Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.

The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.

First report of Fusarium incarnatum causing fruit rot of litchi in China.

Litchi (Litchi chinensis Sonn.) is an indigenous tropical and subtropical fruit in Southern China with an attractive appearance, delicious taste, and good nutritional value (Jiang et al. 2003). In March 2020, brown rots were observed on nearly ripe litchi fruits (cv. Guihuaxiang) in an orchard of Lingshui county, Hainan province of China (18.615877° N, 109.948871° E). About 5% fruits were symptomatic in the field, and the disease caused postharvest losses during storage. The initial infected fruits had no obvious symptoms on the outer pericarp surfaces, but appeared irregular, brown to black-brown lesions in the inner pericarps around the pedicels. Then lesions expanded and became brown rots. Small tissues (4 mm × 4 mm) of fruit pericarps were cut from symptomatic fruits, surface-sterilized in 1% sodium hypochlorite for 3 min, rinsed in sterilized water three times, plated on potato dextrose agar (PDA) and incubated at 28℃ in the darkness. Morphologically similar colonies were isolated from 85% of 20 samples after 4 days of incubation. Ten isolates were purified using a single-spore isolation method. The isolates grown on PDA had abundant, fluffy, whitish to yellowish aerial mycelia, and the reverse side of the Petri dish was pale brown. Morphological characteristics of conidia were further determined on carnation leaf-piece agar (CLA) (Leslie et al. 2006). Macroconidia were straight to slightly curved, 3- to 5-septates with a foot-shaped basal cell, tapered at the apex, 2.70 to 4.43 µm × 18.63 to 37.58 µm (3.56 ± 0.36 × 28.68 ± 4.34 µm) (n = 100). Microconidia were fusoid to ovoid, 0- to 1-septate, 2.10 to 3.57 µm × 8.18 to 18.20 µm (2.88 ± 0.34 × 11.71 ± 1.97 µm) (n = 100). Chlamydospores on hyphae singly or in chains were globose, subglobose, or ellipsoidal. Based on cultural features and morphological characteristics, the fungus was identified as a Fusarium species (Leslie et al. 2006). To further confirm the pathogen, DNA was extracted from the 7-day-old aerial mycelia of three isolates (LZ-1, LZ-3, and LZ-5) following Chohan et al. (2019). The sequences of the internal transcribed spacer region of rDNA (ITS), translation elongation factor-1 alpha (tef1) gene, and histone H3 (his3) gene were partially amplified using primers ITS1/ITS4, EF1-728F/EF1-986R, and CYLH3F/CYLH3R, respectively (Funnell-Harris et al. 2017). The nucleotide sequences were deposited in GenBank (ITS: 515 bp, MW029882, 533 bp, MW092186, and 465 bp, MW092187; tef1: 292 bp, MW034437, 262 bp, MW159143, and 292 bp, MW159141; his3: 489 bp, MW034438, 477 bp, MW159142, and 474 bp, MW159140). The ITS, tef1, and his3 genes showed 99-100% similarity with the ITS (MH979697), tef1 (MH979698), and his3 (MH979696) genes, respectively of Fusarium incarnatum (TG0520) from muskmelon fruit. The phylogenetic analysis of the tef1 and his3 gene sequences showed that the three isolates clustered with F. incarnatum. Pathogenicity tests were conducted by spraying conidial suspension (1×106 conidia/ml) on wounded young fruits in the orchid. Negative controls were sprayed with sterilized water. Fruits were bagged with polythene bags for 24 hours and then unbagged for 10 days. Each treatment had 30 fruits. The inoculated fruits developed symptoms similar to those observed in the orchard and showed light brown lesions on the outer pericarp surfaces and irregular, brown to black-brown lesions in the inner pericarps, while the fruits of negative control remained symptomless. The same fungus was successfully recovered from symptomatic fruits, and thus, the test for the Koch's postulates was completed. F. semitectum (synonym: F. incarnatum) (Saha et al. 2005), F. oxysporum (Bashar et al. 2012), and F. moniliforme (Rashid et al. 2015) have been previously reported as pathogens causing litchi fruit rots in India and Bangladesh. To our knowledge, this is the first report of Fusarium incarnatum causing litchi fruit rot in China.

First report of Cercospora cf. citrulina causing leaf spot of Ipomoea pes-caprae in China.

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2-25.8% of leaves were infected. The initial symptoms were small (1-3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9-115.8 (63.4±22.5) µm × 3.2-5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0-125.5 (60.2±20.1) µm × 2.0-5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.

