The central helicase domain holds the major conformational epitopes of melanoma differentiation-associated gene 5 autoantibodies.

OBJECTIVES: Autoantibodies against MDA5 serve as a biomarker for dermatomyositis (DM) and a risk factor for interstitial lung disease (ILD). MDA5 is a protein responsible for sensing RNA virus infection and activating signalling pathways against it. However, little is known about antigen epitopes on MDA5 autoantibodies. We aimed to determine the interaction of the MDA5 autoantibody-antigen epitope. METHODS: Cell-based assays (CBAs), immunoprecipitation-immunoblot assays, and various immunoblotting techniques were used in the study. RESULTS: We demonstrate that DM patient autoantibodies recognize MDA5 epitopes in a native conformation-dependent manner. Furthermore, we identified the central helicase domain formed by Hel1, Hel2i, Hel2, and pincer (3Hel) as the major epitopes. As proof of principle, the purified 3Hel efficiently absorbed MDA5 autoantibodies from patient sera through immunoprecipitation-immunoblot assay. CONCLUSION: Our study uncovers the nature of antigen epitopes on MDA5 and provides guidance for diagnosis and targeted therapeutic approach development.

Transplantation of hESCs-Derived Neural Progenitor Cells Alleviates Secondary Damage of Thalamus After Focal Cerebral Infarction in Rats.

Human embryonic stem cells-derived neural progenitor cells (hESCs-NPCs) transplantation holds great potential to treat stroke. We previously reported that delayed secondary degeneration occurs in the ventroposterior nucleus (VPN) of ipsilateral thalamus after distal branch of middle cerebral artery occlusion (dMCAO) in adult male Sprague-Dawley (SD) rats. In this study, we investigate whether hESCs-NPCs would benefit the neural recovery of the secondary damage in the VPN after focal cerebral infarction. Permanent dMCAO was performed with electrocoagulation. Rats were randomized into Sham, dMCAO groups with or without hESCs-NPCs treatment. HESCs-NPCs were engrafted into the peri-infarct regions of rats at 48 h after dMCAO. The transplanted hESCs-NPCs survive and partially differentiate into mature neurons after dMCAO. Notably, hESCs-NPCs transplantation attenuated secondary damage of ipsilateral VPN and improved neurological functions of rats after dMCAO. Moreover, hESCs-NPCs transplantation significantly enhanced the expression of BDNF and TrkB and their interaction in ipsilateral VPN after dMCAO, which was reversed by the knockdown of TrkB. Transplantated hESCs-NPCs reconstituted thalamocortical connection and promoted the formation of synapses in ipsilateral VPN post-dMCAO. These results suggest that hESCs-NPCs transplantation attenuates secondary damage of ipsilateral thalamus after cortical infarction, possibly through activating BDNF/TrkB pathway, enhancing thalamocortical projection, and promoting synaptic formation. It provides a promising therapeutic strategy for secondary degeneration in the ipsilateral thalamus post-dMCAO.

Prognostic Value of Lymph Node Necrosis at Different N Stages in Patients with Nasopharyngeal Carcinoma.

Background: Lymph node necrosis (LNN), including retropharyngeal nodal necrosis and cervical nodal necrosis, which is related to radiotherapy/ chemotherapy resistance, is a common phenomenon in nasopharyngeal carcinoma (NPC). This study was to assess the prognostic value of LNN at different N stages in NPC patients. Materials and Methods: In total, 1,665 newly diagnosed NPC patients at stage TxN1-3M0 from two centers were enrolled. Univariate and multivariate models were constructed to assess the association between LNN and long-term survival outcomes. The propensity score matching method was performed to balance treatment groups for baseline characteristics. Results: Of the 1,665, 540 patients (540/1665, 32.4%) were diagnosed with LNN, of which 54.1% (292/540) patients were at stage N1, 31.3% (169/540) at stage N2, and 14.6% (79/540) at stage N3. Univariate and multivariate analyses indicated LNN as an independent predictor for progression­free survival (PFS), overall survival (OS), distant metastasis-free survival (DMFS), and locoregional relapse-free survival (LRRFS) in stage N1-3 patients (all P<0.001). When patients were analyzed according to stage, similar findings were observed for N1 patients (all P<0.001); for N2 patients, LNN independently predicted PFS (P=0.003), OS (P=0.011), and DMFS (P=0.004), and for stage N3, LNN only independently predicted LRRFS (P=0.019). 123 pairs of patients who received induction chemotherapy plus concurrent chemoradiotherapy or only concurrent chemoradiotherapy were matched, adding induction chemotherapy improved 5-year OS, PFS and LRFFS, but the results were not statistically significant. Conclusions: In NPC patients, LNN could independently predict poor prognosis at all N1-3 stages and at each N stage (N1 to N3). The value of adding induction chemotherapy to concurrent chemoradiotherapy in patients with LNN still requires further prospective studies.

