Transcriptional activity of the long control region in human papillomavirus type 33 intratype variants.

BACKGROUND: High-risk human papillomaviruses (HPVs) are responsible for the development of cervical and other anogenital cancers. Intratype sequence variants of certain high-risk HPV types (e.g. 16, 18 and 31) are thought to have different oncogenic potential, partly due to nucleotide sequence variation in the viral long control region (LCR). The LCR has an important role in the regulation of viral replication and transcription. The purpose of this study was to explore sequence variation in the LCR of HPV 33 intratype variants in Hungary and to see whether there are differences in the transcriptional activities of the variants. METHODS: The complete HPV 33 LCR was amplified from HPV 33 positive cervical samples. After sequencing the LCR variants, multiple sequence alignment and phylogenetic analyses were carried out. Representative HPV 33 LCR sequence variants were selected for cloning and functional analysis. After transient transfection of HeLa cells, luciferase reporter assays were used to analyse the transcriptional activities of different LCR variants. RESULTS: Altogether 10 different variants were identified by sequence analysis of the HPV 33 LCR. The results of phylogenetic analysis showed that 3 variants belonged to sublineage A1, while the other 7 variants clustered with sublineage A2. Variants belonging to sublineage A2 had significantly lower transcriptional activities than variants belonging to sublineage A1. Within sublineage A2, the two variants analysed had significantly different transcriptional activities, which was shown to be caused by the A7879G variation. CONCLUSIONS: Nucleotide variation in the HPV 33 LCR can result in altered transcriptional activity of the intratype variants. Our results can help to understand the correlation between LCR polymorphism and the oncogenic potential of HPV 33 variants.

Coordinated action of human papillomavirus type 16 E6 and E7 oncoproteins on competitive endogenous RNA (ceRNA) network members in primary human keratinocytes.

BACKGROUND: miRNAs and lncRNAs can regulate cellular biological processes both under physiological and pathological conditions including tumour initiation and progression. Interactions between differentially expressed diverse RNA species, as a part of a complex intracellular regulatory network (ceRNA network), may contribute also to the pathogenesis of HPV-associated cancer. The purpose of this study was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by the E6 and E7 oncoproteins of HPV16, and construct a corresponding ceRNA regulatory network of coding and non-coding genes to suggest a regulatory network associated with high-risk HPV16 infections. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes. METHODS: Small and large RNA deep sequencing were performed to detect expression changes of miRNAs, lncRNAs and mRNAs in primary human keratinocytes expressing HPV16 E6, E7 or both oncoproteins. The relationships between lncRNAs, miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. GO and KEEG pathway enrichment analysis was performed using DAVID v6.8. RESULTS: We revealed that 85 miRNAs in 21 genomic clusters and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. We constructed a ceRNA network with members of 15 lncRNAs - 43 miRNAs - 358 mRNAs with significantly altered expressions. GO and KEGG functional enrichment analyses identified numerous cancer related genes, furthermore we recognized common miRNAs as key regulatory elements in biological pathways associated with tumorigenesis driven by HPV16. CONCLUSIONS: The multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 host cells occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network.

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota.

Effects of nutraceuticals on the intestinal microbiota are receiving increased attention; however, there are few studies investigating their effects on broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial examining the interplay between natural biologically active compounds such as carotenoids, anthocyanins, fermentable oligosaccharides, and synbiotics and the gastrointestinal tract microbiota. Our feeding program was applied to an intensive production system with a flock of 1,080 Ross 308 broilers. Aging induced significant changes through the feeding experiment. Nutraceuticals were shown to modulate broiler intestinal diversity and differentially enriched Lactobacillus, Enterococcus, Campylobacter, and Streptococcus in the core microbiome during the different stages of broiler rearing. Additionally, they did not remarkably affect animal growth performance; nevertheless, a positive correlation was found between body weight and Corynebacteriales and Pseudomonadales Furthermore, a diet high in carotenoid, fermentable oligosaccharide, and anthocyanin contents affected the number of beneficial genera such as Faecalibacterium, Lactobacillus, Blautia, and Ruminococcus With this comprehensive trial, we revealed that nutraceuticals induced modulations in broiler gastrointestinal tract microbiota. We believe that plant-derived immunostimulants, recycled from plant food waste products, can supplement antibiotic-free broiler meat production.IMPORTANCE In this trial, nutraceuticals were manufactured from waste products of food industry processing of Hungarian red sweet pepper and sour cherry and incorporated into the diet of poultry to investigate their effects on broilers' growth and the broiler gastrointestinal tract microbiota. To avoid the generation of food waste products, we believe that this approach can be developed into a sustainable, green approach that can be implemented in commercial antibiotic-free poultry to provide safe and high-quality meat.

