Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

UMP-CMP kinase 2 inhibits ZIKV replication through activation of type I IFN signaling pathway.

Cytidine/uridine monophosphate kinase 2 (UMP-CMP kinase 2, CMPK2) has been reported as an antiviral interferon-stimulated gene (ISG). We previously observed that the expression of CMPK2 was significantly upregulated after Zika Virus (ZIKV) infection in A549 cells. However, the association and the underlying mechanisms between CMPK2 induction and ZIKV replication remain to be determined. We investigated the induction of CMPK2 during ZIKV infection and the effect of CMPK2 on ZIKV replication in A549, U251, Vero, IFNAR-deficient U5A and its parental 2fTGH cells, Huh7 and its RIG-I-deficient derivatives Huh7.5.1 cells. The activation status of Jak-STAT signaling pathway was determined by detecting the phosphorylation level of STAT1, the activity of interferon stimulated response element (ISRE) and the expression of several interferon stimulated genes (ISGs). We found that ZIKV infection induced CMPK2 expression through an IFNAR and RIG-I dependent manner. Overexpression of CMPK2 inhibited while CMPK2 knockdown promoted ZIKV replication in A549 and U251 cells. Mechanically, we found that CMPK2 overexpression increased IFNß expression and activated Jak/STAT signaling pathway as shown by the increased level of p-STAT1, enhanced activity of ISRE, and the upregulated expression of downstream ISGs. These findings suggest that ZIKV infection induced CMPK2 expression, which inhibited ZIKV replication and serves as a positive feedback regulator for IFN-Jak/STAT pathway.

HIV coinfection exacerbates HBV-induced liver fibrogenesis through a HIF-1α- and TGF-ß1-dependent pathway.

BACKGROUND & AIMS: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-ß1 signaling. METHODS: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. RESULTS: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-ß1 (TGF-ß1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-ß1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-ß1-induced fibrogenesis. CONCLUSIONS: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-ß1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. IMPACT AND IMPLICATIONS: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-ß1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.

METTL3 promotes osteogenic differentiation of human umbilical cord mesenchymal stem cells by up-regulating m6A modification of circCTTN.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising seed cells in bone tissue engineering. circRNA and N6-methyladenosine (m6A) RNA methylation play important roles in osteogenic differentiation. Here, we investigated the potential relevance of a critical circRNA, hsa_circ_0003376 (circCTTN), and methyltransferase-like 3 (METTL3) in osteogenic differentiation of hUCMSCs. METHODS: Expression of circCTTN after hUCMSC osteogenic induction was detected by qRT-PCR. Three databases (RMBase v2.0, BERMP, and SRAMP) were used to predict m6A sites of circCTTN. RNA was enriched by methylated RNA immunoprecipitation (MeRIP), followed by quantitative real-time polymerase chain reaction to detect m6A level of circCTTN after METTL3 overexpression and osteogenic induction. RNA pull-down, Western blotting, and protein mass spectrometry were performed to investigate the potential mechanisms by which METTL3 promoted m6A modification of circCTTN. Bioinformatic analyses based on database (STRING) search and co-immunoprecipitation were used to analyze the proteins that interacted with METTL3. RESULTS: Overexpression of METTL3 promoted osteogenic differentiation of hUCMSCs and increased m6A level of circCTTN. Two potential m6A modification sites of circCTTN were predicted. No direct interaction between METTL3 and circCTTN was observed. Thirty-one proteins were pulled down by probes specific for circCTTN, including NOP2, and two m6A reading proteins, EIF3A and SND1. Bioinformatics analysis and co-immunoprecipitation showed that METTL3 interacted with EIF3A indirectly through NOP2. CONCLUSIONS: METTL3 promotes the osteogenic differentiation of hUCMSCs by increasing the m6A level of circCTTN. However, METTL3 does not bind directly to circCTTN. METTL3 interacts with circCTTN indirectly through NOP2 and EIF3A.

Hsa_circ_0007321 regulates Zika virus replication through miR-492/NFKBID/NF-κB signaling pathway.

