Neuronal ferroptosis plays a key role in neurologic deficits post intracerebral hemorrhage (ICH). However, the endogenous regulation of rescuing ferroptotic neurons is largely unexplored. Here, we analyzed the integrated alteration of metabolomic landscape after ICH using LC-MS and MALDI-TOF/TOF MS, and demonstrated that aconitate decarboxylase 1 (Irg1) and its product itaconate, a derivative of the tricarboxylic acid cycle, were protectively upregulated. Deficiency of Irg1 or depletion of neuronal Irg1 in striatal neurons was shown to exaggerate neuronal loss and behavioral dysfunction in an ICH mouse model using transgenic mice. Administration of 4-Octyl itaconate (4-OI), a cell-permeable itaconate derivative, and neuronal Irg1 overexpression protected neurons in vivo. In addition, itaconate inhibited ferroptosis in cortical neurons derived from mouse and human induced pluripotent stem cells in vitro. Mechanistically, we demonstrated that itaconate alkylated glutathione peroxidase 4 (GPx4) on its cysteine 66 and the modification allosterically enhanced GPx4’s enzymatic activity by using a bioorthogonal probe, itaconate-alkyne (ITalk), and a GPx4 activity assay using phosphatidylcholine hydroperoxide. Altogether, our research suggested that Irg1/itaconate-GPx4 axis may be a future therapeutic strategy for protecting neurons from ferroptosis post ICH.

Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.

This study aims to characterize the majority of immune cell subsets in peripheral blood mononuclear cells in children with Mycoplasma pneumoniae pneumonia (MPP) by a 21-color flow cytometry panel. Patients who met the predetermined eligibility criteria for pneumonia diagnosis were recruited for the research study. Multi-color flow cytometry was conducted on the peripheral blood mononuclear cells of each patient group, which were then subjected to dimensionality reduction and cluster analysis. In our study, the proportion of activated CD4 + T cell and naïve CD8 + T in children with MPP was higher than that of children with non-MPP, and the proportion of CD8 + T cell and central memory CD8 + T cell in MPP children was lower. Central memory CD4 + T cell and activated CD4 + T cell in the severe MPP were higher than those in the mild MPP. The highest proportions of CD8 + T cell, CD8 + Tn cell, activated CD8 + T cell, and total activated T cell were observed in the pulmonary consolidation-mucous group when compared to the pulmonary consolidation-necrosis and bronchiolitis groups. In the pulmonary consolidation-necrosis group, the proportions of central memory CD4 + T cell and T helper 17 cell were higher than those in pulmonary consolidation-mucous and bronchiolitis groups. In the bronchiolitis group, the percentages of CD4 + T cell, naïve CD4 + T cell, and T helper 2 cell were higher than those in pulmonary consolidation-mucous and the pulmonary consolidation-necrosis groups. The T lymphocyte subsets were different among various groups, offering new insights into the immune system of pediatric patients with Mycoplasma pneumoniae pneumonia.

Data on epidemiology trends of paediatric tuberculosis (TB) are limited in China. So, we investigated the clinical and epidemiological profiles in diagnosed TB disease and TB infection patients at Beijing Children's Hospital. Of 3 193 patients, 51.05% had pulmonary TB (PTB) and 15.16% had extrapulmonary TB (EPTB). The most frequent forms of EPTB were TB meningitis (39.05%), pleural TB (29.75%), and disseminated TB (10.33%). PTB patients were significantly younger and associated with higher hospitalization frequency. Children aged 1-4 years exhibited higher risk of PTB and TB meningitis, and children aged 5-12 years had higher risk of EPTB. The proportion of PTB patients increased slightly from 40.9% in 2012 to 65% in 2019, and then decreased to 17.8% in 2021. The percentage of EPTB cases decreased from 18.3% in 2012 to 15.2% in 2019, but increased to 16.4% in 2021. Among EPTB cases, the largest increase was seen in TB meningitis. In conclusion, female and young children had higher risk of PTB in children. TB meningitis was the most frequent forms of EPTB among children, and young children were at high risk of TB meningitis. The distribution of different types of EPTB differed by age.

