Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer-stabilized in the prefusion conformation and fused with SpyCatcher-could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.

Although circular RNAs (circRNAs) have recently garnered interest as disease markers, they have been relatively poorly studied as a biomarker in colorectal cancer (CRC). In this study, we aimed to screen the exosome-derived circRNAs in CRC and explore their potential as diagnostic and prognostic biomarkers of CRC METHODS: Exosomes were extracted from the plasma using a kit and validated by immunoblotting, transmission electron microscopy, and particle size analysis. The microarray datasets were employed to identify differentially-expressed circRNAs from plasma exosomes. Real-time quantitative reverse transcription PCR (RT-qPCR) verified the results of the microarray analysis, and Receiver operating characteristic (ROC) curve revealed the diagnostic ability of a single circRNA. The Starbase combined with microT, miRmap, and RNA22 were used to establish a circRNA-miRNA-mRNA network. Gene ontology, Kyoto Encyclopedia of Genes, Genomes pathway enrichment analysis, and Gene Set Enrichment Analysis were applied to determine potential functions of the identified mRNAs RESULTS: Comparing the microarray of plasma exosome-derived circRNAs and the microarray downloaded from the GEO database, 15 candidate circRNAs with up-regulated expression were identified. RT-qPCR verified that hsa_circ_0003270 (circGAPVD1) was upregulated in CRC plasma exosomes. ROC analysis showed that circGAPVD1 in plasma exosomes has potential diagnostic value for CRC. The sensitivity and specificity of circGAPVD1 in the diagnosis of CRC were found to be 75.64 and 71.79%, respectively (area under ROC = 0.7662). Furthermore, the lymph node metastasis and TNM staging of patients were positively correlated with high expression of circGAPVD1. Combined with the ENCORI database and GEO datasets, we identified the circGAPVD1-related ceRNA network. The enrichment analysis revealed that key nodes in the ceRNA network participate in many important signaling pathways such as protein post-translational modifications CONCLUSION: Our results revealed the diagnostic efficiency of circGAPVD1 in plasma exosomes. The highly expressed circGAPVD1 is expected to be a novel diagnostic marker for CRC.

Immunization with SARS-CoV-2 spike elicits diverse antibodies, but can any of these neutralize broadly? Here, we report the isolation and characterization of antibody WS6, from a mouse immunized with mRNA encoding the SARS-CoV-2 spike. WS6 bound diverse beta-coronavirus spikes and neutralized SARS-CoV-2 variants, SARS-CoV, and related sarbecoviruses. Epitope mapping revealed WS6 to target a region in the S2 subunit, which was conserved among SARS-CoV-2, MERS-CoV, and hCoV-OC43. The crystal structure at 2-Å resolution of WS6 with its S2 epitope revealed recognition to center on a conserved helix, which was occluded in both prefusion and post-fusion spike conformations. Structural and neutralization analyses indicated WS6 to neutralize by inhibiting fusion, post-viral attachment. Comparison of WS6 to other antibodies recently identified from convalescent donors or mice immunized with diverse spikes indicated a stem-helical supersite - centered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156 - to be a promising target for vaccine design.

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike elicits diverse antibodies, but it is unclear if any of the antibodies can neutralize broadly against other beta-coronaviruses. Here, we report antibody WS6 from a mouse immunized with mRNA encoding the SARS-CoV-2 spike. WS6 bound diverse beta-coronavirus spikes and neutralized SARS-CoV-2 variants, SARS-CoV, and related sarbecoviruses. Epitope mapping revealed WS6 to target a region in the S2 subunit, which was conserved among SARS-CoV-2, Middle East respiratory syndrome (MERS)-CoV, and hCoV-OC43. The crystal structure at 2 Å resolution of WS6 revealed recognition to center on a conserved S2 helix, which was occluded in both pre- and post-fusion spike conformations. Structural and neutralization analyses indicated WS6 to neutralize by inhibiting fusion and post-viral attachment. Comparison of WS6 with other recently identified antibodies that broadly neutralize beta-coronaviruses indicated a stem-helical supersite-centered on hydrophobic residues Phe1148, Leu1152, Tyr1155, and Phe1156-to be a promising target for vaccine design.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 μg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of β-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

Multidonor antibodies are of interest for vaccine design because they can in principle be elicited in the general population by a common set of immunogens. For influenza, multidonor antibodies have been observed against the hemagglutinin (HA) stem, but not the immunodominant HA head. Here, we identify and characterize a multidonor antibody class (LPAF-a class) targeting the HA head. This class exhibits potent viral entry inhibition against H1N1 A/California/04/2009 (CA09) virus. LPAF-a class antibodies derive from the HV2-70 gene and contain a "Tyr-Gly-Asp"-motif, which occludes the HA-sialic acid binding site as revealed by a co-crystal structure with HA. Both germline-reverted and mature LPAF antibodies potently neutralize CA09 virus and have nanomolar affinities for CA09 HA. Moreover, increased frequencies for LPFA-a class antibodies are observed in humans after a single vaccination. Overall, this work highlights the identification of a multidonor class of head-directed influenza-neutralizing antibodies and delineates the mechanism of their recurrent elicitation in humans.

