Molecular investigation of some DNA viruses in mucosal melanoma: Case-control study.

The association between viral infections and both cutaneous and mucosal melanoma (MM) has not been fully investigated. Here, we assessed the prevalence of the DNA of a broad range of viruses in 31 MMs and 15 biopsies of healthy mucosa (HM) using molecular methods. The parvoviruses CuV and B19V, herpesviruses HSV1, HSV2, EBV, HHV6, and HHV8, polyomavirus MCPyV, and α-HPVs were not detected, or rarely found, in MMs, and in HM, of the digestive, respiratory, and female genital tract. The overall prevalence of ß-HPV in MMs was not significantly higher compared to that in HM (70.9% and 53.3% respectively; p = 0.514). However, the number of MMs positive for ß-HPV types belonging to Species 3 and 5 and for some viral types belonging to Species 1, 2, 3, and 5 were significantly higher compared with HM (p < 0.05). Moreover, compared to HM, the MM samples contained a significantly higher number of ß-HPV types, mainly belonging to Species 1, 3, and 5 (p < 0.05). Our data, although suggesting a role for certain ß-HPV types in MM oncogenesis, require additional investigation in larger populations to support this hypothesis.

Parvovirus B19 induces cellular senescence in human dermal fibroblasts: putative role in systemic sclerosis-associated fibrosis.

OBJECTIVE: Emerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in ageing or in SSc. Recently we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/pro-inflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis. METHODS: Primary cultures of fibroblasts were infected with B19V and analysed for the acquisition of senescence markers, such as morphological modifications, senescence-associated ß-galactosidase (SA-ß-gal) activity, DNA damage response and expression of senescence-associated secretory phenotype (SASP)-related factors. RESULTS: We demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-ß-gal activity similar to that observed in SSc skin fibroblasts. They also developed an SASP-like phenotype characterized by mRNA expression and release of some pro-inflammatory cytokines, along with activation of the transcription factor nuclear factor κB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significantly higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. An increased level and nuclear localization of γH2AX, a hallmark of DNA damage response, were also found. CONCLUSIONS: B19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process.

Insights into the knowledge of complex diseases: Environmental infectious/toxic agents as potential etiopathogenetic factors of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease secondary to three cardinal pathological features: immune-system alterations, diffuse microangiopathy, and fibrosis involving the skin and internal organs. The etiology of SSc remains quite obscure; it may encompass multiple host genetic and environmental -infectious/chemical-factors. The present review focused on the potential role of environmental agents in the etiopathogenesis of SSc based on epidemiological, clinical, and laboratory investigations previously published in the world literature. Among infectious agents, some viruses that may persist and reactivate in infected individuals, namely human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and parvovirus B19 (B19V), and retroviruses have been proposed as potential causative agents of SSc. These viruses share a number of biological activities and consequent pathological alterations, such as endothelial dysfunction and/or fibroblast activation. Moreover, the acute worsening of pre-existing interstitial lung involvement observed in SSc patients with symptomatic SARS-CoV-2 infection might suggest a potential role of this virus in the overall disease outcome. A variety of chemical/occupational agents might be regarded as putative etiological factors of SSc. In this setting, the SSc complicating silica dust exposure represents one of the most promising models of study. Considering the complexity of SSc pathogenesis, none of suggested causative factors may explain the appearance of the whole SSc; it is likely that the disease is the result of a multifactorial and multistep pathogenetic process. A variable combination of potential etiological factors may modulate the appearance of different clinical phenotypes detectable in individual scleroderma patients. The in-deep investigations on the SSc etiopathogenesis may provide useful insights in the broad field of human diseases characterized by diffuse microangiopathy or altered fibrogenesis.

Parvovirus B19 activates in vitro normal human dermal fibroblasts: a possible implication in skin fibrosis and systemic sclerosis.

