HPV16 Genomes: In Silico Analysis of E6 and E7 Oncoproteins in 20 South American Variants.

Background: Human papillomavirus (HPV) is the main risk factor for the development of squamous cell cervical cancer, and E6 oncoprotein and E7 oncoprotein are important components of the viral genome and its oncogenic potential. It is known that different viral variants of HPV16 have different pathology and impact on the development of neoplasia, although few studies have been performed on South American variants. Objective: Therefore, the present study aimed to analyze in silico the genomic diversity of HPV16 in 20 complete genome variants of South America in the National Center for Biotechnology Information (NCBI) database. Methods: We performed a descriptive study to characterize the polymorphic regions of the E6 and E7 genes in HPV16 variants, using software for genomic data and single nucleotide polymorphism (SNP) analysis and others for phylogenetic analysis. Results: The variants analyzed included six SNPs linked to cancer (A131G, G145T, C335T, T350G, C712A, and T732C) and significant variation (798 nucleotide substitutions). Despite this, the variants showed low genetic diversity. Eighteen variants of unclear significance (VUS) were identified, 10 of which were in the coding E6 regions and 8 in the coding E7 regions. The prevalence of lineage D variants is of concern due to their pathology in cervical cancer and requires more research and epidemiological vigilance regarding their prevalence in the population. Conclusion: The data obtained in this study may contribute to future research on South American variants of HPV16, their pathogenicity, and the development of treatments.

Microfluid biosensor for detection of hpv in patient scraping samples: Determining E6 and E7 oncogenes.

E6 and E7 oncogenes are pivotal in the carcinogenic transformation in HPV infections and efficient diagnostic methods can ensure the detection and differentiation of HPV genotype. This study describes the development and validation of an electrochemical, label-free genosensor coupled with a microfluidic system for detecting the E6 and E7 oncogenes in cervical scraping samples. The nanostructuring employed was based on a cysteine and graphene quantum dots layer that provides functional groups, surface area, and interesting electrochemical properties. Biorecognition tests with cervical scraping samples showed differentiation in the voltammetric response. Low-risk HPV exhibited a lower biorecognition response, reflected in ΔI% values of 82.33 % ± 0.29 for HPV06 and 80.65 % ± 0.68 for HPV11 at a dilution of 1:100. Meanwhile, high-risk, HPV16 and HPV18, demonstrated ΔI% values of 96.65 % ± 1.27 and 93 % ± 0.026, respectively, at the same dilution. Therefore, the biorecognition intensity followed the order: HPV16 >HPV18 >HPV06 >HPV11. The limit of detection and the limit of quantification of E6E7 microfluidic LOC-Genosensor was 26 fM, and 79.6 fM. Consequently, the E6E7 biosensor is a valuable alternative for clinical HPV diagnosis, capable of detecting the potential for oncogenic progression even in the early stages of infection.

In vitro comparison of viral replication and cytopathology induced by SARS-CoV-2 variants.

A myriad of coronaviruses cause diseases from a common cold to severe lung infections and pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic and many laboratory techniques were examined for virus culture and basic and applied research. Understanding the replication kinetics and characterizing the effect the virus has on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will help give us a clear understanding of their characteristic differences in pathogenicity. In this study we compared the cytopathic effect and replication of Wild-Type (USA/WA1), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on five different cell lines; VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. This data will aid researchers with experimental planning and viral pathogenicity analysis and provide a baseline for testing any future variants.

HPV18 E6/E7 activates Ca2+ influx to promote the malignant progression of cervical cancer by inhibiting Ca2+ binding protein 1 expression.