The continuous development of multidrug-resistant (MDR) Gram-negative bacteria poses a serious risk to public health on a worldwide scale. Colistin is used as the last-line antibiotic for the treatment of MDR pathogens, and colistin-resistant (COL-R) bacterial emergence thus has the potential to have a severe adverse impact on patient outcomes. In this study, synergistic activity was observed when colistin and flufenamic acid (FFA) were combined and used for the in vitro treatment of clinical COL-R Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii strains, as shown by checkerboard and time-kill assays. Crystal violet staining and scanning electron microscopy revealed the synergistic action of colistin-FFA against biofilms. When used to treat murine RAW264.7 macrophages, this combination did not induce any adverse toxicity. Strikingly, the survival rates of bacterially infected Galleria mellonella larvae were improved by such combination treatment, which was also sufficient to reduce the measured bacterial loads in a murine thigh infection model. Mechanistic propidium iodide (PI) staining analysis further demonstrated the ability of these agents to alter bacterial permeability in a manner that enhanced the efficacy of colistin treatment. Together, these data thus demonstrate that colistin and FFA can be synergistically combined to combat the spread of COL-R Gram-negative bacteria, providing a promising therapeutic tool with the potential to protect against COL-R bacterial infections and improve patient outcomes. IMPORTANCE Colistin is a last-line antibiotic used for the treatment of MDR Gram-negative bacterial infections. However, increasing resistance to it has been observed during clinical treatment. In this work, we assessed the efficacy of the combination of colistin and FFA for the treatment of COL-R bacterial isolates, demonstrating that the combined treatment has effective antibacterial and antibiofilm activities. Due to its low cytotoxicity and good therapeutic effects in vitro, the colistin-FFA combination may be a potential candidate for research into a resistance-modifying agent to combat infections caused by COL-R Gram-negative bacteria.

Background: cerebral ischemia/reperfusion (I/R) injury is an important complication of ischemic stroke, and autophagy is one of the mechanisms of it. In this study, we aimed to determine the role and mechanism of autophagy in cerebral I/R injury. Methods: the oxygen and glucose deprivation/reoxygenation (OGD/R) method was used to model cerebral I/R injury in HT22 cells. CCK-8 and LDH were conducted to detect viability and damage of the cells, respectively. Apoptosis was measured by flow cytometry and Tunel staining. Autophagic vesicles of HT22 cells were assessed by transmission electron microscopy. Western blotting analysis was used to examine the protein expression involving AMPK/DDiT4/mTOR axis and autophagy-related proteins. 3-Methyladenine and rapamycin were, respectively, used to inhibit and activate autophagy, compound C and AICAR acted as AMPK inhibitor and activator, respectively, and were used to control the starting link of AMPK/DDiT4/mTOR axis. Results: autophagy was activated in HT22 cells after OGD/R was characterized by an increased number of autophagic vesicles, the expression of Beclin1 and LC3II/LC3I, and a decrease in the expression of P62. Rapamycin could increase the viability, reduce LDH leakage rate, and alleviate cell apoptosis in OGD/R cells by activating autophagy. 3-Methyladenine played an opposite role to rapamycin in OGD/R cells. The expression of DDiT4 and the ratio of p-AMPK/AMPK were increased after OGD/R in HT22 cells. While the ratio of p-mTOR/mTOR was reduced by OGD/R, AICAR effectively increased the number of autophagic vesicles, improved viability, reduced LDH leakage rate, and alleviated apoptosis in HT22 cells which suffered OGD/R. However, the effects of compound C in OGD/R HT22 cells were opposite to that of AICAR. Conclusions: autophagy is activated after OGD/R; autophagy activator rapamycin significantly enhanced the protective effect of autophagy on cells of OGD/R. AMPK/DDiT4/mTOR axis is an important pathway to activate autophagy, and AMPK/DDiT4/mTOR-mediated autophagy significantly alleviates cell damage caused by OGD/R.

