Effect of cold plasma on periodontal wound healing-an in vitro study.

OBJECTIVES: Cold atmospheric plasma (CAP), a room temperate ionized gas, seems to be a possible way to enhance tissue recovery. An in vitro study was conducted to investigate the influence of medical CAP on the regenerative capacity of human periodontal ligament (PDL) cells. MATERIAL AND METHODS: Human PDL cells were subjected to CAP at various intensities, distances, and durations. The effects of CAP on a number of specific markers were studied at transcriptional level using real-time PCR. Additionally, an in vitro wound healing assay was applied to PDL cell monolayers either in the presence or absence of CAP by using JuLI™ Br Live Cell Analyzer and software. Finally, cell viability of CAP-treated cells was analyzed by an XTT assay. RESULTS: CAP treatment enhanced significantly the expression of the cytokines tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, interleukin (IL)-1ß, IL-6, IL-8, collagen (COL)1α, and matrix metalloproteinase (MMP)1, as well as the proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), but downregulated apoptotic markers Apaf1 and p53. Additionally, the in vitro wound healing rate was significantly enhanced after CAP application. Moreover, CAP treatment resulted in a significantly increased cell viability in the XTT assay. CONCLUSION: This in vitro study shows that CAP has regulatable effects on markers of periodontal wound healing thereby underlining the potential use of CAP as a benefit treatment strategy. CLINICAL RELEVANCE: Our study demonstrates the application of CAP in the treatment of oral pathologies suggesting a promising future treatment approach.

[Is supplemental ear acupuncture for surgical tooth removal with local anesthesia effective? : A pilot study]. / Ist die Supplementierung der Ohrakupunktur bei operativer Zahnentfernung in Lokalanästhesie effektiv? : Eine Pilotstudie.

BACKGROUND: The application of ear acupuncture can contribute to a reduction of acute pain. Data on the application of ear acupuncture following oral surgery in odontology is insufficient. OBJECTIVE: This study investigated the effectiveness of ear acupuncture as an auxiliary analgesic treatment in addition to local anesthesia for operative tooth removal. METHODS: In this prospective open non-randomized pilot study (in accordance with the CONSORT publication) 2 cohorts of 50 patients each with the indications for an operative tooth removal either with or without the application of ear acupuncture in addition to local anesthesia with articain were observed. Patients were allocated to the groups according to their preference. Pain intensity while resting and while chewing was recorded as the primary parameter for a period of 10 days. The secondary parameters were the subjective experience of anxiety and symptoms, such as headaches, dizziness and nausea. RESULTS: The two groups did not differ significantly with respect to demographic variables or the use of local anesthetics. At the various measurement intervals, pain intensity while resting or chewing differed significantly between the two groups (ANOVA, p = 0.004, p = 0.007, respectively). Furthermore, the experience of anxiety (ANOVA, p = 0.0001), the number of patients taking analgesics (χ2-test, p = 0.017) and the total postoperative consumption of analgesics (t-test, 0.001) revealed significant differences. In both groups the numerical rating scales (NRS) for postoperative headaches, dizziness and nausea were low. DISCUSSION AND CONCLUSION: Despite a potential bias and methodological limitations of the study design, the results of this investigation suggest that ear acupuncture influences the experience of pain and anxiety in the postoperative period after tooth removal. As a treatment method with low adverse effects ear acupuncture can contribute to postoperative pain control, especially in patients with preoperative anxiety.

[Participation in Preventive Medical Examinations for Children in Saxony-Anhalt (Germany) at the Time of School Entry Medical Examination]. / Inanspruchnahme von Früherkennungsunter-suchungen bei Kindern aus Sachsen-Anhalt zum Zeitpunkt der Schuleingangsuntersuchung.

OBJECTIVE: The aim of this study is to present data concerning children's participation in the German preventive medical examinations for children ("U2" to "U8") in accordance with sections 26 and 92 of the German Social Code (Book V) as retrospectively recorded by the Public Health Service ("Öffentlicher Gesundheitsdienst") of the German Province of Saxony-Anhalt during school entry medical examinations. Also we wanted to analyse the additional variables recorded per child in the areas of social factors, diagnostic findings and levels of therapeutic care in connection with their degree of participation in the preventive medical examinations. METHODOLOGY: The statistical analysis of 73 628 anonymised data sets from the health monitoring system of the German Province of Saxony-Anhalt that were collected by the 14 health authorities in Saxony-Anhalt during school entry medical examinations between 2008 and 2012. An analysis of the data for 20 variables per child was performed with regard to the influence of their degree of participation in the U2 to U8 medical examinations using differences in frequency in the examination groups and checking the significance of these differences by means of the chi-squared test. RESULTS: 99-96% of children in Saxony-Anhalt underwent the 5 preventive medical examinations U2-U6. As the children get older, the participation rates decrease (U2=98.7% down to U8=88.5%). By the time the school entry medical examinations are carried out (at an average age of 63 months), 83% of the children have -undergone all 7 preventive medical examinations for children, while 0.4% have not -undergone one single "U" examination. A child's gender has no influence on its parents' decision as to whether or not it should undergo the examinations. The results also reveal that children who attend day care -facilities are significantly more likely to have undergone all of the U examinations (84.8%) than children who are cared for at home (55.1%). CONCLUSION: The retrospective comprehensive collection of data concerning the children's degree of participation in preventive medical examinations using the school entry medical examination is suitable for identifying connections between participation rates and the social factors, diagnostic findings and levels of therapeutic care of the children in question.

