LncRNAs expression profile in a family household cluster of COVID-19 patients.

More than 3 years after the start of SARS-CoV-2 pandemic, the molecular mechanisms behind the viral pathogenesis are still not completely understood. Long non-coding RNAs (lncRNAs), well-known players in viral infections, can represent prime candidates for patients' risk stratification. The purpose of the current study was to investigate the lncRNA profile in a family cluster of COVID-19 cases with different disease progression, during the initial wave of the pandemic and to evaluate their potential as biomarkers for COVID-19 evolution. LncRNA expression was investigated in nasopharyngeal swabs routinely collected for diagnosis. Distinct expression patterns of five lncRNAs (HOTAIR, HOTAIRM1, TMEVPG1, NDM29 and snaR) were identified in all the investigated cases, and they were associated with disease severity. Additionally, a significant increase in the expression of GAS5-family and ZFAS1 lncRNAs, which target factors involved in the inflammatory response, was observed in the sample collected from the patient with the most severe disease progression. An lncRNA prognostic signature was defined, opening up novel research avenues in understanding the interactions between lncRNAs and SARS-CoV-2.

New Cobalt (II) Complexes with Imidazole Derivatives: Antimicrobial Efficiency against Planktonic and Adherent Microbes and In Vitro Cytotoxicity Features.

Three novel Co(II) complexes of the type [Co(C4H5O2)2L2] (where C4H5O2 is methacrylate anion; L = C3H4N2 (imidazole; HIm) (1), C4H6N2 (2-methylimidazole; 2-MeIm) (2), C5H8N2 (2-ethylimidazole; 2-EtIm) (3)) have been synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopic techniques, thermal analysis and single crystal X-ray diffraction. X-ray crystallography revealed for complexes (1) and (2) distorted trigonal bipyramid stereochemistry for Co(II), meanwhile for complex (3) evidenced that the unit cell comprises three molecular units with interesting structural features. In each unit, both stereochemistry adopted by metallic ion and coordination modes of carboxylate anions are different. The screening of antimicrobial activity revealed that Candida albicans planktonic cells were the most susceptible, with minimal inhibitory concentration (MIC) values of 7.8 µg/mL for complexes (1) and (2) and 15.6 µg/mL for complex (3). Complexes (1) and (2) proved to be more active than complex (3) against the tested bacterial strains, both in planktonic and biofilm growth state, with MIC and minimal biofilm eradication concentration (MBEC) values ranging from 15.6 to 62.5 µg/mL, the best antibacterial effects being noticed against Staphylococcus aureus and Pseudomonas aeruginosa. Remarkably, the MBEC values obtained for the four tested bacterial strains were either identical or even lower than the MIC ones. The cytotoxicity assay indicated that the tested complexes affected the cellular cycle of HeLa, HCT-8, and MG63 cells, probably by inhibiting the expression of vimentin and transient receptor potential canonical 1 (TRPC1). The obtained biological results recommend these complexes as potential candidates for the development of novel anti-biofilm agents.

Human papillomaviruses' proteins with clinical utility.

Cervical cancer, the fourth leading cause of cancer-associated deaths among women worldwide, is associated with human papilloma virus (HPV) infection. Despite the prophylactic HPV vaccination and the implementation of cervical and HPV-based screening programs, a significant increase in cervical cancer incidence is estimated by the year 2020. Thus, further development of diagnostic tools that allow detection and risk assesment in genital HPV infection is necessary. A special interest is focused on the HPV viral proteins whose expression might be of use either as primary screening tool or in conjunction with other markers (cellular proteins, HPV DNA, PAP test).

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

Exploring Microbiota Diversity in Cervical Lesion Progression and HPV Infection through 16S rRNA Gene Metagenomic Sequencing.

(1) Background: Cervical cancer is a significant health concern, with the main cause being persistent infection with high-risk Human Papillomavirus (hrHPV). There is still no evidence for why viral persistence occurs in some women, but recent studies have revealed the interplay between cervical microbiota and hrHPV. This research aimed to characterize the cervicovaginal microbiota in cervical lesion progression and HPV infection status. (2) Methods: This study included 85 cervical specimens from women from the north-eastern region of Romania. DNA was isolated from cervical secretion for HPV genotyping and 16S ribosomal RNA gene NGS sequencing. (3) Results: Our study revealed a distinct pattern within the studied group when considering Lactobacillus species, which differs from findings reported in other populations. Specifically, the presence of Lactobacillus iners coupled with the absence of Lactobacillus crispatus alongside Atopobium spp., Prevotella spp., and Gardnerella spp. could serve as defining factors for severe cervical lesions. The results also showed a significant association between microbiota diversity, HPV infection, and cervical lesion progression. (4) Conclusions: As the microbiota profile seems to vary among different populations and individuals, a deeper comprehension of its composition has the potential to develop personalized detection and treatment approaches for cervical dysplasia and cancer.

