E2 Protein Research Articles

Abstract PR003: Preventing and treating HPV-related Cancer with mRNA Therapy Expressing A DC-targeting Antigen

Abstract Persistent human papillomavirus (HPV) infection is linked to several malignancies, highlighting the need for effective therapeutic vaccines. In this study, we tested a lipid nanoparticle-encapsulated mRNA vaccine expressing tHA-mE7-mE6. By mutating the E6 and E7 proteins to reduce their tumorigenicity and fusing them with a truncated influenza hemagglutinin protein (tHA) for better antigen uptake, we improved the vaccine’s efficacy. The tHA-mE7-mE6 mRNA vaccine showed superior results compared to mE7-mE6 mRNA, achieving complete tumor regression and preventing new tumors in an E6 and E7 positive model. It elicited a strong CD8+ T-cell response, with antigen-specific CD8+ T-cells found in the spleen, blood, and tumors. Additionally, the therapy increased DC and NK cell infiltration into tumors. Overall, this study demonstrates that the tHA-mE7-mE6 mRNA vaccine induces a potent anti-tumor immune response, showing promise for treating HPV-induced cancers and preventing recurrence. Citation Format: William Jia. Preventing and treating HPV-related Cancer with mRNA Therapy Expressing A DC-targeting Antigen [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR003.

Abstract A065: Estimation of HPV16-induced cervical cancer cells in routine Thin-prep cytology samples of cancer patients

Abstract Persistent infection with human papillomavirus has been implicated in the etiology of cervical cancer and sustained expression of its E7 oncoprotein has been rendered suggestive of malignant transformation. However, there are limited E7 protein-based detection assays. This might be due to paucity of data on steady-state expression level of E7 protein and the number of HPV-induced cervical cancer cells in routine Thin-prep cytology samples. So, the objective of the present study was to identify and estimate HPV16-induced cervical cancer cells in cytology samples of cancer patients, with the goal to generate a quantitative immunological method in the cytological diagnosis of cervical lesions. We collected 25 Thin-prep cytology samples of cervical cancer patients (PAP test confirmed) from Obstetrics and Gynecology department, AIIMS, New Delhi. Samples were conditioned to enrich cellular components and were then quantified using hemocytometer for total cell count, PAP staining for cancerous cell count and immunofluorescence using HPV16 E7 antibody for HPV16-induced cancerous cell count. The method devised efficiently found an average number of 7 × 103 cells/ ml to be E7 positive, out of total 27 x 104 cells/ ml. Although PAP test detected 71 x 103 cells/ ml to be abnormal or cancerous, only 10% of them were found to be HPV16 transformed, suggesting HPV E7 as the primary marker for detecting transformed cells and thereby, identifying patients at-risk and prompting a better prognosis. This simple and efficient method deduced allows optimizing the number of HPV16-induced cervical cancer cells from cytology samples which directly correlates to the amount of HPV-16 major oncoprotein- E7 expression, and the disease severity. Citation Format: Srishty Raman, Pranay Tanwar, Neerja Bhatla, Subhash Chandra Yadav. Estimation of HPV16-induced cervical cancer cells in routine Thin-prep cytology samples of cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A065.

A Study Into Systemic and Oral Levels of Proinflammatory Biomarkers Associated With Endpoints After Active Non-Surgical Periodontal Therapy.

