type, 2 cases of infracardiac type, and 6 cases of mixed type. Chromosomal analysis was performed in 42 cases, and no obvious abnormalities were found. Among the 64 TAPVC fetuses, 37 were induced labor, and 27 were followed up until term birth. Among the 27 TAPVC cases, 2 cases accepted palliative care, 2 cases were referred to another hospital for treatment and lost to follow-up, while the remaining 23 cases underwent primary repair surgery. One case died within 6 months after the operation due to low cardiac output syndrome, while the other 22 cases were followed up for (2.1±0.3) years with good outcomes (2 cases underwent a second surgery within 1 year after the first operation due to anastomotic stenosis or pulmonary vein stenosis). CONCLUSIONS: TAPVC fetuses can achieve good outcomes with integrated management during the perinatal period.

Glucagon-Like Peptide-1 (GLP-1) Rescue Diabetic Cardiac Dysfuntions in Human iPSC-Derived Cardiomyocytes.

Glucagon-like peptide-1 (GLP-1) can improve cardiac function and cardiovascular outcomes in diabetic cardiomyopathy; however, the beneficial effect of GLP-1 on human diabetic cardiomyocytes (DCMs) and its mechanism have not been fully elucidated. Here, the DCMs model by human-induced pluripotent stem cells-derived cardiomyocytes is developed. Two subtypes of GLP-1, GLP-17-36 and GLP-19-36 , are evaluated for their efficacy on the DCMs model. Diabetogenic condition is sufficient to induce most characteristics of diabetic cardiomyopathy in vitro, such as cardiac hypertrophy, lipid accumulation, impaired calcium transients, and abnormal electrophysiological properties. GLP-17-36 and GLP-19-36 can restore cardiomyocyte hypertrophic phenotype, impaired calcium transient frequency, abnormal action potential amplitude, depolarization, and repolarization velocity. Interestingly, RNA-seq reveals different pathways altered by GLP-17-36 and GLP-19-36 , respectively. Differentially expressed gene analysis reveals that possible targets of GLP-17-36 involve the regulation of mitotic nuclear division and extracellular matrix-receptor interaction, while possible targets of GLP-19-36 involve kinetochore assembly, and the complement and coagulation cascades. This study demonstrates the therapeutic effects of GLP-1 on human DCMs and provides a novel platform to unveil the cellular mechanisms of diabetic cardiomyopathy, shedding light on discovering better targets for novel therapeutic interventions.

A Pseudo-Siamese Feature Fusion Generative Adversarial Network for Synthesizing High-Quality Fetal Four-Chamber Views.

Four-chamber (FC) views are the primary ultrasound(US) images that cardiologists diagnose whether the fetus has congenital heart disease (CHD) in prenatal diagnosis and screening. FC views intuitively depict the developmental morphology of the fetal heart. Early diagnosis of fetal CHD has always been the focus and difficulty of prenatal screening. Furthermore, deep learning technology has achieved great success in medical image analysis. Hence, applying deep learning technology in the early screening of fetal CHD helps improve diagnostic accuracy. However, the lack of large-scale and high-quality fetal FC views brings incredible difficulties to deep learning models or cardiologists. Hence, we propose a Pseudo-Siamese Feature Fusion Generative Adversarial Network (PSFFGAN), synthesizing high-quality fetal FC views using FC sketch images. In addition, we propose a novel Triplet Generative Adversarial Loss Function (TGALF), which optimizes PSFFGAN to fully extract the cardiac anatomical structure information provided by FC sketch images to synthesize the corresponding fetal FC views with speckle noises, artifacts, and other ultrasonic characteristics. The experimental results show that the fetal FC views synthesized by our proposed PSFFGAN have the best objective evaluation values: SSIM of 0.4627, MS-SSIM of 0.6224, and FID of 83.92, respectively. More importantly, two professional cardiologists evaluate healthy FC views and CHD FC views synthesized by our PSFFGAN, giving a subjective score that the average qualified rate is 82% and 79%, respectively, which further proves the effectiveness of the PSFFGAN.

FAM3A Ameliorates Brain Impairment Induced by Hypoxia-Ischemia in Neonatal Rat.

Hypoxia-ischemia (HI) during crucial periods of brain formation can lead to changes in brain morphology, propagation of neuronal stimuli, and permanent neurodevelopmental impairment, which can have profound effects on cognitive function later in life. FAM3A, a subgroup of family with sequence similarity 3 (FAM3) gene family, is ubiquitously expressed in almost all cells. Overexpression of FAM3A has been evidenced to reduce hyperglycemia via the PI3K/Akt signaling pathway and protect mitochondrial function in neuronal HT22 cells. This study aims to evaluate the protective role of FAM3A in HI-induced brain impairment. Experimentally, maternal rats underwent uterine artery bilateral ligation to induce neonatal HI on day 14 of gestation. At 6 weeks of age, cognitive development assessments including NSS, wire grip, and water maze were carried out. The animals were then sacrificed to assess cerebral mitochondrial function as well as levels of FAM3A, TNF-α and IFN-γ. Results suggest that HI significantly reduced FAM3A expression in rat brain tissues, and that overexpression of FAM3A through lentiviral transduction effectively improved cognitive and motor functions in HI rats as reflected by improved NSS evaluation, cerebral water content, limb strength, as well as spatial learning and memory. At the molecular level, overexpression of FAM3A was able to promote ATP production, balance mitochondrial membrane potential, and reduce levels of pro-inflammatory cytokines TNF-α and IFN-γ. We conclude that FAM3A overexpression may have a protective effect on neuron morphology, cerebral mitochondrial as well as cognitive function. Created with Biorender.com.