OBJECTIVE: Fibrosis is the most characteristic pathological hallmark of SSc, a connective tissue disease characterized by vascular and immunological abnormalities, inflammation and enhanced extracellular matrix production, leading to progressive fibrosis of skin and internal organs. We previously demonstrated that parvovirus B19 (B19V) can infect normal human dermal fibroblasts (NHDFs) and that B19V persists in SSc fibroblasts. In this study, we investigated whether parvovirus B19V is able to activate in vitro NHDFs and to induce in these cells some phenotypic features similar to that observed in the SSc fibroblasts. METHODS: We preliminarily analysed the time course of B19V infection in cultured NHDFs, then we investigated the ability of B19V to induce cell migration, invasive phenotype and mRNA expression of some profibrotic and/or proinflammatory genes. RESULTS: We confirmed our previous findings that B19V infects NHDFs, but the infection is not productive. After incubation with B19V, NHDFs showed a significant increase of both migration and invasiveness, along with mRNA expression of different profibrotic genes (α-SMA, EDN-1, IL-6, TGF-ß1 receptors 1 and 2, Col1α2), some genes associated with inflammasome platform (AIM2, IFI16, IL-1ß, CASP-1) and genes for metalloprotease (MMP 2, 9 and 12). CONCLUSION: These data suggest that B19V can activate dermal fibroblasts and may have a role in the pathogenesis of fibrosis. B19V-induced fibroblast migration and invasiveness could be due to the B19V-associated MMP9 overexpression and activation. Moreover, the up-regulation of MMP12, typical of SSc, could link the B19V infection of fibroblasts to the anti-angiogenic process.

Sequence-dependent response of DNA to torsional stress: a potential biological regulation mechanism.

Torsional restraints on DNA change in time and space during the life of the cell and are an integral part of processes such as gene expression, DNA repair and packaging. The mechanical behavior of DNA under torsional stress has been studied on a mesoscopic scale, but little is known concerning its response at the level of individual base pairs and the effects of base pair composition. To answer this question, we have developed a geometrical restraint that can accurately control the total twist of a DNA segment during all-atom molecular dynamics simulations. By applying this restraint to four different DNA oligomers, we are able to show that DNA responds to both under- and overtwisting in a very heterogeneous manner. Certain base pair steps, in specific sequence environments, are able to absorb most of the torsional stress, leaving other steps close to their relaxed conformation. This heterogeneity also affects the local torsional modulus of DNA. These findings suggest that modifying torsional stress on DNA could act as a modulator for protein binding via the heterogeneous changes in local DNA structure.

Analyzing DNA curvature and its impact on the ionic environment: application to molecular dynamics simulations of minicircles.

We propose a method for analyzing the magnitude and direction of curvature within nucleic acids, based on the curvilinear helical axis calculated by Curves+. The method is applied to analyzing curvature within minicircles constructed with varying degrees of over- or under-twisting. Using the molecular dynamics trajectories of three different minicircles, we are able to quantify how curvature varies locally both in space and in time. We also analyze how curvature influences the local environment of the minicircles, notably via increased heterogeneity in the ionic distributions surrounding the double helix. The approach we propose has been integrated into Curves+ and the utilities Canal (time trajectory analysis) and Canion (environmental analysis) and can be used to study a wide variety of static and dynamic structural data on nucleic acids.

Human herpesviruses respiratory infections in patients with acute respiratory distress (ARDS).

Acute respiratory distress syndrome (ARDS) is today a leading cause of hospitalization in intensive care unit (ICU). ARDS and pneumonia are closely related to critically ill patients; however, the etiologic agent is not always identified. The presence of human herpes simplex virus 1, human cytomegalovirus and Epstein-Barr virus in respiratory samples of critically ill patients is increasingly reported even without canonical immunosuppression. The main aim of this study was to better understand the significance of herpesviruses finding in lower respiratory tract of ARDS patients hospitalized in ICU. The presence of this group of herpesviruses, in addition to the research of influenza viruses and other common respiratory viruses, was investigated in respiratory samples from 54 patients hospitalized in ICU, without a known microbiological causative agent. Moreover, the immunophenotype of each patient was analyzed. Herpesviruses DNA presence in the lower respiratory tract seemed not attributable to an impaired immunophenotype, whereas a significant correlation was observed between herpesviruses positivity and influenza virus infection. A higher ICU mortality was significantly related to the presence of herpesvirus infection in the lower respiratory tract as well as to impaired immunophenotype, as patients with poor outcome showed severe lymphopenia, affecting in particular T (CD3+) cells, since the first days of ICU hospitalization. In conclusion, these results indicate that herpesviruses lower respiratory tract infection, which occurs more frequently following influenza virus infection, can be a negative prognostic marker. An independent risk factor for ICU patients with ARDS is an impaired immunophenotype.