To analyse whether some selected inflammatory biomarkers collected from venous blood and gingival crevicular fluid (GCF) were associated with the outcome of non-surgical periodontal therapy. Two-hundred and nine patients affected by periodontitis were enrolled in the study, who had undergone steps I and II therapy as well as a non-surgical re-instrumentation (NSRI) of periodontal pockets after 6 months. Serum (SE), plasma (PL) and GCF samples were quantitatively analysed for the following inflammatory biomarkers: active matrix metalloproteinase-8 (aMMP-8), prostaglandin E2 (PGE2) and surfactant protein D (SP-D). Therapy outcomes were evaluated using a 'treat-to-target' endpoint (T2T) at the patient level, defined as ≤ 4 sites with pocket depth ≥ 5 mm. Patients presented with 23 ± 6 teeth (mean ± SD) at baseline. After steps I and II therapy, 41.6% of the patients reached T2T and after NSRI 47.4%. Univariate analysis identified a potential association between high levels of PL-SP-D and more favourable treatment outcomes. Multivariate binary logistic regression adjusted for sex, mean baseline probing depth, diabetes and current smoking status confirmed an independent relationship between baseline PL-SP-D and the T2T after steps I and II therapy (aOR 0.432, p = 0.011), implying that a higher level PL-SP-D at baseline is associated with a > 50% reduced risk of failing T2T. However, no such association was found for PL-SP-D and NSRI. Higher baseline PL-SP-D levels might be associated with more favourable treatment outcomes after steps I and II therapy. This may be due to its role in the regulation of neutrophil function. However, further investigation is required to confirm this hypothesis. If proven, PL-SP-D could play a role as a biomarker for identifying individuals who respond differentially to primary therapy.

A Self-Amplifying Human Papillomavirus 16 Vaccine Candidate Delivered by Tobacco Mosaic Virus-Like Particles.

Virus-like particles (VLPs) are naturally occurring delivery platforms with potential for mRNA vaccines that can be used as an alternative to lipid nanoparticles. Here we describe a self-amplifying mRNA vaccine based on tobacco mosaic virus (TMV) expressing a mutated E7 protein from human papillomavirus 16 (HPV16). E7 is an early gene that plays a central role in viral replication and the oncogenic transformation of host cells, but nononcogenic mutant E7 proteins can suppress this activity. Immunization studies involving the delivery of self-amplifying mutant E7 mRNA packaged with TMV coat proteins confirmed the elicitation of E7-specific IgG antibodies. Additional in vitro splenocyte proliferation and cytokine profiling assays indicated the activation of humoral and cellular immune responses. We conclude that TMV particles are suitable for the delivery of mRNA vaccines and can preserve their integrity and functionality in vitro and in vivo.

Modulation of epithelial homeostasis by HPV using Notch and Wnt.

Highly conserved signalling pathways such as Notch and Wnt are essential in the regulation of differentiation and proliferation processes during adult tissue homeostasis. Human papillomaviruses (HPVs) have evolved with humans to manipulate these signalling pathways to establish a basal reservoir of infected cells by limiting HPV-infected keratinocyte differentiation whilst ensuring that differentiating cells are in a replication-competent state. Here, we focus on the canonical Notch and Wnt signalling pathways and their crosstalk to ensure cell-fate lineage determination during epithelial homeostasis. We then examine how HPVs use their E6 and E7 proteins to inhibit differentiation and maintain stem-like characteristics using Notch and Wnt in HPV-infected cells. Determining the functions of E6 and E7 in the maintenance of the infected cell reservoir, and the molecular crosstalk between Notch and Wnt is vital for our understanding of HPV persistence, and may represent an important factor in the development of therapeutic agents for HPV-associated disease.

Comparison of the efficacy of aescin and diclofenac sodium in the management of postoperative sequelae and their effect on salivary Prostaglandin E2 and serum C-reactive protein levels after surgical removal of impacted mandibular third molar: a randomized, double-blind, controlled clinical trial.

