Pulmonary fibrosis is a progressive and fatal disorder. In our previous study, we found that the Yiqihuoxue formula (YQHX), a prescription of Traditional Chinese Medicine, had a curative effect on scleroderma, a typical fibrotic disease. The aim of this study was to determine the key ingredient mediating the therapeutic effects of YQHX and to examine its effect on pulmonary fibrosis, including its mechanism. Luciferase reporter assays showed that the most important anti-fibrotic component of the YQHX was Salviae miltiorrhiza (SM). Experiments performed using a bleomycin-instilled mouse model of pulmonary fibrosis showed that Salvianolic acid B (SAB), the major ingredient of SM, had strong anti-inflammatory and anti-fibrotic effects through its inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition. Furthermore, SAB suppressed TGF-β-induced myofibroblastic differentiation of MRC-5 fibroblasts and TGF-β-mediated epithelial-to-mesenchymal transition of A549 cells by inhibiting both Smad-dependent signaling and the Smad-independent MAPK pathway. Taken together, our results suggest that SM is the key anti-fibrotic component of the YQHX and that SAB, the major ingredient of SM, alleviates experimental pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF-β signaling pathway. Together, these results suggest that SAB potently inhibits pulmonary fibrosis.

Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P < 3.65 × 10(-5)). Eight independent SNPs overlapped across Han Chinese, European and U.S. populations, and APOE and 5q33.3 were replicated as longevity loci. Integrated analysis indicates four pathways (starch, sucrose and xenobiotic metabolism; immune response and inflammation; MAPK; calcium signaling) highly associated with longevity (P ≤ 0.006) in Han Chinese. The association with longevity of three of these four pathways (MAPK; immunity; calcium signaling) is supported by findings in other human cohorts. Our novel finding on the association of starch, sucrose and xenobiotic metabolism pathway with longevity is consistent with the previous results from Drosophilia. This study suggests protective mechanisms including immunity and nutrient metabolism and their interactions with environmental stress play key roles in human longevity.

Macrophage inflammatory protein-1 (MIP-1), MIP-1α (CCL3) and MIP-1β (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1α and MIP-1β form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1α and MIP-1β form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1α, thus depolymerization mutations enhance MIP-1α to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1α ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.

The Th17 cytokine, IL-22, regulates host immune responses to extracellular pathogens. Whether IL-22 plays a role in viral infection, however, is poorly understood. We report here that Il22(-/-) mice were more resistant to lethal West Nile virus (WNV) encephalitis, but had similar viral loads in the periphery compared to wild type (WT) mice. Viral loads, leukocyte infiltrates, proinflammatory cytokines and apoptotic cells in the central nervous system (CNS) of Il22(-/-) mice were also strikingly reduced. Further examination showed that Cxcr2, a chemokine receptor that plays a non-redundant role in mediating neutrophil migration, was significantly reduced in Il22(-/-) compared to WT leukocytes. Expression of Cxcr2 ligands, cxcl1 and cxcl5, was lower in Il22(-/-) brains than wild type mice. Correspondingly, neutrophil migration from the blood into the brain was attenuated following lethal WNV infection of Il22(-/-) mice. Our results suggest that IL-22 signaling exacerbates lethal WNV encephalitis likely by promoting WNV neuroinvasion.

Cell division is closely related to telomerase activity (hTERT mRNA). Lower expression of lymphocitic hTERT mRNA may easily cause cell aging, which is not beneficial to maintaining a durable lymphocyte division. To date, there is no study to investigate IFNα therapy on hTERT mRNA expression in PBMCs of patients with chronic hepatitis B (CHB). We quantitatively detected hTERT mRNA from study subjects and made each hTERT mRNA normalized (NhTERT mRNA). Mean NhTERT mRNA level was lower in either CHB group, but it significantly increased in IFNα-treated group compared with CHB control group, and a longer duration of IFNα therapy could increase the level. Moreover, the mean NhTERT mRNA in subgroup with HBeAg loss was significantly higher than that in subgroup without. NhTERT mRNA was markedly correlated with CD3(+) T lymphocyte count and CD4(+)/CD8(+) ratio. The results showed that IFNα therapy could upregulate the expression of hTERT mRNA in PBMCs.