Analyzing ion distributions around DNA.

We present a new method for analyzing ion, or molecule, distributions around helical nucleic acids and illustrate the approach by analyzing data derived from molecular dynamics simulations. The analysis is based on the use of curvilinear helicoidal coordinates and leads to highly localized ion densities compared to those obtained by simply superposing molecular dynamics snapshots in Cartesian space. The results identify highly populated and sequence-dependent regions where ions strongly interact with the nucleic and are coupled to its conformational fluctuations. The data from this approach is presented as ion populations or ion densities (in units of molarity) and can be analyzed in radial, angular and longitudinal coordinates using 1D or 2D graphics. It is also possible to regenerate 3D densities in Cartesian space. This approach makes it easy to understand and compare ion distributions and also allows the calculation of average ion populations in any desired zone surrounding a nucleic acid without requiring references to its constituent atoms. The method is illustrated using microsecond molecular dynamics simulations for two different DNA oligomers in the presence of 0.15 M potassium chloride. We discuss the results in terms of convergence, sequence-specific ion binding and coupling with DNA conformation.

Unraveling the sequence-dependent polymorphic behavior of d(CpG) steps in B-DNA.

We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3'-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change.

µABC: a systematic microsecond molecular dynamics study of tetranucleotide sequence effects in B-DNA.

We present the results of microsecond molecular dynamics simulations carried out by the ABC group of laboratories on a set of B-DNA oligomers containing the 136 distinct tetranucleotide base sequences. We demonstrate that the resulting trajectories have extensively sampled the conformational space accessible to B-DNA at room temperature. We confirm that base sequence effects depend strongly not only on the specific base pair step, but also on the specific base pairs that flank each step. Beyond sequence effects on average helical parameters and conformational fluctuations, we also identify tetranucleotide sequences that oscillate between several distinct conformational substates. By analyzing the conformation of the phosphodiester backbones, it is possible to understand for which sequences these substates will arise, and what impact they will have on specific helical parameters.

No detectable truncating mutations in large T antigen (LT-Ag) sequence of Merkel cell polyomavirus (MCPyV) DNA obtained from porocarcinomas.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC). In tumor cells the MCPyV large T antigen (LT-Ag) is frequently found truncated and this is considered a major tumor-specific signature. The role of MCPyV in other, non-MCC tumours, is little known. Viral DNA and/or tumour-specific mutations have been sometimes detected in different tumours, but such data are not unequivocal and the involvement of the virus in the tumorigenesis is not clear. In a previous study, we demonstrated a significantly higher prevalence of MCPyV DNA in formalin fixed paraffin embedded (FFPE) porocarcinoma tissues compared to the normal skin. In the present study, we investigated the presence of truncating mutations in MCPyV LT-Ag coding region in porocarcinoma specimens. Using several overlapped PCR primer pairs, the complete LT-Ag sequence from two biopsies were obtained. No truncating mutations were detected. The lack of truncating mutations in LT-Ag sequence does not seem to support the role of MCPyV in porocarcinoma oncogenesis. However, an oncogenetic mechanism, different from that proposed for MCC and not associated with the LT-Ag mutations/deletions, cannot be excluded. Further studies of more sequences coding for LT-Ag would be needed to verify this hypothesis.

CURVES+ web server for analyzing and visualizing the helical, backbone and groove parameters of nucleic acid structures.

Curves+, a revised version of the Curves software for analyzing the conformation of nucleic acid structures, is now available as a web server. This version, which can be freely accessed at http://gbio-pbil.ibcp.fr/cgi/Curves_plus/, allows the user to upload a nucleic acid structure file, choose the nucleotides to be analyzed and after optionally setting a number of input variables, view the numerical and graphic results online or download files containing a set of helical, backbone and groove parameters that fully describe the structure. PDB format files are also provided for offline visualization of the helical axis and groove geometry.

