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Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S 349 and T 359 . In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes., Competing Interests: Declaration of interests S.D. is co-owner of siTOOLs Biotech, Martinsried, Germany, without relation to this work. The other authors disclose no conflicts of interest. This study is part of the PhD thesis of V.R.

Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy.

Human cytomegalovirus (HCMV) has been implicated in the development of human malignancies, for instance in colon cancer. Proteasome inhibitors were developed for cancer therapy and have also been shown to influence HCMV infection. The aim of this study was to investigate if proteasome inhibitors have therapeutic potential for colon carcinoma and how this is influenced by HCMV infection. We show by immunofluorescence and flow cytometry that the colon carcinoma cell line Caco-2 is susceptible to HCMV infection. Growth curve analysis as well as protein expression kinetics and quantitative genome analysis further confirm these results. HCMV has an anti-apoptotic effect on Caco-2 cells by inhibiting very early events of the apoptosis cascade. Further investigations showed that HCMV stabilizes the membrane potential of the mitochondria, which is typically lost very early during apoptosis. This stabilization is resistant to proteasome inhibitor Bortezomib treatment, allowing HCMV-infected cells to survive apoptotic signals. Our findings indicate a possible role of proteasome inhibitors in colon carcinoma therapy.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses., Competing Interests: The authors have declared that no competing interests exist.

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a l arge t umor antigen (LT-Ag) that, together with a s mall t umor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G 2 We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G 2 arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells. IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G 2 arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP)., (Copyright © 2020 Siebels et al.)

A key step in replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. The enzymes involved in this process are the terminases. The HCMV terminase complex consists of two terminase subunits, the ATPase pUL56 and the nuclease pUL89. A potential third component pUL51 has been proposed. Even though the terminase subunit pUL89 has been shown to be essential for DNA packaging and interaction with pUL56, it is not known how pUL89 mechanistically achieves sequence-specific DNA binding and nicking. To identify essential domains and invariant amino acids vis-a-vis nuclease activity and DNA binding, alanine substitutions of predicted motifs were analyzed. The analyses indicated that aspartate 463 is an invariant amino acid for the nuclease activity, while argine 544 is an invariant aa for DNA binding. Structural analysis of recombinant protein using electron microscopy in conjunction with single particle analysis revealed a curvilinear monomer with two distinct domains connected by a thinner hinge-like region that agrees well with the predicted structure. These results allow us to model how the terminase subunit pUL89's structure may mediate its function., Competing Interests: The authors have declared that no competing interests exist.

Thin Au films (~5 nm) are known to form island-like structures with small gaps between the islands, which produce intense electric field "hot spots" under visible illumination. In this work, we perform finite difference time domain (FDTD) simulations based on experimentally observed high resolution transmission electron microscope (HRTEM) images of these films in order to study the nature of the "hot spots" in more detail. Specifically, we study the dependence of the electric field intensity in the hot spots on the surrounding film environment and on the size of the nanogaps. From our simulations, we show that the surrounding film contributes significantly to the electric field intensity at the hot spot by focusing energy to it. Widening of the gap size causes a decrease in the intensity at the hot spot. However, these island-like nanoparticle hot spots are far less sensitive to gap size than nanoparticle dimer geometries, studied previously. In fact, the main factor in determining the hot spot intensity is the focusing effect of the surrounding nano-islands. We show that these random Au island films outperform more sophisticated geometries of spherical nanoparticle clusters that have been optimized using an iterative optimization algorithm.

Acute, subacute, and chronic toxicity studies, carcinogenicity bioassays, and reproductive and genetic toxicology studies were performed with quinapril, an ACE inhibitor used in the treatment of hypertension. Acute toxicity is minimal in rodents, and repeated dosing elicits gastric irritation, juxtaglomerular apparatus (JGA) hypertrophy and hyperplasia and tubular degenerative changes in the kidney, and reduced red cell parameters and heart weights in rodents and/or dogs. Other manifestations of toxicity, including hepatic lesions in dogs, reduced offspring weights in rats, marked sensitivity of the rabbit, and clastogenic effects at cytotoxic doses in the in vitro V79 chromosome aberration assay, have been reported with other drugs of this class.

The ability of the physiologically essential divalent metals calcium and magnesium to inhibit the tumorigenic activities of lead and nickel towards the lungs of strain A mice was investigated. The tumorigenic salts lead(II) subacetate and nickel(II) acetate were injected i.p. at their maximal tolerated doses (0.04 mmol/kg/injection of each metal) for a total of 24 injections, whenever possible. Calcium(II) acetate and magnesium(II) acetate were administered in the same preparation along with the lead and nickel salts at molar doses of approximately 1, 3, 10, and 30 times the maximal tolerated dose of the tumorigen. The animals were sacrificed 30 weeks after the first injection, and the lung tumors were counted. The lead and nickel salts, administered alone, each produced a significant increase in the observed number of lung adenomas per mouse. When administered with any of the doses of calcium acetate or magnesium acetate tested, neither lead subacetate nor nickel acetate showed any significant tumorigenic activity. Calcium acetate alone (total dose, 11 mmol/kg of body weight) appeared to yield a significant rise in lung adenomas observed. The results indicate an antagonism between magnesium and calcium and the tumorigenic metals nickel and lead.