Objectives: To evaluate the diagnostic value of targeted next generation sequencing (tNGS) in childhood tuberculosis (TB) and compare the accuracy with Xpert MTB/RIF method. Methods: Children aged ≤18 years with symptoms suggestive of TB during July 2021 to December 2022 at Beijing Children's Hospital were included, and the performances of tNGS and Xpert were evaluated. Results: A total of 103 children with suspected TB were recruited, including 72 discharge diagnosis of TB and 31 non-TB cases. The mean age was 7.37 ± 4.77 years, and 62.1 % were male. The most common type of specimens was gastric aspirate (GA) (59, 57.3 %). Among all the 72 TB patients, tNGS showed higher sensitivity than Xpert, but the difference was not significant (34.7 %, 25/72 vs 20.8 %, 15/72; P = 0.063). The specificities of tNGS and Xpert were 87.1 % (27/31) and 96.8 % (30/31), respectively (P = 0.162). Among different types of specimen, the highest sensitivity of tNGS on sputum and pus was observed (80.0 %, 4/5), followed by pleural effusion (50.0 %, 2/4). One rifampin resistance and one protionamide resistance were detected in bacteriologically confirmed TB by tNGS. Conclusion: tNGS had a higher sensitivity but lower specificity compared to Xpert in diagnosis of children TB. tNGS yielded higher sensitivity than Xpert on gastric aspirate and sputum and pus.

The hippocampus is essential for learning and memory, but it also plays an important role in regulating emotional behavior, as hippocampal excitability and plasticity affect anxiety and fear. Brain synaptic plasticity may be regulated by tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a known protein inhibitor of extracellular matrix (ECM), and the expression of TIMP1 in the hippocampus can be induced by neuronal excitation and various stimuli. However, the involvement of Timp1 in fear learning, anxiety, and hippocampal synaptic function remains to be established. Our study of Timp1 function in vivo revealed that Timp1 knockout mice exhibit anxiety-like behavior but normal fear learning. Electrophysiological results suggested that Timp1 knockout mice showed hyperactivity in the ventral CA1 region, but the basic synaptic transmission and plasticity were normal in the Schaffer collateral pathway. Taken together, our results suggest that deletion of Timp1 in vivo leads to the occurrence of anxiety behaviors, but that Timp1 is not crucial for fear learning.

BACE1 is the rate-limiting enzyme for β-amyloid (Aβ) production and therefore is considered a prime drug target for treating Alzheimer’s disease (AD). Nevertheless, the BACE1 inhibitors failed in clinical trials, even exhibiting cognitive worsening, implying that BACE1 may function in regulating cognition-relevant neural circuits. Here, we found that parvalbumin-positive inhibitory interneurons (PV INs) in hippocampal CA1 express BACE1 at a high level. We designed and developed a mouse strain with conditional knockout of BACE1 in PV neurons. The CA1 fast-spiking PV INs with BACE1 deletion exhibited an enhanced response of postsynaptic N-methyl-D-aspartate (NMDA) receptors to local stimulation on CA1 oriens, with average intrinsic electrical properties and fidelity in synaptic integration. Intriguingly, the BACE1 deletion reorganized the CA1 recurrent inhibitory motif assembled by the heterogeneous pyramidal neurons (PNs) and the adjacent fast-spiking PV INs from the superficial to the deep layer. Moreover, the conditional BACE1 deletion impaired the AMPARs-mediated excitatory transmission of deep CA1 PNs. Further rescue experiments confirmed that these phenotypes require the enzymatic activity of BACE1. Above all, the BACE1 deletion resets the priming of the fear memory extinction. Our findings suggest a neuron-specific working model of BACE1 in regulating learning and memory circuits. The study may provide a potential path of targeting BACE1 and NMDAR together to circumvent cognitive worsening due to a single application of BACE1 inhibitor in AD patients.

Spontaneous intracerebral hemorrhage (ICH) is a devastating disease with high morbidity and mortality worldwide. We have previously shown that ferroptosis contributes to neuronal loss in ICH mice. The overload of iron and dysfunction of glutathione peroxidase 4 (GPx4) promote neuronal ferroptosis post-ICH. However, how epigenetic regulatory mechanisms affect the ferroptotic neurons in ICH remains unclear. In the current study, hemin was used to induce ferroptosis in N2A and SK-N-SH neuronal cells to mimic ICH. The results showed that hemin-induced ferroptosis was accompanied by an increment of global level of trimethylation in histone 3 lysine 9 (H3K9me3) and its methyltransferase Suv39h1. Transcriptional target analyses indicated that H3K9me3 was enriched at the promoter region and gene body of transferrin receptor gene 1 ( Tfr1) and repressed its expression upon hemin stimulation. Inhibition of H3K9me3 with inhibitor or siRNA against Suv39h1 aggravated hemin- and RSL3-induced ferroptosis by upregulating Tfr1 expression. Furthermore, Suv39h1-H3K9me3 mediated repression of Tfr1 contributes to the progression of ICH in mice. These data suggest a protective role of H3K9me3 in ferroptosis post ICH. The knowledge gained from this study will improve the understanding of epigenetic regulation in neuronal ferroptosis and shed light on future clinical research after ICH.