Mounting studies have shown that the oncoproteins E6 and E7 encoded by the human papillomavirus (HPV) genome are essential in HPV-induced cervical cancer (CC). Ca2+ binding protein 1 (CABP1), a downstream target of HPV18-positive HeLa cells that interferes with E6/E7 expression, was identified through screening the GEO Database (GSE6926). It was confirmed to be down-regulated in CC through TCGA prediction and in vitro detection. Subsequent in vitro experiments revealed that knocking down E6/E7 inhibited cell proliferation, migration, and invasion, whereas knocking down CABP1 promoted these processes. Simultaneously knocking down CABP1 reversed these effects. Additionally, the results were validated in vivo. Previous studies have indicated that CABP1 can regulate Ca2+ channels, influencing Ca2+ influx and tumor progression. In this study, it was observed that knocking down CABP1 enhanced Ca2+ inflow, as demonstrated by flow cytometry and confocal microscopy. Knocking down E6/E7 inhibited these processes, whereas simultaneously knocking down E6/E7 and CABP1 restored the inhibitory effect of knocking down E6/E7 on Ca2+ inflow. To further elucidate that E6/E7 promotes CC progression by inhibiting CABP1 expression and activating Ca2+ influx, BAPTA/AM treatment was administered during CABP1 knockdown. It was discovered that Ca2+ chelation could reverse the effect of CABP1 knockdown on CC cells. In conclusion, our results offer a novel target for the diagnosis and treatment of HPV-induced CC.

Genome composition-based deep learning predicts oncogenic potential of HPVs.

Human papillomaviruses (HPVs) account for more than 30% of cancer cases, with definite identification of the oncogenic role of viral E6 and E7 genes. However, the identification of high-risk HPV genotypes has largely relied on lagged biological exploration and clinical observation, with types unclassified and oncogenicity unknown for many HPVs. In the present study, we retrieved and cleaned HPV sequence records with high quality and analyzed their genomic compositional traits of dinucleotide (DNT) and DNT representation (DCR) to overview the distribution difference among various types of HPVs. Then, a deep learning model was built to predict the oncogenic potential of all HPVs based on E6 and E7 genes. Our results showed that the main three groups of Alpha, Beta, and Gamma HPVs were clearly separated between/among types in the DCR trait for either E6 or E7 coding sequence (CDS) and were clustered within the same group. Moreover, the DCR data of either E6 or E7 were learnable with a convolutional neural network (CNN) model. Either CNN classifier predicted accurately the oncogenicity label of high and low oncogenic HPVs. In summary, the compositional traits of HPV oncogenicity-related genes E6 and E7 were much different between the high and low oncogenic HPVs, and the compositional trait of the DCR-based deep learning classifier predicted the oncogenic phenotype accurately of HPVs. The trained predictor in this study will facilitate the identification of HPV oncogenicity, particularly for those HPVs without clear genotype or phenotype.

Correlation analysis of human papillomavirus E6/E7 mRNA detection with diagnosis, prognosis and recurrence risk in patients with cervical epithelioma.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) is an important precursor of cervical cancer. Early detection and treatment can reduce the incidence of cervical cancer. AIM: To investigate the detection rate of human papillomavirus (HPV) E6/E7 mRNA in cervical tissue of patients with different types of epithelial cell neoplasia (CIN) and its relationship with CIN progression and diagnosis. METHODS: One hundred women with HPV infection detected by cervical exfoliation cytology between January 2022 and January 2023 were retrospectively selected. These patients were graded CIN based on colposcopy and cervical pathology. The positive expression rates of HPV E6/E7 mRNA and HPV [polymerase chain reaction (PCR)-reverse dot crossing] were compared among all groups. Patients with HPV E6/E7 mRNA expression in the grade 1 CIN group were followed up for 1 yr. The relationship between atypical squamous epithelium and high malignant epithelial neoplasia was investigated by univariate and multivariate analysis. RESULTS: The diagnostic sensitivity, specificity, and sensitivity of PCR-reverse point hybridization technology for secondary CIN were 70.41%, 70.66%, and 0.714, respectively. Sensitivity and specificity for secondary CIN were 752% and 7853%, respectively, the area under the curve value was 0.789. Logistic Multifactorial model analysis revealed that the HPV positive rates and the HPV E6/E7 mRNA positive rates were independent risk factors of CIN grade I (P < 0.05). In CIN grade I patients with positive for HPV E6/E7 mRNA, in its orientation to grade CIN patients, in its orientation to grade CIN patients, at 69.2%, compared with patients negative for HPV E6/E7 mRNA (30.8%), significant difference (P < 0.05). CONCLUSION: HPV E6/E7 mRNA and HPV (PCR-reverse dot hybrid) positive expression have a close relationship with CIN-grade disease progression and is an independent risk factor for high-grade CIN lesions.