Lactate dehydrogenase virus is the third in a series of viruses which have been examined for the capacity to alter chemically induced mouse lung tumorigenesis. This virus was given to strain A mice by i.p. injection either 4 weeks before, on the same days as, 4 weeks after, or 8 weeks after the s.c. injection of urethan (either 0.25 or 1.0 mg/g). The pulmonary adenoma response to urethan was suppressed in all of the lactate dehydrogenase-infected mice, with a maximum suppression of 30 to 40% when the virus was given simultaneously with or 4 weeks after urethan. As with murine sarcoma virus and reovirus, it is postulated that this suppression of chemically induced mouse lung tumorigenesis is due to virally induced alterations in the immune response of the mouse to chemically induced tumors.

The effect of commercial saccharin preparations on urethan-induced mouse lung tumorigenesis was assessed by gavaging groups of male strain A mice with 1-g/kg doses of each saccharin preparation on a daily basis 5 days/week. Gavage was initiated 1 week before i.p. injection of either a low (0.1 mg/g) or a high (1 mg/g) dose of urethan and continued until the mice were sacrificed 16 weeks after urethan administration. The average number of surface lung tumors per mouse for each group of mice was determined and was compared statistically with the appropriate control group. The commercial saccharin preparations did not produce an elevated lung tumor response when administered alone. One of the four saccharin preparations enhanced the lung tumor response to urethan when given in conjunction with the low dose of urethan, but this enhancement was not statistically significant. At the high urethan dose, all saccharin preparations produced a statistically significant enhancement of the lung tumor response to urethan.

The effect of formaldehyde (HCHO) inhalation on total cytochrome P450 in the lungs of Sprague-Dawley rats was assessed after single and repeated exposures to 0, 0.5, 3, and 15 ppm HCHO. Whole-body exposures were conducted in dynamic, monitored exposure systems for 6 hr/day, 5 days/week, for periods of exposure of 1 day, 4 days, 12 weeks, or 24 weeks. Lung microsomal fractions were prepared and total protein and cytochrome P450 were measured 18 hr after the end of exposure at each time point. Two separate sets of exposure studies were conducted, thus duplicating all measurements for each dose group and at each time point. There were no detectable levels of total lung P450 in any of the rats that received a single 6-hr exposure to all three HCHO doses, while control lung P450 levels were similar to that found for 4-day and 12-week control rats. After 4 days of repeated exposures, however, there was a highly significant, reproducible, and dose-dependent increase in lung P450 levels relative to controls, with the 0.5, 3, and 15 ppm groups demonstrating 387, 1026, and 1123% of control values, respectively. Lung P450 levels remained elevated at all HCHO concentrations through 12 and 24 weeks of exposure, although the percentage difference between exposed and control rats continually dropped throughout the course of long-term repeated exposures. While HCHO-exposed rats did have decreased total body weight relative to controls, lung microsomal protein and lung weight of nearly all of the HCHO-exposed rats was not significantly different from the controls. The initial inactivation of lung P450 after a single HCHO exposure is apparently a transient phenomenon, with dose-dependent induction of the total P450 levels in the lung as the pattern of response to repeated exposures to inhaled HCHO.

The i.p. injection of caffeine (8, 20, and 40 mg/kg) 3 times weekly for 8 weeks suppressed the development of spontaneous pulmonary adenomas in strain A mice. The same caffeine injection scheme suppressed urethan (0.25 and 1.0 mg/g)-induced lung tumor development when caffeine treatment started 1 week before urethan administration, but this suppression was not significant when caffeine treatment was initiated 1 week after urethan injection. The most pronounced suppression of lung tumor formation occurred when caffeine was given as only two injections 3 hr before and 3 hr after urethan administration. The incorporation of [3H]thymidine into lung tissue DNA of caffeine-treated mice was impaired at the time of urethan administration. Also, caffeine partially antagonized the effects of urethan on lung tissue, as measured by [3H]thymidine incorporation studies. One interpretation of these results is that caffeine-induced suppression of DNA synthesis interferes with pulmonary adenoma induction by decreasing the affinity of lung tissue DNA for urethan. The finding that chronic caffeine treatment produced continued suppression of [3H]thymidine incorporation into lung tissue DNA suggests that caffeine-induced inhibition of spontaneous pulmonary adenoma formation is due to a general suppression of lung DNA-synthetic activity.