The aim of present study was to evaluate the potential effects of Rhodiola crenulata oral liquid (RCOL) on exhaustive exercise (EE)-induced fatigue in mice. Male Institute of Cancer Research mice from five treatment groups (n=10 per group) were orally administered with sterilized water for the Control and EE groups and/or RCOL at doses of 1.02, 3.03 and 6.06 ml/kg/day, once daily for 2 weeks. Anti-fatigue activity was subsequently evaluated by measuring the levels of creatine kinase (CK), lactic acid (LA), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidative capability (T-AOC). Histopathology was assessed using hematoxylin and eosin staining. Ultrastructures of mitochondria were observed by transmission electron microscopy. Energy supply capacity was assessed using citrate synthase (CS), succinate dehydrogenase (SDH), Na+-K+-ATPase, and liver and quadriceps glycogen content assays. Expression levels of mRNA and protein associated with mitophagy in the skeletal muscle were measured by reverse transcription-quantitative PCR and western blotting, respectively. RCOL was observed to markedly inhibit fatigue-induced oxidative stress by increasing the activities of SOD, CAT and T-AOC, whilst reducing the accumulation of LA, CK, LDH and MDA. Histological analysis of the quadriceps femoris tissue suggested increased numbers of muscle fibers in the RCOL groups compared with those in the EE group. RCOL administration was found to reverse EE-induced mitochondrial structural damage and alleviated defects inflicted onto the energy supply mechanism by increasing CS, SDH, Na+-K+-ATPase and glycogen levels. Additionally, RCOL reduced the protein expression of PTEN-induced kinase 1 (PINK1), Parkin, microtubule-associated proteins 1A/1B light chain 3, sequestosome 1 and ubiquitin, whilst lowering the gene expression of PINK1 and Parkin. Taken together, results from the present study clarified the anti-fatigue effect of RCOL, where the underlying mechanism may be associated with increased antioxidant activity, enhanced energy production and the inhibition of mitophagy by suppressing the PINK1/Parkin signaling pathway.

Multidrug-resistant bacteria pose a tremendous challenge to public health worldwide. Many bacteria resistant to last-resort antibiotics due to antibiotic misuse have been recently reported, which may give rise to serious infections without effective treatment. Therefore, it is imperative to develop novel antimicrobial strategies. Natural phenols are known to increase bacterial membrane permeability and are potential candidates for the development of new antimicrobial agents. In this study, gold nanoparticles (Au NPs) carrying natural phenols were synthesized to combat bacteria resistant to last-resort antibiotics. Transmission electron microscopy, dynamic light scattering, zeta potential, and UV-visible spectra were used to characterize the synthesized Au NPs, which showed good monodispersity and uniform particle size. Evaluation of antibacterial activity using the broth microdilution method revealed that thymol-decorated gold nanoparticles (Thymol_Au NPs) had a broad antibacterial spectrum and higher bactericidal effects than last-resort antibiotics against last-resort-antibiotic-resistant bacteria. Considering the underlying antibacterial mechanism, the results showed that Thymol_Au NPs destroyed bacterial cell membranes. Further, Thymol_Au NPs were effective in treating mouse abdominal infections and exhibited acceptable biocompatibility without any significant toxicity in cell viability and histopathological assays, respectively, at most bactericidal concentrations. However, attention should be paid to changes in white blood cells, reticulocyte percentages, and superoxide dismutase activity during Thymol_Au NP treatment. In conclusion, Thymol_Au NPs have the potential for treating clinical infections caused by bacteria resistant to last-resort antibiotics. IMPORTANCE Excessive use of antibiotics can lead to bacterial resistance and the development of multidrug-resistant bacteria. Antibiotic misuse can also promote resistance against last-resort antibiotics. It is thus crucial to develop alternatives to antibiotics to retard the development of multidrug resistance. In recent years, the use of several nanodosage forms of antibacterial drugs has been investigated. These agents kill bacteria through a variety of mechanisms and avoid the problem of resistance. Among them, Au NPs, which are safer to use for medical applications than other metal nanoparticles, have attracted interest as potential antibacterial agents. To combat bacterial resistance to last-resort antibiotics and mitigate the problem of antimicrobial resistance, it is important and meaningful to develop antimicrobial agents based on Au NPs.