Reconsidering the technical aspects and quality management background of faecal microbiota transplantation due to the novel coronavirus pandemic / A székletmikrobiota-transzplantáció technológiájának és minoségirányítási hátterének újragondolása a SARS-CoV-2 víruspandémia kapcsán.

Összefoglaló. A székletmikrobiota-transzplantáció (faecalismikrobiota-transzplantáció - FMT) a Clostridioides difficile fertozés (CDI) kezelésében nemzetközileg széles körben elfogadott, megfelelo szakmai háttér mellett végezve biztonságos, potenciálisan életmento, költséghatékony, valamint a hospitalizációs ido és az orvos-beteg találkozások jelentos redukálására képes eljárás. Az FMT elvégzésére egyes országokban magas szintu minoségirányítási háttérrel muködo, célfeladatra szervezodött donor- és székletbankok rendezkedtek be. Máshol, így például hazánkban, az eljáráshoz az egyértelmu jogi szabályozási környezet, a standardizált technológiai háttér és a finanszírozás hiánya miatt nem egységes a hozzáférés. Régóta idoszeru továbbá, hogy a heterogén, nemegyszer háztartási eszközökkel elokészített beavatkozások helyett a nemzetközi és legújabban már a hazai ajánlásokban is megfogalmazott, a betegbiztonságot legjobban garantáló elvárások mellett történjen a széklettranszplantáció. Az új koronavírus (SARS-CoV-2) okozta pandémia megjelenése eroteljes szakmai érv országos szinten az FMT minoségirányítási környezetének és technológiai hátterének újragondolására, mert a SARS-CoV-2 egyszerre jelent kockázatot a CDI miatt kórházban kezelt sérülékeny betegpopulációnak, és egyben veszélyezteti az FMT biztonságosságát mind a recipiens, mind pedig az eljárást végzo egészségügyi személyzet tekintetében. Ezekre a szakmai és társadalmi kihívásokra reagálva, a széles köru beteghozzáférés és a legmagasabb szintu betegbiztonság garantálására, a Debreceni Egyetemen új eljárásrendet dolgoztunk ki az FMT végzésére. Ezen eljárásrendnek a COVID-19-pandémia miatt módosított, a fagyasztottgraftbank üzemeltetése és a rendszerszemlélet tekintetében releváns elemeit ismertetjük. Javasolt, hogy országos szinten hasonló, megfelelo minoségirányítási és technológiai környezettel, a SARS-CoV-2-fertozés kizárását is integráló donorszurési rendszerrel, továbbá fagyasztottgraft-banki háttérrel muködo laboratóriumok vegyenek részt a széklettranszplantációk végzésében. Felmerül továbbá, hogy az eljárást a számos analógia és a donor-recipiens koncepció alapján a sejt- és szövettranszplantációkra vonatkozó szabályozórendszer keretei közé ajánlott beágyazni. Orv Hetil. 2020; 161(44): 1858-1871. Summary. Stool transplantation (faecal microbiota transplantation - FMT) is a widely accepted, potentially life-saving, cost-effective medical intervention for the treatment of Clostridioides difficile infection (CDI), which has an acceptable safety profile if performed with an appropriate professional background. FMT can significantly reduce hospitalization time and the number of patient visits. National donor and stool banks with high-standard quality management systems were established in certain countries for supporting the procedures. In other regions, including Hungary, patient access is not uniform due to the lack of clear legal regulations, standardized technology or financial reimbursement. It has been expected for a long time to replace the heterogenous techniques, occasionally utilizing household equipment with a technology providing improved patient safety and fulfilling international and recently published local FMT guidelines. The emergence of the novel coronavirus (SARS-CoV-2) pandemic is a very powerful argument in favour of urgently reconsidering the quality management and technological background of FMT procedures. SARS-CoV-2 is a major threat to the vulnerable patients suffering from CDI and also impose risks for the recipient and healthcare personnel involved in carrying out the transplantation. New FMT guidelines were implemented at the University of Debrecen to address these professional and public challenges, to provide wide patient access and to guarantee the highest achievable patient safety. Relevant elements of this new protocol are presented, focusing on a systemic quality management approach, on the operation of a frozen stool bank and on a modified donor screening algorithm taking the risks of COVID-19 into consideration. We suggest that laboratories with proper quality assurance and technological conditions, implementing SARS-CoV-2 donor screening and operating a frozen graft bank should participate in faecal microbiota transplantations. It is also recommended that, based on the analogies and the similar donor-recipient concept, FMT should be embedded under the organ tissue and cell transplantation polices in Hungary. Orv Hetil. 2020; 161(44): 1858-1871.

Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells.

The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an unexpected difficulty experienced when trying to clone HPV33 long control region (LCR) variants in Escherichia coli. Standard cloning strategies proved to be inappropriate to clone HPV33 LCR variants in the forward orientation into a eukaryotic reporter vector (pGL2-Basic). However, by slight modification of culture conditions (incubation at 25 °C instead of 37 °C), constructs containing the HPV33 LCR variants in the forward orientation were obtained. Transformation experiments performed with different HPV33 LCR constructs indicated that there is a sequence element in the 5' LCR of HPV33 causing temperature-dependent toxic effect in E. coli. Sequence analysis revealed the presence of an open reading frame (ORF) in the 5' part of HPV33 LCR potentially encoding a 116-amino acid polypeptide. Protein structure prediction suggested that this putative protein might have a structural similarity to transmembrane proteins. Even a low-level expression of this protein may cause significant toxicity in the host bacteria. In silico analysis of the LCR of HPV33 and some other HPV types belonging to the species Alphapapillomavirus 9 (HPV31, 35 and 58) seemed to support the assumption that the ORFs found in the 5' LCR of these HPVs are protein-coding sequences. Further studies should be performed to prove that these putative proteins are really expressed in the infected host cells and to identify their function.

Patient-related factors, antibiotic prescribing and antimicrobial resistance of the commensal Staphylococcus aureus and Streptococcus pneumoniae in a healthy population - Hungarian results of the APRES study.

BACKGROUND: Antimicrobial resistance (AMR) is an increasing public health problem worldwide. We studied some patient-related factors that might influence the antimicrobial resistance. and whether the volume of antibiotic prescribing of the primary care physicians correlate with the antibiotic resistance rates of commensal nasal Staphylococcus aureus and Streptococcus pneumoniae. METHODS: The socio-demographic questionnaires, the antibiotic prescription and resistance data of commensal nasal S. aureus and S. pneumoniae were collected in the 20 participating Hungarian practices of the APRES study. Multivariate logistic regression analyses were performed on the patient-related data and the antimicrobial resistance of the S. aureus and S. pneumoniae on individual, patient level. Ecological analyses were performed with Spearman's rank correlations at practice level, the analyses were performed in the whole sample (all practices) and in the cohorts of primary care practices taking care of adults (adult practices) or children (paediatric practices). RESULTS: According to the multivariate model, age of the patients significantly influenced the antimicrobial resistance of the S. aureus (OR = 0.42, p = 0.004) and S. pneumoniae (OR = 0.89, p < 0.001). Living with children significantly increased the AMR of the S. pneumoniae (OR = 1.23, p = 0.019). In the cohorts of adult or paediatric practices, neither the age nor other variables influenced the AMR of the S. aureus and S. pneumoniae. At practice level, the prescribed volume of penicillins significantly correlated with the resistance rates of the S. aureus isolates to penicillin (rho = 0.57, p = 0.008). The volume of prescribed macrolides, lincosamides showed positive significant correlations with the S. pneumoniae resistance rates to clarithromycin and/or clindamycin in all practices (rho = 0.76, p = 0.001) and in the adult practices (rho = 0.63, p = 0.021). CONCLUSIONS: The age is an important influencing factor of antimicrobial resistance. The results also suggest that there may be an association between the antibiotic prescribing of the primary care providers and the antibiotic resistance of the commensal S. aureus and S. pneumoniae. The role of the primary care physicians in the appropriate antibiotic prescribing is very important to avoid the antibiotic resistance.