Multistep drug intercalation: molecular dynamics and free energy studies of the binding of daunomycin to DNA.

Atomic-scale molecular dynamics and free energy calculations in explicit aqueous solvent are used to study the complex mechanism by which a molecule can intercalate between successive base pairs of the DNA double helix. We have analyzed the intercalation pathway for the anticancer drug daunomycin using two different methods: metadynamics and umbrella sampling. The resulting free energy pathways are found to be consistent with one another and point, within an equilibrium free energy context, to a three-step process. Daunomycin initially binds in the minor groove of DNA. An activated step then leads to rotation of the drug, coupled with DNA deformation that opens a wedge between the base pairs, bends DNA toward the major groove, and forms a metastable intermediate that resembles structures seen within the interfaces between DNA and minor-groove-binding proteins. Finally, crossing a small free energy barrier leads to further rotation of daunomycin and full intercalation of the drug, reestablishing stacking with the flanking base pairs and straightening the double helix.

Pattern of HPV infection in basal cell carcinoma and in perilesional skin biopsies from immunocompetent patients.

BACKGROUND: The association between human papillomavirus (HPV) infection and non-melanoma skin cancers (NMSCs) such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) is not yet fully understood. We analysed the prevalence and spectrum of cutaneous beta-HPV types and mucosal/genital HPV types in paired biopsies (tumour and corresponding perilesional skin) obtained from 50 BCC immunocompetent patients. A small group of SCC patients (n=9) was also included. We also evaluated some previously postulated risk factors for HPV infection in NMSC patients. RESULTS: All biopsies were negative for mucosal/genital HPV types. Overall, beta-HPV DNA was detected more often in SCC compared to BCC patients (78% vs 55% of total samples). The frequency of infection increased with the patient's age [OR=4.88 (95% CI 1.29-18.39)]. There was no significant correlation between beta-HPV positivity and sex, skin type and UV exposure. The prevalence of beta-HPV species 1 types was significantly higher than those belonging to other beta-HPV species in biopsies from BCC (p=0.022) but not from SCC subjects (p=0.091). There was no significant difference in the overall prevalence of beta-HPV infection and the number of viral types between tumour lesions and perilesional skin. BCC samples were significantly more likely to be infected with beta-HPV species 1 types compared to perilesional skin (p=0.036) and showed a higher frequency of mixed infections (p=0.028). CONCLUSIONS: These findings demonstrate that beta-HPV types belonging to species 1 are the most common HPV types detected in the skin of BCC patients. Moreover beta-1-HPV types and mixed infections are significantly more frequent in tumour samples than in healthy perilesional skin. Our results suggest that beta-1-HPVs as well as co-infection with more than one viral type could be important in NMSC and in particular in BCC.Further studies aimed to compare the biological activity of viral types in tumours and in healthy skin (viral replication and expression, interference of infection with cellular functions) are necessary to understand the role of HPV infection in skin cancer.

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA.

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein-DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50-100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.

Local and global effects of strong DNA bending induced during molecular dynamics simulations.

DNA bending plays an important role in many biological processes, but its molecular and energetic details as a function of base sequence remain to be fully understood. Using a recently developed restraint, we have studied the controlled bending of four different B-DNA oligomers using molecular dynamics simulations. Umbrella sampling with the AMBER program and the recent parmbsc0 force field yield free energy curves for bending. Bending 15-base pair oligomers by 90 degrees requires roughly 5 kcal mol(-1), while reaching 150 degrees requires of the order of 12 kcal mol(-1). Moderate bending occurs mainly through coupled base pair step rolls. Strong bending generally leads to local kinks. The kinks we observe all involve two consecutive base pair steps, with disruption of the central base pair (termed Type II kinks in earlier work). A detailed analysis of each oligomer shows that the free energy of bending only varies quadratically with the bending angle for moderate bending. Beyond this point, in agreement with recent experiments, the variation becomes linear. An harmonic analysis of each base step yields force constants that not only vary with sequence, but also with the degree of bending. Both these observations suggest that DNA is mechanically more complex than simple elastic rod models would imply.