Surgical removal of an impacted third molar is one of the most common oral surgical procedures performed in dental offices. The postoperative phase is often associated with severe inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) are usually prescribed to manage postoperative discomfort. NSAIDs have been associated with gastrointestinal bleeding, renal function disturbances, and platelet count reductions. Thus, the present study demonstrates the utility of aescin in managing postoperative discomfort after the surgical removal of impacted mandibular third molars.This study aimed to correlate and compare the impact of aescin and diclofenac on salivary PGE2 levels and serum C-reactive protein levels after surgical extraction of the mandibular third molar. The study will also evaluate and compare the effectiveness of individual drug therapy in managing postoperative pain, swelling and mouth opening. The planned study is a single-center, double-blind, randomized, parallel, prospective clinical trial. Each patient will be prescribed either diclofenac sodium 150 mg/day or aescin (escin) 120 mg/day to be taken orally in divided doses for five days after surgically removing the impacted mandibular third molar.Pain will be assessed using a visual analog scale. Facial swelling and mouth opening will be recorded using a metric scale with standardized reference points. ELISA (enzyme-linked immunosorbent assay (ELISA) will be employed to measure salivary Prostaglandin E2 and serum C-reactive protein levels. All parameters will be recorded preoperatively (T0) on the second postoperative day (T1) and fifth postoperative day (T2). The proposed study is expected to show a clinically acceptable response to the administration of aescin for the management of postoperative discomfort compared to diclofenac sodium after third molar surgery.The proposed study is expected to positively manipulate the levels of salivary Prostaglandin E2 and serum C-reactive protein, which are reliable inflammatory markers.The outcome of this study may provide an efficacious and safe alternative to conventional nonsteroidal anti-inflammatory drugs for managing postoperative discomfort following third molar surgery.

A vaccine candidate based on baculovirus displaying chikungunya virus E1-E2 envelope confers protection against challenge in mice.

Chikungunya fever is a re-emerging mosquito-borne disease caused by the chikungunya virus (CHIKV) and produces acute arthritis that can progress to chronic disease with arthralgia. The first approved live-attenuated chikungunya vaccine has only recently become available for use in humans in the USA, but the access in endemic regions remains unmet. Here, we exploited the baculovirus display technology to develop a vectored vaccine candidate that exposes the CHIKV membrane proteins E1 and E2 on the baculovirus surface. Using recombinant baculovirus as vector vaccines has both productive and regulatory advantages: they are safe for handling and easy to produce in high titers and are non-pathogenic and non-replicative in mammals but have strong adjuvant properties by inducing humoral and cellular immune responses. CHIKV E1 and E2 envelope proteins with their own signal and transmembrane sequences were expressed on the surface of budded baculovirus virions. Immunization of C57BL/6 mice with non-adjuvanted recombinant baculovirus induced IgG antibodies against E2 with a predominant IgG2c subtype, neutralizing antibodies and a specific IFN-γ CD8+ T-cell response. Immunization with a second dose significantly boosted the antibody response, and mice immunized with two doses of the vaccine candidate were completely protected against challenge with CHIKV showing no detectable viremia or signs of disease. Altogether, baculovirus display of CHIKV envelope proteins served as an efficient vaccine platform against CHIKV.IMPORTANCEThe global spread of chikungunya virus (CHIKV) has disproportionately impacted the Americas that experienced a fourfold increase in 2023 in cases and deaths compared with the same period in 2022. The disease is characterized by acute fever and debilitating joint pain that can become chronic. Despite the socioeconomic burden related to the high morbidity rates of CHIKV infection, a vaccine for CHIKV is currently approved only in the USA. Vaccines are the most effective preventive measure against viral diseases, and advances in the development of different vaccine platforms such as nucleic acids and viral vectors have prompted the rapid deployment of vaccines to contain the COVID-19 pandemic. Here, we report the use of baculovirus display as a strategy for the design of a novel vaccine that provides sterilizing immunity in a mouse model of chikungunya disease. Our results encourage further research regarding the potential of baculovirus as platforms for human vaccine design.

In Silico Drug Designing for the Treatment of Cervical Cancer

Cervical cancer is a significant global health issue, especially in developing countries where limited resources and access to health services pose challenges. Human papillomavirus (HPV) is the primary cause of cervical cancer and plays a crucial role in its development. Despite the availability of prophylactic vaccines, effective treatment options for patients with pre-existing HPV infections or HPV-induced carcinomas remain elusive. High-risk (HR) HPV E6 and E7 oncoproteins serve as biomarkers in cervical cancer progression. Research has focused on these proteins as potential targets for novel therapeutics and to find a novel compound that can overcome the problem of drug ineffectiveness in cancerous cells as they become multi-drug resistant to the existing drugs and treatments. Computational-aided methods, including in silico approaches, have been valuable in discovering and developing small molecules that can block or inhibit the action of the E6 oncoproteins. This review discusses existing small molecules explored as HPV E6 protein blockers and outlines future perspectives in cervical cancer therapy. In molecular docking studies, it is identified that four compounds exhibit the properties of a lead molecule, possess more negative binding energy, and form a highly stable ligand-receptor complex. These four compounds will help in future research and development. Advancing these compounds through further studies will help develop potential and effective drugs for treating cervical cancer.