First report of Colletotrichum siamense causing anthracnose on Erythrina crista-galli in China.

Erythrina crista-galli L. (Fabaceae) is a popular ornamental plant in tropical and subtropical regions of South Asia. In October 2019, anthracnose-like lesions were observed on the leaves of E. crista-galli planted in Haikou, China. 5-30% of leaves were infected. At first, the circular spots of 1-2 mm in diameter were reddish-brown on the leaves, and then enlarged to circular, subcircular or irregular spots with reddish-brown center and surrounded by a diffuse yellow margin. Neighboring spots sometimes coalesced. Under continuously wet or humid conditions, the lesions expanded quickly, and became gray, subcircular or irregular spots covered by grayish-white mycelium and orange-pink conidial masses. Diseased leaves eventually fell off the trees. To identify the pathogen, diseased leaves were sampled from four gardens. Leaf tissues (5×5 mm) were cut from the margins of typical symptomatic lesions, surface-sterilized in 1% sodium hypochlorite for 1 min, plated on potato dextrose agar (PDA) medium, and incubated at 28.0±0.5℃ in the dark. Similar fungal colonies were obtained from all plated tissues after 3 days. The single-conidium colonies of all isolates were white to pale gray and cottony with visible orange conidial masses. Conidia were one-celled, aseptate, hyaline, straight, cylindrical to fusiform with obtuse ends, and ranged from 14.2-18.6 µm (16.4 µm)× 3.8-5.4 µm (4.7 µm) (n=100). After germination, conidia formed single, brown, oval or slightly irregular appressoria ranging from 8.0 to 11.8 µm (9.6 µm), and from 4.8 to 6.0 µm (5.4 µm). Sexual stage was absent. These characteristics of conidia and appressoria were matched with C. siamense belonging to the C. gloeosporioides complex (Prihastuti et al. 2009; Yang et al. 2009; Weir et al. 20012; Hu et al. 2015). To accurately identify the species, DNA was extracted from four purified isolates (JG-1, JG-3-1, SWS-1-3, SWS-2-1) （Fu et al. 2019）. The internal transcribed spacer of rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT) and chitin synthase (CHS) genes were amplified and sequenced. The nucleotide sequences were all deposited in GenBank (ITS: MT229427-MT229430, GAPDH: MT250821-MT250824, CAL: MT258893-MT258896, ACT: MT258897-MT258900 and CHS: MT258901-MT258904). Multi-locus phylogenetic analyses (ITS, GAPDH, CAL, ACT and CHS) (Weir et al. 2012) showed that the four isolates were clustered with C. siamense, which was in accordance with BLAST results. Pathogenicity tests of the four isolates were repeated three times on detached leaves (Ji et al. 2019). The conidial suspension (1×106 conidia/mL) was prepared using the conidia from 10-day-old cultures grown on PDA. Two 20-µL drops of conidial suspension were inoculated on non-wounded young healthy leaves, and each isolate was inoculated on 10 leaves. Two 20-µL drops of sterile water were inoculated on non-wounded young healthy leaves as control. The samples were maintained in containers at a relative humidity of 90± 5 per cent inside and 28℃ with a 12-h photoperiod. Gray, subcircular spots similar to the field disease symptoms were observed on the all inoculated leaves after 7 days, whereas no visible symptoms appeared on the non-inoculated leaves. The pathogen was re-isolated from inoculated leaves thus fulfilling Koch's postulates. C. gloeosporioides has been previously reported as a pathogen causing leaf spot on Erythrina (E. indica var. picta, E. variegata var. orientalis) in Guam in 1983 and Brazil in 2012. (Russo et al. 1983; Oliveira et al. 2012). To our knowledge, this is the first report of C. siamense causing leaf spot of E. crista-galli in China.

An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM.

In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface. Our results demonstrate that the IAAG grid effectively enriches PA-tagged target proteins and overcomes preferred orientation issues. Furthermore, we demonstrate the utility of our IAAG strategy for on-grid purification of low-abundance target complexes from cell lysates, enabling atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification, feasible for high-resolution cryo-EM. This approach holds potential for broader applicability in both cryo-EM and cryo-electron tomography (cryo-ET).

Revealing underlying regulatory mechanisms of LINC00313 in Osimertinib-resistant LUAD cells by ceRNA network analysis.

