Propranolol Inhibits the Growth of Cervical Cancer Cells by Inhibiting Cyclic Guanosine Monophosphate/Protein Kinase G (cGMP/PKG) Pathway

This study assesses the therapeutic effect of propranolol on cervical cancer and its mechanism. Propranolol’s effect on cervical cancer was evaluated by MTT, Western blotting, flow cytometry and colony formation. By searching Drug Bank and String, cGMP/PKG signaling might be downstream targets of propranolol for subsequent analysis. Our results found that propranolol could significantly inhibit Hela and SiHA cell vitality and clone formation in a dose dependent manner. Further, Annexin V-PE/7-AAD Apoptosis Detection assay showed that propranolol could increase Hela and SiHA cell apoptosis. Finally, propranolol attenuated the phosphorylation level of VASP at Ser239 which is critical for PKG activation. In conclusion, propranolol suppressed cervical cancer cell proliferation via inhibition of cGMP/PKG signaling, which provides an affordable and effective method for cervical cancer remedy.

T2Candida Assay in the Diagnosis of Intraabdominal Candidiasis: A Prospective Multicenter Study

The T2Candida magnetic resonance assay is a direct-from-blood pathogen detection assay that delivers a result within 3–5 h, targeting the most clinically relevant Candida species. Between February 2019 and March 2021, the study included consecutive patients aged >18 years admitted to an intensive care unit or surgical high-dependency unit due to gastrointestinal surgery or necrotizing pancreatitis and from whom diagnostic blood cultures were obtained. Blood samples were tested in parallel with T2Candida and 1,3-β-D-glucan. Of 134 evaluable patients, 13 (10%) were classified as having proven intraabdominal candidiasis (IAC) according to the EORTC/MSG criteria. Two of the thirteen patients (15%) had concurrent candidemia. The sensitivity, specificity, positive predictive value, and negative predictive value, respectively, were 46%, 97%, 61%, and 94% for T2Candida and 85%, 83%, 36%, and 98% for 1,3-β-D-glucan. All positive T2Candida results were consistent with the culture results at the species level, except for one case of dual infection. The performance of T2Candida was comparable with that of 1,3-β-D-glucan for candidemic IAC but had a lower sensitivity for non-candidemic IAC (36% vs. 82%). In conclusion, T2Candida may be a valuable complement to 1,3-β-D-glucan in the clinical management of high-risk surgical patients because of its rapid results and ease of use.

Evaluation and modelling of the performance of an automated SARS-CoV-2 antigen assay according to sample type, target population and epidemic trends

The Lumipulse® G SARS-CoV-2 Ag assay performance was evaluated on prospectively collected saliva and nasopharyngeal swabs (NPS) of recently ill in- and outpatients and according to the estimated viral load. Performances were calculated using RT-PCR positive NPS from patients with symptoms ≤ 7 days and RT-PCR negative NPS as gold standard. In addition, non-selected positive NPS were analyzed to assess the performances on various viral loads. This assay yielded a sensitivity of 93.1% on NPS and 71.4% on saliva for recently ill patients. For NPS with a viral load > 103 RNA copies/mL, sensitivity was 96.4%. A model established on our daily routine showed fluctuations of the performances depending on the epidemic trends but an overall good negative predictive value. Lumipulse® G SARS-CoV-2 assay yielded good performance for an automated antigen detection assay on NPS. Using it for the detection of recently ill patient or to screen high-risk patients could be an interesting alternative to the more expensive RT-PCR.

Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32

Background One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. Methodology/Principal findings A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4–6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. Conclusions/Significance The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

A sample-to-answer electrochemical biosensor system for biomarker detection

We interfaced with a painless blood collection device and integrated on-chip blood-to-plasma separation with an electronic bead-based biomarker detection assay to enable true sample-to-answer detection of biomarkers.

Circular RNA CircPVT1 Inhibits 5-Fluorouracil Chemosensitivity by Regulating Ferroptosis Through MiR-30a-5p/FZD3 Axis in Esophageal Cancer Cells

