PddCas: A Polydisperse Droplet Digital CRISPR/Cas-Based Assay for the Rapid and Ultrasensitive Amplification-Free Detection of Viral DNA/RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays have been an emerging diagnostic technology for pathogen diagnosis. In this work, we developed a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) for the rapid and ultrasensitive amplification-free detection of viral DNA/RNA with minimum instruments. LbaCas12a and LbuCas13a were used for the direct detection of viral DNA and RNA, respectively. The reaction mixtures were partitioned with a common vortex mixer to generate picoliter-scale polydisperse droplets in several seconds. The limit of detection (LoD) for the target DNA and RNA is approximately 100 aM and 10 aM, respectively, which is about 3 × 104-105 fold more sensitive than corresponding bulk CRISPR assays. We applied the PddCas to successfully detect severe acute respiratory syndrome coronavirus (SARS-CoV-2) and human papillomavirus type 18 (HPV 18) in clinical samples. For the 23 HPV 18-suspected cervical epithelial cell samples and 32 nasopharyngeal swabs for SARS-CoV-2, 100% sensitivity and 100% specificity were demonstrated. The dual-gene virus detection with PddCas was also established and verified. Therefore, PddCas has potential for point-of-care application and is envisioned to be readily deployed for frequent testing as part of an integrated public health surveillance program.

Circulating exosomal long non-coding RNA H19 as a potential novel diagnostic and prognostic biomarker for gastric cancer.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are involved in carcinogenesis and could be used as diagnostic biomarkers. Our study aimed to elucidate the clinical role of serum exosomal lncRNA H19 in gastric cancer (GC). METHODS: In this prospective clinical study, we determined serum exosomal lncRNA H19 levels in 81 patients with GC and analysed the correlations between serum lncRNA H19 levels and clinical characteristics. Receiver operating characteristics (ROC) curves were constructed to determine the diagnostic performance of exosomal lncRNA H19 in GC. RESULTS: Serum exosomal lncRNA H19 levels were significantly upregulated in patients with GC both before and after surgery compared with healthy controls. Furthermore, serum exosomal lncRNA H19 levels were significantly decreased after compared with before surgery in patients with GC. Preoperative lncRNA H19 levels were significantly correlated with TNM stage. The area under the ROC curve (AUC) value for exosomal lncRNA H19 was 0.849, which was significantly higher than the AUC values for cancer antigens 19-9 and 72-4 and carcinoembryonic antigen, either alone or combined. CONCLUSIONS: These results suggest that circulating exosomal lncRNA H19 may be a potential biomarker with diagnostic and prognostic value in GC.

MDP up-regulates the gene expression of type I interferons in human aortic endothelial cells.

Muramyldipeptide (MDP), the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2). Here, we obtained evidence that the treatment of human aortic endothelial cells (HAECs) with MDP up-regulated the gene expression of type I interferons in a dose- and time-dependent manner. MDP also up-regulated the expression of the receptor NOD2, suggesting that MDP may induce a positive feedback response. The up-regulation of interferons was not dependent on the TNFa signaling, as HAECs did not express TNFa with the stimulation of MDP, and TNFa neutralizing antibody did not decrease the induction of IFNs induced by MDP. RT-PCR results showed that HAECs expressed the gene transcripts of interferon regulatory factor (IRF) 1, 2, 3, 9. The western blot results showed that MDP induced the phosphorylation of IRF3. These results suggested that MDP induced the up-regulation of gene transcript of interferons through the activation of IRF3 signaling pathway. Meanwhile, MDP induced the gene expression of pro-inflammatory cytokines, including IL-1ß, IL-8, and MCP-1. Taken together, these results suggested that HAECs may play roles in the anti-infection immune response and in the induction of innate immunity.

Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells.

Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 µM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.

The functional expression of TLR3 in EPCs impairs cell proliferation by induction of cell apoptosis and cell cycle progress inhibition.