BACKGROUND: Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), is the preferred treatment for EGFR-mutated lung cancer. However, acquired resistance inevitably develops. While non-coding RNAs have been implicated in lung cancer through various functions, the molecular mechanisms responsible for osimertinib resistance remain incompletely elucidated. METHODS: RNA-sequencing technology was employed to determine differentially expressed lncRNAs (DE-lncRNAs) and mRNAs (DE-mRNAs) between H1975 and H1975OR cell lines. Starbase 2.0 was utilized to predict DE-lncRNA and DE-mRNA interactions, constructing ceRNA networks. Subsequently, functional and pathway enrichment analysis were performed on target DE-mRNAs to identify pathways associated with osimertinib resistance. Key target DE-mRNAs were then selected as potential risk signatures for lung adenocarcinoma (LUAD) prognostic modeling using multivariate Cox regression analyses. The Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry staining were used for result validation. RESULTS: Functional analysis revealed that the identified DE-mRNAs primarily enriched in EGFR-TKI resistance pathways, especially in the PI3K/Akt signaling pathway, where their concerted actions may lead to osimertinib resistance. Specifically, upregulation of LINC00313 enhanced COL1A1 expression by acting as a miR-218-5p sponge, triggering an upstream response that activates the PI3K/Akt pathway, potentially contributing to osimertinib resistance. Furthermore, the expressions of LINC00313 and COL1A1 were validated by qRT-PCR, and the activation of the PI3K/Akt pathway was confirmed by immunohistochemistry staining. CONCLUSIONS: Our results suggest that the LINC00313/miR-218-5p/COL1A1 axis potentially contributes to osimertinib resistance through the PI3K/Akt signaling pathway, providing novel insights into the molecular mechanisms underlying acquired osimertinib resistance in LUAD. Additionally, our study may aid in the identification of potential therapeutic targets for overcoming resistance to osimertinib.

Neoadjuvant chemo-immunotherapy with camrelizumab plus nab-paclitaxel and cisplatin in resectable locally advanced squamous cell carcinoma of the head and neck: a pilot phase II trial.

Neoadjuvant chemoimmunotherapy has emerged as a potential treatment option for resectable head and neck squamous cell carcinoma (HNSCC). In this single-arm phase II trial (NCT04826679), patients with resectable locally advanced HNSCC (T2‒T4, N0‒N3b, M0) received neoadjuvant chemoimmunotherapy with camrelizumab (200 mg), nab-paclitaxel (260 mg/m2), and cisplatin (60 mg/m2) intravenously on day one of each three-week cycle for three cycles. The primary endpoint was the objective response rate (ORR). Secondary endpoints included pathologic complete response (pCR), major pathologic response (MPR), two-year progression-free survival rate, two-year overall survival rate, and toxicities. Here, we report the perioperative outcomes; survival outcomes were not mature at the time of data analysis. Between April 19, 2021 and March 17, 2022, 48 patients were enrolled and received neoadjuvant therapy, 27 of whom proceeded to surgical resection and remaining 21 received non-surgical therapy. The ORR was 89.6% (95% CI: 80.9, 98.2) among 48 patients who completed neoadjuvant therapy. Of the 27 patients who underwent surgery, 17 (63.0%, 95% CI: 44.7, 81.2) achieved a MPR or pCR, with a pCR rate of 55.6% (95% CI: 36.8, 74.3). Treatment-related adverse events of grade 3 or 4 occurred in two patients. This study meets the primary endpoint showing potential efficacy of neoadjuvant camrelizumab plus nab-paclitaxel and cisplatin, with an acceptable safety profile, in patients with resectable locally advanced HNSCC.

Does increasing public expenditure on sports promote regional sustainable development: Evidence from China.

In the post-COVID era, how to improve the level of regional sustainable development has attracted much attention. And the vigorous development of the sports economy may be closely related to the regional sustainable development. This paper explores the impact and mechanism analysis of government sports public expenditure on regional sustainable development from the perspective of sports economic development. The study found that China's sustainable development presents obvious ladder-like characteristics and highlights the regional imbalance and inadequacy of regional green and coordinated development. And the government's increase in public expenditure on sports can significantly promote regional sustainable development and improve the level of regional green and coordinated development. With the continuous improvement of the regional economic development, the effect of sports public expenditure continues to increase. It can be seen from this that implementing the strategy of strengthening the country through sports under the government's guidance is an essential guarantee for the public health and quality of life and the sustainable development of the economy and society. Additionally, the development level of market finance is also an important driving factor for the government's public expenditure on sports to improve the level of sustainable development in the region. From the mechanism analysis, the government activates the local residents' consumption level by increasing the public expenditure on sports, thus promoting regional sustainable development.