Heterotrimeric G protein Gα13 is known to transmit G protein-coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα13 in cell migration, however, remains unclear. Recently we found that Gα13 interacts with the cytoplasmic domain of integrin β3 subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα13 and the cytoplasmic domain of the integrin β1 subunit plays a critical role in β1-dependent cell migration. Point mutation of either glutamic acid in the Gα13-binding (767)EKE motif in β1 or treatment with a peptide derived from the Gα13-binding sequence of β1 abolished Gα13-β1 interaction and inhibited β1 integrin-dependent cell spreading and migration. We further show that the Gα13-β1 interaction mediates β1 integrin-dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα13 in mediating GPCR-dependent RhoA activation. These data indicate that Gα13 plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on β1 integrin ligands.

A growing body of evidence shows that the development and progression of gastric cancer (GC) is mainly associated to the presence of gastric cancer stem-like cells (GCSLCs). However, it is unclear how GCSLC population is maintained. This study aimed to explore the role of capillary morphogenesis gene 2 (CMG2) in GCSLC maintenance and the relevance to GC progression. We found that CMG2 was highly expressed in GC tissues and the expression levels were associated with the invasion depth and lymph node metastasis of GC, and inversely correlated with the survival of GC patients. Sorted CMG2High GC cells preferentially clustered in CD44High stem-like cell population, which expressed high levels of stemness-related genes with increased capabilities of self-renewal and tumorigenicity. Depletion of CMG2 gene resulted in reduction of GCSLC population with attenuated stemness and decrease of invasive and metastatic capabilities with subdued epithelial-mesenchymal transition phenotype in GC cells. Mechanistically, CMG2 interacted with LRP6 in GCSLCs to activate a Wnt/β-catenin pathway. Thus, our results demonstrate that CMG2 promotes GC progression by maintaining GCSLCs and can serve as a new prognostic indicator and a target for human GC therapy.

Induced expression of serum amyloid A (SAA) is a hallmark of many inflammatory diseases, but whether SAA exacerbates inflammation or protects tissues against injury remains unclear. In dextran sulfate sodium (DSS)-induced colitis, SAA3 is the predominant isoform of inducible SAA proteins that also include SAA1 and SAA2, and mice with genetic deletion of Saa3 exhibits increased production of proinflammatory cytokines, decreased expression of IL-22 along with aggravated epithelium disruption, and reduced colon length compared with wild-type littermates. Colonic neutrophils have been identified as a major source of IL-22 in these mice. Administration of exogenous SAA3 as recombinant protein to Saa3-/- mice improves neutrophil IL-22 production, colonic epithelial integrity, and secretion of the antimicrobial peptides Reg3β and Reg3γ. Stimulation of mouse bone marrow neutrophils with mouse SAA3 or human SAA1 leads to expansion of IL-22-producing neutrophils. Unlike previously reported IL-22 induction through IL-23, the SAA3-induced neutrophil IL-22 expression utilizes a TLR2-dependent mechanism that does not depend on IL-23. Adoptive transfer of the SAA3-treated neutrophils to Saa3-/- mice ameliorates DSS-induced colitis and improves colonic epithelial integrity. These findings suggest that in the DSS-induced mouse colitis model, SAA isoforms are expressed to different extent in colon and deletion of Saa3 renders these mice more susceptible to DSS-induced injury. The presence of SAA3 in the inflamed colon mucosal serves to protect epithelial barrier in part through expansion of IL-22-producing neutrophils. It is speculated that SAA3 stimulation of autologous neutrophils may have therapeutic potential for inflammatory bowel disease.

Despite the successful application of all-trans retinoic acid (ATRA) in multiple myeloma treatment, ATRA-induced chemoresistance in the myeloma patients is very common in clinic. In this study, we evaluated the effect of ATRA on the expression of apurinic endonuclease/redox factor-1 (Ape/Ref-1) in the U266 and RPMI-8226 myeloma cells to explore the chemoresistance mechanism involved. ATRA treatment induced upregulation of Ape/Ref-1 via a noncanonical signaling pathway, leading to enhanced pro-survival activity counteracting melphalan (an alkylating agent). ATRA rapidly activated p38-MSK (mitogen- and stress activated protein kinase) cascade to phosphorylate cAMP response element-binding protein (CREB). Phosphorylated CREB was recruited to the Ape/Ref-1 promoter to evoke the gene expression. The stimulation of ATRA on Ape/Ref-1 expression was attenuated by either p38-MSK inhibitors or overexpression of dominant-negative MSK1 mutants. Moreover, ATRA-mediated Ape/Ref-1 upregulation was correlated with histone modification and activation of CBP/p300, an important cofactors for CREB transcriptional activity. C646, a competitive CBP/p300 inhibitor, abolished the upregulation of Ape/Ref-1 induced by ATRA. Intriguingly, CBP rather than p300 played a dominant role in the expression of Ape/Ref-1. Hence, our study suggests the existence of a noncanonical mechanism involving p38-MSK-CREB cascade and CBP induction to mediate ATRA-induced Ape/Ref-1 expression and acquired chemoresistance in myeloma cells.