Targeted degradation of the HPV oncoprotein E6 reduces tumor burden in cervical cancer.

Human Papilloma Virus (HPV)-related cancers are a global health burden, yet there are no targeted therapies available for chronically infected patients. The HPV protein E6 is essential for HPV-mediated tumorigenesis and immune evasion, making it an attractive target for antiviral drug development. In this study, we developed an E6-targeting Proteolysis Targeting Chimera (PROTAC) that inhibits the growth of HPV(+) tumors. To develop E6 antagonists, we generated a panel of nanobodies targeting E6 proteins derived from the oncogenic HPV16 subtype. The highest affinity E6 nanobody, A5, was fused to Von Hippel Lindau protein (VHL) to generate a PROTAC that degrades E6 (PROTAC E6 ). Mutational rescue experiments validated specific degradation via the CRL2 VHL E3 ligase. Intralesional administration of the PROTAC E6 using a clinically viable DNA vaccine reduced tumor burden in an immunocompetent mouse model of HPV(+) cancer. The inhibitory effect of the PROTAC E6 was abrogated by CD4 + and CD8 + T-cell depletion, indicating that the antitumor function of the PROTAC E6 relies in part on a host immune response. Overall, these results suggest that the targeted degradation of E6 inhibits its oncogenic function and stimulates a robust immune response against HPV(+) tumors, opening new opportunities for virus-specific therapies in the treatment of HPV-related cancers.

Rubella virus assembly requirements and evolutionary relationships with novel rubiviruses.

Rubella virus (RuV) is an enveloped virus that only infects humans, where transplacental infection can cause miscarriage or congenital birth defects. We identified a potential late domain, 278PPAY281, at the C terminus of the E2 envelope protein. However, rather than this domain recruiting the cellular ESCRT machinery as predicted, our data indicate that E2 Y281 promotes a critical interaction of the E2 endodomain with the capsid protein, leading to capsid's localization to the Golgi where virus budding occurs. Revertant analysis demonstrated that two substitutions on the E2 protein could partially rescue virus growth and Cp-Golgi localization. Both residues were found at the corresponding positions in Ruhugu virus E2, supporting the close evolutionary relationship between RuV and Ruhugu virus, a recently discovered rubivirus from bats.

Targeting Human Papillomavirus 33 E2 DNA Binding Domain With Polyphenols: Unveiling Interactions Through Biophysical and In Silico Methods.

The human papillomavirus (HPV) 33 is a high-risk strain that causes lesions with potential cancerous outcomes. Its E2 protein regulates the viral protein transcription and life cycle maintenance. The DNA binding domain (DBD) of the E2 protein plays a crucial role in the viral life cycle. The DBD region of the E2 protein is particularly interesting for targeting and finding potential inhibitors to inhibit its function or dimerization. Given the limited research on HPV 33 and its proteins, the present work delved into the interaction of two natural polyphenolic compounds, resveratrol, and baicalein, with the E2 DBD of HPV 33 using biophysical and in silico studies. Fluorescence studies of the E2 DBD-polyphenol complexes showed fluorescence quenching with a binding constant of the order of 106 M-1. Circular dichroism data reveal conformational changes upon binding with the polyphenols, possibly due to distinct binding sites of the E2 DBD. Differential scanning calorimetry exhibited higher melting temperatures for the two complexes than alone DBD, suggesting the complexes' stability. ITC experiment suggested favorable binding reactions with kd values in the micromolar range. Molecular docking and dynamic simulation studies revealed that the resveratrol binds to the helical region and baicalein near the central dimeric interface of E2 DBD with a good binding affinity, forming a stable protein-ligand complex during the run of 100 ns simulation. Therefore, the current study identifies both polyphenolic compounds as promising candidates for potential antiviral drug development.