Rationale: Emerging evidence demonstrates that insufficient migration and invasion of trophoblasts play critical roles in the pathogenesis of recurrent spontaneous abortion (RSA). Cell-to-cell communication at the maternal-fetal interface is essential to maintain the invasion and migration of trophoblasts. M1 macrophages, important immune cellular components at the maternal-fetal interface, have been reported to be elevated in decidua tissues from patients with RSA. Recent studies indicate that M1 macrophages modulate trophoblast biological behaviors; however, the underlying mechanisms remain poorly understood. Methods: Extracellular vesicles (EVs) were isolated from the supernatant of M1 macrophages inducted from THP-1 cells (M1-EVs) by ultracentrifugation, identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting, and their miRNA profile was characterized by miRNA sequencing. Scratch wound healing and transwell assays were used to investigate the effect of M1-EVs on trophoblast migration and invasion. RT-PCR, western blotting, and luciferase reporter assays were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of M1-EVs on embryo absorption in mice. Results: M1 macrophages suppressed trophoblast EMT to reduce their migration and invasion abilities in vitro by secreting EVs. Through miRNA sequencing, miR-146a-5p and miR-146b-5p were identified as the most upregulated miRNAs in trophoblasts treated with M1-EVs. Further functional experiments showed that M1-EVs inhibited trophoblast migration and invasion by transferring miR-146a-5p and miR-146b-5p. Mechanistically, EV miR-146a-5p and miR-146b-5p inhibited EMT of trophoblasts by directly suppressing TNF receptor-associated factor 6 (TRAF6) expression at the post-transcriptional level. Furthermore, M1-EVs aggravated embryo absorption in mice. Clinically, expression of miR-146a-5p, miR-146b-5p, and TRAF6 were aberrant in placental villous tissues from patients with RSA, and negative correlations were found between miR-146a-5p/miR-146b-5p and TRAF6 expression levels. Conclusions: Our findings indicate that miR-146a-5p and miR-146b-5p derived from EVs play important roles in intercellular communication between M1 macrophages and trophoblasts, illuminating a novel mechanism in M1 macrophage regulation of trophoblasts and their role in RSA.

Long non-coding RNAs (lncRNAs) in extracellular vesicles (EVs) are considered to be novel non-invasive biomarkers for gastric cancer (GC). lncRNA colon cancer-associated transcript 1 (CCAT1) is aberrantly expressed in certain types of cancer. However, the role of EV lncRNA CCAT1 in patients with GC remains unclear. The current study aimed to assess the expression levels of lncRNA CCAT1 in the serum EVs of patients with GC and evaluate its potential clinical value. EVs were isolated from serum using a commercial kit and ultracentrifugation, and were identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting. Serum EV lncRNA CCAT1 levels in patients with GC, chronic gastritis or atypical hyperplasia and healthy control subjects were detected by reverse transcription-quantitative PCR. Additionally, lncRNA CCAT1 was detected in GC and adjacent non-cancerous tissue samples. Serum EVs were successfully isolated and identified in all patients. The results revealed that serum EV lncRNA CCAT1 levels in patients with GC were significantly higher compared with those in healthy controls, patients with chronic gastritis or atypical hyperplasia (all P<0.05). Additionally, EV lncRNA CCAT1 expression levels were significantly different among various groups based on the depth of invasion, distant metastasis and the Tumor-Node-Metastasis stage. The area under the curve (AUC) value of EV lncRNA CCAT1 was 0.890 [95% confidence interval (CI), 0.826-0.937] with 79.6% sensitivity and 92.6% specificity. The combination of EV lncRNA CCAT1 and carcinoembryonic antibody produced an AUC value of 0.910 (95% CI, 0.849-0.951) with the sensitivity and specificity of 80.5 and 92.6%, respectively. In addition, lncRNA CCAT1 was determined to be stable in serum EVs. The expression levels of lncRNA CCAT1 in GC tissue were positively correlated with those in serum EVs, and high levels of lncRNA CCAT1 were associated with a low disease-free survival rate in patients with GC. The results of the present study demonstrated that serum EV lncRNA CCAT1 levels were upregulated in patients with GC compared with those healthy subjects and patients with other illnesses, and may therefore be used as a novel biomarker for this type of cancer.

Soluble 'SOSIP'-stabilized HIV-1 envelope glycoprotein (Env) trimers elicit dominant antibody responses targeting their glycan-free base regions, potentially diminishing neutralizing responses. Previously, using a nonhuman primate model, we demonstrated that priming with fusion peptide (FP)-carrier conjugate immunogens followed by boosting with Env trimers reduced the anti-base response. Further, we demonstrated that longer immunization intervals further reduced anti-base responses and increased neutralization breadth. Here, we demonstrate that long trimer-boosting intervals, but not long FP immunization intervals, reduce the anti-base response. Additionally, we identify that FP priming before trimer immunization enhances antibody avidity to the Env trimer. We also establish that adjuvants Matrix M and Adjuplex further reduce anti-base responses and increase neutralizing titers. FP priming, long trimer-immunization interval, and an appropriate adjuvant can thus reduce anti-base antibody responses and improve Env-directed vaccine outcomes.