Oxidative stress is one of the pathological mechanisms of Alzheimer’s disease (AD), and ferroptosis has been determined to be involved in neurodegenerative diseases such as AD. Senegenin (Sen) prevents oxidative damage in nerve cells via a mechanism that may be highly related to ferroptosis. However, the mechanism of ferroptosis pathway involvement in AD is unclear. In this study, we established a model of PC12 cytotoxic injury induced by Aβ25–35, and we detected the level of oxidative damage, MMP, and ferroptosis-related protein expression. The results showed that, compared with control group, the level of ROS increased, GPX activities decreased, and MDA levels increased in Aβ25–35 group. Aβ25–35 could induce mitochondrial depolarization in PC12 cells and Fer-1 could not reverse this damage. WB revealed that Aβ25–35 group had increased ACSL4 and PEBP1 proteins, and decreased GPX4 protein. After adding Sen in the model, the level of oxidative damage was reduced, and mitochondrial depolarization was reversed compared with Aβ25–35 group. WB suggested that the expression of ACSL4 and PEBP1 proteins decreased, and the expression of GPX4 protein increased by Sen treatment. In conclusion, we found that Sen exhibits strong neuroprotective activity against Aβ25–35 induced oxidative damage and lipid metabolic associated with ferroptosis. Inhibiting nerve cell ferroptosis might facilitate the future development of strategies to AD.

Background The nucleotide oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) in dorsal root ganglion (DRG) contributes to pain hypersensitivity in multiple neuropathic pain models, but the function of the NLRP3 in diabetic neuropathic pain (DNP) and the regulation mechanism are still largely unknown. Epigenetic regulation plays a vital role in the controlling of gene expression. Ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is a DNA demethylase that contributes to transcriptional activation. TET2 is also involved in high glucose (HG)-induced pathology. Methods DNP was induced in mice via the intraperitoneal injection of streptozotocin (STZ) for five consecutive days and the mechanical threshold was evaluated in STZ-diabetic mice by using von Frey hairs. The expression level of the NLRP3 pathway and TET2 in DRG were determined through molecular biology experiments. The regulation of the NLRP3 pathway by TET2 was examined in in vitro and in vivo conditions. Results In the present research, we first established the DNP model and found that NLRP3 pathway was activated in DRG. The treatment of NLRP3 inhibitor MCC950 alleviated the mechanical allodynia of DNP mice. Then we revealed that in STZ-diabetic mice DRG, the genomic DNA was demethylated, and the expression of DNA demethylase TET2 was increased evidently. Using RNA-sequencing analysis, we found that the expression of Txnip , a gene that encodes a thioredoxin-interacting protein (TXNIP) which mediates NLRP3 activation, was elevated in the DRG after STZ treatment. In addition, knocking down of TET2 expression in DRG using TET2 -siRNA suppressed the mRNA expression of Txnip and subsequently inhibited the expression/activation of NLRP3 inflammasome in vitro and in vivo as well as relieved the pain sensitivity of DNP animals. Conclusion The results suggested that the upregulation of the TXNIP/NLRP3 pathway by TET2 in DRG was involved in the pain hypersensitivity of the DNP model.

RhoGDIα is an inhibitor of RhoGDP dissociation that involves in Aβ metabolism and NFTs production in Alzheimer's disease (AD) by regulating of RhoGTP enzyme activity. Our previous research revealed that RhoGDIα, as the target of Polygala saponin (Sen), might alleviate apoptosis of the nerve cells caused by hypoxia/reoxygenation (H/R). To further clarify the role of RhoGDIα in the generation of NFTs, we explored the relationship between RhoGDIα and Tau. We found out that RhoGDIα and Tau can bind with each other and interact by using coimmunoprecipitation (Co-IP) and GST pulldown methods in vitro. This RhoGDIα-Tau partnership was further verified by using immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) approaches in PC12 cells. Using the RNA interference (RNAi) technique, we found that the RhoGDIα may be involved in an upstream signaling pathway for Tau. Subsequently, in Aβ25-35- and H/R-induced PC12 cells, forced expression of RhoGDIα via cDNA plasmid transfection was found to reduce the hyperphosphorylation of Tau, augment the expression of bcl-2 protein, and inhibit the expression of Bax protein (reducing the Bax/bcl-2 ratio) and the activity of caspase-3. In mouse AD and VaD models, forced expression of RhoGDIα via injection of a viral vector (pAAV-EGFP-RhoGDIα) into the lateral ventricle of the brain alleviated the pathological symptoms of AD and VaD. Finally, GST pulldown confirmed that the binding sites on RhoGDIα for Tau were located in the range of the ΔC33 fragment (aa 1–33). These results indicate that RhoGDIα is involved in the phosphorylation of Tau and apoptosis in AD and VaD. Overexpression of RhoGDIα can inhibit the generation of NFTs and delay the progress of these two types of dementia.

