Characterization of nuclear polyhedrosis viruses obtained from Adoxophyes orana and from Barathra brassicae

ln infectivity experiments some A. orana larvae died after being inoculated with an inoculum containing NW isolated from B. brassicae. The polyhedra formed upon infection occluded single virus particles, whereas the inoculum contained polyhedra with bundles of virus particles. This change could be explained either by activation of a virus in A. orana, which is singly embedded, or the inoculum from B. brassicae had infected A. orana and consequently the inclusion of virus particles in outer membranes is controlled by the hosts. This thesis describes studies performed to discriminate between both possibilities. Therefore, the first task was to characterize the virus particles from B. brassicae and A. orana NPV and their polyhedra by different techniques (Chapter 1).The properties of the NW of A. orana and of B. brassicae as observed with the electron microscope and polyacrylamide gel electrophoresis are similar to those found for many other NPVs. The polyhedra of both NPVs differ in size and shape. Most of the A. orana polyhedra are globular and range in diameter from 1-2 μm. Most of the B. brassicae polyhedra are hexagonal or pentagonal in outline and range in diameter from 1.5-4 μm. Analysis of polyhedral protein by polyacrylamide gel electrophoresis shows the presence of two polypeptides of molecular weight 28,000 and 54,000 Daltons.Treatment of the polyhedra of both viruses with sodium carbonate ruptures the polyhedral membrane and the virus particles and polyhedral proteins are released. The virus particles of A. orana polyhedra are singly embedded in the polyhedral matrix and have a size of 250 x 60 nm. The multiply embedded virus particles of B. brassicae have a size of 347 x 113 nm. Analysis of the viral proteins by SDS-polyacrylamide gel electrophoresis showed that NW of A. orana has 5 polypeptides of 68,000, 48,000, 39,000, 32-34,000, and 28,000 Daltons, respectively. Those of the NPV of B. brassicae were 69,000, 57,000, 46,000, 34-39,000, and 28,000 Daltons, respectively.In the polyhedral membrane fractions of both polyhedra one polypeptide of molecular weight of 28,000 Daltons as estimated by polyacrylamide gel electrophoresis, was found.Due to proteolytic activity associated with the polyhedra, which is evident after dissociation of the polyhedra, it was difficult to establish the number of polyhedral proteins and their molecular weight (Chapter 2). The electrophoretic pattern of polyhedral proteins of A.orana and B. brassicae polyhedra dissociated in alkali differed from those proteins obtained by other means. Six to seven polypeptides with molecular weights between 28,000 and 8,000 Daltons were found after incubation at pH 10.5. After inactivation of the enzyme only two polypeptides with molecular weights of 28,000 and 26,000 Daltons were observed. When the polyhedral proteins were analysed without incubation at pH 10.5 also two proteins were found, but their molecular weight was 54,000 and 28,000 Daltons.On the basis of the results described in Chapters 1 and 2 it can be concluded that the virus particles of B. brassicae and A.orana NPV differ with respect to size, the way of occlusion, and the form and size of the polyhedra involved. Protein analysis by polyacrylamide gel electrophoresis reveal some difference in molecular weight of viral protein but no significant difference in the protein composition of their polyhedra. Further analyses of amino acid composition and sequence of these proteins is necessary to elucidate possible differences.To differentiate further between both viruses their genomes were analysed (Chapter 3). Both genomes are circular double-stranded DNA molecules. The molecular weights of A.orana and of B. brassicae NPV-DNAs are 6.7 x 10 7and 8.9 x 10 7Daltons, respectively as determined by electron microscopy and by renaturation kinetic analysis. The renaturation also indicated that both genomes contain only unique sequences. The buoyant density in CsCl of the NPV-DNA of A. orana and of B. brassicae is 1.694 and 1.696 g/cm 3, respectively. These values are in good agreement with (G+C) contents of 34.5 and 37%, respectively as determined by thermal denaturation. The digestion of the A. orana and of the B. brassicae NPV-DNA with endonuclease Eco RI resulted in completely different electrophoretic patterns. Also in experiments oncompetition hybridization no homology between these genomes was found. The conclusion of these studies is that these two NPVs can be clearly differen tiated by their DNA properties.In order to study the occurrence of viral DNA in uninfected larvae the DNA of A. orana and B. brassicae was isolated and the complexity studied (Chapter 4). The genomes of A.orana and of B. brassicae differ in their kinetic complexity as estimated from the reassociation data on hyperchromicity, but they are both relatively small and show remarkable similarity in the extent of intragenome homology. A haploid cell of A. orana has a DNA equivalent of 4.2 x 10 10and that of B. brassicae of 8.4 x 10 10Daltons. The intragenome homology was estimated to be 10 and 9% for A.orana and B. brassicae genome, respectively. The (G+C) content, estimated by thermal denaturation, was found to be 36.2% for the A. orana genome and 35.8% for the B. brassicae genome.The results obtained during rearing of insects from surface-sterilized eggs and from untreated eggs showed that the NPV of A.orana and of B. brassicae can be transmitted to the progeny of these insects on the outside of the eggs (transovum) as well as inside the eggs (transovarially) (Chapter 5). Evidence for transovarial transmission was also obtained from reassociation of viral DNA with the host DNA of homologous insects reared from surface-sterilized eggs. These experiments revealed the presence of viral sequences in host DNA: 0.03 and about 2.5 viral copies for the diploid quantity of the A. orana and of the B. brassicae host DNA, respectively.Results obtained in infectivity experiments with insects in various developmental stages showed that transstadial transmission is a prerequisite for generation-to-generation transmission.The presence of a latent virus infection in both insects could also be demonstrated in cross-inoculation experiments (Chapter 6). When the larvae of A. orana and of B. brassicae were inoculated with polyhedra of the reciprocal species, the number of larvae containing polyhedra increased compared with that of the control. Comparison of the restriction endonuclease Eco RI pattern of DNA isolated from polyhedra used as inocula with that from polyhedra obtained after cross-inoculation indicated that both viruses are not cross-infective but that they activate a latent virus infection in both insects. Because the cross- inoculation experiments were done under laboratory conditions (as aseptic as possible), it could be concluded that the B. brassicae NPV is not suitable for biological control of A. orana in the field, because this virus is not cross-infective.</em