Chronic stress-induced inflammatory responses occur in part via danger-associated molecular pattern (DAMP) molecules, such as high mobility group box 1 protein (HMGB1), but the receptor(s) underlying DAMP signaling have not been identified.

Injury of oligodendrocytes (OLs) induces demyelination, and patients with neurodegenerative diseases exhibit demyelination concomitantly with neurological deficit and cognitive impairment. Oligodendrocyte progenitor cells (OPCs) are present in the adult central nervous system (CNS), and they can proliferate, differentiate, and remyelinate axons after damage. However, remyelination therapies are not in clinical use. Multiple sclerosis (MS) is a major demyelinating disease in the CNS. Mesenchymal stromal cells (MSCs) have demonstrated therapeutic promise in animal models and in clinical trials of MS. Exosomes are nanoparticles generated by nearly all cells and they mediate cell-cell communication by transferring cargo biomaterials. Here, we hypothesize that exosomes harvested from MSCs have a similar therapeutic effect on enhancement of remyelination as that of MSCs. In the present study we employed exosomes derived from rhesus monkey MSCs (MSC-Exo). Two mouse models of demyelination were employed: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS; and 2) cuprizone (CPZ) diet model, a toxic demyelination model. MSC-Exo or PBS were intravenously injected twice a week for 4 weeks, starting on day 10 post immunization in EAE mice, or once a week for 2 weeks starting on the day of CPZ diet withdrawal. Neurological and cognitive function were tested, OPC differentiation and remyelination, neuroinflammation and the potential underlying mechanisms were investigated using immunofluorescent staining, transmission electron microscopy and Western blot. Data generated from the EAE model revealed that MSC-Exo cross the blood brain barrier (BBB) and target neural cells. Compared with the controls (p < 0.05), treatment with MSC-Exo: 1) significantly improved neurological outcome; 2) significantly increased the numbers of newly generated OLs (BrdU+/APC+) and mature OLs (APC+), and the level of myelin basic protein (MBP); 3) decreased amyloid-β precursor protein (APP)+ density; 4) decreased neuroinflammation by increasing the M2 phenotype and decreasing the M1 phenotype of microglia, as well as their related cytokines; 5) inhibited the TLR2/IRAK1/NFκB pathway. Furthermore, we confirmed that the MSC-Exo treatment significantly improved cognitive function, promoted remyelination, increased polarization of M2 phenotype and blocked TLR2 signaling in the CPZ model. Collectively, MSC-Exo treatment promotes remyelination by both directly acting on OPCs and indirectly by acting on microglia in the demyelinating CNS. This study provides the cellular and molecular basis for this cell-free exosome therapy on remyelination and modulation of neuroinflammation in the CNS, with great potential for treatment of demyelinating and neurodegenerative disorders.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

Autapses are synaptic contacts of a neuron's axon onto its own dendrite and soma. In the neocortex, self-inhibiting autapses in GABAergic interneurons are abundant in number and play critical roles in regulating spike precision and network activity. Here we examine whether the principal glutamatergic pyramidal cells (PCs) also form functional autapses. In patch-clamp recording from both rodent and human PCs, we isolated autaptic responses and found that these occur predominantly in layer-5 PCs projecting to subcortical regions, with very few in those projecting to contralateral prefrontal cortex and layer 2/3 PCs. Moreover, PC autapses persist during development into adulthood. Surprisingly, they produce giant postsynaptic responses (∼5 fold greater than recurrent PC-PC synapses) that are exclusively mediated by AMPA receptors. Upon activation, autapses enhance burst firing, neuronal responsiveness and coincidence detection of synaptic inputs. These findings indicate that PC autapses are functional and represent an important circuit element in the neocortex.

Several influenza antibodies with broad group 2 neutralization have recently been isolated. Here, we analyze the structure, class, and binding of one of these antibodies from an H7N9 vaccine trial, 315-19-1D12. The cryo-EM structure of 315-19-1D12 Fab in complex with the hemagglutinin (HA) trimer revealed the antibody to recognize the helix A region of the HA stem, at the supersite of vulnerability recognized by group 1-specific and by cross-group-neutralizing antibodies. 315-19-1D12 was derived from HV1-2 and KV2-28 genes and appeared to form a new antibody class. Bioinformatic analysis indicated its group 2 neutralization specificity to be a consequence of four key residue positions. We specifically tested the impact of the group 1-specific N33 glycan, which decreased but did not abolish group 2 binding of 315-19-1D12. Overall, this study highlights the recognition of a broad group 2-neutralizing antibody, revealing unexpected diversity in neutralization specificity for antibodies that recognize the HA stem supersite.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development.