HCR-Assisted RTF-EXPAR-Based Lateral Flow Analysis for Sensitive Detection of H1N1 Influenza Virus.

The H1N1 influenza virus is a significant pathogen responsible for seasonal influenza, and its frequent outbreaks pose substantial challenges to global public health. The present study successfully developed a lateral flow analysis platform that integrates reverse transcription-free exponential amplification reaction (RTF-EXPAR) and hybridization chain reaction (HCR) processes with functionalized quantum dots for the direct detection of H1N1 influenza virus RNA, eliminating the need for reverse transcription. The fluorescence signal on the band recorded with a smartphone can be utilized for the quantitative determination of the target. Interestingly, the dual signal amplification strategy exhibits high sensitivity with a remarkably low detection limit of 10 aM. Moreover, this platform exhibits excellent flexibility and universality, where the various pathogens can be determined by replacing the specific nucleic acid fragments in RTF-EXPAR. The aforementioned advantages reveal its huge potential in the early diagnosis of H1N1 influenza virus infection and developing point-of-care testing (POCT) equipment for nucleic acid analysis.

Association between genetic polymorphisms of inflammatory response genes and the risk of ovarian cancer.

BACKGROUND/PURPOSE: Inflammation plays an important role in promoting ovarian tumorigenesis and cancer progression. However, the relationship between polymorphisms in inflammatory response genes and risk of ovarian cancer remains poorly understood. In this study, we investigated the association of PPARG Pro12Ala, IL6-174G/C, E-selectin S128R, NFKB1-94 ins/del, NFKBIA-826C/T, and ICAM-1 K469E polymorphisms with ovarian cancer risk in a Chinese population. METHODS: Genotyping of the polymorphisms was performed on 687 cases and 687 controls employing the PCR-RFLP technique, and the logistic regression model was used to measure the risk association. RESULTS: A significantly increased risk association was observed for the heterozygous genotypes of PPARG [odds ratio (OR) = 1.52, 95% confidence interval (CI) = 1.01-2.29] and E-selectin (OR = 1.77, 95% CI = 1.07-2.93) polymorphisms, as well as the homozygous ins/ins genotype of NFKB1 polymorphism (OR = 1.39, 95% CI = 1.00-1.92). By contrast, ICAM-1 KE genotype was associated with a decreased ovarian cancer risk (OR = 0.77, 95% CI = 0.60-0.98). In addition, the NFKB1 del/del + NFKBIA TT combination was also found to be associated with a decreased ovarian cancer risk, with OR = 0.12 (95% CI = 0.01-0.95). The associations of the NFKB1 and ICAM-1 polymorphisms replicated the findings of previous reports, assuring the reliability of the results obtained. CONCLUSION: NFKB1 and ICAM-1 polymorphisms could serve as useful ovarian cancer risk prediction biomarkers for the Chinese population, while the utility of PPARG and E-selectin polymorphisms as biomarkers requires further confirmation in independent ovarian cancer cohorts.

Research progress of microfluidics-based food safety detection.

High demands for food safety detection and analysis have been advocated with people's increasing living standards. Even though numerous analytical testing techniques have been proposed, their widespread adoption is still constrained by the high limit of detection, narrow detection ranges, and high implementation costs. Due to their advantages, such as reduced sample and reagent consumption, high sensitivity, automation, low cost, and portability, using microfluidic devices for food safety monitoring has generated significant interest. This review provides a comprehensive overview of the latest microfluidic detection platforms (published in recent 4 years) and their applications in food safety, aiming to provide references for developing efficient research strategies for food contaminant detection and facilitating the transition of these platforms from laboratory research to practical field use.

Multiplexed detection of respiratory pathogens using a portable device combining a CREM strategy.

Rapid and precise detection of respiratory pathogens is crucial for clinical diagnosis and treatment of respiratory infections. In this study, the multiplex and visual detection of respiratory pathogens is facilitated by specifically designed engineered CRISPR RNA (en-crRNA) to activate the trans-cleavage activity of Cas12a, along with a homemade portable device. The en-crRNA comprised an original crRNA and a DNA reporter molecule that is labelled with both a fluorophore and a quencher. Moreover, the DNA is partially complementary to the variable region of the original crRNA. The proof of concept was demonstrated by simultaneously identifying distinct respiratory pathogens with a detection limit of 102 copies per µL. The visual discrimination was subsequently achieved using a homemade portable device that was seamlessly integrated with a smartphone. The specificity of the strategy was validated by comparing with qPCR assays for clinical sample detection, demonstrating exceptional accuracy with areas under the ROC curves of 0.98 for all targets. The research provides a promising avenue for the development of rapid, specific, and on-site detection techniques aimed at multiplex identification of respiratory pathogens.

On-site detection of OTA and AFB1 based on branched hybridization chain reaction coupled with lateral flow assay.