IMPORTANCE: Over the past decade, increasing evidence has shown that circular RNAs (circRNAs) play important regulatory roles in viral infection and host antiviral responses. However, reports on the role of circRNAs in Zika virus (ZIKV) infection are limited. In this study, we identified 45 differentially expressed circRNAs in ZIKV-infected A549 cells by RNA sequencing. We clarified that a downregulated circRNA, hsa_circ_0007321, regulates ZIKV replication through targeting of miR-492 and the downstream gene NFKBID. NFKBID is a negative regulator of nuclear factor-κB (NF-κB), and we found that inhibition of the NF-κB pathway promotes ZIKV replication. Therefore, this finding that hsa_circ_0007321 exerts its regulatory role on ZIKV replication through the miR-492/NFKBID/NF-κB signaling pathway has implications for the development of strategies to suppress ZIKV and possibly other viral infections.

Mitophagy Activation Targeting PINK1 Is an Effective Treatment to Inhibit Zika Virus Replication.

Mitophagy is a selective degradation mechanism that maintains mitochondrial homeostasis by eliminating damaged mitochondria. Many viruses manipulate mitophagy to promote their infection, but its role in Zika virus (ZIKV) is unclear. In this study, we investigated the effect of mitophagy activation on ZIKV replication by the mitochondrial uncoupling agent niclosamide. Our results demonstrate that niclosamide-induced mitophagy inhibits ZIKV replication by eliminating fragmented mitochondria, both in vitro and in a mouse model of ZIKV-induced necrosis. Niclosamide induces autophosphorylation of PTEN-induced putative kinase 1 (PINK1), leading to the recruitment of PRKN/Parkin to the outer mitochondrial membrane and subsequent phosphorylation of ubiquitin. Knockdown of PINK1 promotes ZIKV infection and rescues the anti-ZIKV effect of mitophagy activation, confirming the role of ubiquitin-dependent mitophagy in limiting ZIKV replication. These findings demonstrate the role of mitophagy in the host response in limiting ZIKV replication and identify PINK1 as a potential therapeutic target in ZIKV infection.

Vaccination increased host antiviral gene expression and reduced COVID-19 severity during the Omicron variant outbreak in Fuyang City, China.

BACKGROUND: The differences in host antiviral gene expression and disease severity between vaccinated and non-vaccinated coronavirus disease 2019 (COVID-19) patients are not well characterized. We sought to compare the clinical characteristics and host antiviral gene expression patterns of vaccinated and non-vaccinated cohorts at the Second People's Hospital of Fuyang City. METHODS: In this case-control study, we retrospectively analyzed 113 vaccinated patients with a COVID-19 Omicron variant infection, 46 non-vaccinated COVID-19 patients, and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We compared host antiviral gene expression profiles between healthy controls and COVID-19 patients who were either vaccinated or non-vaccinated at the time of infection. RESULTS: In the vaccinated group, most patients were asymptomatic, with only 42.9 % of patients developing fever. Notably, no patients had extrapulmonary organ damage. In contrast, 21.4 % of patients in the non-vaccinated group developed severe/critical (SC) disease and 78.6 % had mild/moderate (MM) disease, with fever occurring in 74.2 % patients. We found that Omicron infection in COVID-19 vaccinated patients was associated with significantly increased expression of several important host antiviral genes including IL12B, IL13, CXCL11, CXCL9, IFNA2, IFNA1, IFNγ, and TNFα. CONCLUSION: Vaccinated patients infected with the Omicron variant were mostly asymptomatic. In contrast, non-vaccinated patients frequently developed SC or MM disease. Older patients with SC COVID-19 also had a higher occurrence of mild liver dysfunction. Omicron infection in COVID-19 vaccinated patients was associated with the activation of key host antiviral genes and thus may play a role in reducing disease severity.

