Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted pathogen known for causing genital herpes. Glycoprotein B (gB) of HSV-2 plays a crucial role in viral entry and infection. However, its post-translational modifications are not well understood. This study investigated the palmitoylation of HSV-2 gB, identifying cysteine residue 8 (C8) as a critical palmitoylation site. Using an acyl-biotin exchange assay, we confirmed that gB was indeed palmitoylated, and treatment with 2-bromopalmitate significantly reduced this modification. Further analysis revealed that ZDHHC14 interacts with gB and facilitated its palmitoylation. We also identified APT2 as a negative regulator of gB palmitoylation. Importantly, palmitoylation enhanced gB's localization to the plasma membrane, whereas the C8S mutation substantially impaired this localization. Functionally, palmitoylation of gB enhanced the infective efficiency of HSV-2 pseudotyped particles (pp) and live virus, while both palmitoyltransferase and APT2 inhibition affected the entry efficiency of HSV-2pp. Our findings demonstrate that the palmitoylation of gB is crucial for its localization to the membrane and for facilitating efficient HSV-2 infection, highlighting palmitoylation as a promising target for antiviral interventions and providing new insights into the pathogenesis of HSV-2.
Graphical Two very different DNA aptamers have been reported for dopamine, one derived from an RNA aptamer (named Apt1) and other obtained via direct aptamer selection (named Apt2). In this study, we used four homogeneous binding assays to compare these two DNA dopamine aptamers. Apt1 does not appear to be able to bind dopamine specifically, while Apt2 showed specific binding and could be used for developing related biosensors. Dopamine is an essential neurotransmitter and its detection is important for bioanalytical chemistry. Two very different DNA aptamers have been reported for dopamine, one derived from an RNA aptamer (named Apt1) and other obtained via direct aptamer selection (named Apt2). In this study, we used four homogeneous binding assays to compare these two DNA dopamine aptamers. Thiazole orange (TO) fluorescence assay indicated that the Apt2 specifically bound with dopamine with a K d of 2.37 μM, which was consistent with that from the isothermal titration calorimetry (ITC) assay. However, Apt1 had much less TO fluorescence change and also no signal from ITC. By labeling the two ends of the two aptamers by a fluorophore and a quencher, the aptamer beacons showed binding of dopamine only for Apt2. Finally, the label-free AuNP-based colorimetric assay showed no difference between these two aptamer sequences, and even non-binding random DNA showed the same response, indicating that AuNPs were not a good probe for detecting dopamine. According to the data, Apt1 does not appear to be able to bind dopamine specifically, while Apt2 showed specific binding and could be used for developing related biosensors.
For the rapid and sensitive detection of vascular endothelial growth factor 165 (VEGF165) in clinical blood samples, a microfluidic sensing chip that integrates a centrifugal separation pretreatment unit and a composite nanosensing film was proposed in this paper. An efficient sensing strategy and method was established. The blood sample was first separated and extracted by centrifugal force on the centrifugal microfluidic chip within 5 min after injection. The separated plasma can be automatically transferred through the designed microchannels to the detection area integrated electrodes for subsequent differential pulse voltammetric detection. The Au NPs/MCH/Apt2 sensing film was constructed on the surface of the Au working electrode. A sandwich sensing strategy based on “double aptamers” and “nanoprobe” for VEGF165 detection was established, by which the synthetic Apt1/PThi/Au NP nanoprobe was applied to capture VEGF165 in plasma and bind to the sensing film. By this method, the detection limit of VEGF165 in whole blood was 0.67 pg/mL and the linear range was between 1 pg and 10 ng, which met the needs of clinical VEGF165 detection. It was illustrated that the proposed methodology based on the centrifugal microfluidic chip had potential application prospects in the development of the point-of-care testing fields.
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