A novel rare variant of CNPY3 from familial NMOSD impairs the TLR-mediated immune response.

Toll-like receptors (TLRs) are a class of proteins that play essential roles in innate and adaptive immune responses. Recently, accumulating evidence has demonstrated that impairments in the TLR signalling pathway contribute to the development and progression of neuroimmune diseases, such as neuromyelitis optica spectrum disorder (NMOSD). However, the cellular and molecular mechanisms are still largely unknown. In this study, we report a novel variant, C52Y, of canopy FGF signalling regulator 3 (CNPY3) from patients with familial NMOSD and demonstrate that this variant shows a stronger interaction with GP96 and TLRs than with wild-type CNPY3. We find that C52Y has dominant negative effects on TLR4 surface expression. Importantly, the TLR4 surface expression level is decreased in RAW264.7 cells infected with the C52Y virus upon LPS stimulation. We further demonstrate that bone marrow-derived macrophages (BMDMs) from CNPY3C52Y/+ transgenic mice secrete less tumour necrosis factor (TNF) and interleukin (IL)-6 than BMDMs from wild-type mice upon stimulation with LPS. These data suggest that impairment of TLR trafficking may contribute to the development of neuroimmune disorders.

Seratrodast, a thromboxane A2 receptor antagonist, inhibits neuronal ferroptosis by promoting GPX4 expression and suppressing JNK phosphorylation.

More than 30 % of individuals with epilepsy are refractory to currently available drugs, highlighting the urgent need to develop novel candidate drugs. Accumulating evidence implicates the key role of ferroptosis in the pathophysiology of epileptic seizuresand its potential as a new drug target. Drug repurposing is a promising strategy for the rapid generation of new candidate drugs from the market drugs with new therapeutic indications, such as the best-selling drug thalidomide. Herein, we reported the discovery of Seratrodast, a market drug of thromboxane A2 receptor antagonist as a new ferroptosis inhibitor (IC50: 4.5 µmol·L-1). Seratrodast could reduce lipid ROS production, regulate the system xc-/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis, and inhibit JNK phosphorylation and p53 expression. In addition, Seratrodast elevated GPX4 expression and decreased JNK phosphorylation in pentylenetetrazole-induced seizures in mice. Seratrodast increased the latency of seizures and reduced seizure duration in pentylenetetrazole-induced seizures. Our results suggest Seratrodast might be either a ferroptosis inhibitor or a novel lead compound for further optimization of novel drug discovery.

Physical exercise promotes integration of grafted cells and functional recovery in an acute stroke rat model.

Human neural progenitor cell (hNPC) transplantation holds great potential to treat neurological diseases. However, hNPC grafts take a long time to differentiate into mature neurons due to their intrinsically prolonged developmental timetable. Here, we report that postoperative physical exercise (PE), a prevailing rehabilitation intervention, promotes the neuronal commitment, maturation, and integration of engrafted hNPCs, evidenced by forming more synapses, receiving more synaptic input from host neurons, and showing higher neuronal activity levels. More important, NPC transplantation, combined with PE, shows significant improvement in both structural and behavioral outcomes in stroke-damaged rats. PE enhances ingrowth of blood vessels around the infarction region and neural tract reorganization along the ischemic boundary. The combination of NPC transplantation and postoperative PE creates both a neurotrophic/growth factor-enriched proneuronal microenvironment and an ideal condition for activity-dependent plasticity to give full play to its effects. Our study provides a potential approach to treating patients with stroke injury.

Hypoproliferative human neural progenitor cell xenografts survived extendedly in the brain of immunocompetent rats.