Functionalized protein microparticles targeting hACE2 as a novel preventive strategy for SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), resulting in a serious burden on public health and social economy worldwide. SARS-CoV-2 infection is mainly initialized in the nasopharyngeal cavity through the binding of viral spike (S) protein to human angiotensin-converting enzyme 2 (hACE2) receptors which are widely expressed in many human cells. Thus, blockade of the interaction between viral S protein and hACE2 receptor in the primary entry site is a promising prevention strategy for the management of COVID-19. Here we showed protein microparticles (PMPs) decorated with hACE2 could bind and neutralize SARS-CoV-2 S protein-expressing pseudovirus (PSV) and protect host cells from infection in vitro. In the hACE2 transgenic mouse model, administration of intranasal spray with hACE2-decorated PMPs markedly decreased the viral load of SARS-CoV-2 in the lungs though the inflammation was not attenuated significantly. Our results provided evidence for developing functionalized PMPs as a potential strategy for preventing emerging air-borne infectious pathogens, such as SARS-CoV-2 infection.

Role of noncoding RNAs in liver fibrosis.

Liver fibrosis is a wound-healing response following chronic liver injury caused by hepatitis virus infection, obesity, or excessive alcohol. It is a dynamic and reversible process characterized by the activation of hepatic stellate cells and excess accumulation of extracellular matrix. Advanced fibrosis could lead to cirrhosis and even liver cancer, which has become a significant health burden worldwide. Many studies have revealed that noncoding RNAs (ncRNAs), including microRNAs, long noncoding RNAs and circular RNAs, are involved in the pathogenesis and development of liver fibrosis by regulating signaling pathways including transforming growth factor-ß pathway, phosphatidylinositol 3-kinase/protein kinase B pathway, and Wnt/ß-catenin pathway. NcRNAs in serum or exosomes have been reported to tentatively applied in the diagnosis and staging of liver fibrosis and combined with elastography to improve the accuracy of diagnosis. NcRNAs mimics, ncRNAs in mesenchymal stem cell-derived exosomes, and lipid nanoparticles-encapsulated ncRNAs have become promising therapeutic approaches for the treatment of liver fibrosis. In this review, we update the latest knowledge on ncRNAs in the pathogenesis and progression of liver fibrosis, and discuss the potentials and challenges to use these ncRNAs for diagnosis, staging and treatment of liver fibrosis. All these will help us to develop a comprehensive understanding of the role of ncRNAs in liver fibrosis.

Refractory acute promyelocytic leukemia with PLZF/RARa rearrangement: a case report and literature review.

Acute promyelocytic leukemia patients with PLZF-RARa rearrangement have no obvious differentiation-inducing effect on retinoic acid, have a poor response to traditional chemotherapy, and have poor overall prognosis. A case of acute promyelocytic leukemia with PLZF / RARa rearrangement reported in this article was treated with induction chemotherapy with arsenic trioxide combined with a new anthracycline (idarubicin) cytotoxic chemotherapy. The patient achieved complete response in the bone marrow. After the first induction, and achieved molecular remission after the second consolidation chemotherapy. At present, the patient was followed up for 40 months after hematological and cytogenetic remission, and the PLZF / RARa real-time PCR test was continuously negative.

CRISPR/Cas12a-Enhanced Loop-Mediated Isothermal Amplification for the Visual Detection of Shigella flexneri.

Shigella flexneri is a serious threat to global public health, and a rapid detection method is urgently needed. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is widely used in gene editing, gene therapy, and in vitro diagnosis. Here, we combined loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a to develop a novel diagnostic test (CRISPR/Cas12a-E-LAMP) for the diagnosis of S. flexneri. The CRISPR/Cas12a-E-LAMP protocol conducts LAMP reaction for S. flexneri templates followed by CRISPR/Cas12a detection of predefined target sequences. LAMP primers and sgRNAs were designed to the highly conserved gene hypothetical protein (accession: AE014073, region: 4170556-4171,068) of S. flexneri. After the LAMP reaction at 60°C for 20 min, the pre-loaded CRISPR/Cas12a regents were mixed with the LAMP products in one tube at 37°C for 20 min, and the final results can be viewed by naked eyes with a total time of 40 min. The sensitivity of CRISPR/Cas12a-E-LAMP to detect S. flexneri was 4 × 100 copies/µl plasmids and without cross-reaction with other six closely related non-S. flexneri. Therefore, the CRISPR/Cas12a-E-LAMP assay is a useful method for the reliable and quick diagnosis of S. flexneri and may be applied in other pathogen infection detection.