NSCLC patients with rare EGFR Ex19del/G724S mutation showed good response to afatinib combined with chemotherapy treatment: A two-case report.

EGFR G724S mutation in exon 18 has been shown to be resistant to both first- and third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). However, we found a rare mutation of EGFR Ex19del/G724S in two patients with lung cancer who demonstrated a favorable response to the combination of afatinib and chemotherapy. Identified by next-generation sequencing (NGS), EGFR G724S was found from a primary and a secondary tumor biopsy, respectively. Treated with afatinib combined with chemotherapy, both patients responded well and achieved progression-free survival. Analysis of acquired mutations developed during treatment using afatinib revealed that the emergence of EGFR T790M or ALK fusion was the potential mechanism of afatinib resistance. Our study lends credence to treatment using afatinib combined with chemotherapy as a viable option for patients with Ex19del/G724S.

Enhanced antibacterial activity of acid treated MgO nanoparticles on Escherichia coli.

Acid treatment is one of the effective methods that directly modifies surface physical and chemical properties of inorganic materials, which improves the materials' application potential. In this work, the surface modified MgO nanoparticles (NPs) were prepared through a facile acid-treatment method at room temperature. Compared with the untreated sample, the surviving Escherichia coli (E. coli, ATCC 25922) colonies of the modified MgO NPs decreased from 120 to 54 (102 CFU mL-1). The enhanced antibacterial activity may be due to the improvement of oxygen vacancies and absorbed oxygen (OA) content (from 41.6% to 63.1%) as confirmed by electron spin resonance (ESR) and X-ray photoelectron spectroscopy (XPS). These findings revealed that the acid treatment method could directly modify the surface of MgO NPs to expose more oxygen vacancies, which would promote reactive oxygen species (ROS) generation. The membrane tube and single ROS scavenging results further indicated that the increased antibacterial ability originated from the synergetic effect of ROS damage (especially ˙O2 -) and direct contact between H-MgO NPs and E. coli.

Partial Response to Pyrotinib Plus Capecitabine in an Advanced Breast Cancer Patient with HER2 Amplification and R157W Mutation After Anti-HER2 Treatment: A Case Report and Literature Review.

Human epidermal growth factor receptor2 (HER2) overexpression/amplification is associated with high malignancy, rapid disease progression and poor overall survival in breast cancer. The application of anti-HER2 drugs has greatly improved the survival of patients with HER2-positive breast cancer, but drug resistance issues affect the long-term efficacy. The HER2 mutation is considered to be one of the reasons for resistance to anti-HER2 therapy, and there is currently no standard treatment. We report for the first time the detection of HER2 amplification with R157W mutation by second-generation sequencing (NGS) in a 57-year-old hormone receptor-negative, HER2-positive woman with advanced breast cancer who was resistant to multi-line anti-HER2 therapies. She subsequently received pyrotinib combined with capecitabine treatment and achieved partial response. The small-molecule pan-HER family irreversible inhibitor pyrotinib combined with capecitabine has shown a promising effect in the treatment of HER2 mutation-induced resistance, but the molecular mechanism and efficacy need to be further verified.

Engineering a sustained release vaccine with a pathogen-mimicking manner for robust and durable immune responses.

Sustained release vaccine carriers can facilitate an increased interaction time between the antigen and immune system to strengthen immune responses, but their promotion on adaptive immune responses, especially cellular immunity, are still unfavorable. Herein, we report a sustained antigen delivery vector, which carries abundant antigens, a nucleic acid adjuvant and pathogen-associated molecular patterns to simulate a natural pathogen to reinforce immune responses. Specifically, murine colorectal cancer cells MC38 lysate and Toll-like receptor 9 agonist CpG are loaded into yeast derived ß-glucan particles (GPs). After vaccination, these particles can form a vaccine depot that continuously release the antigen similar to the traditional aluminum hydroxide gel, but recruit more immune cells and induce more cytokine secretion at the injection site. Stronger antibody responses, Th1 and Th17 biased cellular immunity and immune memory are achieved compared with aluminum hydroxide gel. More importantly, treatment with these particles significantly suppress tumor growth in a therapeutic tumor model. This work shed light on the efficacy of combining sustained antigen release with pathogen-mimicking manner in vaccine design.