Isolation, Identification, and Biological Activities of a New Chlorin e6 Derivative.

Chlorin e6 is a well-known photosensitizer used in photodynamic diagnosis and therapy. A method for identifying and purifying a novel process-related impurity during the synthesis of chlorin e6 has been developed. Its structure was elucidated using NMR and HRMS. This new impurity is formed from chlorophyll b rather than chlorophyll a, which is the source of chlorin e6. The intermediates formed during chlorin e6 synthesis were monitored using HPLC-mass spectrometry. This new impurity was identified as rhodin g7 71-ethyl ester, the structure of which remains unknown to date. The cytotoxic effects of this novel compound in both dark and light conditions were studied against five cancer cell lines (HT29, MIA-PaCa-2, PANC-1, AsPC-1, and B16F10) and a normal cell line (RAW264.7) and compared to those of chlorin e6. Upon irradiation using a laser at 0.5 J/cm2, rhodin g7 71-ethyl ester demonstrated higher cytotoxicity (2-fold) compared to chlorin e6 in the majority of the cancer cell lines. Furthermore, this new compound exhibited higher dark cytotoxicity compared to chlorin e6. Studies on singlet oxygen generation, the accumulation in highly vascular liver tissue, and the production of reactive oxygen species in MIA-PaCa-2 cancer cells via rhodin g7 71-ethyl ester correspond to its higher cytotoxicity as a newly developed photosensitizer. Therefore, rhodin g7 71-ethyl ester could be employed as an alternative or complementary agent to chlorin e6 in the photodynamic therapy for treating cancer cells.

Comparison of preclinical efficacy of immunotherapies against HPV-induced cancers.

INTRODUCTION: Persistent infections with the human papilloma viruses, HPV16 and HPV18, are associated with multiple cancers. Although prophylactic vaccines that induce HPV-neutralizing antibodies are effective against primary infections, they have no effect on HPV-mediated malignancies against which there is no approved immuno-therapy. Active research is ongoing on immunotherapy of these cancers. AREAS COVERED: In this review, we compared the preclinical efficacy of vaccine platforms used to treat HPV-induced tumors in the standard model of mice grafted with TC-1 cells, which express the HPV16 E6 and E7 oncoproteins. We searched for the key words, 'HPV,' 'vaccine,' 'therapy,' 'E7,' 'tumor,' 'T cells' and 'mice' for the period from 2005 to 2023 in PubMed and found 330 publications. Among them, we selected the most relevant to extract preclinical antitumor results to enable cross-sectional comparison of their efficacy. EXPERT OPINION SECTION: We compared these studies for HPV antigen design, immunization regimen, immunogenicity, and antitumor effect, considering their drawbacks and advantages. Among all strategies used in murine models, certain adjuvanted proteins and viral vectors showed the strongest antitumor effects, with the use of lentiviral vectors being the only approach to result in complete tumor eradication in 100% of experimental individuals while providing the longest-lasting memory.

Persistent infections with the human papilloma virus HPV16 and HPV18 gentoypes can cause multiple cancers.Prophylactic anti-HPV vaccines show no efficacy against persistent HPV infections or already malignant tissues.No immunotherapy against HPV-induced cancers has been thus far approved for use in humans.Active research is ongoing on immunotherapy of HPV-induced malignancies.We compared the efficacy of the immunotherapy strategies developed against HPV-induced cancers in the standard murine TC-1 tumor model since 2005.Certain adjuvanted proteins and viral vectors induce the strongest effects against HPV-induced tumors.Lentiviral vectors, able to induce the longest-lasting T-cell immune memory, give rise to full eradication of large solid tumors in 100% of mice.