Toll-like receptor 3 (TLR3), a member of the TLR family that recognizes double-stranded RNA (dsRNA), plays an important role in antiviral immunity. TLR3 is widely expressed in various cells and the activation of TLR3 induces cell apoptosis in some cells. However, the effect of TLR3 on cell proliferation in endothelial progenitor cells (EPCs) is unclear. In this study, we found that EPCs expressed high levels of TLR1, 3, 4, and 6 and low levels of TLR2, 5, 7, 8, and 10. The treatment of EPCs with TLR3 agonist Poly I:C up-regulated the expression of cytokines IL-1ß, IL-6, IL-8, TNF-α, IFN-α, and IFN-ß, indicating that EPCs expressed functional TLR3. Moreover, Poly I:C treatment induced cell cycle progress inhibition and cell apoptosis, leading to the inhibition of cell proliferation. Further studies indicated that IL-1ß was involved in TLR3-induced cell proliferation inhibition, as IL-1ß inhibited cell proliferation in a dose-dependent manner, and the IL-1ß receptor type I (IL-1R1)-neutralizing antibody ameliorated Poly I:C-induced cell proliferation inhibition. Taken together, these results suggest that Poly I:C impairs cell proliferation by inducing cell cycle progress inhibition and cell apoptosis via TLR3 in EPCs.

P2Y11 impairs cell proliferation by induction of cell cycle arrest and sensitizes endothelial cells to cisplatin-induced cell death.

Extracellular ATP mediates a wide range of physiological effects, including cell proliferation, differentiation, maturation, and migration. However, the effect of ATP on cell proliferation has been contradictory, and the mechanism is not fully understood. In the current study, we found that extracellular ATP significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Treatment with ATP did not induce cell apoptosis but instead induced cell cycle arrest in S phase. ATP induced the phosphorylation of ERK1/2, but the ERK inhibitors, U0126 and PD9809, did not regulate the inhibition of cell proliferation induced by ATP. However, ATP-induced inhibition of cell proliferation was blocked by suramin, a nonspecific antagonist of the P2Y receptors, and endothelial cells expressed P2Y11, a P2Y receptor that specifically binds ATP. Moreover, the down-regulation of P2Y11 by RNA interference not only reversed the inhibition of cell proliferation but also ameliorated cell cycle arrest in S phase. In addition, P2Y11 sensitized endothelial cells to cisplatin-induced cell death by down-regulation of the expression of Bcl-2. Taken together, these results suggest that extracellular ATP impairs cell proliferation by triggering signaling to induce cell cycle arrest and sensitizes cell to death via P2Y11 in endothelial cells.

Toll-like receptor 3 (TLR3) induces apoptosis via death receptors and mitochondria by up-regulating the transactivating p63 isoform alpha (TAP63alpha).

Toll-like receptor 3 (TLR3), a member of the pathogen recognition receptors, is widely expressed in various cells and has been shown to activate immune signaling pathways by recognizing viral double-stranded RNA. Recently, it was reported that the activation of TLR3 induced apoptosis in some cells, but the detailed molecular mechanism is not fully understood. In this study, we found that in endothelial cells polyinosinic-polycytidylic acid (poly(I-C)) induced dose- and time-dependent cell apoptosis, which was elicited by TLR3 activation, as TLR3 neutralization and down-regulation repressed the apoptosis. Poly(I-C) induced the activation of both caspases 8 and 9, indicating that TLR3 triggered the signaling of both the extrinsic and intrinsic apoptotic pathways. Poly(I-C) up-regulated tumor necrosis factor-related apoptosis-inducing ligand and its receptors, death receptors 4/5, resulting in initiating the extrinsic pathway. Furthermore, poly(I-C) down-regulated anti-apoptotic protein, B cell lymphoma 2 (Bcl-2), and up-regulated Noxa, a key Bcl-2 homology 3-only antagonist of Bcl-2, leading to the priming of the intrinsic pathway. A p53-related protein, the transactivating p63 isoform α (TAp63α), was induced by TLR3 activation and contributed to the activation of both the intrinsic and extrinsic apoptotic pathways. Both the cells deficient in p63 gene expression by RNA interference and cells that overexpressed the N-terminally truncated p63 isoform α (ΔNp63α), a dominant-negative variant of TAp63α, by gene transfection, survived TLR3 activation. Taken together, TAp63α is a crucial regulator downstream of TLR3 to induce cell death via death receptors and mitochondria.

The nuclear localization of CAPON in hippocampus and cerebral cortex neurons after lipopolysaccharide stimulation.