Enhanced anti-Escherichia coli properties of Fe-doping in MgO nanoparticles.

Hetero-elements doping is an effective way to modify the composition and nanostructure of metal oxides. These modifications could lead to changes in physical and chemical properties correspondingly. In this study, Fe-doped MgO nanoparticles (NPs) were synthesized by simple calcination method in air. The antibacterial activity of MgO NPs against Escherichia coli (E. coli, ATCC 25922) was significantly improved as shown by the bactericidal efficacy test results. According to X-ray diffraction (XRD) results, Fe was successfully doped into MgO lattice and mainly adopted interstitial doping. The Fe-doping led to increased oxygen vacancies and OA content (from 13.5% to 41.3%) on MgO surface, which may have facilitated the reactive oxygen species (ROS) generation and bacteria death. The wrinkled and sunken E. coli surface after contact with Fe-doped MgO NPs also confirmed the existence of adsorption damage mechanism. Thus, the antibacterial activity enhancement against E. coli was originated from the synergistic effect of increased ROS concentration and the interaction with Fe-doped MgO NPs.

Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM.

The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glycoprotein interacts with the human ACE2 receptor to mediate viral entry into host cells. We report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7- and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Our findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti-SARS-CoV-2 vaccines and therapeutics.

Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen-antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections.

The pore size of mesoporous silica nanoparticles regulates their antigen delivery efficiency.

Subunit vaccines generally proceed through a 4-step in vivo cascade-the DUMP cascade-to generate potent cell-mediated immune responses: (1) drainage to lymph nodes; (2) uptake by dendritic cells (DCs); (3) maturation of DCs; and (4) Presentation of peptide-MHC I complexes to CD8+ T cells. How the physical properties of vaccine carriers such as mesoporous silica nanoparticles (MSNs) influence this cascade is unclear. We fabricated 80-nm MSNs with different pore sizes (7.8 nm, 10.3 nm, and 12.9 nm) and loaded them with ovalbumin antigen. Results demonstrated these MSNs with different pore sizes were equally effective in the first three steps of the DUMP cascade, but those with larger pores showed higher cross-presentation efficiency (step 4). Consistently, large-pore MSNs loaded with B16F10 tumor antigens yielded the strongest antitumor effects. These results demonstrate the promise of our lymph node-targeting large-pore MSNs as vaccine-delivery vehicles for immune activation and cancer vaccination.

Undetectable circulating tumor DNA levels correlate with low risk of recurrence/metastasis in postoperative pathologic stage I lung adenocarcinoma patients.

OBJECTIVES: The application of circulating tumor DNA (ctDNA) monitoring after resection in pathologic(p) stage I lung adenocarcinoma (LUAD) patients remains controversial and it is of great clinical interest to decipher the difference of genetic features between ground-glass opacity (GGO) and solid nodules (non-GGO) subgroups. We aim to assess the utility of ctDNA in tracking early recurrence or metastasis following surgery and reveal the genetic differences between GGO and non-GGO. MATERIALS AND METHODS: Tumor tissues and matched postoperative plasma samples were collected from a total of 82 (p)stage I LUAD patients. Comprehensive genomic profiling was performed using capture-based hybrid next generation sequencing by targeting 422 cancer relevant genes. RESULTS: EGFR and TP53 represent commonly mutated genes in this cohort of (p)stage I lung adenocarcinoma, followed by alterations in ALK, PIK3CA, STK11 and MYC. For a median follow-up period of 22.83 months after surgery, 65 out of 67 ctDNA-negative patients remained progression-free, while 3 out of 15 ctDNA-positive patients progressed [P = 0.040; positive predictive value = 0.20, 95 % confidence interval (CI), 0.04-0.48; negative predictive value = 0.97, 95 % CI, 0.9-1]. With time-dependent Cox regression analysis, we observed that ctDNA positivity significantly correlated with increased probability of early tumor recurrence or metastasis (P = 0.02, HR=8.5). Further comparison between GGO and non-GGO subgroups indicated the frequency of TP53 mutations in non-GGO was markedly higher than that in GGO (47 % vs 21 %, P < 0.05). Pathway analysis showed the epigenetic regulation pathway was more frequently affected in GGO subgroup, while impaired apoptosis/cell cycle pathway was more enriched in non-GGO LUADs. CONCLUSIONS: Our longitudinal ctDNA monitoring data showed that undetectable ctDNA may predict low risk of tumor recurrence or metastasis in postoperative (p)stage I LUAD patients, while it requires further investigation on how robust the positive ctDNA results could predict tumor relapse in these patients. CLINICAL REGISTRATION NUMBER: NCT03172156.