Mycotoxins are widely prevalent in various agricultural commodities, whose excessive consumption can pose significant risks to human health. In this study, we developed a facile mycotoxin detection platform based on branched hybridization chain reaction coupled with lateral flow assay. Ochratoxin A/Aflatoxin B1 bind to aptamers triggering the release of initiators, which leads to bHCR amplification and forms three-dimensional dendritic DNA nanostructures. Using the functionalized quantum dots as a fluorescent label, by leveraging smartphones and handheld ultraviolet lamps, the qualitative and quantitative detection of OTA and AFB1 can be achieved with a significantly enhanced sensitivity level, surpassing that of commercial test strips by 2-3 orders of magnitude. The visual detection limits for OTA and AFB1 were 30 pg/mL and 4 pg/mL, respectively. This approach eliminates the necessity for enzyme catalysis or the preparation and purification of antibodies and/or hapten, thereby reducing testing expenses and streamlining operational procedures. Moreover, substituting aptamer and nucleic acid sequences can effectively expand the scope of detection targets. Consequently, the as-proposed strategy exhibits great potential as a versatile technique, suitable for various analytical scenarios due to its sensitivity, accuracy, simplicity, and portability.

Advancements of CRISPR technology in public health-related analysis.

Pathogens and contaminants in food and the environment present significant challenges to human health, necessitating highly sensitive and specific diagnostic methods. Traditional approaches often struggle to meet these requirements. However, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized nucleic acid diagnostics. The present review provides a comprehensive overview of the biological sensing technology based on the CRISPR/Cas system and its potential applications in public health-related analysis. Additionally, it explores the enzymatic cleavage capabilities mediated by Cas proteins, highlighting the promising prospects of CRISPR technology in addressing bioanalysis challenges. We discuss commonly used CRISPR-Cas proteins and elaborate on their application in detecting foodborne bacteria, viruses, toxins, other chemical pollution, and drug-resistant bacteria. Furthermore, we highlight the advantages of CRISPR-based sensors in the field of public health-related analysis and propose that integrating CRISPR-Cas biosensing technology with other technologies could facilitate the development of more diverse detection platforms, thereby indicating promising prospects in this field.

A portable CRISPR-Cas12a triggered photothermal biosensor for sensitive and visual detection of Staphylococcus aureus and Listeria monocytogenes.

The detection of foodborne pathogens is crucial for ensuring the maintenance of food safety. In the present study, a portable CRISPR-Cas12a triggered photothermal biosensor integrating branch hybrid chain reaction (bHCR) and DNA metallization strategy for sensitive and visual detection of foodborne pathogens was proposed. The sheared probes were utilized to block the locker probes, which enabled preventing the assembly of bHCR in the absence of target bacteria, while target bacteria can activate the cleavage of sheared probes through CRISPR-Cas12a. Therefore, the locker probes functioned as initiating chains, triggering the formation of the branching double-stranded DNA consisting of H1, H2, and H3. The silver particles, which were in situ deposited on the DNA structure, functioned as a signal factor for conducting photothermal detection. Staphylococcus aureus and Listeria monocytogenes were selected as the foodborne pathogens to verify the analytical performance of this CRISPR-Cas12a triggered photothermal sensor platform. The sensor exhibited a sensitive detection with a low detection limit of 1 CFU/mL, while the concentration ranged from 100 to 108 CFU/mL. Furthermore, this method could efficiently detect target bacteria in multiple food samples. The findings demonstrate that this strategy can serve as a valuable reference for the development of a portable platform enabling quantitative analysis, visualization, and highly sensitive detection of foodborne bacteria.

BMI1 induces ubiquitination and protein degradation of Nod-like receptor family CARD domain containing 5 and suppresses human leukocyte antigen class I expression to induce immune escape in non-small cell lung cancer.

The Nod-like receptor (NLR) family CARD domain containing 5 (NLRC5) has been reported as an activator of human leukocyte antigen (HLA) class I that is responsible for immune activity in cancer treatment. This work focuses on the role of BMI1 proto-oncogene (BMI1) in the NLRC5-HLA class I axis and in immune escape in non-small cell lung cancer (NSCLC). First, immunoblot analysis and/or reverse transcription-quantitative polymerase chain reaction were performed, which identified decreased NLRC5 and HLA class I levels in NSCLC tissues and cell lines. NSCLCs were co-cultured with activated CD8+ T cells. Overexpression of NLRC5 in NSCLC cells elevated the expression of HLA class I and increased the activity of T cells and IL-2 production, and it reduced the PD-1/PD-L1 levels. The ubiquitination and immunoprecipitation assays confirmed that BMI1 bound to NLRC5 to induce is ubiquitination and protein degradation. Downregulation of BMI1 in NSCLC cells elevated NLRC5 and HLA class I levels, and consequently promoted T cell activation and decreased PD-1/PD-L1 levels in the co-culture system. However, overexpression of BMI1 in cells led to inverse trends. In summary, this study demonstrates that BMI1 induces ubiquitination and protein degradation of NLRC5 and suppresses HLA class I expression, which potentially helps immune escape in NSCLC.