OBJECTIVE: In the brain, nitric oxide (NO) is a retrograde signalling molecule that transfers information from post- to pre-synaptic nerve endings. NO has been shown to be an important inflammatory mediator responding to lipopolysaccharide (LPS). It has been stated that the constitutive NO synthase isoforms (nNOS and eNOS) may also contribute to the inflammation. CAPON, a nNOS regulator, helps regulate nNOS stability, localization and possibly expression during synapse formation as well as muscle re-innervation. Recently, it has been reported that CAPON is associated with psychiatric illness. So we speculated that the CAPON expression-induced physiological changes may be mediated by modifications of NOS-NO signalling pathways. But little is known about the role of CAPON during the inflammation in the central nervous system, so we investigated the expression of CAPON in the brain treated with LPS. METHODS AND RESULTS: Real-time PCR and Western blot showed that the expression of CAPON increased at mRNA and protein levels in the brain after LPS stimulation. Immunocytochemistry staining revealed that CAPON localized in the nuclei of neurons in the brain after peritoneal injection with LPS in vivo. The same phenomenon was also shown in primary cultured neurons in vitro incubated with LPS for 36 h. In addition, we found that CAPON had a colocalization with phosphorylated nNOS Ser847 but not with nNOS in hippocampus and cerebral cortex by double immunofluorescence. CONCLUSION: CAPON localized in the nuclei of neurons in hippocampus and cerebral cortex after LPS treatment. Because CAPON competed with PSD-95 for binding nNOS in neurons and nNOS was activated to produce NO through the NMDA- NMDAR pathway, we hypothesized that CAPON might play a proactive role in the process of inflammation by transferring from cytoplasm to the nucleus and through the NMDA-nNOS signal pathway. Further studies are required to clarify the mechanism of the nuclear localization of CAPON and the possible relationship with nNOS/NO signalling.

Elevated expression of CAPON and neuronal nitric oxide synthase in the sciatic nerve of rats following constriction injury.

Neuronal nitric oxide synthase (nNOS) has been implicated in peripheral nerve lesions and regeneration. The CAPON adaptor protein interacts with the PDZ domain of nNOS, helping to regulate nNOS activity at post-synaptic sites in neurones, but it is not known whether its expression is altered in sciatic nerves after chronic nerve constriction injury. In the present study, the spatiotemporal expression of CAPON was determined in chronically constricted rat sciatic nerves. Similar to the level of protein expression, CAPON mRNA was significantly up-regulated for almost 5weeks following sciatic nerve injury. Immunohistochemistry demonstrated that increased CAPON was found mainly in S-100-positive Schwann cells. In addition, co-immunoprecipitation demonstrated an interaction between CAPON and nNOS in Schwann cells and the interaction was enhanced in injured sciatic nerves. CAPON may be involved in peripheral nerve regeneration through regulation of nNOS activity.

The expression of functional Toll-like receptor 4 is associated with proliferation and maintenance of stem cell phenotype in endothelial progenitor cells (EPCs).

Endothelial dysfunction is involved in various cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) contribute to re-endothelialization and neo-vascularization, and the increase of EPCs in peripherial circulation benefits the prognosis of cardiovascular disease. However, little is known about the biological stimuli that initiate the proliferation and the maintenance of stem cell phenotype of EPCs. Here we reported that human umbilical vein blood derived EPCs expressed gene transcripts coding for Toll-like receptor (TLR) 1-6, TLR8-10, TLR4 co-receptor CD14, and myeloid differentiation factor 88 (MyD88), a TLR adaptor molecule. Protein expression of TLR2, 4, CD14, and MyD88 was also detected by FACS or Western blot. The activation of TLR4 by LPS modulated the expression of TLRs, induced the phosphorylation of NF-kappaB, P38, and ERK42/44, and up-regulated the gene expression of cytokines IL-8, IFN-alpha, IFN-beta, and TNF-alpha, suggesting EPCs expressed functional TLR4. Unexpectedly LPS treatment failed to induce apoptosis in EPCs, but instead promoted cell proliferation of EPCs. Furthermore, the treatment of EPCs with LPS up-regulated stem cell markers AC133 and CD34 in both mRNA and protein levels, and down-regulated the protein expression of differential marker eNOS. These results suggested that TLR4 functions to maintain the stem cell phenotype of EPCs and enlarge its population, which reveals a novel aspect of the multiple-faced TLR biology, and may open new prospects for using TLR4 agonists to promote the production of EPCs for clinical use.