Formyl peptide receptors (FPRs) are G protein-coupled chemoattractant receptors expressed mainly in phagocytic leukocytes. High expression of FPRs has also been detected in several cancers but the functions of FPR1 in tumor invasion and metastasis is poorly understood. In this study, we investigated the expression of FPRs in primary human colorectal cancer (CRC) and analyzed the association of FPRs expression with clinicopathological parameters. The levels of FPRs mRNA, especially those of FPR1, were significantly higher in colorectal tumors than in distant normal tissues and adjacent non-tumor tissues. FPR1 mRNA expression was also associated with tumor serosal infiltration. FPR1 protein expression was both in the colorectal epitheliums and tumor infiltrating neutrophils/macrophages. Furthermore, the functions of FPR1 in tumor invasion and tissue repair were investigated using the CRC cell lines SW480 and HT29. Higher cell surface expression of FPR1 is associated with significantly increased migration in SW480 cells compared with HT29 cells that have less FPR1 membrane expression. Finally, genetic deletion of fpr1 increased the survival rate of the resulting knockout mice compared with wild type littermates in a mouse model of colitis-associated colorectal cancer. Our data demonstrate that FPR1 may play an important role in tumor cell invasion in CRC patients.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal interstitial lung disease characterized by consistent pulmonary inflammation. Although protein kinase C delta (PKCδ) is involved in broad scope cellular response, the role of PKCδ in IPF is complicated and has not been fully defined yet. Here, we reported that PKCδ deficiency (PKCδ-/-) aggravated bleomycin (BLM)-induced pulmonary fibrosis and inflammation. Upon challenge with BLM, the pulmonary capillary permeability, immune cell infiltration, inflammatory cytokine production, and collagen deposition were enhanced in PKCδ-/- mice compared to that in PKCδ+/+ mice. In response to poly(I:C) stimulation, PKCδ deficient macrophages displayed an increased production of IL-1β, IL-6, TNF-α, and IL-33, which were associated with an enhanced NF-κB activation. Furthermore, we found that PKCδ could directly bind to and phosphorylate A20, an inhibitory protein of NF-κB signal. These results suggested that PKCδ may inhibit the NF-κB signaling pathway via enhancing the stability and activity of A20, which in turn attenuates pulmonary fibrosis, suggesting that PKCδ is a promising target for treating pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible lung disease with limited therapeutic strategies. Lycorine (LYC), an alkaloid isolated from Amaryllidaceae family plants, exhibits effective anti-inflammatory, antiviral, and anti-tumor activities. In this study, we attempted to determine the effect of LYC on bleomycin (BLM)-induced IPF and NLRP3 inflammasome activation. Our results demonstrated that the LYC treatment ameliorated BLM-induced pulmonary fibrosis and inflammation in mice. LYC inhibited active Caspase-1 expression and lactate dehydrogenase (LDH) release during BLM-induced acute lung injury (ALI) in mice. Furthermore, our in vitro assay showed that LYC inhibited LPS/Nigericin- or LPS/ATP-induced NACHT, LRP and PYD domains-containing protein 3 (NLRP3) inflammasome activation, and pyroptosis in bone marrow-derived macrophages (BMDMs). Mechanically, LYC could disturb the interaction of NLRP3 with apoptosis-associated speck-like protein containing a CARD (ASC) by targeting the pyrin domain (PYD) on Leu9, Leu50, and Thr53. Our findings indicate that LYC ameliorated BLM-induced pulmonary fibrosis by inhibiting NLRP3 inflammasome activation and pyroptosis through targeting the PYD domain of ASC. Thus, LYC might be a potential therapeutic agent for pulmonary inflammation and fibrosis.