MiR-103a-3p Promotes Zika Virus Replication by Targeting OTU Deubiquitinase 4 to Activate p38 Mitogen-Activated Protein Kinase Signaling Pathway.

Background: MicroRNAs (miRNAs) play critical roles in regulating virus infection and replication. However, the mechanism by which miRNA regulates Zika virus (ZIKV) replication remains elusive. We aim to explore how the differentially expressed miR-103a-3p regulates ZIKV replication and to clarify the underlying molecular mechanism. Methods: Small RNA sequencing (RNA-Seq) was performed to identify differentially expressed miRNAs in A549 cells with or without ZIKV infection and some of the dysregulated miRNAs were validated by quantitative real time PCR (qRT-PCR). The effect of miR-103a-3p on ZIKV replication was examined by transfecting miR-103a-3p mimic or negative control (NC) into A549 cells with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and expression levels of ZIKV NS5 mRNA and NS1 protein were detected by qRT-PCR and Western blot, respectively. The potential target genes for miR-103a-3p were predicted by four algorithms and further validated by mutation analysis through luciferase reporter assay. The predicated target gene OTU deubiquitinase (DUB) 4 (OTUD4) was over-expressed by plasmid transfection or silenced by siRNA transfection into cells prior to ZIKV infection. Activation status of p38 MAPK signaling pathway was revealed by looking at the phosphorylation levels of p38 (p-p38) and HSP27 (p-HSP27) by Western blot. Results: Thirty-five differentially expressed miRNAs in ZIKV-infected A549 cells were identified by RNA-Seq analysis. Five upregulated and five downregulated miRNAs were further validated by qRT-PCR. One of the validated upregulated miRNAs, miR-103a-3p significantly stimulated ZIKV replication both at mRNA (NS5) and protein (NS1) levels. We found p38 MAPK signaling was activated following ZIKV infection, as demonstrated by the increased expression of the phosphorylation of p38 MAPK and HSP27. Blocking p38 MAPK signaling pathway using SB203580 inhibited ZIKV replication and attenuated the stimulating effect of miR-103a-3p on ZIKV replication. We further identified OTUD4 as a direct target gene of miR-103a-3p. MiR-103a-3p over-expression or OTUD4 silencing activated p38 MAPK signaling and enhanced ZIKV replication. In contrast, OTUD4 over-expression inhibited p38 MAPK activation and decreased ZIKV replication. In addition, OTUD4 over-expression attenuated the stimulating effect of miR-103a-3p on ZIKV replication and activation of p38 MAPK signaling. Conclusion: Zika virus infection induced the expression of miR-103a-3p, which subsequently activated p38 MAPK signaling pathway by targeting OTUD4 to facilitate ZIKV replication.

Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Blood Safety.

Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it has spread rapidly around the world and caused a serious global social crisis. During the epidemic, the blood collection and supply industry have been greatly impacted, due to the sharply dropped blood donors and transfusion transmission risk of SARS-CoV-2. Many infected individuals are asymptomatic and they may donate blood without awareness of the infection or before symptoms appear. In addition, viral RNAs have been detected in the blood of some patients infected with SARS-CoV-2. Although no infectious SARS-CoV-2 virus was found in the blood nor the blood components, there is a risk of transmission through blood transfusion which may endanger blood safety, especially during the pandemic period. This review briefly introduces the biological characteristics, epidemiology of SARS-CoV-2, with a particular focus on SARS-CoV-2 infection and blood safety.

Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-ß1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.

[Interaction between SARS-CoV-2 and Host Innate Immunity].

Coronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic since its outbreak in 2019, presenting serious threats to public health and the health of the people. As one of the main components of the host innate immune system, type-Ⅰ interferon (IFN) plays a critical role in the defense against viral infections. The battle between the virus and the host innate immune system determines the disease progression. It has been reported that SARS-CoV-2 inhibits IFN production and suppresses the activation of IFN signaling pathway through its interactions with the host innate immune system. Then, the weakened or delayed response of type-Ⅰ interferon causes the disturbance of host immune responses, which is one of the important reasons why SARS-CoV-2 causes such high morbidity and mortality. Herein, we reviewed and discussed the interaction between SARS-CoV-2 viral proteins and the host innate immune system, especially the interaction with type-Ⅰ IFN pathway, to provide new insights into the mechanisms of viral evasion of host immune response and new perspectives and strategies for treating COVID-19 with IFN.

