Reprogramming of skin keratinocytes by the E7 oncoprotein of HPV8

The human papillomavirus type 8 (HPV8), an oncogenic member of the genus betapapillomavirus (betaHPV), is associated with the development of actinic keratoses and squamous cell carcinoma of the skin. The viral early proteins E6 and E7 are oncoproteins which are known to interact with important regulatory mechanisms of infected keratinocytes. However, the molecular details how this occurs in case of HPV8-E7 is still only poorly understood. Previous research of our group had already shown that HPV8-E7 plays a pivotal role in inducing and regulating keratinocyte proliferation and invasion. It was also shown that the extracellular matrix protein fibronectin and the integrin α3β1, expressed on HPV8 positive keratinocytes, is key in regulating the invasive behavior of these cells. Furthermore, we had shown that fibronectin is not only overexpressed in HPV8-E7 positive keratinocytes, but also found to be enriched in the peri-tumoral stroma of betaHPV-positive skin cancer. Therefore, the overall aim of this thesis was to arrive at a deeper understanding how HPV8-E7 reprograms the cell and how it hijacks vital cellular processes and how this interference may ultimately contribute to malignant transformation processes in HPV8 infected keratinocytes. To extend our knowledge of how these events occur transcriptomic, total and phospho-proteomics as well as protein-protein interaction assays were employed. cDNA microarray results led to the observation that global gene expression changes induced by HPV8-E7 are mainly accomplished through binding sites within the gene promoters for the transcription factors Sp1/Sp3. In transient transfection assays we could confirm that HPV8-E7 stimulates the activity of promoters of cellular genes which contain Sp1/Sp3 binding sites, including the fibronectin gene promoter. Interestingly the HPV8-E7L23A mutant, which is not able to trigger keratinocyte invasion was unable to activate the fibronectin promoter (Kirschberg*, Heuser* et al., 2019). By using the same cDNA microarray results GADD34 (growth arrest and DNA damage-inducible protein 34) and GDF15 (growth/differentiation factor 15) were identified as novel downstream genes targeted by E7. In addition, proteomic analyses of HPV8-E7 positive keratinocytes cultivated on a fibronectin matrix showed that the presence of E7 led to hyper- or hypo-phosphorylation of cellular proteins. Here, we also revealed that hypo-phosphorylated proteins are mainly associated with DNA damage repair and DNA replication, whereas hyper-phosphorylated proteins are apparently important for cytoskeletal organization and also include proteins associated with cell polarity. Therefore, these observations reveal that signals downstream of fibronectin resulting in hyper-phosphorylation of proteins are critical for cell invasion. Interestingly, among the identified up-stream kinases were the proto-oncogenic Src-kinase family members Lyn and Fyn, which are over-activated in the presence of HPV8-E7 (Kirschberg et al., 2021). Looking at direct E7 interacting proteins, NuMa (nuclear mitotic apparatus protein 1) and ATP5B (ATP synthase F1 subunit beta) were identified as novel HPV8-E7 interacting partners. E7 appears to co-localize with NuMa in mitotic cells. Curiously, in a small subset of these cells E7 was only found at one whereas NuMa was found at both spindle poles. This could implicate that the binding of E7 to NuMa at only one spindle pole might also affect the ratio of symmetric / asymmetric cell division of epidermal progenitor cells, which could explain the already known enrichment of stem-cell-like cancer cells in HPV8 positive primary keratinocyte cultures (Oswald*, Kirschberg* et al., 2019). The observed interaction of E7 and ATP5B was most intriguing as betaHPV E7 had been mainly known as a nuclear and cytoplasmic protein. The ability to bind ATP5B is not exclusive for HPV8 but could also be shown for low-risk (HPV11) and high-risk (HPV16) mucosa -infecting HPV. The association of E7 with ATP5B was accompanied by an increase in mitochondrial energy production. In particular E7 positive keratinocytes showed a stark increase in their spare respiratory capacity, allowing the cells to meet their heightened energy demands. The influence of ATP5B manipulation was further analyzed in oropharyngeal cancer patient groups which included both HPV16 positive and HPV-negative tumors. Interestingly, HPV-positive cancers showed a far more favorable outcome compared to HPV-negative tumors if ATP5B expression was high (Kirschberg et al., 2020). Having identified these new interaction partners, namely NuMa and ATP5B, it appears that E7 exerts its oncogenic functions in various cellular compartments. Virtually all so far published studies on the oncogenic functions of E7 were conducted in cells expressing the viral oncoproteins individually. Interestingly, when HPV8-E7 is co-expressed with HPV8-E6 other oncogenic functions appear to become active, leading to the CHK1 (checkpoint kinase-1) protein degradation. This disruption of CHK1 was mediated by manipulating the autophagic pathway that was so far not known to be a degradation pathway used by HPV oncoproteins (Akgül, Kirschberg et al., 2019)

Chemical gastritis after chronic bromazepam intake: a case report

<p>Abstract</p> <p>Background</p> <p>We describe a rare case of diffuse macroscopic discoloration and chemical gastritis due to chronic bromazepam intake. The chemical composition of pharmaceuticals has to be considered at endoscopy and it is evident that some chemical substances damage the epithelial tissue and lead to clinical symptoms.</p> <p>Case Presentation</p> <p>Endoscopy was performed in an 82-year-old patient due to gastroesophageal reflux symptoms and epigastric pain. Gastroscopy showed a hiatal hernia and a scarred duodenal bulb. More striking was the yellow-brownish discoloration of the gastric and the duodenal mucosa. The gastric antrum and the duodenal bulb showed local discoloration that could not be rinsed off. The medical history indicated that bromazepam (6 mg) had been used daily as a sleeping aid in the previous two years. The histopathological findings showed appearances of chemical gastritis. Within the lamina propria and on the epithelial surface there were granules. There was no foreign body reaction to these granules. Corpus mucosa showed a mild chronic gastritis.</p> <p>Conclusions</p> <p>If discoloration of the mucosa at endoscopy is seen, a careful drug history must be sought. This is the first case in literature that shows a chemical gastritis after bromazepam intake.</p