An aluminum adjuvant-integrated nano-MOF as antigen delivery system to induce strong humoral and cellular immune responses.

Metal-organic frameworks (MOFs) have high surface area, tunable pore size, and high loading capacity, making them promising for drug delivery. However, their synthesis requires organic solvents, high temperature and high pressure that are incompatible with biomacromolecules. Zeolitic imidazole frameworks (ZIF-8) which forms through coordination between zinc ions and 2-methylimidazole (MeIM) have emerged as an advanced functional material for drug delivery due to its unique features such as high loading and pH-sensitive degradation. In this study, we took advantage of a natural biomineralization process to create aluminum-containing nanoZIF-8 particles for antigen delivery. Without organic solvents or stabilizing agent, nanoparticles (ZANPs) were synthesized by a mild and facile method with aluminum, model antigen ovalbumin (OVA) and ZIF-8 integrated. A high antigen loading capacity (%) of 30.6% and a pH dependent antigen release were achieved. A Toll-like receptor 9 agonist cytosine-phosphate-guanine oligodeoxynucleotides (CpG) was adsorbed on the surface of ZANPs (hereafter CpG/ZANPs) to boost the immune response. After subcutaneous injection in vivo, CpG/ZANPs targeted lymph nodes (LNs), where their cargo was efficiently internalized by LN-resident antigen-presenting cells (APCs). ZANPs decomposition in lysosomes released antigen into the cytoplasm and enhanced cross-presentation. Moreover, CpG/ZANPs induced strong antigen-specific humoral and cytotoxic T lymphocyte responses that significantly inhibited the growth of EG7-OVA tumors while showing minimal cytotoxicity. We demonstrate that ZANPs may be a safe and effective vehicle for the development of cancer vaccines.

Urine-Based Biomarkers for Alzheimer's Disease Identified Through Coupling Computational and Experimental Methods.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder contributing to nearly 70% of dementia cases. However, no diagnostic protein biomarkers are available in urine. In this study, we combined computational and experimental methods to identify urinary biomarkers for AD. First, by analyzing brain tissue-based gene expression data of AD, 2,754 differentially expressed genes were identified, 559 of which were predicted to encode urine-excretory proteins that might act as candidate protein biomarkers of AD. GO enrichment analyses implied that they were mainly involved in microtubule-based process, myelin sheath, and calcium ion binding, suggesting that they might be associated with AD pathogenesis. In order to verify these proteins in urine, an iTRAQ experiment was carried out to analyze urine samples from AD patients and healthy controls, and 15 proteins were detected. Based on the expression changes of these proteins, 4 proteins were chosen for further validation by ELISA experiment, and SPP1, GSN, and IGFBP7 were found to be differentially expressed in the urine of AD patients. After a literature survey, we found that they were involved in AD pathophysiology and might serve as new urine biomarkers for AD. To our knowledge, this is the first time that urine biomarkers for AD were identified by combining computational and experimental methods. Furthermore, this is the first time SPP1, GSN, and IGFBP7 have been reported as potential urine protein biomarkers for AD. Therefore, our findings might provide significant guidance for finding early biomarkers of AD in urine.

Architecture and subunit arrangement of the complete Saccharomyces cerevisiae COMPASS complex.

Methylation of histone H3 lysine 4 (H3K4) is catalyzed by the multi-component COMPASS or COMPASS-like complex, which is highly conserved from yeast to human, and plays essential roles in gene expression and transcription, cell cycle progression, and DNA repair. Here we present a cryo-EM map of the complete S. cerevisiae COMPASS complex. Through tag or Fab labeling strategy combined with cryo-EM 3D reconstruction and cross-linking and mass spectrometry (XL-MS) analysis, we uncovered new information on the subunit arrangement: Cps50, Cps35, and Cps30 were determined to group together to form the face region in the head of the complex, and Cps40 and the N-terminal portion of Set1 reside on the top of the head. Our map reveals the location of the active center and a canyon in the back of the head. Together, our study provides the first snapshot of the complete architecture of yeast COMPASS and a picture of its subunit interaction network, which could facilitate our understanding of the COMPASS machinery and its functionality.