Epigenetic approaches for cervical neoplasia screening (Review).

Human papillomavirus (HPV) infection is the leading cause of cervical cancer. The Papanicolaou cytology test is the usually employed type of screening for this infection; however, its sensibility is limited. Only a small percentage of women infected with high-risk HPV develop cervical cancer with an array of genetic and epigenetic modifications. Thus, it is necessary to develop rapid, reproducible and minimally invasive technologies for screening. DNA methylation has gained attention as an alternative method for molecular diagnosis and prognosis in HPV infection. The aim of the present review was to highlight the potential of DNA methylation in cervical neoplasia screening for clinical applications. It was observed that the methylation human and viral genes was correlated with high-grade lesions and cancer. Methylation biomarkers have shown a good capacity to discriminate between high-grade lesions with a transformative potential and cervical cancer, being able to detect these modifications at an early stage. With further research, the epigenetic profiles and subtypes of the tumors could be elaborated, which would aid in therapy selection by opening avenues in personalized precision medicine. Response to therapy could also be evaluated through such methods and the accessibility of liquid biopsies would allow a constant monitoring of the patient's status without invasive sampling techniques.

The association between lncRNA H19 and EZH2 expression in patients with EBV-positive laryngeal carcinoma.

OBJECTIVE: Laryngeal cancer is the second most common malignancy in the head and neck, with Epstein-Barr virus infection as a risk factor. Our aim is to evaluate correlations between the expression of lncRNA H19 and EBV infection in laryngeal cancer and H19 involvement in neoplastic progression through EZH2 association. MATERIALS AND METHODS: 30 paired laryngeal tissue specimens (neoplastic and non-neoplastic) were included in the study. Nucleic acid isolation and cDNA synthesis was performed according to the manufacturer's protocol. EBV DNA and expression of lytic (BZLF1) and latent (LMP1) forms of infection were assessed in PCR assays; expression levels of H19 and EZH2 were quantified in qRT-PCR. Data was analysed using GraphPad Prism 5.0. RESULTS: Higher H19 relative expression in neoplastic vs paired non-neoplastic samples was found (p < 0.0001) with a significant increase in EBV DNA positive neoplasms (p = 0.0434). An inverse correlation between H19 and EZH2 expression levels was noticed in EBV positive cases. Additionally, increased levels of H19 in LMP1 positive samples compared with those positive for BZLF1 was found (p = 0.0593). CONCLUSIONS: lncRNA H19 and EZH2 significantly contribute to the development of laryngeal carcinoma, being correlated with EBV infection markers.

Alterations of regulatory factors and DNA methylation pattern in thyroid cancer.