Cardiovascular manifestations account for marked morbi-mortality in autosomal dominant polycystic kidney disease (ADPKD). Pkd1- and Pkd2-deficient mice develop cardiac dysfunction, however the underlying mechanisms remain largely unclear. It is unknown whether impairment of polycystin-1 cleavage at the G-protein-coupled receptor proteolysis site, a significant ADPKD mutational mechanism, is involved in this process. We analyzed the impact of polycystin-1 cleavage on heart metabolism using Pkd1V/V mice, a model unable to cleave this protein and with early cardiac dysfunction. Pkd1V/V hearts showed lower levels of glucose and amino acids and higher lipid levels than wild-types, as well as downregulation of p-AMPK, p-ACCβ, CPT1B-Cpt1b, Ppara, Nppa and Acta1. These findings suggested decreased fatty acid β-oxidation, which was confirmed by lower oxygen consumption by Pkd1V/V isolated mitochondria using palmitoyl-CoA. Pkd1V/V hearts also presented increased oxygen consumption in response to glucose, suggesting that alternative substrates may be used to generate energy. Pkd1V/V hearts displayed a higher density of decreased-size mitochondria, a finding associated with lower MFN1, Parkin and BNIP3 expression. These derangements were correlated with increased apoptosis and inflammation but not hypertrophy. Notably, Pkd1V/V neonate cardiomyocytes also displayed shifts in oxygen consumption and p-AMPK downregulation, suggesting that, at least partially, the metabolic alterations are not induced by kidney dysfunction. Our findings reveal that disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice, expanding the understanding of heart dysfunction associated with Pkd1 deficiency and likely with human ADPKD.

Endogenous retroviruses (ERVs) are transposable elements that cause host genome instability and usually play deleterious roles in disease such as tumorigenesis. Recent advances also suggest that this "enemy within" may encode a viral mimic to induce antiviral immune responses through viral sensors. Here, through whole-genome transcriptome analysis with RNA sequencing (RNA-Seq), we discovered that a full-length ERV-derived long noncoding RNA (lncRNA), designated lnc-EPAV (ERV-derived lncRNA positively regulates antiviral responses), was a positive regulator of NF-κB signaling. lnc-EPAV expression was rapidly upregulated by viral RNA mimics or RNA viruses to facilitate the expression of RELA, an NF-κB subunit that plays a crucial role in antiviral responses. Transcriptome analysis of lnc-EPAV-silenced macrophages showed that lnc-EPAV was critical for RELA target gene expression and innate immune responses. Consistently, lnc-EPAV-deficient mice exhibited reduced expression of type I interferons (IFNs) and, consequently, increased viral loads and mortality following lethal RNA virus infection. Mechanistically, lnc-EPAV promoted expression of RELA by competitively binding to and displacing SFPQ, a transcriptional repressor of Rela Altogether, our work demonstrates an alternative mechanism by which ERVs regulate antiviral immune responses.IMPORTANCE Endogenous retroviruses are transposable genetic elements comprising 8% to 10% of the human and mouse genomes. Although most ERVs have been inactivated due to deleterious mutations, some are still transcribed. However, the biological functions of transcribed ERVs are largely unknown. Here, we identified a full-length ERV-derived lncRNA, designated lnc-EPAV, as a positive regulator of host innate immune responses. We found that silencing lnc-EPAV impaired virus-induced cytokine production, resulting in increased viral replication in cells. The lnc-EPAV-deficient mice exhibited enhanced susceptibility to viral challenge. We also found that lnc-EPAV regulated expression of RELA, an NF-κB subunit that plays a critical role in antiviral responses. ERV-derived lncRNA coordinated with a transcription repressor, SFPQ, to control Rela transcription. Our report provides new insights into the previously unrecognized immune gene regulatory mechanism of ERV-derived lncRNAs.