Prevalence of hepatitis C virus hypervariable region 1 insertions and their role in antibody evasion.

Chronic hepatitis C virus (HCV) infection afflicts around 50 million people globally, causing ~250,000 deaths yearly. An effective vaccine needs to overcome high viral diversity and HCV's ability to evade neutralizing antibodies (NAbs). Rapid antigenic drift in the N-terminal motif of envelope protein E2, named hypervariable region 1 (HVR1), is critically involved in NAb evasion via an incompletely understood mechanism involving viral entry factors. The canonical length of HVR1 is 27 amino acids, but insertions of 2-4 amino acids was described in patients infected with genotype 1b. We aimed at determining whether HVR1 insertions may be underreported due to extreme HVR1 variability. We observed a 0.7% HVR1 insertion prevalence in routine NGS patient contigs. Thus, we performed direct sequence analysis of E1E2 sequences from 131 HCV infected patients. Interestingly, we observed that 3% of patients harbored viruses (genotype 1a, 2b, 3a) with dominant HVR1 insertions. Insertion of longer non-canonical HVR1s into HCV cell culture recombinants frequently caused loss of fitness. However, culture-viable viruses with HVR1 insertions were fully viable in vivo. Interestingly, in adapted genotype 1b recombinants with HVR1 insertions, we found internal HVR1 deletions, that increased antibody sensitivity, which surprisingly correlated more with reduced LDLr than reduced SR-BI dependency, indicating a role of LDLr in NAb evasion. Conversely, HVR1 insertions had no effect on receptor dependency, however, they modulated epitope-specific NAb sensitivity. HVR1 insertion prevalence and NAb sensitivity modulation indicate they represent a mechanism by which HCV evades emerging NAbs during infection.

Molecular interactions of plant-derived inhibitors from Euphorbia hirta against binding sites of HPV E6 and E7 oncoproteins - An in-silico analysis

Although no cure for Human Papilloma virus (HPV) exists at the moment, effective vaccines are present that can prevent the most harmful strains of the virus from infecting people. While potential cures like therapeutic vaccines, immunotherapy, and gene therapy are still in the early stages of development and may take years to become widely available, we focussed on using traditional Siddha-based medicines (phytochemicals of Euphorbia hirta L.) as a prospect through in-silico docking studies. Twelve phytochemicals from Euphorbia hirta L. were selected to investigate the antiviral properties against HPV. These ligand structures were obtained from PubChem database, prepared for docking with two major HPV targets i.e., E6 and E7 oncoproteins obtained from PDB, using AutoDock tools. The analysis revealed the potential of lobeline and euphol ligands effectively binding to the active sites of E6 and E7 proteins respectively. Using the ADMET lab 2.0 server, the ligands were evaluated for their commercial drug potential by considering their absorption, distribution, metabolism, excretion, and toxicity properties.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

Generation and epitope mapping of novel neutralizing monoclonal antibodies against glycoprotein E2 of CSFV

Classical swine fever virus (CSFV) is a highly contagious and economically important pathogen threatening pig industry worldwide, the envelope glycoprotein E2 of CSFV is the dominant antigen inducing strong antiviral neutralizing immunity. In this study, 7 monoclonal antibodies (mAbs) with neutralizing potency were generated using E2 protein of CSFV Shimen strain (SM) expressed by eukaryotic cells. Their reactivity with 116 CSFV strains in cell cultures and E2 proteins of 10 subgenotypes in western blots showed different CSFV spectrums they recognized. Of them, three (HCL-001, HCL-005 and HCL-010) reacted with all CSFV subgenotypes, while HCL-014 and HCL-002 reacted with most CSFV strains, except for some variants in genotype 2.3. In contrast, mAb HCL-009 reacted only with a few subgenotype 1.1 strains including SM, field strains and some vaccine strains. Interestingly, mAb HCL-018 reacted only with SM and field subgenotype 1.1 strains, not with any vaccine strains. Further epitope mapping using chimeric and site-directed mutated E2 proteins showed that HCL-001, HCL-005 and HCL-010 recognized a conservative epitope motif 143SPT145,L147, and HCL-002 recognized a conformational epitope with key aa motifs of 95GDD97,157RX(D/E)K(R)XFXXR164. HCL-014 recognized a new conservative epitope with key aa motifs of 41D,58XNVVXRR64. HCL-009 and HCL-018 recognized the epitope with key aa motifs of 36D,40ND41,45KXI47 and 69LHXGXLLT76, respectively. Taken together, present study has provided not only new insights into the antigenic structure of E2 protein, but also key reagents for antigenic characterization of CSFV strains and development of antibody assay for evaluation of the vaccination efficacy.

Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

Redox Status and Protein Glutathionylation in Binase-Treated HPV16-Positive SiHa Carcinoma Cells

Human papillomavirus type 16 (HPV16) belongs to viruses of the high-risk type and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus, such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of the cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the level of reactive oxygen species (ROS) and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis in HPV-16-positive SiHa cells of cervical squamous cell carcinoma,. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.e., decreases ROS and reduced glutathione (GSH) levels, suppresses the expression of the E6 oncoprotein, activates the expression of the p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the integrity of the mitochondrial membrane is a signal for activation of the mitochondrial apoptosis pathway.

Design, Synthesis, Characterization, Molecular Docking Studies, In vitro and In vivo Cervical Cancer Activity of Novel N-Substituted Pyrazole Derivatives

The aim of this study is to design, synthesize, characterize few novel N-substituted pyrazole derivatives and evaluated for their anticancer capabilities; these compounds were selected based on their ability to inhibit the HPV E6 protein. The IR, 1H & 13C NMR, mass spectral and elemental analysis were used to determine the structural details of the newly synthesized substances. The molecular docking was performed using Auto Dock Vina, and the protein-ligand interaction was analyzed using Discovery Studio. The sulforhodamine B (SRB) assay was used to determine the in vitro anticancer activity against the HeLa cell line. Body weight analysis, mean survival time and % increase in life span approaches were used to assess in vivo anticancer effectiveness in Swiss albino mice bearing Dalton’s Lymphoma Ascites (DAL) tumor model. Synthesized compounds have excellent docking energies, according to docking experiments. Compounds I, II and III were selected for anticancer activity in vivo DAL-bearing mice based on the findings of their in vitro and SRB assays exhibiting the antiproliferative activity. Pyrazole derivatives, compounds I and III, demonstrated significant potential as anticancer agents.

Daphnia stress response to environmental concentrations of chloramphenicol—multi-omics approach

Commonly used medicines, when discarded or improperly disposed of, are known to contaminate freshwater ecosystems. Pharmaceuticals can be toxic and mutagenic, and can modify freshwater organisms, even at environmentally relevant concentrations. Chloramphenicol (CAP) is an antibiotic banned in Europe. However, it is still found in surface waters around the world. The aim of this study was to evaluate the impact of chloramphenicol contamination in freshwater on the model organism Daphnia magna. Specific life history parameters, proteome, and host-associated microbiome of four D. magna clones were analyzed during a three-generation exposure to CAP at environmental concentrations (32 ng L−1). In the first generation, no statistically significant CAP effect at the individual level was detected. After three generations, exposed animals were smaller at first reproduction and on average produced fewer offspring. The differences in D. magna’s life history after CAP treatment were in accordance with proteome changes. D. magna’s response to CAP presence indicates the high stress that the tested organisms are under, e.g., male production, upregulation of ubiquitin-conjugating enzyme E2 and calcium-binding protein, and downregulation of glutathione transferase. The CAP-exposed D. magna proteome profile confirms that CAP, being reactive oxygen species (ROS)-inducing compounds, contributes to structural changes in mitochondria. Microbiome analysis showed a significant difference in the Shannon index between control and CAP-exposed animals, the latter having a more diverse microbiome. Multilevel analyses, together with long exposure in the laboratory imitating conditions in a polluted environment, allow us to obtain a more complete picture of the impact of CAP on D. magna.