Inflammation plays an important role in the occurrence and development of neuropathic pain. Immune-responsive gene 1 (IRG1) decarboxylates cis -aconitate to produce itaconate in the mitochondria. Itaconate serves as an immunomodulator of macrophages and represses inflammation in infectious diseases. Recently, a study showed that an itaconate derivative inhibits neuroinflammation and reduces chronic pain in mice. However, the function and molecular mechanisms of endogenous itaconate in neuropathic pain have not been fullyelucidated. In this study, the content of itaconate in the ipsilateral spinal cord after nerve-injured mice was detected with mass spectrometry. The Irg1 -/- mouse was constructed to determine the role of endogenous itaconate in the chronic constriction nerve injury (CCI) model. The analgesic effect of exogenous itaconate was assessed with intraperitoneal and intrathecal administration in both male and female CCI mice. The spinal application of 4-OI also reduced the evoked responses of wide dynamic range neurons in CCI mice. The potential analgesic mechanism of itaconate was explored through molecular biology experiments and verified in Interleukin (IL)-10 -/- mice. We found the levels of itaconate and IRG1 in the spinal cord significantly increased after CCI. Irg1 deficiency aggravated the mechanical and heat hypersensitivity, while the exogenous administration of the itaconate derivative 4-OI alleviated the neuropathic pain in male and female CCI mice. Mechanistically, the treatment of 4-OI increased the level of IL-10 and activates STAT3/β-endorphin pathway in the spinal cord, and the analgesia effect of itaconate was impaired in IL-10 -/- mice. Finally, we showed that the upregulation of IL-10 induced by 4-OI was mainly from spinal neurons through Nrf2 pathway. This study demonstrated the analgesic effect of endogenous and exogenous itaconate in the neuropathic pain model, suggesting that the spinal IL-10/STAT3/β-endorphin pathway might mediate the analgesia effect of itaconate.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

BACKGROUND Wilms tumor (WT) is the most common primary renal malignancy in children. Autophagy plays dual roles in the promotion and suppression of various cancers. OBJECTIVE The goal of our study was to develop a novel autophagy-related gene (ARG) prognostic nomogram for WT. METHODS The Cancer Genome Atlas (TCGA) database was used. We screened the expression profiles of ARGs in 136 WT patients. The differentially expressed prognostic ARGs were evaluated by multivariate Cox regression analysis and survival analysis. A novel prognostic nomogram based on the ARGs and clinical characteristics was established using multivariate Cox regression analysis. RESULTS First, 69 differentially expressed ARGs were identified in WT patients. Then, multivariate Cox regression analysis was used to determine 4 key prognostic ARGs (CC3CL1, ERBB2, HIF-α and CXCR4) in WT. According to their ARG expression levels, the patients were clustered into high- and low-risk groups. Next, survival analysis indicated that high-risk patients had significantly poorer overall survival than low-risk patients. The results of functional enrichment analysis suggested that autophagy may play a tumor-suppressive role in the initiation of WT. Finally, a prognostic nomogram with a Harrell's concordance index (C-index) of 0.841 was used to predict the survival probability of WT patients by integrating clinical characteristics and the 4-ARG signature. The calibration curve indicated its excellent predictive performance. CONCLUSION In summary, the ARG signature could be a promising biomarker for monitoring the outcomes of WT. We established a novel nomogram based on the ARG signature, which accurately predicts the overall survival of WT patients.

Reactive astrocytes play a complex role in multiple sclerosis, and the astrocytes reactivity is an important factor in the pathogenesis of pain. It is of great significance to explore the genesis and development mechanism of pain in the early stage of multiple sclerosis (MS) for early intervention of the disease. This study aims to explore astrocyte reactivity at different stages of the experimental autoimmune encephalomyelitis (EAE) model, a mouse model of MS, and the role of astrocytes in the pain in the early stage of the EAE. In this study, we demonstrated that spinal dorsal horn astrocytes were activated in the pre-clinical stage of EAE mice, and the inhibition of spinal cord astrocyte reactivity effectively alleviates pain symptoms in EAE mice. On the other hand, spinal cord microglia were not directly participated in the early EAE pain. Moreover, the ion channel LRRC8A mediated the reactivity of spinal dorsal horn astrocytes by regulating the STAT3 pathway, therefore playing a role in the early pain of EAE.