Expression of Septin4 in Schistosoma japonicum-infected mouse livers after praziquantel treatment.

BACKGROUND: Septin4 (SEPT4) exists widely in human tissues and is related to mechanical stability, actin dynamics, membrane trafficking, viral replication and apoptosis. Data from many studies have suggested that SEPT4 plays a significant role in liver fibrosis. SEPT4 is down-regulated in the model of CCl4 and BDL treated liver fibrosis. However, it is up-regulated and peaked at 12 weeks post-infection (p.i.), and then decreased subsequently in Schistosoma japonicum (S. japonicum) egg-induced liver fibrosis. The aim of this study was to observe the dynamic alteration of SEPT4 after the treatment of praziquantel (PZQ) in ICR mice infected with S. japonicum. METHODS: Expression of SEPT4 was determined by western blot, immunofluorescence and qRT-PCR. And pro-inflammatory cytokines IL-6 and TNF-α were detected by qRT-PCR. The number of eggs, the diameter of egg granulomas and fibrosis-associated genes were also measured. RESULTS: Our results showed that the granulomatous inflammation was reduced, whereafter the expression of SEPT4 on hepatic stellate cells (HSCs) was decreased after PZQ anti-schistosome therapy. And the variation tendency of SEPT4 had positive correlation with the inflammatory response in the area of S. japonicum egg granulomas. CONCLUSIONS: Based on these findings, the inhibition of the expression of the SEPT4 by PZQ might be due to alleviation of the inflammatory response at the chronic and advanced stage of S. japonicum infection.

Schistosoma japonicum soluble egg antigens induce apoptosis and inhibit activation of hepatic stellate cells: a possible molecular mechanism.

Hepatic stellate cells play a key role in the development of hepatic fibrosis. Activated hepatic stellate cells can be reversed to a quiescent-like state or apoptosis can be induced to reverse fibrosis. Some studies have recently shown that Schistosoma mansoni eggs could suppress the activation of hepatic stellate cells and that soluble egg antigens from schistosome eggs could promote immunocyte apoptosis. Hence, in this study, we attempt to assess the direct effects of Schistosoma japonicum soluble egg antigens on hepatic stellate cell apoptosis, and to explore the mechanism by which the apoptosis of activated hepatic stellate cells can be induced by soluble egg antigens, as well as the mechanism by which hepatic stellate cell activation is inhibited by soluble egg antigens. Here, it was shown that S. japonicum-infected mouse livers had increased apoptosis phenomena and a variability of peroxisome proliferator-activated receptor γ expression. Soluble egg antigens induce morphological changes in the hepatic stellate cell LX-2 cell line, inhibit cell proliferation and induce cell-cycle arrest at the G1 phase. Soluble egg antigens also induce apoptosis in hepatic stellate cells through the TNF-related apoptosis-inducing ligand/death receptor 5 and caspase-dependent pathways. Additionally, soluble egg antigens could inhibit the activation of hepatic stellate cells through peroxisome proliferator-activated receptor γ and the transforming growth factor ß signalling pathways. Therefore, our study provides new insights into the anti-fibrotic effects of S. japonicum soluble egg antigens on hepatic stellate cell apoptosis and the underlying mechanism by which the liver fibrosis could be attenuated by soluble egg antigens.

Expression of Septin4 in human hepatic stellate cells LX-2 stimulated by LPS.

Septin4, a member of polymerizing GTP-binding proteins family, is reported to be involved in cytoskeletal organization in mitosis, apoptosis, fibrosis, and other cellular processes. Since various Septin4 expression patterns were reported in different diseases, this study aimed to investigate Septin4 expression in human LX-2 cell line stimulated by lipopolysaccharides (LPS) and attempted to clarify the relationship between Septin4 and hepatic inflammatory injury and fibrosis. In this subject, human stellate cell line LX-2 was stimulated by LPS. The expression of Septin4 was analyzed by Western blot and quantitative real-time PCR. To observe the relationship among Toll-like receptor 4 (TLR4), TGF-ß, and Septin4, proteins from the anti-TLR4 antibody blocked cells, as well as the TGF-ß-induced cells, were analyzed by the method of Western blot. As the results, LPS could induce the alteration of α-smooth muscle actin and Septin4 expression in LX-2 cells. Septin4 expression was regulated by LPS stimulation through TLR4 and TGF-ß pathway. These results therefore suggest that Septin4 may be involved in the process of activation of hepatic stellate cells by LPS stimulation. Further work would focus on the function of Septin4 in hepatic inflammatory injury and fibrosis.