BACKGROUND: There is a huge controversy about whether xenograft or allograft in the "immune-privileged" brain needs immunosuppression. In animal studies, the prevailing sophisticated use of immunosuppression or immunodeficient animal is detrimental for the recipients, which results in a short lifespan of animals, confounds functional behavioral readout of the graft benefits, and discourages long-term follow-up. METHODS: Neuron-restricted neural progenitor cells (NPCs) were derived from human embryonic stem cells (ESCs, including H1, its gene-modified cell lines for better visualization, and HN4), propagated for different passages, and then transplanted into the brain of immunocompetent rats without immunosuppressants. The graft survivals, their cell fates, and HLA expression levels were examined over time (up to 4 months after transplantation). We compared the survival capability of NPCs from different passages and in different transplantation sites (intra-parenchyma vs. para- and intra-cerebroventricle). The host responses to the grafts were also investigated. RESULTS: Our results show that human ESC-derived neuron-restricted NPCs survive extendedly in adult rat brain parenchyma with no need of immunosuppression whereas a late-onset graft rejection seems inevitable. Both donor HLA antigens and host MHC-II expression level remain relatively low with little change over time and cannot predict the late-onset rejection. The intra-/para-cerebroventricular human grafts are more vulnerable to the immune attack than the intrastriatal counterparts. Prevention of graft hyperplasia by using hypoproliferative late passaged human NPCs further significantly extends the graft survival time. Our new data also shows that a subpopulation of host microglia upregulate MHC-II expression in response to the human graft, but fail to present the human antigen to the host immune system, suggestive of the immune-isolation role of the blood-brain barrier (BBB). CONCLUSIONS: The present study confirms the "immune privilege" of the brain parenchyma and, more importantly, unveils that choosing hypoproliferative NPCs for transplantation can benefit graft outcome in terms of both lower tumor-genic risk and the prolonged survival time without immunosuppression.

Characterization and generation of human definitive multipotent hematopoietic stem/progenitor cells.

Definitive hematopoiesis generates hematopoietic stem/progenitor cells (HSPCs) that give rise to all mature blood and immune cells, but remains poorly defined in human. Here, we resolve human hematopoietic populations at the earliest hematopoiesis stage by single-cell RNA-seq. We characterize the distinct molecular profiling between early primitive and definitive hematopoiesis in both human embryonic stem cell (hESC) differentiation and early embryonic development. We identify CD44 to specifically discriminate definitive hematopoiesis and generate definitive HSPCs from hESCs. The multipotency of hESCs-derived HSPCs for various blood and immune cells is validated by single-cell clonal assay. Strikingly, these hESCs-derived HSPCs give rise to blood and lymphoid lineages in vivo. Lastly, we characterize gene-expression dynamics in definitive and primitive hematopoiesis and reveal an unreported role of ROCK-inhibition in enhancing human definitive hematopoiesis. Our study provides a prospect for understanding human early hematopoiesis and a firm basis for generating blood and immune cells for clinical purposes.

Compound AD16 Reduces Amyloid Plaque Deposition and Modifies Microglia in a Transgenic Mouse Model of Alzheimer's Disease.

Microglial dysfunction is involved in the pathological cascade of Alzheimer's disease (AD). The regulation of microglial function may be a novel strategy for AD therapy. We previously reported the discovery of AD16, an antineuroinflammatory molecule that could improve learning and memory in the AD model. Here, we studied its properties of microglial modification in the AD mice model. In this study, AD16 reduced interleukin-1ß (IL-1ß) expression in the lipopolysaccharide-induced IL-1ß-Luc transgenic mice model. Compared with mice receiving placebo, the group treated with AD16 manifested a significant reduction of microglial activation, plaque deposition, and peri-plaques microgliosis, but without alteration of the number of microglia surrounding the plaque. We also found that AD16 decreased senescent microglial cells marked with SA-ß-gal staining. Furthermore, altered lysosomal positioning, enhanced Lysosomal Associated Membrane Protein 1 (LAMP1) expression, and elevated adenosine triphosphate (ATP) concentration were found with AD16 treatment in lipopolysaccharide-stimulated BV2 microglial cells. The underlying mechanisms of AD16 might include regulating the microglial activation/senescence and recovery of its physiological function via the improvement of lysosomal function. Our findings provide new insights into the AD therapeutic approach through the regulation of microglial function and a promising lead compound for further study.

Transplantation of neural progenitor cells generated from human urine epithelial cell-derived induced pluripotent stem cells improves neurological functions in rats with stroke.