Korrelation von patient related outcome measures und Ganganalyseparametern nach Knieendoprothese

Der Endoprothetische Kniegelenksersatz ist eine etablierte Methode zur kausalen Therapie der manifesten Gonarthrose. Ziel des operativen Eingriffs ist die Wiederherstellung einer schmerzfreien Belastbarkeit und Gelenkbeweglichkeit, sowie eine Verbesserung der Lebensqualität. Für die Bewertung der Funktionsverbesserung, sowie die damit einhergehende Verbesserung der Lebensqualität, nehmen sogenannte Patient related outcome measures eine tragende Rolle ein. Nachteile bei der Anwendung von PROMs sind zum Einen die geringe Objektivität der Ergebnisse und zum Anderen die hohe Anzahl von Patienten, welche in eine Studie eingeschlossen und befragt werden müssen, um statistisch signifikante Effekte auf die Patient related outcome measures zu erfassen. Mit dem Messinstrument der Ganganalyse kann eine Beurteilung des Outcomes hingegen anhand objektiver Parameter des spezifischen Gangbildes eines Patienten erfolgen. Im Rahmen des endoprothetischen Gelenkersatz konnte durch verschiedene Forschungsgruppen ein pathologisches Gangbild festgestellt werden. Um eine Verbesserung des Gangbildes nach Implantation eines bikondylären Oberflächenersatz zu erzielen ist die stetige Weiterentwicklung der präoperativen Diagnostik, der Operationstechniken, des Prothesendesigns und der Rehamaßnahmen nötig. Bislang war in der Behandlung von Patienten nach Knie- Totalendoprothese unklar, welche Gangparameter optimiert werden müssten, um den größten klinischen Benefit, gemessen an Patient related outcome measures, zu erzielen. Diese Dissertation identifiziert drei Parameter, welche im Rahmen der postoperativen Rehabilitation gezielt korrigiert werden könnten, um in Zukunft das Outcome von Patienten nach Knie Totalendoprothese weiter zu verbessern

BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes

Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.Peer reviewe

Computer-aided recording of automatic endoscope washing and disinfection processes as an integral part of medical documentation for quality assurance purposes

<p>Abstract</p> <p>Background</p> <p>The reprocessing of medical endoscopes is carried out using automatic cleaning and disinfection machines. The documentation and archiving of records of properly conducted reprocessing procedures is the last and increasingly important part of the reprocessing cycle for flexible endoscopes.</p> <p>Methods</p> <p>This report describes a new computer program designed to monitor and document the automatic reprocessing of flexible endoscopes and accessories in fully automatic washer-disinfectors; it does not contain nor compensate the manual cleaning step. The program implements national standards for the monitoring of hygiene in flexible endoscopes and the guidelines for the reprocessing of medical products. No FDA approval has been obtained up to now. The advantages of this newly developed computer program are firstly that it simplifies the documentation procedures of medical endoscopes and that it could be used universally with any washer-disinfector and that it is independent of the various interfaces and software products provided by the individual suppliers of washer-disinfectors.</p> <p>Results</p> <p>The computer program presented here has been tested on a total of four washer-disinfectors in more than 6000 medical examinations within 9 months.</p> <p>Conclusions</p> <p>We present for the first time an electronic documentation system for automated washer-disinfectors for medical devices e.g. flexible endoscopes which can be used on any washer-disinfectors that documents the procedures involved in the automatic cleaning process and can be easily connected to most hospital documentation systems.</p

Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

The hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR) on the eight (A-H) standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI) and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%), 25 cases (50%), and 14 cases (28%) respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

Photoinduced electron transfer reactions of alpha-cyclopropyl- and alpha-epoxy ketones. Tandem fragmentation-cyclization to bi-, tri-, and spirocyclic ketones

Kirschberg T, Mattay J. Photoinduced electron transfer reactions of alpha-cyclopropyl- and alpha-epoxy ketones. Tandem fragmentation-cyclization to bi-, tri-, and spirocyclic ketones. The Journal of Organic Chemistry. 1996;61(25):8885-8896.Reductive photoinduced electron transfer (PET) reactions have been performed with various bicyclic alpha-cyclopropyl-substituted ketones and tertiary amines. The reaction resulted in a regioselective cleavage of one cyclopropyl bond under formation of an exocyclic radical with an endocyclic enolate unit. In the case of bicyclic ketones with an unsaturated side chain, various bicyclic, spirocyclic, and tricyclic products are accessible via radical cyclization, depending on the position of the alkenyl substituent. In addition to triethylamine, N-silylated amines have also been used as electron donors, leading to a variety of compounds, among them are silylated fragmentation products, indicating that a proton is transferred from not only the amine radical cation but also the cationic silyl group. The intramolecular Paterno-Buchi reaction has also been studied for cyclopropane derivatives of the jasmone type leading to tetracyclic oxetanes. Finally, alpha-epoxy-substituted ketones have been investigated under PET conditions, yielding ring-opened products