The technique of flow cytometry was used to monitor the cell-cycle distribution of DNA and RNA in selected tissues of rats that were subchronically exposed to formaldehyde (HCHO) inhalation. Male Sprague-Dawley rats inhaled HCHO vapor concentrations of 0, 0.5, 3, or 15 ppm for 6 hr/day, 5 days/week, for up to 24 weeks. Simultaneous two-parameter measurements were made on a Phywe ICP 22 pulse cytophotometer by use of an acridine orange stain of the DNA and RNA of each cell sampled. No significant changes relative to the controls were determined in the percentage S and G2 + M phases of the DNA from sample tissues of the HCHO-exposed animals. Increases of 50 to 60% in the RNA content of G1 cells (RI) in the alveolar macrophages were seen after 1 week of exposure at all three HCHO doses. This effect was diminished after subchronic exposure. No HCHO-related effect was observed, though, in the RI of the rat bone marrow cells at any time point. However, the observed changes in RNA content were of rather limited magnitude relative to the cell-cycle perturbation induced by known cytotoxic agents in this and other flow cytometry studies. These modest and transient RI increases therefore probably did not reflect any significant effect on cell viability or cell proliferation in the affected tissues.

An in vitro-in vivo system for screening potentially effective drugs against solid tumors is described. Drug toxicity to plateau-phase pulmonary adenoma cells is used as an in vitro screen for potential activity against solid tumors, since both plateau phase cultured cell populations and solid tumors are composed predominantly of nondividing cells. The effect of drugs with in vitro activity on the rate of appearance of urethan-induced adenomas on the lung surface of strain A mice in vivo is used to assess drug efficacy in the treatment of solid tumors, taking into consideration drug toxicity to and drug metabolism by the host. Arabinosylcytosine and hydroxyurea were ineffective against plateau phase cells in vitro, even at high concentrations (5 to 10 mg/ml), and did not affect pulmonary adenoma growth in vivo, even at toxic doses (arabinosylcytosine, 80 mg/kg; hydroxyurea, 800 mg/kg), as would be expected with these cell cycle-active drugs. Adriamycin, an effective agent against human solid tumors, was cytotoxic to plateau phase cultured cells (0% survivors at 1 mug/ml), and a dose of 2 mg/kg completely inhibited pulmonary adenoma growth in mice. Thus, this pulmonary adenoma bioassay would appear to effectively select for drugs which may be active against solid tumors in humans.

The influence of metronidazole, misonidazole, and desmethylmisonidazole on the induction of lung adenomas in the strain A mouse was examined. Two dose levels of the hypoxic cell sensitizers, 0.2 and 0.6 mg/g, were used either alone or in combination with 900 rads of gamma-radiation in a fractionated dose schedule of twice a week for 3 weeks. In the groups of mice which received hypoxic cell sensitizers only, the prevalence and the mean number of lung tumors per mouse were somewhat increased (p less than 0.10) in the group receiving the higher dose (0.6 mg/g) of misonidazole but was not significantly different from results for the control animals in the other two sensitizer groups. The combination of hypoxic cell sensitizer and radiation did not show any significant enhancement of lung tumor response when compared with the group which received radiation only. The dose of radiation used in this study significantly enhanced lung tumor formation in mice when compared with that in the control group. Thus, under the experimental exposure conditions used in this investigation, which were somewhat similar to the exposure conditions occurring in clinical treatment, each of the hypoxic cell sensitizers tested failed to sensitize significantly the mice to the carcinogenic effects of gamma-radiation.

The production of lung adenomas in strain A following multiple i.p. injections of selected organic water contaminants was investigated. Of the 16 contaminants tested, only bromoform produced a pulmonary adenoma response that was significantly greater than the pulmonary adenoma response of vehicle-treated control mice.

The Salmonella reversion test was used to measure the mutagenic activities of urine concentrates from individuals preparing cancer chemotherapy agents for i.v. administration. Longitudinal studies were performed in which the total urine produced in 24-hr periods was collected, starting on a Sunday at 7 p.m. after a duty-free weekend and extending over an 8-day period. There was no detectable increase in mutagenic activity in the urine concentrates of three pharmacy administrators who had no contact with these drugs. All six individuals admixing drugs in open-faced, horizontal laminar flow hoods displayed a 2-fold increase in mutagenesis by the fourth day with peak values of 2.7- to 24-fold occurring on Days 5 and 6, reduced values by Day 7, and a return to the spontaneous level by Day 8. When four of the six positive individuals in the preceding experiment admixed comparable amounts of chemotherapeutic drugs in a closed-face, vertical laminar flow hood, no increase in mutagenic activity was detected in their urine concentrates over the 8-day period.