Human parvovirus PARV4 DNA in tissues from adult individuals: a comparison with human parvovirus B19 (B19V).

BACKGROUND: PARV4 is a new member of the Parvoviridae family not closely related to any of the known human parvoviruses. Viremia seems to be a hallmark of PARV4 infection and viral DNA persistence has been demonstrated in a few tissues. Till now, PARV4 has not been associated with any disease and its prevalence in human population has not been clearly established. This study was aimed to assess the tissue distribution and the ability to persist of PARV4 in comparison to parvovirus B19 (B19V). RESULTS: PARV4 and B19V DNA detection was carried out in various tissues of individuals without suspect of acute viral infection, by a real time PCR and a nested PCR, targeting the ORF2 and the ORF1 respectively. Low amount of PARV4 DNA was found frequently (>40%) in heart and liver of adults individuals, less frequently in lungs and kidneys (23,5 and 18% respectively) and was rare in bone marrow, skin and synovium samples (5,5%, 4% and 5%, respectively). By comparison, B19V DNA sequences were present in the same tissues with a higher frequency (significantly higher in myocardium, skin and bone marrow) except than in liver where the frequency was the same of PARV4 DNA and in plasma samples where B19V frequency was significantly lower than that of PARV4 CONCLUSIONS: The particular tropism of PARV4 for liver and heart, here emerged, suggests to focus further studies on these tissues as possible target for viral replication and on the possible role of PARV4 infection in liver and heart diseases. Neither bone marrow nor kidney seem to be a common target of viral replication.

Magnitude and direction of DNA bending induced by screw-axis orientation: influence of sequence, mismatches and abasic sites.

DNA-bending flexibility is central for its many biological functions. A new bending restraining method for use in molecular mechanics calculations and molecular dynamics simulations was developed. It is based on an average screw rotation axis definition for DNA segments and allows inducing continuous and smooth bending deformations of a DNA oligonucleotide. In addition to controlling the magnitude of induced bending it is also possible to control the bending direction so that the calculation of a complete (2-dimensional) directional DNA-bending map is now possible. The method was applied to several DNA oligonucleotides including A(adenine)-tract containing sequences known to form stable bent structures and to DNA containing mismatches or an abasic site. In case of G:A and C:C mismatches a greater variety of conformations bent in various directions compared to regular B-DNA was found. For comparison, a molecular dynamics implementation of the approach was also applied to calculate the free energy change associated with bending of A-tract containing DNA, including deformations significantly beyond the optimal curvature. Good agreement with available experimental data was obtained offering an atomic level explanation for stable bending of A-tract containing DNA molecules. The DNA-bending persistence length estimated from the explicit solvent simulations is also in good agreement with experiment whereas the adiabatic mapping calculations with a GB solvent model predict a bending rigidity roughly two times larger.

Human parvovirus B19 (B19V) infection in systemic sclerosis patients.

BACKGROUND: Our previous reports suggested a possible association between parvovirus B19 (B19V) infection and systemic sclerosis (SSc), based on higher prevalence of B19V DNA in SSc patients in respect to controls. METHODS: In the present study, to further evaluate the differences in the pattern of B19 infection in SSc, skin biopsies and bone marrow samples from patients and controls were analysed for B19V DNA detection, genotyping and viral expression. RESULTS: B19V DNA was detected in skin biopsies from 39/49 SSc patients and from 20/28 controls. Bone marrow showed positive in 17/29 SSc patients, 5/21 haematological patients and 0/10 healthy controls. Genotype 1 was more frequent in skin and bone marrow from patients than from controls. Simultaneous persistence of 2 genotypes was detected in SSc skin and bone marrow samples, never in controls. Viral mRNA for capsid protein was detected in the skin of genotype 1-positive patients and not in control skins. CONCLUSION: The results outline some differences in the rate of persistence of B19V DNA, in the simultaneous persistence of 2 genotypes and in the pattern of viral expression among SSc patients and controls.