A full genome tiling array enhanced the inspection and quarantine of SARS-CoV-2.

As the worldwide spreading epidemic of SARS-CoV-2, quick inspection and quarantine of passengers for SARS-CoV-2 infection are essential for controlling the spread of SARS-CoV-2, especially the cross-border transmission. This study reports a SARS-CoV-2 genome sequencing method based on a re-sequencing tiling array successfully used in border inspection and quarantine. The tiling array chip has four cores, with one core of 240,000 probes dedicated to the whole genome sequencing of the SAR-CoV-2 genome. The assay protocol has been improved to reduce the detection time to within one day and can detect 96 samples in parallel. The detection accuracy has been validated. This fast and simple procedure is also of low cost and high accuracy, and it is particularly suitable for the rapid tracking of viral genetic variants in custom inspection applications. Combining these properties means this method has significant application potential in the clinical investigation and quarantine of SARS-CoV-2. We used this SARS-CoV-2 genome re-sequencing tiling array to inspect and quarantine China's entry and exit ports in the Zhejiang Province. From November 2020 to January 2022, we observed the gradual shift of SARS-CoV-2 variants from the D614G type to the Delta Variant, and then to the dominance of the Omicron variant recently, consistently with the global emergency pattern of the new SARS-CoV-2 variant.

Description of an integrated management system for invasive mosquitoes at entry-exit ports in Zhejiang, China.

BACKGROUND: As mosquitoes are one of the most harmful creatures in the world, recent high-frequency interceptions of invasive mosquito species have emphasized the need to enhance the biological security of the Zhejiang Province in China. As such, an integrated management system should be implemented to monitor the vectors of mosquito-borne diseases during data digitization and the processing of permanent E-forms and provide an online one-stop identification service. METHODS: This system is a semi-open network built on the latest Microsoft.NET Framework, Active Server Page.NET (ASP.NET) and Internet Information Services (IIS) for the Windows 2000 service as a basic infrastructure platform. This creates a physical separation between the data input as the back-page intranet and the online automated Lucid identification as the front-page internet through the digital interchange platform and security firewall. RESULTS: This system mainly comprises three core modules: automated statistical analysis of operational data, online vector identification and digital specimen storage management, in addition to accessory modules. The joint analysis of invasive and native data collected between 2011 and 2017 at 14 surveillance points in the Zhejiang Province, excluding Ningbo Port, provided insights into the geographical differences in species abundance and the dynamic nature of seasonal interception within the statistical analysis module. Most importantly, multi-access keys to mosquitoes based on Lucid software were loaded in the module for vector identification. Subscribers can utilize this procedure for the online identification of 2 subfamilies, 10 genera and 33 mosquitoes by selecting any typical morphological feature in the classification system that matches the current images at hand. CONCLUSIONS: Our report suggests that this system can enhance the ability to master the basic information on invasive mosquitoes and satisfy the increasing requirements for public health safety in the integrated management of vector-borne diseases.

A cluster of Zika virus infection among travellers returning to China from Samoa: a case tracing study.

Background: A febrile man, who returned to China after a 9-day travel in Fiji and Samoa, was detected to be infected with Zika virus (ZIKV) at the port by Shenzhen Entry-Exit Inspection and Quarantine Bureau on 14 February 2016. Methods: The patient and his 32 travelling companions were traced for ZIKV infection. A standardised questionnaire was used to obtain the information on demographics, clinical manifestations and exposure history. Their samples were tested for ZIKV by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The positive samples were subjected to viral culture and genome sequencing. Findings: Four of the 33 travellers were confirmed to be infected by ZIKV through qRT-PCR and viral culture, with an overall infection rate of 12%. Interestingly, one case (Patient 3) where high viremia levels were tested 4 days prior to symptoms. In addition, a 7-year-old girl was identified to have ZIKV infection on 17 February, but never had any manifestation. ZIKV was isolated from the four imported cases. Phylogenetic analysis based on whole genome sequences revealed that these isolates were similar to each other and close to the strain causing the French Polynesia outbreak in 2013. Interpretation: The travellers should be informed of the high risk for ZIKV infection during their stay in areas with active transmission and measures to prevent mosquito bites.

Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis.

Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis; hence, perturbed Notch signaling may contribute to tumorigenesis. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in Africa and Asia. The mechanisms that orchestrate the multiple oncogenic insults required for initiation and progression of HCC are not clear. We constitutively overexpressed active Notch1 in human HCC to explore the effects of Notch1 signaling on HCC cell growth and to investigate the underlying molecular mechanisms. We show here that overexpression of Notch1 was able to inhibit the growth of HCC cells in vitro and in vivo. Biochemical analysis revealed the involvement of cell cycle regulated proteins in Notch1-mediated G(0)/G(1) arrest of HCC cells. Compared with green fluorescent protein (GFP) control, transient transfection of Notch1 ICN decreased expression of cyclin A (3.5-fold), cyclin D1 (2-fold), cyclin E (4.5-fold), CDK2 (2.8-fold), and the phosphorylated form of retinoblastoma protein (3-fold). Up-regulation of p21(waf/cip1) protein expression was observed in SMMC7721-ICN cells stably expressing active Notch1 but not in SMMC7721-GFP cells, which only express GFP. Furthermore, a 12-fold increase in p53 expression and an increase (4.8-fold) in Jun-NH(2)-terminal kinase activation were induced in SMMC7721-ICN cells compared with SMMC7721-GFP cells. In contrast, expression of the antiapoptotic Bcl-2 protein could not be detected in SMMC7721-ICN cells. These findings suggest that Notch1 signaling may participate in the development of HCC cells, affecting multiple pathways that control both cell proliferation and apoptosis.

Up-regulation of TLR9 gene expression by LPS in mouse macrophages via activation of NF-kappaB, ERK and p38 MAPK signal pathways.

Toll-like receptors (TLR) are critical in the activation of macrophages by bacterial products. It has been shown that TLR2 and TLR4 mediate lipopolysaccharide (LPS) and lipoproteins signal transduction, respectively. Regulation of TLR2 and TLR4 expression by LPS was considered to be one of the mechanisms to control the overall responses of immune cells to bacteria. However, little is known about whether the other members of TLR family are regulated by LPS. Recently, TLR9 was demonstrated to be essential for CpG DNA signaling. Given the effective immune modulation by CpG DNA, regulation of TLR9 expression might play important role in controlling the overall responses of immune cells to bacteria. In this study, regulation of TLR9 gene expression in mouse macrophage cell line RAW264.7 by LPS was investigated. Semiquantitative RT-PCR was performed to determine gene expression of TLR9. Following LPS stimulation, TLR9 gene expression was upregulated within 1 h and reached peak level at about 3 h. LPS stimulation activated NF-kappaB, ERK and p38 MAPK signal pathways. Pretreatment of macrophages with inhibitors of NF-kappaB, ERK and p38 MAPK signal pathways inhibited LPS-induced upregulation of TLR9 mRNA expression. Our results demonstrated that LPS stimulation could upregulate gene expression of TLR9 via NF-kappaB, ERK, and p38 MAPK signal pathways in macrophages, indicating that macrophages with increased TLR9 expression induced by LPS might respond to invading bacteria more effectively.

Intratumoral expression of MIP-1beta induces antitumor responses in a pre-established tumor model through chemoattracting T cells and NK cells.

Direct intratumoral introduction of therapeutic or regulatory genes is a developing technology with potential application for cancer gene therapy. Macrophage inflammatory protein-1 beta (MIP-1beta) is a chemokine which can chemoattract immune cells such as T cells. In the present study, murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus (AdhMIP-1beta) carrying the human MIP-1beta gene. 24 h post-transfection, hMIP-1beta levels reached approximately 980 pg/ml in supernatants of 10(6) hMIP-1beta-transfected CT26 cells. Moreover, the supernatants exhibited chemotactic activity for CD8(+) T cells, CD4(+) T cells, NK cells and immature DCs. Intratumoral injection of AdhMIP-1beta significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice. Intratumoral hMIP-1beta gene transfer also induced powerful tumor-specific CTL responses in vivo. The therapeutic effects of hMIP-1beta gene therapy were greatly reduced following in vivo depletion of both CD4(+) and CD8(+) T cells, but were unaffected by depletion of single T cell subsets. Immune cell depletion experiments also revealed that NK cells played an important role in hMIP-1beta-induced antitumor responses. These results suggest that intratumoral expression of hMIP-1beta has the potential effect to induce host antitumor immunity and may prove to be a useful form of cancer gene therapy.

Notch1 signaling sensitizes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human hepatocellular carcinoma cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression.

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.

Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism.

Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via the phosphoinositide 3-kinase (PI-3K) pathway by catalyzing the PI-3K product PtdIns-3,4,5P3 (phosphatidylinositol-3,4,5-triphosphate) into PtdIns-3,4P2. However, the role of SHIP1 in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response remains unclear. Here we demonstrate that SHIP1 negatively regulates LPS-induced inflammatory response via both phosphatase activity-dependent and -independent mechanisms in macrophages. SHIP1 becomes tyrosine phosphorylated and up-regulated upon LPS stimulation in RAW264.7 macrophages. SHIP1-specific RNA-interfering and SHIP1 overexpression experiments demonstrate that SHIP1 inhibits LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by negatively regulating the LPS-induced combination between TLR4 and myeloid differentiation factor 88 (MyD88); activation of Ras (p21(ras) protein), PI-3K, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK); and degradation of IkappaB-alpha. SHIP1 also significantly inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation in TLR4-reconstitited COS7 cells. Although SHIP1-mediated inhibition of PI-3K is dependent on its phosphatase activity, phosphatase activity-disrupted mutant SHIP1 remains inhibitory to LPS-induced TNF-alpha production. Neither disrupting phosphatase activity nor using the PI-3K pathway inhibitor LY294002 or wortmannin could significantly block SHIP1-mediated inhibition of LPS-induced ERK1/2, p38, and JNK activation and TNF-alpha production, demonstrating that SHIP1 inhibits LPS-induced activation of MAPKs and cytokine production primarily by a phosphatase activity- and PI-3K-independent mechanism.

Ras participates in CpG oligodeoxynucleotide signaling through association with toll-like receptor 9 and promotion of interleukin-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor 6 complex formation in macrophages.

CpG oligodeoxynucleotides (ODN) activate immune cells to produce immune mediators by Toll-like receptor 9 (TLR9)-mediated signal transduction, which activates mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) through the MyD88/IRAK/TRAF6 kinases cascade. However, the precise mechanisms of CpG ODN activation of immune cells have not been fully elucidated. The small GTP-binding protein Ras mediates MAPK activation in response to a variety of stimuli. Up to now, it is not clear whether Ras plays a role in CpG ODN signaling. In the present study, we found that the dominant-negative version of Ras (RasN17) and specific Ras inhibitor, FTI-277, inhibited CpG ODN-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by murine macrophage cell line RAW264.7. While overexpression of wild-type Ras enhanced CpG ODN-induced extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and NF-kappaB activation, overexpression of RasN17 inhibited CpG ODN-induced ERK, JNK, and NF-kappaB activation. RasN17 overexpression also inhibited CpG ODN-induced IRAK1/TRAF6 complex formation. Further studies revealed that CpG ODN activated Ras in a time- and dose-dependent manner, and Ras associated with TLR9 in a CpG ODN-dependent manner. Most interestingly, activation of Ras preceded the association of Ras with TLR9, giving rise to a possibility that Ras activation might not be dependent on the interaction between Ras and TLR9. Our data demonstrate for the first time that Ras can be activated by CpG ODN in macrophages, and Ras is involved in CpG ODN signaling as an early event by associating with TLR9 and promoting IRAK1/TRAF6 complex formation, and MAPK and NF-kappaB activation.

Psoriatic lesional keratinocytes promote the maturation of human monocyte-derived Langerhans cells.

BACKGROUND: Langerhans cells (LCs) are important antigen-presenting cells in the epidermis and may play a key role in the pathogenesis of psoriasis. It has been proven that LCs isolated from psoriatic lesions are abnormal. However, the mechanism of the abnormality has not been reported so far. OBJECTIVE: In the present study, we investigated the effect of psoriatic lesional keratinocytes on the maturation of LCs. METHODS: Monocytes isolated from healthy peripheral blood could differentiate into LCs in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL) 4 and transforming growth factor beta1 for 5 days. Then, human monocyte-derived LCs were cultured with supernatants from psoriatic lesional keratinocytes for another 2 days. Their phenotypes and phagocytic capacity were analyzed by flow cytometry. IL-12 secreted by LCs was determined by ELISA. RESULTS: Supernatants from psoriatic lesional keratinocytes could up-regulate the expression of HLA-DR and CD86 on LCs more significantly than supernatants from healthy keratinocytes, but less powerfully than lipopolysaccharide. The levels of IL-12 secreted by LCs also increased. In contrast, the expression of CD1a on LCs and their phagocytic capacity were reduced. CONCLUSION: Human monocyte-derived LCs cultured with supernatants from psoriatic lesional keratinocytes displayed the characteristics of maturation. This suggests that psoriatic lesional keratinocytes might secrete some factors that could promote the maturation of LCs, which may play important roles in immune reactions related to psoriasis.

Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells.

Toll-like receptors (TLR) are sentinel receptors capable of recognizing pathogen-associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG-containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram-positive and Gram-negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up-regulation of co-stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up-regulated following LPS stimulation. The up-regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor-alpha induced by LPS plus CpG ODN. While inhibition of extracellular signal-related kinase and NF-kappaB activation suppressed the up-regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up-regulation of TLR2 and TLR4 mRNA expression but enhanced the up-regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up-regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.