IRF2 inhibits ZIKV replication by promoting FAM111A expression to enhance the host restriction effect of RFC3.

BACKGROUND: Although interferon regulatory factor 2 (IRF2) was reported to stimulate virus replication by suppressing the type I interferon signaling pathway, because cell cycle arrest was found to promote viral replication, IRF2-regulated replication fork factor (FAM111A and RFC3) might be able to affect ZIKV replication. In this study, we aimed to investigate the function of IRF2, FAM111A and RFC3 to ZIKV replication and underlying mechanism. METHODS: siIRF2, siFAM111A, siRFC3 and pIRF2 in ZIKV-infected A549, 2FTGH and U5A cells were used to explore the mechanism of IRF2 to inhibit ZIKV replication. In addition, their expression was analyzed by RT-qPCR and western blots, respectively. RESULTS: In this study, we found IRF2 expression was increased in ZIKV-infected A549 cells and IRF2 inhibited ZIKV replication independent of type I IFN signaling pathway. IRF2 could activate FAM111A expression and then enhanced the host restriction effect of RFC3 to inhibit replication of ZIKV. CONCLUSIONS: We speculated the type I interferon signaling pathway might not play a leading role in regulating ZIKV replication in IRF2-silenced cells. We found IRF2 was able to upregulate FAM111A expression and thus enhance the host restriction effect of RFC3 on ZIKV.

WarmStart colorimetric loop-mediated isothermal amplification for the one-tube, contamination-free and visualization detection of Shigella flexneri.

OBJECTIVES: Shigella flexneri (S. flexneri) is prevalent worldwide and the most common Shigella in many countries, causing highly contagious diarrhea, which seriously threatens public health. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) for the rapid, accurate, and visualization detection of S. flexneri. METHODS: According to the screened specific genes of S. flexneri, three groups of LAMP primers were designed and evaluated, and the colorimetric LAMP reaction volume was optimized. The specificity of the colorimetric LAMP was validated by 20 S. flexneri and 96 non-S. flexneri clinical isolates. In addition, the sensitivity of the developed assay was evaluated by the serial 10-fold dilutions of plasmid DNA. RESULTS: A colorimetric LAMP assay was developed based on the specific S. flexneri hypothetical protein gene (Accession: AE014073 Region: 4170556.4171068). The colorimetric LAMP method had good specificity for detecting S. flexneri and enabled detection of S. flexneri within 30 minutes, with a plasmid detection limit of 7*10° copies/µL. The results of amplification could be easily identified by color. CONCLUSIONS: This colorimetric LAMP assay could be used for rapid and accurate diagnosis of S. flexneri infection, especially in remote hospitals and laboratories with under-equipped medical facilities, and in situations where an urgent diagnosis is needed.

USP18 Mediates Interferon Resistance of Dengue Virus Infection.

Previous studies demonstrated that dengue virus (DENV) infection developed resistance to type-I interferons (IFNα/ß). The underlying mechanism remains unclear. USP18 is a negative regulator of IFNα/ß signaling, and its expression level is significantly increased following DENV infection in cell lines and patients' blood. Our previous study revealed that increased USP18 expression contributed to the IFN-α resistance of Hepatitis C Virus (HCV). However, the role of USP18 in DENV replication and resistance to IFN-α is elusive. In this current study, we aimed to explore the role of USP18 in DENV-2 replication and resistance to IFN-α. The level of USP18 was up-regulated by plasmid transfection and down-regulated by siRNA transfection in Hela cells. USP18, IFN-α, IFN-ß expression, and DENV-2 replication were monitored by qRT-PCR and Western blot. The activation of the Jak/STAT signaling pathway was assessed at three levels: p-STAT1/p-STAT2 (Western blot), interferon-stimulated response element (ISRE) activity (Dual-luciferase assay), and interferon-stimulated genes (ISGs) expression (qRT-PCR). Our data showed that DENV-2 infection increased USP18 expression in Hela cells. USP18 overexpression promoted DENV-2 replication, while USP18 silence inhibited DENV-2 replication. Silence of USP18 potentiated the anti-DENV-2 activity of IFN-α through activation of the IFN-α-mediated Jak/STAT signaling pathway as shown by increased expression of p-STAT1/p-STAT2, enhanced ISRE activity, and elevated expression of some ISGs. Our data indicated that USP18 induced by DENV-2 infection is a critical host factor utilized by DENV-2 to confer antagonism on IFN-α.