Inhibition of nitric oxide-induced nuclear localization of CAPON by NMDA receptor antagonist in cultured rat primary astrocytes.

Astrocytes play a key role in regulating aspects of inflammation in the central nervous system. It was observed that nNOS had located in the nucleus of cultured cerebral cortical astrocytes of 7 days. In the present study, we found that carboxy-terminal PDZ ligand of nNOS (CAPON) mainly located in the nucleus of astrocytes stimulated with NO donor sodium nitroprusside (SNP) or GSNO or N-methyl-d-aspartate (NMDA) receptor agonist-NMDA. However, originally, it was localized mostly in the cytoplasm of normal astrocytes. Immunocytochemistry showed that nNOS was co-localized with CAPON in the nucleus of astrocytes stimulated with SNP. In addition to the nuclear localization, treatment with SNP increased the mRNA and protein expression of CAPON. When SNP was removed from media, CAPON accumulated in nucleus transported back to cytoplasm. MK801, an inhibitor of NMDA receptor, was able to reverse the nuclear localization of CAPON resulted from SNP, suggesting that there is a functional relationship of NO with NMDA receptor in the regulation of the nuclear localization of CAPON. These findings provide a new insight in the understanding of the physical and pathological significances of CAPON/nNOS/NMDA receptor.

Altered gene expression of NIDD in dorsal root ganglia and spinal cord of rats with neuropathic or inflammatory pain.

Nitric oxide and nitric oxide synthases are key players in synaptic plasticity events in spinal cord (SC), which underlies the chronic pain states. To date, little is known about the molecular mechanisms regulating the activity of nitric oxide synthases in nociceptive systems. The present study was aimed at the determination of the gene expression of nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD), a recently identified protein regulating nNOS enzyme activity, in rat SC and dorsal root ganglia (DRG) and studying its regulation in states of nociceptive hypersensitivity in a rat model of neuropathic or inflammatory pain. It was found that NIDD mRNA was predominantly expressed in nociceptive primary neurons and in neurons of the spinal dorsal horn (DH) and the number of NIDD-positive neurons in the corresponding DRG or SC increased significantly following induction of chronic hyperalgesia. Meanwhile, remarkable changes of nNOS were detected under such pain conditions. Our data suggest a potential role for NIDD in the maintenance of thermal pain hypersensitivity possibly via regulating the nNOS activity.

Changes in mRNA for CAPON and Dexras1 in adult rat following sciatic nerve transection.

Peripheral nerve transection has been implicated to cause a production of neuronal nitric oxide synthase (nNOS), which may influence a range of post-axotomy processes necessary for neuronal survival and nerve regeneration. Carboxy-terminal post synaptic density protein/Drosophila disc large tumor suppressor/zonula occuldens-1 protein (PDZ) ligand of neuronal nitric oxide synthase (CAPON), as an adaptor, interacts with nNOS via the PDZ domain helping regulate nNOS activity at postsynaptic sites in neurons. And Dexras1, a small G protein mediating multiple signal transductions, has been reported to form a complex with CAPON and nNOS. A role for the physiologic linkage by CAPON of nNOS to Dexras1 has suggested that NO-mediated activation of Dexras1 is markedly enhanced by CAPON. We investigated the changes in mRNA for CAPON, Dexras1 and nNOS in the sciatic nerve, dorsal root ganglia and lumbar spinal cord of adult rat following sciatic axotomy by TaqMan quantitative real-time PCR and in situ hybridization combined with immunofluorescence. Signals of mRNA for CAPON and Dexras1 were initially expressed in these neural tissues mentioned, transiently increased at certain time periods after sciatic axotomy and finally recovered to the basal level. It was also found that nNOS mRNA underwent a similar change pattern during this process. These results suggest that CAPON as well as Dexras1 may be involved in the different pathological conditions including nerve regeneration, neuron loss or survival and even pain process, possibly via regulating the nNOS activity or through the downstream targets of Dexras1.