PURPOSE: DNA methylation plays an important role in thyroid oncogenesis. The aim of this study was to investigate the connection between global and local DNA methylation status and to establish the levels of important DNA methylation regulators (TET family and DNMT1) in thyroid tumours: follicular adenoma-FA, papillary thyroid carcinoma-PTC (classic papillary thyroid carcinoma-cPTC and papillary thyroid carcinoma follicular variant fvPTC). METHODS: Global DNA methylation profile in thyroid tumours tissue (41 paired samples) was assessed by 5-methylcytosine and 5-hydroxymethylcytosine levels evaluation (ELISA), along with TETs and DNMT1 genes expression quantification. Also, it was investigated for the first time TET1 and TET2 promoter's methylation in thyroid tumours. BRAF V600E mutation and RET/PTC translocation testing were performed on all investigated samples. In vitro studies upon DNA methylation in K1 thyroid cancer cells were performed with demethylating agents (5-AzaC and vitamin C). RESULTS: TET1 and TET2 displayed a significantly reduced gene expression level in PTC, while DNMT1 gene presented a high level of expression. PTC samples presented increased levels of 5-methylcytosine and low levels of 5-hydroxymethylcytosine. 5-methylcytosine levels were associated with TET1/TET2 expression levels. TET1 gene expression was significantly lower in patients positive for BRAF mutation and with RET/PTC rearrangement. TET2 gene was found hypermethylated in thyroid carcinoma patients overall, especially in PTC-follicular variant samples (p= 0.0002), where TET2 gene expression levels were significantly reduced (p= 0.0031). Furthermore, the data indicate for all thyroid cancer patients a good sensitivity (81.08%) and specificity (86.49%) regarding the use of TET1 (p< 0.0001), and TET2 (71.79%, 64.10%, p= 0.0001) hypermethylation as biomarkers for thyroid oncogenesis. CONCLUSIONS: These results suggest that TET1/TET2 gene expression and methylation may serve as potential diagnostic tools for thyroid neoplasia. Our study showed that the methylation of TET1 increases in malignant thyroid tumours. fvPTC patients presented lower methylation levels compared to cPTC and could be a discriminatory factor between two cancer types and benign lesions. TET2 is a poorer discriminator between FA and fvPTC, but it can be useful for cPTC identification. K1-cells treated with demethylating agents showed a demethylation effect, especially upon TET2 gene. The cumulative effect of L-AA and 5-AzaC proved to have a potent combined demethylating effect on genes promoter's activation and could open new perspectives for thyroid cancer therapy.

Methylation of tumour suppressor genes associated with thyroid cancer.

BACKGROUND: Thyroid carcinoma is the most common endocrine malignancy worldwide. Changes in DNA methylation can cause silencing of normally active genes, especially tumour suppressor genes (TSG) or activation of normally silent genes. OBJECTIVE: The aim of this study is to evaluate the degree of promoter methylation for a panel of markers for thyroid neoplasms and to establish their relationship with thyroid oncogenesis. METHODS: To generate a comprehensive DNA methylation signature of TSGs involved in thyroid neoplasia, we use Human TSG EpiTect Methyl II Signature PCR Array-Qiagen for 24 samples (follicular adenomas and papillary thyroid carcinomas) compared with normal thyroid tissue. We extended the evaluation for three TSGs (TP73, WIF1, PDLIM4) using qMS-PCR. Statistical analysis was performed with GraphPad Prism. RESULTS: We noted four important genes NEUROG1, ESR1, RUNX3, MLH1, which presented methylated promoter in tumour samples compared to normal. We found new characteristic of thyroid tumours: methylation of TP73, WIF1 and PDLIM4 TSGs, which can contribute to thyroid neoplasia. A significant correlation between BRAF V600E mutation and RET/PTC rearrangements with TIMP3 and CDH13, RARB methylation, respectively was observed. CONCLUSIONS: TSGs promoter hypermethylation is a hallmark of cancer and a test that uses methylation quantification method is suitable for diagnosis and prognosis of thyroid cancer.

High-risk human papillomaviruses distribution in Romanian women with negative cytology.

INTRODUCTION: Romania has the highest incidence and mortality rate of cervical cancer in Europe. The objective was to estimate the prevalence of high-risk human papillomavirus (hrHPV) genotypes and to evaluate the role of certain socio-behavioral factors in acquiring viral infection, in a cohort of Romanian women with negative Pap. METHODOLOGY: In a prevalence study 611 women (aged 17-58 years) with negative Pap, with no known history of atypical cytology and valid HPV test were included. Each participant completed a questionnaire containing data on socio-behavioral factors. From 344 women aged between 30-58 years, 63 were randomly selected for a second examination (conventional cytology and HPV detection and genotyping) after twelve months. RESULTS: Of the 611 women, 19.80% were HPV positive, 14.73% infected with hrHPV. Differences in the prevalence of hrHPV (17.60% versus 12.50%) as single (13.01% vs 9.01%) and multiple infections (9.71% vs 3.49%) were noted between women under the age of 30 and above. Among socio-behavioral factors, marital status and multiple sexual partners correlate with HPV and hrHPV infection. At follow-up, from 34 HPV negative cases, 10 changed to positive (5 hrHPV), while 2 developed abnormal cytology. Out of the 29 HPV positive cases, 12 cleared the HPV infection and 17 retested positive of which 4 worsened their cytology. CONCLUSIONS: In Romania, HPV infection is common in women with negative cytology. HPV genotyping is of epidemiological importance because the distribution of hrHPV types can determine the impact of prophylactic vaccines and the necessity of HPV testing as screening method.