On-site leakage locating of underground natural gas pipeline based on Ne tracer by miniature time-of-flight mass spectrometry.

Natural gas pipeline leakage seriously endangers people's lives and properties, and there is an urgent need for on-site, rapid, and accurate locating the leakage point of the underground natural gas pipeline. Here, we added neon gas to natural gas pipelines as a tracer gas, and used a miniature time-of-flight mass spectrometry (mini-TOFMS) to on-site detect neon gas to quickly locate the leak point of underground natural gas pipelines. The mini-TOFMS used capillary tube sampling to directly analyze the leaked neon gas without sample preparation, and the analysis time of a single sample was only 60 s, which was less than one-seventeenth that of traditional off-line gas chromatography (GC) method. The mini-TOFMS exhibited a linear response range from 69 ppmv to 3.0 × 105 ppmv with the limit of detection (LOD, S/N = 3) of 19.0 ppmv. The correlation of GC and mini-TOFMS for Ne quantitative analysis was as high as 0.98. The performance of the newly designed method with the mini-TOFMS was demonstrated by on-site locating the underground natural gas pipeline leakage point in the experimental station. And leakage point of the natural gas pipeline, especially for those pipelines with different gas pressure buried under the same road, can be found more efficiently and accurately.

Atypical Legionella GTPase effector hijacks host vesicular transport factor p115 to regulate host lipid droplet.

The intracellular bacterial pathogen Legionella pneumophila uses hundreds of effector proteins to manipulate multiple processes of the host cells to establish a replicative niche known as Legionella-containing vacuole (LCV). Biogenesis of the LCV has been known to depend on host small guanosine triphosphatases (GTPases), but whether bacterial effector GTPases are also involved remains unknown. Here, we show that an ankyrin repeat containing effector LegA15 localizes directly in host lipid droplets (LDs), leading to Golgi apparatus fragmentation of the host cells by hijacking the host vesicular transport factor p115. LegA15 is a GTPase with a unique catalytic mechanism, unlike any eukaryotic small GTPases. Moreover, the effector LegA15 co-opts p115 to modulate homeostasis of the host LDs in its GTPase-dependent manner. Together, our data reveal that an atypical GTPase effector regulates the host LDs through impeding the vesicle secretion system of the host cells for intracellular life cycle of Legionella.

Assembly and purification of AvrSr35-induced Sr35 resistosome and determination of its structure by cryo-EM.

Sr35, a coiled-coil nucleotide-binding leucine-rich repeat receptor (CC-NLR) from the wheat species Triticum monococcum can directly recognize the pathogen avirulence factor AvrSr35 and confers immunity against wheat stem rust caused by Puccinia graminis f.sp. tritici race Ug99. Assembly of a stable Sr35 resistosome induced by AvrSr35 in vitro is usually limited by protein expression and low assembly efficiency. Here, we describe the expression and purification of AvrSr35 and Sr35, in vitro assembly of Sr35 resistosome for structure determination by cryo-EM. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2022).

A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein.

The emerging SARS-CoV-2 variants, commonly with many mutations in S1 subunit of spike (S) protein are weakening the efficacy of the current vaccines and antibody therapeutics. This calls for the variant-proof SARS-CoV-2 vaccines targeting the more conserved regions in S protein. Here, we designed a recombinant subunit vaccine, HR121, targeting the conserved HR1 domain in S2 subunit of S protein. HR121 consisting of HR1-linker1-HR2-linker2-HR1, is conformationally and functionally analogous to the HR1 domain present in the fusion intermediate conformation of S2 subunit. Immunization with HR121 in rabbits and rhesus macaques elicited highly potent cross-neutralizing antibodies against SARS-CoV-2 and its variants, particularly Omicron sublineages. Vaccination with HR121 achieved near-full protections against prototype SARS-CoV-2 infection in hACE2 transgenic mice, Syrian golden hamsters and rhesus macaques, and effective protection against Omicron BA.2 infection in Syrian golden hamsters. This study demonstrates that HR121 is a promising candidate of variant-proof SARS-CoV-2 vaccine with a novel conserved target in the S2 subunit for application against current and future SARS-CoV-2 variants.

Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. The direct recognition mechanism of pathogen effectors by coiled-coil NLRs (CNLs) remains unclear. We demonstrate that the Triticum monococcum CNL Sr35 directly recognizes the pathogen effector AvrSr35 from Puccinia graminis f. sp. tritici and report a cryo-electron microscopy structure of Sr35 resistosome and a crystal structure of AvrSr35. We show that AvrSr35 forms homodimers that are disassociated into monomers upon direct recognition by the leucine-rich repeat domain of Sr35, which induces Sr35 resistosome assembly and the subsequent immune response. The first 20 amino-terminal residues of Sr35 are indispensable for immune signaling but not for plasma membrane association. Our findings reveal the direct recognition and activation mechanism of a plant CNL and provide insights into biochemical function of Sr35 resistosome.