Oligodendrocyte progenitor cells (OPCs) differentiate to myelin-producing mature oligodendrocytes and enwrap growing or demyelinated axons during development and post central nervous diseases. Failure of remyelination owing to cell death or undifferentiation of OPCs contributes to severe neurologic deficits and motor dysfunction. However, how to prevent the cell death of OPCs is still poorly understood, especially in hemorrhagic diseases. In the current study, we injected autologous blood into the mouse lateral ventricular to study the hemorrhage-induced OPC cell death in vivo. The integrity of the myelin sheath of the corpus callosum was disrupted post intraventricular hemorrhage (IVH) assessed by using magnetic resonance imaging, immunostaining, and transmission electron microscopy. Consistent with the severe demethylation, we observed massive cell death of oligodendrocyte lineages in the periventricular area. In addition, we found that ferroptosis is the major cell death form in Hemin-induced OPC death by using RNA-seq analysis, and the mechanism was glutathione peroxidase 4 activity reduction-resulted lipid peroxide accumulation. Furthermore, inhibition of ferroptosis rescued OPC cell death in vitro, and in vivo attenuated IVH-induced white matter injury and promoted recovery of neurological function. These data demonstrate that ferroptosis is an essential form of OPC cell death in hemorrhagic stroke, and rescuing ferroptotic OPCs could serve as a therapeutic target for stroke and related diseases.

Sirtuin6(SIRT6)属于Sirtuin家族,具有去乙酰化酶和ADP-核糖基转移酶活性,在骨代谢、基因稳定、葡萄糖代谢、脂质代谢、炎性介质调控、肿瘤发生中发挥关键作用.本综述主要总结了SIRT6在骨代谢中的作用及其相关的分子信号通路.

Gestational diabetes mellitus (GDM) increases the risk of fetal heart malformations, though little is known about the mechanism of hyperglycemia-induced heart malformations. Thus, we aimed to reveal the global landscape of miRNAs and mRNAs in GDM-exposed fetoplacental arterial endothelial cells (dAECs) and establish regulatory networks for exploring the pathophysiological mechanism of fetal heart malformations in maternal hyperglycemia. Gene Expression Omnibus (GEO) datasets were used, and identification of differentially expressed miRNAs (DEMs) and genes (DEGs) in GDM was based on a previous sequencing analysis of dAECs. A miRNA-mRNA network containing 20 DEMs and 65 DEGs was established using DEMs altered in opposite directions to DEGs. In an in vivo study, we established a streptozotocin-induced pregestational diabetes mellitus (PGDM) mouse model and found the fetal cardiac wall thickness in different regions to be dramatically increased in the PGDM grouValidation of DEMs and DEGs in the fetal heart showed significantly upregulated expression of let-7e-5p, miR-139-5p and miR-195-5p and downregulated expression of SGOL1, RRM2, RGS5, CDK1 and CENPA. In summary, we reveal the miRNA-mRNA regulatory network related to fetal cardiac development disorders in offspring, which may shed light on the potential molecular mechanisms of fetal cardiac development disorders during maternal hyperglycemia.

Enterobacter cloacae is a well-characterized opportunistic pathogen that is closely associated with various nosocomial infections. The O-antigen, which is one of the most variable constituents on the cell surface, has been used widely and traditionally for serological classification of many gram-negative bacteria. E. cloacae is divided into 30 serotypes, based on its O-antigen diversity. In this study, by using genomic and comparative-genomic approaches, we analyzed the O-antigen gene clusters of 26 E. cloacae serotypes in depth. We also identified the sero-specific gene for each serotype and developed a multiplex polymerase chain reaction (PCR) method. The sensitivity of the assay was 0.1 ng for genomic DNA and 10(3) colony forming units for pure cultures. The assay reliability was evaluated by double-blinded testing with 81 clinical strains. Furthermore, we established a valid, genome-based tool for in silico serotyping of E. cloacae. By screening 431 E. cloacae genomes deposited in GenBank, 304 were classified into current antigenic scheme, and 112 were allocated into 55 putative novel serotypes. Our results represent the first genetic basis of the O-antigen diversity and variation of E. cloacae, providing a rationale for studying the O-antigen associated evolution and pathogenesis of this bacterium. In addition, we extended the current serotyping system for E. cloacae, which is important for detection and epidemiological surveillance purposes for this important pathogen.