The establishment of a multiplex fluorescent polymerase chain reaction coupled with capillary electrophoresis analysis technology enables the simultaneous detection of 16 genotypes of human papillomavirus.

BACKGROUND: The detection and accurate genotyping of human papillomavirus (HPV) infection is critical for preventing and effectively treating cervical cancer. METHODS: A multiplex fluorescent polymerase chain reaction (PCR) coupled with a capillary electrophoresis method was developed for the simultaneous detection of the 16 most prevalent HPV genotypes. Twenty-five pairs of primers were ultimately selected to ensure that both E and L regions of nine HPV genotypes, as well as the E regions of seven HPV genotypes could be accurately amplified. RESULTS: This method enables the simultaneous detection and differentiation of 16 HPV genotypes in a single closed-tube reaction, accurately distinguishing products with molecular weight differences >1 bp through capillary electrophoresis. This method demonstrated exceptional accuracy, specificity, and repeatability with a detection limit of 10 copies/µL for all 16 HPV genotypes. Furthermore, 152 cervical swab specimens were obtained to compare the disparities between this approach and Cobas 4800 HPV detection method. The concordance rate and κ value were 90.1% and 0.802, respectively, indicating a high level of agreement. The established detection method was successfully applied to cervical swab specimens for determining HPV genotypes across all levels of cervical lesions, HPV52, 56, 16, and 59 were found to be most prevalent with infection rates of 10.8%, 9.1%, 6.5%, and 6.2%, respectively. CONCLUSIONS: This study has successfully established a detection method capable of simultaneously identifying 16 HPV genotypes. This approach can be further applied to HPV vaccine research and surveillance, with the potential for broad applications.

A synthetic bivalent peptide ligand of EphB4 with potent agonistic activity.

Interaction between ephrin receptor EphB4 and its ligand EFNB2 mediates bidirectional signaling important for cancer: forward EFNB2-to-EphB4 signaling that is tumor suppressive, and reverse EphB4-to-EFNB2 signaling that promotes angiogenesis important for tumor growth and metastasis. Molecular agents targeting these forward and reverse signals of EphB4-EFNB2 interaction can be used to probe the molecular mechanisms of these complex signaling pathways and develop new anticancer therapeutics. In this study, we applied a bivalent ligand design strategy to synthesize a novel dimeric peptide based on an antagonist TNYL-RAW. The dimeric peptide possessed higher EphB4 receptor binding affinity than the monomeric TNYL-RAW peptide. Interestingly, the dimerization of TNYL-RAW peptide converted a monomeric antagonist of EphB4 to a dimeric agonist. This dimeric agonist promoted EphB4 phosphorylation, internalization and degradation, reduced cancer cell motility, and inhibited tube formation of HUVEC. To investigate the mechanism of action of this bivalent dimeric peptide, FRET experiments and molecular dynamic simulation were conducted and suggested that this bivalent ligand recognizes two EphB4 simultaneously which may promote receptor dimerization and oligomerization. This was further supported by the study of this bivalent ligand containing deletion of critical residues on one of its monomers which impaired its simultaneous binding to two EphB4 and ability to cause EphB4 dimerization and phosphorylation. These results demonstrate the value of this novel bivalent agonist ligand of EphB4 as a probe of the bidirectional signaling of EphB4-EFNB2 and lead for cancer drug development.

Analysis of SARS-CoV-2 variants B.1.617: host tropism, proteolytic activation, cell-cell fusion, and neutralization sensitivity.

SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell-cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.

SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

Na⁺/H⁺ exchanger 1 participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system.

Despite the importance of NHX1 (Na(+)/H(+) exchanger 1) in plant salt tolerance, little is known about its other functions. In this study, intriguingly, it was found that NHX1 participated in plant disease defence against Phytophthora parasitica var. nicotianae (Ppn) in Nicotiana benthamiana. NbNHX1 was originally isolated from N. benthamiana, and characterized. The subcellular localization of NbNHX1 with its C-terminus fused with green fluorescent protein indicated that NbNHX1 localized primarily to the tonoplast. Tobacco rattle virus-induced NbNHX1 silencing led to reduced H(+) efflux from the vacuole to cytoplasts, and decreased Ppn resistance in N. benthamiana. After attack by Ppn, NbNHX1-silenced plants exhibited impaired ability to scavenge reactive oxidative species (ROS) induced by the pathogen. Pea early browning virus-mediated ectopic expression of SeNHX1 (from Salicornia europaea) or AtNHX1 (from Arabidopsis thaliana) both conferred enhanced Ppn resistance to N. benthamiana, with a lower H2O2 concentration after Ppn inoculation. Further investigation of the role of NHX1 demonstrated that transient overexpression of NbNHX1 improved the vacuolar pH and cellular ROS level in N. benthamiana, which was coupled with an enlarged NAD(P) (H) pool and higher expression of ROS-responsive genes. In contrast, NbNHX1 silencing led to a lower pH in the vacuole and a lower cellular ROS level in N. benthamiana, which was coupled with a decreased NAD(P) (H) pool and decreased expression of ROS-responsive genes. These results suggest that NHX1 is involved in plant disease defence; and regulation of vacuolar pH by NHX1, affecting the cellular oxidation state, primes the antioxidative system which is associated with Ppn resistance in tobacco.

Modified noninvasive microtest electrophysiological technology for vacuolar H(+) flux detection.

This paper describes a modified noninvasive microtest electrophysiological technology (NMT) for vacuolar H(+) flux detection. In this NMT system, the vacuole isolation procedure and buffer slope were modified, and the measuring errors from small spherical geometry were corrected. The trends in changes of vacuolar H(+) flux (ΔH(+) flux) after ATP or PP(i) supply calculated by NMT were consistent with the activities of V-ATPase and PPase measured by traditional methods. These findings indicate that our modified NMT is an appropriate method for vacuolar H(+) flux detection.

Transformation of beta-lycopene cyclase genes from Salicornia europaea and Arabidopsis conferred salt tolerance in Arabidopsis and tobacco.

Inhibition of lycopene cyclization decreased the salt tolerance of the euhalophyte Salicornia europaea L. We isolated a ß-lycopene cyclase gene SeLCY from S. europaea and transformed it into Arabidopsis with stable expression. Transgenic Arabidopsis on post-germination exhibited enhanced tolerance to oxidative and salt stress. After 8 and 21 d recovery from 200 mM NaCl treatment, transgenic lines had a higher survival ratio than wild-type (WT) plants. Three-week-old transgenic plants treated with 200 mM NaCl showed better growth than the WT with higher photosystem activity and less H(2)O(2) accumulation. Determination of endogenous pigments of Arabidopsis treated with 200 mM NaCl for 0, 2 or 4 d demonstrated that the transgenic plants retained higher contents of carotenoids than the WT. Furthermore, to compare the difference between SeLCY and AtLCY from Arabidopsis, we used viral vector mediating ectopic expression of SeLCY and AtLCY in Nicotiana benthamiana. Although LCY genes transformation increased the salt tolerance in tobacco, there is no significant difference between SeLCY- and AtLCY-transformed plants. These findings indicate that SeLCY transgenic Arabidopsis improved salt tolerance by increasing synthesis of carotenoids, which impairs reactive oxygen species and protects the photosynthesis system under salt stress, and as a single gene, SeLCY functionally showed no advantage for salt tolerance improvement compared with AtLCY.