As a potentially unlimited autologous cell source, induced pluripotent stem cells (iPSCs) provide a needed option for the application of iPSC-derived neural progenitor cells (NPCs) for regenerative medicine for the treatment of stroke. To enable the application of iPSC technology, it is essential to develop a practical approach to generate iPSC cells under a non-viral, non-integration, feeder-free condition from the most optimal somatic cell type. In this study, we differentiated NPCs from a urine-derived iPSC line (UC-05) which was generated with optimized episomal vectors in a feeder-free culture system. UC-05 can be induced into NPCs efficiently in monolayer cultures using dual SMAD inhibitions, and have the ability to differentiate further into astrocytes and functional neurons in vitro. We then characterized UC-05-derived NPCs upon transplantation into the striatum of adult male rats subjected to transient middle cerebral artery occlusion (tMCAO) reperfusion. While NPCs were grafted into rats 7 days before the MCAO surgery, cells were found to migrate from the grafted side to the lesion side of the brain via corpus callosum 14 days after tMCAO. UC05-derived NPCs were grafted into the striatum 7 days after tMCAO, grafted cells can survive and differentiate into neurons and astrocytes 35 days after transplantation, and synaptic protein SYNAPSIN 1 could also be detected around the grafted human cells. tMCAO rats with NPC engraftment showed better behavior improvement in both postural reflex test and cylinder test compared to control rats engrafted with the cell medium only. Our data indicate that NPCs differentiated from urine-derived iPSCs could act similarly to endogenous neural progenitors in vitro and in vivo. Urine-derived iPSCs could be a potential candidate for cell transplantation therapy in stroke.

Generation of a DAPK1 knockout first (conditional ready) human embryonic stem cell line (ZSSYe001-A) by CRISPR-Cas9 technology.

Death-associated protein kinase 1 (DAPK1) is a Ca2+/calmodulin regulated Ser/Thr kinase involved in various cellular processes including cell death, autophagy and inflammation. Its dysregulation has been linked to tumour metastasis, anti-viral responses, Alzheimer's disease and other neurological disorders. To further investigate the role of DAPK1 in these processes, we generated a DAPK1 knockout first (conditional ready) human embryonic stem (hES) cell line in which the endogenous DAPK1 can be easily restored with expression of FLPe. This cell line provides an ideal model to study the role of DAPK1 in human development and various pathologies related to DAPK1 dysregulation in vitro.

Generation of a PARK2 homozygous knockout induced pluripotent stem cell line (GIBHi002-A-1) with two common isoforms abolished.

Loss of function mutations in PARK2 (encoding PARKIN) cause autosomal recessive Parkinson's disease (PD), which often manifests at a juvenile age. Molecular and biochemical studies show that PARKIN functions as an E3 ubiquitin ligase controlling mitochondrial homeostasis. Yet, the exact mechanisms are unclear due to the use of sub-optimal models including cancer cells and fibroblasts. We have generated a PARK2 knockout (KO) isogenic cell line using a well-characterized induced pluripotent stem cell (iPSC) clone with good differentiation potential. This cell line lacks the expression of all PARKIN isoforms and is valuable for elucidating the role of PARK2 mutations in PD.

Vitamin C-dependent lysine demethylase 6 (KDM6)-mediated demethylation promotes a chromatin state that supports the endothelial-to-hematopoietic transition.

Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.

Retrograde monosynaptic tracing through an engineered human embryonic stem cell line reveals synaptic inputs from host neurons to grafted cells.

Retrograde monosynaptic tracing with EnvA-pseudotyped rabies virus has been employed to identify the afferent and efferent connectivity of transplanted human embryonic stem (hES) cell-derived neurons in animal models. Due to the protracted development of transplanted human neurons in host animals, it is important that those transplanted cells express avian leukosis and sarcoma virus subgroup A receptor (TVA) and rabies glycoprotein G (Rgp) for a period of up to several months to enable identification of the synaptic inputs from host neurons to grafted neurons through this rabies virus-based method. Here, we report the generation of an engineered hES cell line through CRISPR/Cas9-mediated targeting to the AAVS1 locus of an EnvA-pseudotyped rabies virus-based tool for retrograde monosynaptic tracing. This engineered hES cell line, named H1-CAG-GTRgp, expresses GFP, TVA and Rgp. Upon transplantation of H1-CAG-GTRgp-derived neural progenitor cells (NPCs) into the rat brain after traumatic injury, the grafted neurons derived from H1-CAG-GTRgp cells expressed GFP, TVA, and Rgp stably for up to 6 months post-transplantation and received robust synaptic inputs from host neurons in the target regions of the orthotopic neural circuitry. The retrograde monosynaptic tracing hES cell line provides an efficient approach to analyze transplant connectivity for the comprehensive assessment of host-donor cell innervation.

NCoR/SMRT co-repressors cooperate with c-MYC to create an epigenetic barrier to somatic cell reprogramming.