Molecular Actions of Enicostemma hyssopifolium Whole Plant Extract on HPV18-Infected Human Cervical Cancer (HeLa) Cells.

OBJECTIVE: Enicostemma hyssopifolium (E. hyssopifolium) contains several bioactive compounds with anti-cancer activities. This study was performed to investigate the molecular effects of E. hyssopifolium on HPV18-containing HeLa cells. METHODS: The methanol extract of E. hyssopifolium whole plant was tested for cytotoxicity by MTT assay. A lower and higher dose (80 and 160 µg/mL) to IC50 were analyzed for colonization inhibition (Clonogenic assay), cell cycle arrest (FACS analysis), and induction of apoptosis (AO/EtBr staining fluorescent microscopy and FACS analysis) and DNA fragmentation (comet assay). The HPV 18 E6 gene expression in treated cells was analyzed using RT-PCR and qPCR. RESULTS: A significant dose-dependent anti-proliferative activity (IC50 - 108.25±2 µg/mL) and inhibition of colony formation cell line were observed using both treatments. Treatment with 80 µg/mL of extract was found to result in a higher percent of cell cycle arrest at G0/G1 and G2M phases with more early apoptosis, while 160 µg/mL resulted in more cell cycle arrest at SUBG0 and S phases with late apoptosis for control. The comet assay also demonstrated a highly significant increase in DNA fragmentation after treatment with 160 µg/mL of extract (tail moments-19.536 ± 17.8), while 80 µg/mL of extract treatment showed non-significant tail moment (8.152 ± 13.0) compared to control (8.038 ± 12.0). The RT-PCR and qPCR results showed a significant reduction in the expression of the HPV18 E6 gene in HeLa cells treated with 160 µg/mL of extract, while 80 µg/mL did not show a significant reduction. CONCLUSION: The 160 µg/mL methanol extract of E. hyssopifolium demonstrated highly significant anti-cancer molecular effects in HeLa cells.

Genetic variability of human papillomavirus type 18 based on E6, E7 and L1 genes in central China.

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16. METHODS: In this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively. RESULTS: A total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes. CONCLUSION: The sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.

Human papillomavirus-16 E6-positive cervical cancer attenuated by potent 2-(4-biphenylyl)-N-(1-ethyl-4-piperidinyl) acetamide second-generation analogs with improved binding affinity.

Human papillomavirus (HPV) infection, particularly HPV16, is a major contributor to the development of cervical cancer. Given the urgent need for novel therapeutic strategies targeting HPV-associated cancers, this study focuses on characterizing second-generation analogs of a lead compound, as a potential inhibitor of HPV16-E6. Protein-ligand docking, Gibbs binding free energy estimation, and molecular dynamics simulations were conducted. HPV16-infected SiHa and CaSki cell lines were used. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay for proliferation and flow cytometry for target inhibition and apoptosis were employed. Computational and cell proliferation analyses revealed that modifications to E6-855, particularly in the piperidinyl group, enhanced binding affinities against HPV16-E6, with E6-272 demonstrating superior binding properties. Molecular dynamics simulations confirmed the stable binding of E6-272 to HPV16-E6, supported by favorable binding energy estimates. E6-272 inhibited the proliferation of SiHa and CaSki cells with GI50 values of 32.56 and 62.09 nM, respectively. The compound reduced HPV16-E6-positive population, while inducing the early and late phase apoptosis in these cells. Structural alterations at the piperidinyl group of E6-855 identified E6-272 as a promising inhibitor of HPV16-E6 with improved efficacy against HPV16-E6. Further experimental validation of E6-272 and its analogs warrant to advance its clinical utility in combating HPV-associated cancers.

Multifunctional Nanosystem for Dual Anti-Inflammatory and Antibacterial Photodynamic Therapy in Infectious Diabetic Wounds.