BackgroundCircPVT1 is demonstrated to promote cancer progression in esophageal squamous cell carcinoma (ESCC). However, the role and potential functional mechanisms of circPVT1 in regulating 5-fluorouracil (5-FU) chemosensitivity remain largely unknown.MethodsESCC cells resistant to 5-FU were induced with continuous increasing concentrations of 5-FU step-wisely. A cell counting kit-8 assay was used to analyze the viability of ESCC cells. LDH release assay kit was used to evaluate the cytotoxicity. RT-qPCR was used to assess the expression level of non-coding RNAs and cDNAs. Luciferase was used to confirm the interaction between non-coding RNAs and targets. Western blotting was used to detect the expression of downstream signaling proteins. Flow cytometry and ferroptosis detection assay kit were utilized to measure the ferroptosis of ESCC cells.ResultsCircPVT1 was significantly upregulated in ESCC cells resistant to 5-FU. Knockdown of circPVT1 enhanced the 5-FU chemosensitivity of ESCC cells resistant to 5-FU by increasing cytotoxicity and downregulating multidrug-resistant associated proteins, including P-gp and MRP1. Luciferase assay showed that circPVT1 acted as a sponge of miR-30a-5p, and Frizzled3 (FZD3) was a downstream target of miR-30a-5p. The enhanced 5-FU chemosensitivity by circPVT1 knockdown was reversed with miR-30a-5p inhibitor. Besides, the increased 5-FU chemosensitivity by miR-30a-5p mimics was reversed with FZD3 overexpression. Furthermore, knockdown of circPVT1 increased ferroptosis through downregulating p-β-catenin, GPX4, and SLC7A11 while miR-30a-5p inhibition and FZD3 overexpression reversed the phenotype by upregulating p-β-catenin, GPX4, and SLC7A11.ConclusionsThese results suggested a key role for circPVT1 in ESCC 5-FU-chemosensitivity in regulating the Wnt/β-catenin pathway and ferroptosis via miR-30a-5p/FZD3 axis, which might be a potential target in ESCC therapy.

Loquacious Modulates Flaviviral RNA Replication in Mosquito Cells

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins SEC61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both SEC61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3’ end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.Author SummaryThere is a wealth of information that dictates virus-host interactions in flavivirus-infected mammalian cells, yet there is only sparse information on the mechanisms that modulate viral gene expression in the mosquito host. Using a novel RNA-protein detection assay, the interactions of SEC61A1 and Loqs with the dengue viral genome were found to have proviral functions in infected mosquito cells. In particular, Loqs forms complexes with the positive-strand of the viral RNA and the very 3’ end of the negative-strand viral RNA. Further analyses showed that Loqs modulates viral RNA replication of dengue virus and gene amplification of several other flaviviral genomes. These findings argue that Loqs is an essential proviral host factor in mosquitos.

Colorimetric Detection of Organophosphate Pesticides Based on Acetylcholinesterase and Cysteamine Capped Gold Nanoparticles as Nanozyme

Organophosphates (OPs) are neurotoxic agents also used as pesticides that can permanently block the active site of the acetylcholinesterase (AChE). A robust and sensitive detection system of OPs utilising the enzyme mimic potential of the cysteamine capped gold nanoparticles (C-AuNPs) was developed. The detection assay was performed by stepwise addition of AChE, parathion ethyl (PE)-a candidate OP, acetylcholine chloride (ACh), C-AuNPs, and 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the buffer solution. The whole sensing protocol completes in 30–40 min, including both incubations. The Transmission Electron Microscopy (TEM) results indicated that the NPs are spherical and have an average size of 13.24 nm. The monomers of C-AuNPs exhibited intense catalytic activity (nanozyme) for the oxidization of TMB, revealed by the production of instant blue colour and confirmed by a sharp peak at 652 nm. The proposed biosensor’s detection limit and linear ranges were 5.8 ng·mL−1 and 11.6–92.8 ng·mL−1, respectively, for PE. The results strongly advocate that the suggested facile colorimetric biosensor may provide an excellent platform for on-site monitoring of OPs.

Anti-ganglioside Antibodies in Guillain-Barre Syndrome: A Novel Immunoblotting-Panel Assay

Objective: This study aimed to determine the diagnostic efficiency of a novel immunoblotting detection assay for anti-ganglioside antibodies (AGAs) in the Guillain–Barre syndrome (GBS).Method: Serum immunoglobulin (IgG and IgM) of AGAs were measured in 121 participants from a registered cohort study of immune-mediated neuropathies and 29 healthy controls by immunoblotting panel assay. Sensitivity, specificity, and positive predictive value (PPV) of the assay were compared to calculate the diagnostic accuracy.Result: In our cohort, any of the AGAs were positive in 42.4% of the GBS patients. The sensitivity and specificity of AGAs (both IgG and IgM) in the diagnosis of GSB were 42 and 76% while for IgG-AGAs were 35 and 87%. AGAs positivity had a significant association with the AMAN subtype (P = 0.0004), and the sensitivity, specificity of AGAs in AMAN were 86, 69%, respectively with high (AUC = 0.78, p = 0.002) discriminative powers. GM1-IgG AGA was more common and specific to AMAN patients than other GBS forms (p = 0.008).Conclusion: Our novel immunoblotting detection assay could complement GBS diagnosis. IgG-AGAs were more likely to be detected in GBS, and GM1-IgG AGA could assist AMAN diagnosis.