Heterogeneous Host Susceptibility Enhances Prevalence of Mixed-Genotype Micro-Parasite Infections

Dose response in micro-parasite infections is usually shallower than predicted by the independent action model, which assumes that each infectious unit has a probability of infection that is independent of the presence of other infectious units. Moreover, the prevalence of mixed-genotype infections was greater than predicted by this model. No probabilistic infection model has been proposed to account for the higher prevalence of mixed-genotype infections. We use model selection within a set of four alternative models to explain high prevalence of mixed-genotype infections in combination with a shallow dose response. These models contrast dependent versus independent action of micro-parasite infectious units, and homogeneous versus heterogeneous host susceptibility. We specifically consider a situation in which genome differences between genotypes are minimal, and highly unlikely to result in genotype-genotype interactions. Data on dose response and mixed-genotype infection prevalence were collected by challenging fifth instar Spodoptera exigua larvae with two genotypes of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), differing only in a 100 bp PCR marker sequence. We show that an independent action model that includes heterogeneity in host susceptibility can explain both the shallow dose response and the high prevalence of mixed-genotype infections. Theoretical results indicate that variation in host susceptibility is inextricably linked to increased prevalence of mixed-genotype infections. We have shown, to our knowledge for the first time, how heterogeneity in host susceptibility affects mixed-genotype infection prevalence. No evidence was found that virions operate dependently. While it has been recognized that heterogeneity in host susceptibility must be included in models of micro-parasite transmission and epidemiology to account for dose response, here we show that heterogeneity in susceptibility is also a fundamental principle explaining patterns of pathogen genetic diversity among hosts in a population. This principle has potentially wide implications for the monitoring, modeling and management of infectious diseases

Gender and Age Differences in Patients' Satisfaction with Dental Care in the Slovak Republic: A Cross-sectional Questionnaire Study

