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Human papillomavirus (HPV) infection is a necessary but not sufficient condition for the development of cervical cancer. The dysbiotic shift in the cervicovaginal microbiome appears to be a major co-factor in carcinogenesis. New analytical methods, such as next-generation sequencing (NGS), can be used to detect all of the vaginal microorganisms present and therefore identify individual therapeutic options. The relationship of bacterial vaginosis and carcinogenesis, as well as possible indications for the use of microbiome analysis, will be discussed.

Background Tracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective. Aim We demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters. Methods Genomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021. Results Cluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf. Conclusion IGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.

Background Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing. Methods In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform. Results Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population. Conclusions Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population., Tracing of SARS-CoV-2 population transmission chains is still a major challenge. We present an integrated system of genomic surveillance and show it to be capable of detecting infection chains in a large city during ongoing community transmission.

Integration of genomic surveillance with contact tracing provides a powerful tool for the reconstruction of person-to-person pathogen transmission chains. We report two large clusters of SARS-CoV-2 cases (“Delta” clade, 110 cases combined) detected in July 2021 by Integrated Genomic Surveillance in Düsseldorf. Structured interviews and deep contact tracing demonstrated an association to a single SARS-CoV-2 infected return traveller (Cluster 1) and to return travel from Catalonia and other European countries (Cluster 2), highlighting the importance of containing travel-imported SARS-CoV-2 infections.

16S rRNA based analysis is the established standard for elucidating microbial community composition. While short read 16S analyses are largely confined to genus-level resolution at best since only a portion of the gene is sequenced, full-length 16S sequences have the potential to provide species-level accuracy. However, existing taxonomic identification algorithms are not optimized for the increased read length and error rate of long-read data. Here we present Emu, a novel approach that employs an expectation-maximization (EM) algorithm to generate taxonomic abundance profiles from full-length 16S rRNA reads. Results produced from one simulated data set and two mock communities prove Emu capable of accurate microbial community profiling while obtaining fewer false positives and false negatives than alternative methods. Additionally, we illustrate a real-world application of our new software by comparing clinical sample composition estimates generated by an established whole-genome shotgun sequencing workflow to those returned by full-length 16S sequences processed with Emu.

Viral genome sequencing can address key questions about SARS-CoV-2 evolution and viral transmission. Here, we present an integrated system of genomic surveillance in the German city of Düsseldorf, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) analysis of hospital outbreaks, d) integration of public health authority contact tracing data, and e) a user-friendly dashboard application as a central data analysis platform. The generated surveillance sequencing data (n = 320 SARS-CoV-2 genomes) showed that the development of the local viral population structure from August to December 2020 was consistent with European trends, with the notable absence of SARS-CoV-2 variants 20I/501Y.V1/B.1.1.7 and B.1.351 until the end of the local sampling period. Against a background of local surveillance and other publicly available SARS-CoV-2 data, four putative SARS-CoV-2 outbreaks at Düsseldorf University Hospital between October and December 2020 (n = 44 viral genomes) were investigated and confirmed as clonal, contributing to the development of improved infection control and prevention measures. An analysis of the generated surveillance sequencing data with respect to infection clusters in the population based on a greedy clustering algorithm identified five candidate clusters, all of which were subsequently confirmed by the integration of public health authority contact tracing data and shown to be represent transmission settings of particular relevance (schools, care homes). A joint analysis of outbreak and surveillance data identified a potential transmission of an outbreak strain from the local population into the hospital and back; and an in-depth analysis of one population infection cluster combining genetic with contact tracing data enabled the identification of a previously unrecognized population transmission chain involving a martial arts gym. Based on these results and a real-time sequencing experiment in which we demonstrated the feasibility of achieving sample-to-turnaround times of

There are many causes of illness. One could get hit by a car, and the mechanical forces of the impact damage soft tissues and fracture bones. An analogous concept could be established for infectious diseases: a pathogenic microorganism “hits” the body and causes infection and inflammation along with fever, pain, and reduced physical fitness. Akin to the mechanistic forces that break a bone, the cause of infection is a virus or bacterium, and medical practice therefore tries to diagnose the causative pathogen in order to prescribe the correct therapy, be it an antiviral or an antibiotic. Moreover, in analogy to physical forces that cause injury, it is the number of microorganisms that have entered the body, which determines whether an infection will cause disease. ### Different ways to become ill But if we look at infection patterns in whole populations, it becomes obvious that not everybody who is infected by a specific “infective dose” of a particular pathogen becomes ill and shows clinical symptoms. In some cases, people appear immune even against high infective doses, whereas in other circumstances, even non‐pathogenic microorganisms can cause severe infections, which often affect leukemia patients or patients suffering from immunodeficiency. This highlights another crucial factor that determines whether we become ill or not: the immune system. In contrast to an accident, infection depends on more than just physical forces. The immune system, involving numerous specialized cells, receptors, cytokines, antibodies, and so on, adds a layer of complexity that makes it more difficult to establish causality. Its efficiency in clearing the body of pathogens not only depends on its internal state—for instance the presence of memory B cells to produce specific antibodies—but also on external factors, such as the state of nutrition, climate, or stress levels. The relation between exposure to microorganism and infection is therefore not linear but complex or …

Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), induces growth arrest in HeLa cells and causes strong inhibition of the G1 to S transition of the cell cycle in a concentration-dependent manner. The G1 arrest is preceded by suppression of cyclin E and A, inactivation of cdk2, and the complete loss of the viral oncoprotein E7, despite ongoing HPV transcription. As shown by inhibitors specific for cyclooxygenase (COX) 1 and 2 loss of E7 is COX-independent. Moreover, inhibition of the proteasome activity with MG132 partially blocked the ability of sulindac to suppress E7 suggesting that sulindac induces degradation of E7 by the proteasomal pathway. In addition to inhibiting growth, sulindac strongly induces apoptosis, which can be abrogated by using the general caspase inhibitor zVAD-fmk. Unchanged expression of the pro-apoptotic protein Bax and suppression of the anti-apoptotic molecules Bcl-2 and Bcl-x(L) argues for the engagement of the mitochondrial apoptotic pathway. These results support the notion that sulindac is a potent growth inhibitor and inducer of apoptosis on cervical cancer cells in vitro and may offer new perspectives as a chemopreventive or supplementary anti-cervical cancer drug.

We reconstruct genetic determinism as a reductionist thesis to the effect that the molecular properties of cells can be accounted for to a great extent by their genetic outfit. The non‐reductionist arguments offered at this molecular level often use the relationship between structure and function as their point of departure. By contrast, we develop a non‐reductionist argument that is confined to the structural characteristics of biomolecules; no appeal to functions is made. We raise two kinds of objections against the reducibility claim underlying genetic determinism. First, some conceptual distinctions at the protein level cannot be captured on a genetic basis. A one‐to‐many relationship between DNA sequences and proteins emerges from them. Second, the relationship between genes and proteins is characterized by explanatory loops or reciprocal explanatory dependence. The presence of proteins is explained by the transcription from corresponding DNA sequences, and the latter is in turn accounted for by the ...

Histone deacetylase (HDAC) inhibitors induce an intrinsic type of apoptosis in human papillomavirus (HPV)-positive cells by disrupting the mitochondrial transmembrane potential (deltapsim). Loss of deltapsim was only detected in E7, but not in E6 oncogene-expressing cells. HDAC inhibition led to a time-dependent degradation of the pocket proteins pRb, p107 and p130, releasing 'free' E2F-1 following initial G1 arrest. Inhibition of proteasomal proteolysis, but not of caspase activity rescued pRb from degradation and functionally restored its inhibitory effect on the cyclin E gene, known to be suppressed by pRb-E2F-1 in conjunction with HDAC1. Using siRNA targeted against p53, E2F-1 still triggered apoptosis by inducing the E2F-responsive proapoptotic alpha- and beta-isoforms of p73. These data may determine future therapeutic strategies in which HDAC inhibitors can effectively eliminate HPV-positive cells by an apoptotic route that does not rely on the reactivation of the 'classical' p53 pathway through a preceding shut-off of viral gene expression.

Inhibitors of histone deacetylase, such as sodium butyrate, block proliferation of cervical carcinoma cells by inhibiting the G1 to S transition of the cell cycle. The derivative phenylbutyrate (PB), characterized by its higher pharmacological half-life, and its metabolite phenylacetate (PA) were tested for their growth-inhibitory function on cervical cancer cells differing in their HPV type, copy number, and integration sites.Using flow cytometric and Western blot analyses, we show that a 24-h incubation period with PB, but not with PA, was already sufficient to cause a dose-dependent growth arrest by increasing the G1 fraction with a concomitant drop in the S-phase. Consistent with the cell cycle block, only PB, but not PA, induced the cyclin-dependent kinase inhibitors p21(CIP1) and p27(KIP1). The inhibitory effect was not the result of a non-specific cytotoxic effect of PB, since cessation of cellular growth was already completely reversible 5 h after drug removal.Due to its broad growth inhibitory properties on different cervical carcinoma cells in vitro, and its low toxic profile demonstrated in preceding clinical studies, PB may serve as an effective drug in handling pre-cancerous lesions and cervical cancer in patients.