Somatic cell reprogramming by exogenous factors requires cooperation with transcriptional co-activators and co-repressors to effectively remodel the epigenetic environment. How this interplay is regulated remains poorly understood. Here, we demonstrate that NCoR/SMRT co-repressors bind to pluripotency loci to create a barrier to reprogramming with the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC), and consequently, suppressing NCoR/SMRT significantly enhances reprogramming efficiency and kinetics. The core epigenetic subunit of the NCoR/SMRT complex, histone deacetylase 3 (HDAC3), contributes to the effects of NCoR/SMRT by inducing histone deacetylation at pluripotency loci. Among the Yamanaka factors, recruitment of NCoR/SMRT-HDAC3 to genomic loci is mostly facilitated by c-MYC. Hence, we describe how c-MYC is beneficial for the early phase of reprogramming but deleterious later. Overall, we uncover a role for NCoR/SMRT co-repressors in reprogramming and propose a dual function for c-MYC in this process.

PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs.

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.Polycomb repressive complex 2 (PRC2) plays an essential role in development by modifying chromatin but what this means at a cellular level is unclear. Here, the authors show that ablation of PRC2 genes in human embryonic stem cells and in mice results in changes in pluripotency and the primed state of cells.

Generation of Human Liver Chimeric Mice with Hepatocytes from Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Familial hypercholesterolemia (FH) causes elevation of low-density lipoprotein cholesterol (LDL-C) in blood and carries an increased risk of early-onset cardiovascular disease. A caveat for exploration of new therapies for FH is the lack of adequate experimental models. We have created a comprehensive FH stem cell model with differentiated hepatocytes (iHeps) from human induced pluripotent stem cells (iPSCs), including genetically engineered iPSCs, for testing therapies for FH. We used FH iHeps to assess the effect of simvastatin and proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies on LDL-C uptake and cholesterol lowering in vitro. In addition, we engrafted FH iHeps into the liver of Ldlr-/-/Rag2-/-/Il2rg-/- mice, and assessed the effect of these same medications on LDL-C clearance and endothelium-dependent vasodilation in vivo. Our iHep models recapitulate clinical observations of higher potency of PCSK9 antibodies compared with statins for reversing the consequences of FH, demonstrating the utility for preclinical testing of new therapies for FH patients.

Oncogenic Kras expression in postmitotic neurons leads to S100A8-S100A9 protein overexpression and gliosis.

Previous studies suggest that up-regulation of Ras signaling in neurons promotes gliosis and astrocytoma formation in a cell nonautonomous manner. However, the underlying mechanisms remain unknown. To address this question, we generated compound mice (LSL Kras G12D/+;CamKII-Cre) that express oncogenic Kras from its endogenous locus in postmitotic neurons after birth. These mice developed progressive gliosis, which is associated with hyperactivation of Ras signaling pathways. Microarray analysis identified S100A8 and S100A9 as two secreted molecules that are significantly overexpressed in mutant cortices. In contrast to their usual predominant expression in myeloid cells, we found that overexpression of S100A8 and S100A9 in the mutant cortex is primarily in neurons. This neuronal expression pattern is associated with increased infiltration of microglia in mutant cortex. Moreover, purified S100A8-S100A9 but not S100A8 or S100A9 alone promotes growth of primary astrocytes in vitro through both TLR4 and receptor of advanced glycation end product receptors. In summary, our results identify overexpression of S100A8-S100A9 in neurons as an early step in oncogenic Kras-induced gliosis. These molecules expressed in nonhematopoietic cells may be involved in tumorigenesis at a stage much earlier than what has been reported previously.

DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke.

N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extrasynaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292-1304 (NR2B(CT)). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2B(CT) that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca(2+) influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extrasynaptic sites and this interaction acts as a central mediator for stroke damage.

Amino terminus of the SARS coronavirus protein 3a elicits strong, potentially protective humoral responses in infected patients.

The 3a protein of severe acute respiratory syndrome (SARS)-associated coronavirus is expressed and transported to the plasma membrane in tissue cells of infected patients. Its short N-terminal ectodomain was found to elicit strong humoral responses in half of the patients who had recovered from SARS. The ectodomain-specific antibodies from the convalescent-phase plasma readily recognized and induced destruction of 3a-expressing cells in the presence of the human complement system, demonstrating their potential ability to provide immune protection by recognizing and eliminating SARS coronavirus-infected cells that express the target protein. In addition, when coupled to a carrier protein, the ectodomain peptide elicited 3a-specific antibodies in mice and rabbit at high titres. These results showed that the N terminus of the 3a protein is highly immunogenic and elicits potentially protective humoral responses in infected patients. Therefore, the short extracellular domain may be a valuable immunogen in the development of a vaccine for infectious SARS.