Infectious diabetic wounds present a substantial challenge, characterized by inflammation, infection, and delayed wound healing, leading to elevated morbidity and mortality rates. In this work, we developed a multifunctional lipid nanoemulsion containing quercetin, chlorine e6, and rosemary oil (QCRLNEs) for dual anti-inflammatory and antibacterial photodynamic therapy (APDT) for treating infectious diabetic wounds. The QCRLNEs exhibited spherical morphology with a size of 51 nm with enhanced encapsulation efficiency, skin permeation, and localized delivery at the infected wound site. QCRLNEs with NIR irradiation have shown excellent wound closure and antimicrobial properties in vitro, mitigating the nonselective cytotoxic behavior of PDT. Also, excellent biocompatibility and anti-inflammatory and wound healing responses were observed in zebrafish models. The infected wound healing properties in S. aureus-infected diabetic rat models indicated re-epithelization and collagen deposition with no signs of inflammation. This multifaceted approach using QCRLNEs with NIR irradiation holds great promise for effectively combating oxidative stress and bacterial infections commonly associated with infected diabetic wounds, facilitating enhanced wound healing and improved clinical outcomes.

Time to take HPV infection in colorectal cancer patients more seriously.

Background: The association between viral infections and colorectal cancer (CRC) remains an enigma in cancer research. Certain types of Human Papillomaviruses (hr-HPVs), known for their oncogenic properties, have been observed in particular CRC biopsies, further adding to the enigma surrounding this association. Materials and methods: This cross-sectional study was conducted on 40 confirmed cases of CRC adenocarcinoma. The presence and genotyping of HPV DNA in colorectal fresh tissue and urine samples was assessed using an HPV DNA hybridization kit. A subset of serum samples from both CRC cases and healthy volunteers was randomly chosen and subjected to western blot to investigate the presence of HPV16 E6/E7 oncoproteins carried by exosomes. Results: It was observed that 26/40 HPV-positive CRC patients demonstrated 7 times more chance to develop colorectal cancer when compared to those 8/40 normal tissue (odds ratio [OR] = 7.4; confidence interval [CI] 95% = 0.483156-0.793718; p < 0.001). Of 26 HPV-positive CRC patients, 14 urine samples were also showed HPV DNA positivity (p = 0.013). High-risk HPV16 was the most prevalent genotype detected in both 24/40 tumor and 12/40 urine samples (p < 0.001). The tumor sample of a male was HPV45, while another male's urine sample was HPV31. A female CRC patient had HPV83 in tumor and HPV56 in urine. Here, was the first detection of HPV83 in a CRC patient. Notably among 20 randomly selected serum exosome samples, one serum sample concurrently tested positive for both HPV16 E6 and E7 oncoproteins, and one sample tested positive for HPV16 E7 oncoprotein. Conclusion: High risk HPV DNA detection in CRC urine samples supports non-invasive screening tools. Detection of HPV16 E6 and E7 oncoproteins in exosomes from serum samples shows potential for non-invasive diagnostics. HPV's potential role in CRC development is also underscored. HPV vaccination should be implemented in low- and middle-income countries to prevent cancer.

Whole-Genome Characterization of Rotavirus G9P[6] and G9P[4] Strains That Emerged after Rotavirus Vaccine Introduction in Mozambique.