Objective: To analyze the level of quality of healthcare satisfaction/dissatisfaction according to gender and age in terms of time spent with the doctor, communication, general satisfaction, interpersonal and financial aspects, technical quality and access/availability/convenience. Material and Methods: The standardized "Patient Satisfaction Questionnaire" (n=445) was collected in dental clinics in the Slovak Republic in the period of 2014-2015. The study sample was divided into subgroups according to gender (males: n=236; females: n=209) and age (<35 years: n= 243; ≥36 years: n=202). A scale from 1 (maximum dissatisfaction with quality of healthcare) to 5 (maximum satisfaction with quality of healthcare) was evaluated. Results: The highest significant satisfaction level of quality of healthcare (expressed as a mean score) were found in females in the subscale time spent with doctor (<35 y.) (2.90±0.69; p<0.042); the lowest satisfaction level was found in females (<35 y.) in the subscale general satisfaction (2.53±0.36; p=n.s.). Females (≥36y.) were significantly more satisfied with health care quality in the subscale communication than males (2.72±0.34 and 2.62±0.3, respectively; p=0.046). Males (<35 y.) were significantly less satisfied in the subscale technical quality compared to females (2.54±0.27 and 2.65±0.26, respectively; p=0.002). Conclusion: For the future, it is necessary to specify the next reasons for patients’ satisfaction/dissatisfaction with dental care

DNA Damage Response in Cancer Therapy and Resistance: Challenges and Opportunities

Resistance to chemo- and radiotherapy is a common event among cancer patients and a reason why new cancer therapies and therapeutic strategies need to be in continuous investigation and development. DNA damage response (DDR) comprises several pathways that eliminate DNA damage to maintain genomic stability and integrity, but different types of cancers are associated with DDR machinery defects. Many improvements have been made in recent years, providing several drugs and therapeutic strategies for cancer patients, including those targeting the DDR pathways. Currently, poly (ADP-ribose) polymerase inhibitors (PARP inhibitors) are the DDR inhibitors (DDRi) approved for several cancers, including breast, ovarian, pancreatic, and prostate cancer. However, PARPi resistance is a growing issue in clinical settings that increases disease relapse and aggravate patients&rsquo; prognosis. Additionally, resistance to other DDRi is also being found and investigated. The resistance mechanisms to DDRi include reversion mutations, epigenetic modification, stabilization of the replication fork, and increased drug efflux. This review highlights the DDR pathways in cancer therapy, its role in the resistance to conventional treatments, and its exploitation for anticancer treatment. Biomarkers of treatment response, combination strategies with other anticancer agents, resistance mechanisms, and liabilities of treatment with DDR inhibitors are also discussed

The Analysis of Selected Malocclusion Risk Factors: A Pilot Study

Objective: To analyse selected malocclusion risk factors, their exposure time and overall malocclusion risk scores. Material and Methods: The self-prepared questionnaires were collected at dental practitioners’ waiting rooms from 6/2014 to 12/2015. The study group consisted of patients treated by dental braces (n=82; 15.5±4.4 years) and the control group consisted of other patients not treated by dental braces (n=45; 17.6±4.7 years). Data were processed by the statistical program SPSS using descriptive statistics. To verify the hypothesis wad used two sample t-test to compare the average exposure scores and the exposure time between the two groups. To determine associations between categorical variables was used Chi-square test. Statistical significance was set at p-value <0.05. Results: Our results confirmed longer exposure times in all studied malocclusion risk factors, in the case of pacifier sucking the difference was significant (p=0.001). The longest exposure time was found in mouth breathing in the study group (12.2±6.5 years). The lip sucking/chewing cannot be confirmed as a malocclusion risk factor. The study group had higher level of an overall mean risk score (19.8±11.5) compared the control group (16.1±12.1), although not significant. It can be concluded that non-nutritive sucking habits and/or mouth breathing could have damaging effect to normal teeth development. Conclusion: Malocclusions could be preventable, thus we recommend setting up educational programs for dentists and paediatricians as well as for parents focusing on the improvement of oral health knowledge

The Risks of Social Noise Exposure in the Vulnerable Population in Slovakia

The study is aimed to quantify the effects of social noise exposure (personal music players (PMP), events with high noise exposure) and the exposure to the other environmental noise sources in the selected sample of Slovak university students. The validated ICBEN methodology was used to assess noise annoyance. The measurement of ambient noise levels was done using hand-held sound level analyzer. There were 526 university students (143 males and 383 females, average age 23 ± 2.2) enrolled into the study so far, 192 in the exposed housing facility to road traffic noise and 326 in the control housing facility in Bratislava. The social noise exposure was quantified and followed according to the authorized methodology of the study Ohrkan. From the total sample 416 (79.4%) students reported the use of PMP in the last week for the average time of 314 minutes. There was a significant difference in PMP use between the exposed (85.34%) and the control group (76.31%) (p = 0:01). Among PMP users 28.1% exceeded the LAV (lower action value for industry = 80 dB). The results showed the importance of road traffic and the social noise as well and the need for prevention and intervention in these vulnerable groups