Differentially expressed immune response genes in COVID-19 patients based on disease severity.

BACKGROUND: Dysregulated immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are thought to underlie the progression of coronavirus disease 2019 (COVID-19). We sought to further characterize host antiviral and cytokine gene expression in COVID-19 patients based on illness severity. METHODS: In this case-control study, we retrospectively analyzed 46 recovered COVID-19 patients and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We assessed changes in antiviral gene expression between healthy controls and patients with mild/moderate (MM) and severe/critical (SC) disease. RESULTS: We found that type I interferon signaling (IFNA2, TLR8, IFNA1, IFNAR1, TLR9, IRF7, ISG15, APOBEC3G, and MX1) and genes encoding proinflammatory cytokines (IL12B, IL15, IL6, IL12A and IL1B) and chemokines (CXCL9, CXCL11 and CXCL10) were upregulated in patients with MM and SC disease. Moreover, we found that IFNA1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), and Fas-associated protein with death domain (FADD) were significantly downregulated (P < 0.05) in the SC group compared to the MM group. We also observed that microRNA (miR)-155 and miR-130a levels were markedly higher in the MM group compared to the SC group. CONCLUSION: COVID-19 is associated with the activation of host antiviral genes. Induction of the IFN system appears to be particularly important in controlling SARS-CoV-2 infection, as decreased expression of IFNA1, APOBEC3G and FADD genes in SC patients, relative to MM patients, may be associated with disease progression.

High expression of Ran binding protein 1 predicts poor outcomes in hepatocellular carcinoma patients: a Cancer Genome Atlas database analysis.

BACKGROUND: Ran-specific binding protein 1 (RANBP1) is involved in the regulation of the cell cycle, while its role in hepatocellular carcinoma (HCC) is unknown. Therefore, we aimed to demonstrate the association of RANBP1 with clinicopathologic features and potential biological functions in HCC based on The Cancer Genome Atlas (TCGA) data. METHODS: We assessed RANBP1 expression and its correlation with clinicopathologic features and evaluated the prognostic value of RANBP1 with Kaplan-Meier survival analysis and the MethSurv database. Univariate and multivariate Cox regression analyses were conducted to elucidate the factors responsible for prognosis. The identification of a co-expression network and the analysis of related biological events with RANBP1 in HCC were assessed using LinkedOmics. Moreover, gene set enrichment analysis (GSEA) was employed to annotate the biological function of RANBP1. We also explored the correlation between RANBP1 and tumor immune infiltrates using a single sample GSEA (ssGSEA). RESULTS: The expression of RANBP1 was found significantly elevated in HCC and linked to advanced T stage and histopathological grade. Up-regulated RANBP1 expression was linked to poor prognosis. High DNA methylation levels of RANBP1 were significantly linked to very poor overall survival (OS). Co-expression network analysis revealed that RANBP1 was involved in ribosome, spliceosome, deoxyribonucleic acid (DNA) replication, ribonucleic acid (RNA) transport, and cell cycle. GSEA showed enrichment of G2M-checkpoint, Wingless and Int-1 (Wnt) cell signaling, and DNA repair in the RANBP1 high-expression phenotype. By using ssGSEA analysis, the increased RANBP1 expression was positively linked to the immune infiltration level of T helper cell type-1 (Th1) and negatively linked to the immune infiltration levels of T helper cell type-17 (Th17). CONCLUSIONS: Findings suggest that RANBP1 may play a pivotal role in HCC prognosis and can potentially serve as a candidate biosignature and as a therapeutic target for HCC.