Background: Pancreatic cancer comprises not only cancer cells but also a collection of cross-talking noncancerous cells within tumor. Therefore, selective delivery of cytotoxic agents towards cancer cells and limiting the collateral damage to tumor suppressive benign cells, such as effector lymphocytes in the tumor microenvironment, is of great value. Methods: Pancreatic cancer cells harbor oncogenic KRAS which induces a constitutively high level of macropinocytosis. Inspired by such uniquity, we sought to explore the targeting potential of dextran, a biomaterial presumed to be endocytosed in the macropinocytosis dependent manner. Cell entry preference, mechanism and subcellular sorting of dextran with different molecular weights were firstly examined. Triptolide (TP), a potent cytotoxin was then set as the model payload for dextran conjugation. KRAS selectivity and the therapeutic effects of dextran-conjugated TP were investigated via both in vitro cellular studies and in vivo tumor model assessment. Results: Dextran, with a specific molecular weight of 70 kDa rather than other weights, was identified as a robust KRAS-responsive intracellular delivery carrier with enhanced entry upon KRAS mutation. The 70 kDa dextran-conjugated TP (DEX-TP) displayed greater efficacy and cellular deposition efficiency towards KRAS mutant cells than KRAS wild-type cells. Treatment with DEX-TP suppressed tumor progression in KRAS mutant pancreatic cancer orthotopic mouse models with reduced toxicity and significantly extended mouse survival time. Furthermore, the conjugate attained a more favorable therapeutic outcome in the tumor immune microenvironment than the free drug, preserving the fraction of T cells and their effector cytokines. Conclusions: In summary, macropinocytic dextran was able to provide drug delivery selectivity towards KRAS mutant cancer cells and reduce tumor immunity depletion caused by the cytotoxic drug in pancreatic cancer.

Glucocorticoid-induced TNF receptor-related (GITR) can act as a co-stimulatory receptor, representing a potential target for safely enhancing immunotherapy efficacy. GITR is triggered by a GITR ligand or an agonist antibody and activates CD8+ and CD4+ effector T cells, reducing tumor-infiltrating Treg numbers and resulting in activation of immune responses and tumor cell destruction by effector T cells. GITR is an attractive target for immunotherapy, especially in combination therapy with immune checkpoint inhibitors, as is being explored in clinical trials. Using H2L2 transgenic mice encoding the human immunoglobulin variable region and hybridoma technology, we generated a panel of fully human antibodies that showed excellent specific affinity and strong activation of human T cells. After conversion to fully human antibodies and engineering modification, we obtained an anti-GITR antibody hab019e2 with enhanced antitumor activity in a B-hGITR MC38 mouse model compared to Tab9H6V3, an anti-GITR antibody that activates T cells and inhibits Treg suppression from XenoMouse. As a fully human antibody with its posttranslational modification hot spot removed, the hab019e2 antibody exerted more potent therapeutic effects, and may have potential as a novel and developable antibody targeting GITR for follow-up drug studies.

Plexin-domain containing 2 (PLXDC2) has been reported as an oncoprotein in several human malignancies. However, its expression and roles in gastric cancer remain largely unclear. In this study, we found that PLXDC2 was highly expressed in gastric cancer tissues, and the expression levels were positively correlated with clinicopathological features, but negatively with the patients' outcome. Cox regression analysis identified PLXDC2 as an independent prognostic indicator for the patients. Knockdown of PLXDC2 markedly suppressed the in vitro invasion and in vivo metastasis of gastric cancer cells, while overexpression of PLXDC2 resulted in opposite effects. Mechanistically, PLXDC2 enhanced the level of phosphorylated Cortactin (p-Cortactin) by physically interacting with protein tyrosine phosphatase 1B (PTP1B), an important dephosphorylase, to prevent its dephosphorylating of p-Cortactin, thereby promoting the formation of invadopodia. Collectively, our results indicate that PLXDC2 contributes to the invasion and metastasis of gastric cancer by inhibiting PTP1B to facilitate the invadopodium formation, and may serve as a potential prognostic biomarker and a therapeutic target for this disease.

Nef is an accessory protein encoded by human immunodeficiency virus type-1 (HIV-1) and plays important roles in regulating HIV-1 infection and viral replication. Interestingly, HIV-1 Nef can promote degradation of numerous host proteins to disrupt cellular antiviral immune response. However, how HIV-1 Nef is degraded by host factors remains largely unexplored. Here, we identified c-Cbl as a host ubiquitin E3 ligase of HIV-1 Nef. We found that c-Cbl interacts with Nef and reduces protein levels of HIV-1 Nef. Further studies demonstrated that c-Cbl promoted Lys48-linked polyubiquitination of HIV-1 Nef, thus attenuating protein stability of HIV-1 Nef. Importantly, cellular c-Cbl ubiquitinated and degraded Nef proteins produced by HIV-1 NL4-3 virions, and ultimately attenuated HIV-1 virulence for infection of THP1 cells. This study reveals a ubiquitination and proteasome-dependent degradation mechanism of HIV-1 Nef protein, and could provide potential strategies for fighting against HIV-1.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor β-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor β) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.

Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called "Ultrafast flexible thin tube-based droplet digital PCR (utPCR)" that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.