Study of fast and slow consecutive processes by heterogeneous isotope exchange using P-32 radiotracer.

The sorption of phosphate on soils is studied by radioisotopic tracer method. Two consecutive processes with rather different rates were differentiated: namely the heterogeneous isotope exchange between the phosphate in the soil solution and the weakly sorbed phosphate (fast reaction), and the transformation of weakly sorbed phosphate to tightly sorbed phosphate (slow reaction). In this paper, it is shown how the rate constants of these two processes can be determined by a radiotracer with a relatively short half-life.

Down-regulation of increased TRAF6 expression in the peripheral mononuclear cells of patients with primary Sjögren's syndrome by an EBV-EBER1-specific synthetic single-stranded complementary DNA molecule.

AIM: We described earlier a simultaneously increased that the increased expression of miRNA-146a/b was accompanied by an increase in the expression of and TRAF6 and a decrease in the expression of IRAK1 genes in the peripheral mononuclear cells (PBMCs) of patients with primary Sjogren's syndrome (pSS) patients. Recently, the expression of EBV encoded. RNA (EBER) was published in the B cells of salivary glands of in pSS. In the present study, we applied an EBV-EBER1 specific synthetic single stranded complementary DNA molecule (EBV-EBER1-cDNA) to test whether any EBER1 related effect exists also in PBMCs of pSS patients. METHODS: In the PBMCs of pSS patients and healthy controls, we investigated in vitro the effects of a synthetic single stranded EBV-EBER1-cDNA molecule, synthetic double-stranded (ds)RNA polyinosinic-polycytidylic acid [poly (I:C)] and polyadenylic acid potassium salt poly-adenylic acid [poly-(A)] on the expression of TRAF6 gene tested by qRTPCR. The release of interferon -α was detected by ELISA. RESULTS: EBV-EBER1-cDNA resulted in a significant reduction in the expression of TRAF6 in the cells of patients, but in the healthy controls not, whereas the treatments with poly (I:C) and poly-(A) could not reduce the TRAF6 over-expression. No release of EBER1 could be observed in the culture supernatants of patients with pSS. Only the treatment with poly (I:C) resulted in a significant increase of interferon -α release, and only in the heathy controls. No release of EBER1 molecules took place during the culturing of cells. EBV-EBER- cDNA acted functionally on the cells of patients only. CONCLUSION: These findings give a further evidence of the linkage between EBV and pSS, furthermore, they show the possible role of EBV-EBER1 in the induction of increased TRAF6 expression in the peripheral B cells of Sjögren's patients.

The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor.

Phylogenetic and functional analysis of sequence variation of human papillomavirus type 31 E6 and E7 oncoproteins.

High-risk human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as a subset of head and neck cancers. The E6 and E7 oncoproteins of HPV contribute to oncogenesis by associating with the tumour suppressor protein p53 and pRb, respectively. For HPV types 16 and 18, intratypic sequence variation was shown to have biological and clinical significance. The functional significance of sequence variation among HPV 31 variants was studied less intensively. HPV 31 variants belonging to different variant lineages were found to have differences in persistence and in the ability to cause high grade cervical intraepithelial neoplasia. In the present study, we started to explore the functional effects of natural sequence variation of HPV 31 E6 and E7 oncoproteins. The E6 variants were tested for their effects on p53 protein stability and transcriptional activity, while the E7 variants were tested for their effects on pRb protein level and also on the transcriptional activity of E2F transcription factors. HPV 31 E7 variants displayed uniform effects on pRb stability and also on the activity of E2F transcription factors. HPV 31 E6 variants had remarkable differences in the ability to inhibit the trans-activation function of p53 but not in the ability to induce the in vivo degradation of p53. Our results indicate that natural sequence variation of the HPV 31 E6 protein may be involved in the observed differences in the oncogenic potential between HPV 31 variants.