Mozambique introduced the Rotarix® vaccine into the National Immunization Program in September 2015. Following vaccine introduction, rotavirus A (RVA) genotypes, G9P[4] and G9P[6], were detected for the first time since rotavirus surveillance programs were implemented in the country. To understand the emergence of these strains, the whole genomes of 47 ELISA RVA positive strains detected between 2015 and 2018 were characterized using an Illumina MiSeq-based sequencing pipeline. Of the 29 G9 strains characterized, 14 exhibited a typical Wa-like genome constellation and 15 a DS-1-like genome constellation. Mostly, the G9P[4] and G9P[6] strains clustered consistently for most of the genome segments, except the G- and P-genotypes. For the G9 genotype, the strains formed three different conserved clades, separated by the P type (P[4], P[6] and P[8]), suggesting different origins for this genotype. Analysis of the VP6-encoding gene revealed that seven G9P[6] strains clustered close to antelope and bovine strains. A rare E6 NSP4 genotype was detected for strain RVA/Human-wt/MOZ/HCN1595/2017/G9P[4] and a genetically distinct lineage IV or OP354-like P[8] was identified for RVA/Human-wt/MOZ/HGJM0644/2015/G9P[8] strain. These results highlight the need for genomic surveillance of RVA strains detected in Mozambique and the importance of following a One Health approach to identify and characterize potential zoonotic strains causing acute gastroenteritis in Mozambican children.

Cationic Liposomes Carrying HPV16 E6-siRNA Inhibit the Proliferation, Migration, and Invasion of Cervical Cancer Cells.

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.

In-silico study unveils potential phytocompounds in Andrographis paniculata against E6 protein of the high-risk HPV-16 subtype for cervical cancer therapy.

Despite therapeutic advancements, cervical cancer caused by high-risk subtypes of the human papillomavirus (HPV) remains a leading cause of cancer-related deaths among women worldwide. This study aimed to discover potential drug candidates from the Asian medicinal plant Andrographis paniculata, demonstrating efficacy against the E6 protein of high-risk HPV-16 subtype through an in-silico computational approach. The 3D structures of 32 compounds (selected from 42) derived from A. paniculata, exhibiting higher binding affinity, were obtained from the PubChem database. These structures underwent subsequent analysis and screening based on criteria including binding energy, molecular docking, drug likeness and toxicity prediction using computational techniques. Considering the spectrometry, pharmacokinetic properties, docking results, drug likeliness, and toxicological effects, five compounds-stigmasterol, 1H-Indole-3-carboxylic acid, 5-methoxy-, methyl ester (AP7), andrographolide, apigenin and wogonin-were selected as the potential inhibitors against the E6 protein of HPV-16. We also performed 200 ns molecular dynamics simulations of the compounds to analyze their stability and interactions as protein-ligand complexes using imiquimod (CID-57469) as a control. Screened compounds showed favorable characteristics, including stable root mean square deviation values, minimal root mean square fluctuations and consistent radius of gyration values. Intermolecular interactions, such as hydrogen bonds and hydrophobic contacts, were sustained throughout the simulations. The compounds displayed potential affinity, as indicated by negative binding free energy values. Overall, findings of this study suggest that the selected compounds have the potential to act as inhibitors against the E6 protein of HPV-16, offering promising prospects for the treatment and management of CC.

Enhancing Cervical Cancer Screening with 7-Type HPV mRNA E6/E7 Testing on Self-Collected Samples: Multicentric Insights from Mexico.

Cervical cancer remains a significant public health issue, particularly in regions with low screening uptake. This study evaluates the effectiveness of self-sampling and the 7-type HPV mRNA E6/E7 test in improving cervical cancer screening outcomes among a referral population in Mexico. A cohort of 418 Mexican women aged 25 to 65, referred for colposcopy and biopsy due to abnormal cytology results (ASC-US+), participated in this study. Self-samples were analyzed using both the 14-type HPV DNA test and the 7-type HPV mRNA E6/E7 test. The study assessed the sensitivity, specificity, positive predictive value (PPV), and the necessity of colposcopies to detect CIN3+ lesions. Participant acceptability of self-sampling was also evaluated through a questionnaire. The 7-type HPV mRNA E6/E7 test demonstrated equivalent sensitivity but significantly higher specificity (77.0%) and PPV for CIN3+ detection compared to the 14-type HPV DNA test (specificity: 45.8%, p < 0.001). The use of the HPV mRNA test as a triage tool reduced the number of colposcopies needed per CIN3+ case detected from 16.6 to 7.6 (p < 0.001). Self-sampling was highly accepted among participants, with the majority reporting confidence in performing the procedure, minimal discomfort, and willingness to undertake self-sampling at home. Self-sampling combined with the 7-type HPV mRNA E6/E7 testing offers a promising strategy to enhance cervical cancer screening by improving accessibility and ensuring precise diagnostics. Implementing these app roaches could lead to a significant reduction in cervical cancer morbidity and mortality, especially in underserved populations. Future research should focus on the long-term impact of integrating these methods into national screening programs and explore the cost-effectiveness of widespread implementation.

HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

Regional and cellular organization of the autism-associated protein UBE3A/E6AP and its antisense transcript in the brain of the developing rhesus monkey.

Angelman syndrome (AS) is a neurogenetic disorder caused by mutations or deletions in the maternally-inherited UBE3A allele, leading to a loss of UBE3A protein expression in neurons. The paternally-inherited UBE3A allele is epigenetically silenced in neurons during development by a noncoding transcript (UBE3A-ATS). The absence of neuronal UBE3A results in severe neurological symptoms, including speech and language impairments, intellectual disability, and seizures. While no cure exists, therapies aiming to restore UBE3A function-either by gene addition or by targeting UBE3A-ATS-are under development. Progress in developing these treatments relies heavily on inferences drawn from mouse studies about the function of UBE3A in the human brain. To aid translational efforts and to gain an understanding of UBE3A and UBE3A-ATS biology with greater relevance to human neurodevelopmental contexts, we investigated UBE3A and UBE3A-ATS expression in the developing brain of the rhesus macaque, a species that exhibits complex social behaviors, resembling aspects of human behavior to a greater degree than mice. Combining immunohistochemistry and in situ hybridization, we mapped UBE3A and UBE3A-ATS regional and cellular expression in normal prenatal, neonatal, and adolescent rhesus macaque brains. We show that key hallmarks of UBE3A biology, well-known in rodents, are also present in macaques, and suggest paternal UBE3A silencing in neurons-but not glial cells-in the macaque brain, with onset between gestational day 48 and 100. These findings support proposals that early-life, perhaps even prenatal, intervention is optimal for overcoming the maternal allele loss of UBE3A linked to AS.

Enhancement of Tumor Antigen-specific T-cell Responses by Immune Checkpoint Blockade in Human Papillomavirus-related Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically and immunologically distinct from HPV-negative HNSCC. Herein, we investigated the presence of tumor antigens HPV E6/E7 and wild-type p53-specific T-cell responses, and the impact of immune checkpoint blockade in patients with HPV-positive HNSCC. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from patients with HPV-positive HNSCC were stimulated with HPV E6/E7 or wild-type p53-derived peptide mixture and evaluated using the interferon-γ enzyme-linked immunosorbent spot assay. Flow cytometry was performed to analyze the proportion of T-cell subsets and T cells expressing immune checkpoint molecules. RESULTS: HPV E6/E7-specific T cells were detected in 22 (95.7%) of 23 patients, whereas wild-type p53-specific T cells were detected in 3 (15.0%) of 20 patients. Seven (43.8%) of 16 patients exhibited wild-type p53-specific T-cell responses, as determined using whole proteins instead of peptides. Immune checkpoint blockade enhanced wild-type p53-specific T-cell responses in 9 (45.0%) of 20 patients. Flow cytometric analysis of PBMCs revealed that responders exhibiting enhanced wild-type p53-specific T-cell responses following immune checkpoint blockade had a significantly higher proportion of Ki-67+CD4+ T cells, Ki-67+CD8+ T cells, regulatory T cells, PD-1+CD4+ T cells, and TIM-3+CD4+ T cells than non-responders. CONCLUSION: Our findings indicate that tumor antigen-specific T cells are present in the peripheral blood of patients with HPV-positive HNSCC. Blockade of checkpoint pathways can enhance T-cell responses in certain patients, probably via activated T cells, Tregs, and/or exhausted CD4+ T cells.