The oncogenic potential of 'high risk' human papillomaviruses can be mainly attributed to two small proteins called E6 and E7. Even these oncoproteins have a low molecular size, they are highly promiscuous and are capable to interact with a whole variety of host cellular regulator proteins to elicit cellular immortalization and ultimately complete malignant transformation. To avoid reiterations in summarizing the biochemical and molecular biological properties of E6/E7 in terms of their influence on cell cycle control, the present review is mainly an attempt to describe some regulatory principles by which human papillomavirus (HPV) oncoproteins can interfere with apoptosis in order to escape immunological surveillance during progression to cervical cancer. The models derived from these basic cellular and molecular studies are relevant to our understanding of HPV-induced carcinogenesis. Conversely, experimental procedures aimed at relieving apoptosis resistance, can facilitate the eradication of immunologically suspicious cells and may prevent the accumulation of cervical intraepithelial cell abnormalities in future prophylactic or therapeutic approaches.

Sowohl systemtheoretische als auch biomedizinische Neukonzeptionen verandern die gegenwartige Medizin. Deren Grundlage bildet die Komplexitat der Organismen, die die Zerteilung der biologischen Systeme zutage gefordert hat. Die Teile setzen sich dabei nicht einfach so zusammen, sondern bilden Interaktionen und Wechselwirkungsbeziehungen aus, die sich zu emergenten Ebenen organisieren konnen. Nicht die blose Struktur der Teile und Komponenten, sondern die Organisation der Systeme stellt dabei das konstituierende Prinzip des Lebens dar.

Ob dem Leben eine besondere Stellung in der Natur zukommt, steht mit der Frage in Verbindung, was das Lebendige vom Unbelebten unterscheidet. Jacques Monod formulierte drei Eigenschaften, die Lebewesen exklusiv aufweisen: zunachst die Teleonomie, namlich mit einem Plan ausgestattet zu sein, der gleichzeitig durch die eigene Struktur dargestellt und durch die eigene Leistung ausgefuhrt wird; weiterhin die autonome Morphogenese, die von auseren Kraften und Bedingungen freie Bildung der Gestalt, und die Invarianz, also die Fahigkeit, sich unverandert zu reproduzieren.

Wer schon einmal versucht hat, einen kleinen Golfball richtig zu treffen, dass er weit, aber nicht zu weit nach links oder rechts fliegt, oder ihn so zu treffen, dass er nach einer welligen und begrasten Strecke ins Loch fallt, weis, wie viele Details es fur einen guten Schlag zu beachten gibt. Es sind hunderte! Wie halte ich den Schlager, wie schwinge ich mit den Armen, wie bewegt sich die Hufte, wie stehe ich mit den Fusen, was mache ich mit den Handen an welchem Punkt des Schlages, wie halte ich den Kopf? Die Fragen des Golfspieles sind nicht minder komplex. Wohin versucht man den Ball zu spielen auf welcher Bahn, wie sind die Gelandebegebenheiten, wie das Wetter, wie das Gras? Erstaunlich ist, dass das Spiel – uber einfache Regeln – am Ende zu einem klar quantifizierbaren Ergebnis fuhrt, namlich die Schlage pro Bahn. Golf ist also ein Spiel, das trotz komplexester Situationen zu diskreten Ergebnissen fuhrt.

Die Medizin konnte im zuruckliegenden Jahrhundert gewaltige Erfolge erringen. Dazu zahlen die Behandlung von Infektionskrankheiten durch die Einfuhrung der Vakzinierung oder antiinfektiver Substanzen wie dem Penicillin. Diese Erfolgsgeschichte wird auch gegenwartig weitergeschrieben, etwa mit den therapeutischen Optionen in der Behandlung der Immunschwachekrankheit AIDS, die vor nicht allzu langer Zeit meist todlich verlief. Ebenso spektakular waren die Erfolge der Chirurgie mit der Entwicklung neuer Operationsverfahren, der Einfuhrung der Herz-Kreislauf-Maschine oder der erfolgreichen Transplantation des Herzens, dem inzwischen die Verpflanzung weiterer Organe – der Leber, der Lunge, der Hornhaut usw. – folgte.

Histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A arrest human papillomavirus (HPV)-positive carcinoma cells in G1 to S transition of the cell cycle, which is paralleled by an up-regulation of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1 and p27KIP1 as well as the complete loss of cdk2 activity. Although HPV expression was hitherto thought to be required to maintain a proliferative phenotype of these cells, cdk2 suppression is achieved even in the presence of ongoing viral transcription. While CKIs normally cannot exert their cdk2-inhibitory function in the presence of the viral oncoprotein E7, co-immunoprecipitation experiments revealed that E7 binding is prevented. Increase of p27KIP1 correlates with down-regulation of p45SKP2, a component of the ubiquitin-protein ligase SCF(SKP2) controlling the half-life of regulatory proteins during the cell cycle. HDAC inhibition also triggered an E7-dependent degradation of pRb, while the levels of E2F remained unaffected. The presence of free intracellular E2F and the concomitant up-regulation of CKIs during G1 arrest results in a 'conflicting growth situation', which finally renders the cells to undergo apoptosis. These data provide novel molecular insights into how the transforming potential of HPV can be bypassed and open new therapeutical perspectives for the treatment of cervical cancer.

AP-1 represents a transcription factor, which plays a pivotal role in initiating and maintaining the expression of human papillomavirus (HPV) oncoproteins E6 and E7 during HPV-linked carcinogenesis of the uterine cervix. AP-1 stands as a synonym for different proteins such as c-Jun, JunB, JunD, c-Fos, FosB as well as the Fos-related antigens Fra-1 and Fra-2, which can either homo- or heterodimerize to build up a functional transcription complex. AP-1 is mainly considered as a positive regulator, which binds to cognate DNA sequences within the viral upstream regulatory region. By using non-tumorigenic HeLa-fibroblast hybrids ('444'), their tumorigenic segregants ('CGL3') as well as HPV 18 positive HeLa cells as a experimental model system, evidence is provided that AP-1 composition differs considerably between these cell lines. In nuclear extracts obtained from non-tumorigenic cells, Jun-family members (in the order c-Jun>JunD>JunB) were mainly heterodimerized with Fra-1, a protein, known to be involved in the abrogation of AP-1 activity under certain experimental conditions. In contrast, Fra-1 concentration is low in extracts from tumorigenic cells. Conversely, c-Fos, the canonical dimerization partner of Jun proteins is expressed in substantial quantity in HeLa- and 'CGL3' cells, but it is completely absent in AP-1 complexes from non-tumorigenic '444' cells. Ectopical expression of c-fos under a heterologous promoter in '444'-cells induces tumorigenicity and a change of the Jun/Fra-1 ratio towards a constellation initially detected in 'CGL3'-and HeLa cells. Furthermore, conversion to tumorigenicity is accompanied with a resistance against TNF-alpha, a cytokine, capable to selectively suppress HPV 18 transcription in formerly non-malignant cells. These data propose a novel role for AP-1 as an essential component of an inter- and intracellular surveillance mechanism negatively controlling HPV transcription in non-tumorigenic cells.

Inhibitors of histone deacetylase (HDAC) are capable of arresting growth in cervical carcinoma cells in the G1 phase of the cell cycle. Although HPV E6/E7 mRNA steady-state levels appeared to be constant after prolonged treatment, time-course experiments revealed that viral transcription was transiently down-regulated between 7–10 h prior to cdk2 suppression. To test whether transitory suppression was a prerequisite for the biological outcome after HDAC inhibition, we took advantage of two immortalized human keratinocyte cell lines in which E6/E7 oncogene expression was controlled by different regulatory regions. After treatment with sodium butyrate (NaB) or trichostatin A (TSA), HPV16 upstream regulatory region (URR)-directed transcription was down-regulated, showing kinetics similar to those in cervical carcinoma cells. In contrast, β-actin promoter controlled E6/E7 transcription was even temporarily increased and finally declined to levels initially detected in the untreated controls. Both cell lines, however, were arrested in G1 and showed complete suppression of cdk2 activity that was preceded by a strong up-regulation of the cdk2 inhibitors p21CIP1 and p27KIP1. These results demonstrate that growth of HPV16/18-positive cells can be arrested by HDAC inhibitors despite ongoing HPV transcription and thus independently of any potential position effects uncoupling URR-directed gene expression by adjacent cellular promoters or by downstream 3′-polyadenylation sites after viral integration into the host genome during multistep carcinogenesis.

The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

The transcription factor AP-1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP-1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV-positive cells under in vivo conditions. The dissection of AP-1 composition in cervical-carcinoma cells revealed an inverse relationship between the Fos-related antigen Fra-1 and the tumorigenic phenotype. Cervical-carcinoma cell lines were either negative or expressed only low amounts of Fra-1 (jointly with c-Fos) within their AP-1 complexes. Somatic-cell hybridization technique was used to fuse different HPV-positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP-1 composition in electrophoretic mobility super-shift assays showed that the amount of Fra-1 was substantially increased within the AP-1 complex of non-malignant cells. In contrast, Fra-1 was even diminished in malignant hybrids, while c-Fos remained expressed. This correlation suggests that the concentration of Fra-1 within the AP-1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV-positive cells.