[Pilot study of laser-doppler flowmetry measurement of oral mucosa blood flow].

OBJECTIVE: To explore and compare the perfusion pattern of oral mucosa on Han Chinese and Caucasian by laser-doppler flowmetry. METHODS: A cross-sectional study was carried out, in 20 healthy Han Chinese adult subjects (average age: 28.4 years) and 20 healthy Caucasian (average age: 25.3 years) adult subjects, either gender with 10 subjects. Gingival perfusion was evaluated at 8 points (including upper incisor labial gingival, lower incisor labial gingival, palatal mucosa, cheek mucosa) using a laser-doppler flowmetry(O2C, Medizintechnik GmbH, Germany). Each measurement was carried out 25 seconds consisting 5 seconds of fore period and 20 seconds of work period, without pressure. The measurements were taken by two well- trained doctors, each measurement was exammed 3 times by an examiner, and the average value was recorded as final data. Each measurement has 4 parame ters: SpO2(oxygen saturation), rHB (relative amount of hemoglobin), flow (the blood flow of unit interval), and velocity (blood flow velocity). We compared the data by different sites, different genders, and different races. RESULTS: For palatal gingival, the average SpO2 was 77.1%±10.9%, the average rHB 67.8±11.1, and the average flow 194.1±63.7, which presented significant lower values than other oral mucosa. There was no significant difference among other sites. There was some significant difference between the Caucasian and the Han Chinese: the maxillary central incisor oxygen saturation (SpO2) which were averages of 75.6%±8.2% and 70.4%±7.6%; buccal mucosa hemoglobin (rHB) averages of 79.9±5.8 and 83.5±6.6, which had statistical differences. For most measurement points, the oxygen saturation on men was lower than that on women, which had significant difference. CONCLUSION: To investigate microcirculation pattern, oral mucosa can be the good observation site. Laser-doppler flowmetry is a well-documented instrument to survey on microcirculation.There may be differences between the genders in hemoglobin oxygen-binding capacity, which may have some impact on the ability of soft tissue healing. Oral mucosa display more blood perfusion than attached gingival. As the recipient site of gingival graft, maxilla and mandible have slight difference in blood supply.

[Pilot study of laser-doppler flowmetry measurement of oral mucosa blood flow].

OBJECTIVE: To explore and compare the perfusion pattern of oral mucosa on Han Chinese and Caucasian by laser-doppler flowmetry. METHODS: A cross-sectional study was carried out, in 20 healthy Han Chinese adult subjects (average age: 28.4 years) and 20 healthy Caucasian (average age: 25.3 years) adult subjects, either gender with 10 subjects. Gingival perfusion was evaluated at 8 points (including upper incisor labial gingival, lower incisor labial gingival, palatal mucosa, cheek mucosa) using a laser-doppler flowmetry(O2C, Medizintechnik GmbH, Germany). Each measurement was carried out 25 seconds consisting 5 seconds of fore period and 20 seconds of work period, without pressure. The measurements were taken by two well- trained doctors, each measurement was exammed 3 times by an examiner, and the average value was recorded as final data. Each measurement has 4 parame ters: SpO2(oxygen saturation), rHB (relative amount of hemoglobin), flow (the blood flow of unit interval), and velocity (blood flow velocity). We compared the data by different sites, different genders, and different races. RESULTS: For palatal gingival, the average SpO2 was 77.1%±10.9%, the average rHB 67.8±11.1, and the average flow 194.1±63.7, which presented significant lower values than other oral mucosa. There was no significant difference among other sites. There was some significant difference between the Caucasian and the Han Chinese: the maxillary central incisor oxygen saturation (SpO2) which were averages of 75.6%±8.2% and 70.4%±7.6%; buccal mucosa hemoglobin (rHB) averages of 79.9±5.8 and 83.5±6.6, which had statistical differences. For most measurement points, the oxygen saturation on men was lower than that on women, which had significant difference. CONCLUSION: To investigate microcirculation pattern, oral mucosa can be the good observation site. Laser-doppler flowmetry is a well-documented instrument to survey on microcirculation.There may be differences between the genders in hemoglobin oxygen-binding capacity, which may have some impact on the ability of soft tissue healing. Oral mucosa display more blood perfusion than attached gingival. As the recipient site of gingival graft, maxilla and mandible have slight difference in blood supply.

Selective capture of acentric fragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA.

The amplification and overexpression of a number of oncogenes is strongly associated with the progression of a variety of different cancers. We now present a strategy to purify amplified DNA on double minute chromosomes (DMs) to enable analysis of their prevalence and contribution to tumourigenesis. Using cell lines derived from four different tumour types, we have developed a general and rapid method to purify micronuclei that are known to entrap extrachromosomal elements. The isolated DNA is highly enriched in DM sequences and can be used to prepare probes to localize the progenitor single copy chromosomal regions. The capture of DMs by micronuclei appears to be dependent on their lack of a centromere rather than their small size.

A transactivation-deficient mouse model provides insights into Trp53 regulation and function.

The gene Trp53 is among the most frequently mutated and studied genes in human cancer, but the mechanisms by which it suppresses tumour formation remain unclear. We generated mice with an allele encoding changes at Leu25 and Trp26, known to be essential for transcriptional transactivation and Mdm2 binding, to enable analyses of Trp53 structure and function in vivo. The mutant Trp53 was abundant, its level was not affected by DNA damage and it bound DNA constitutively; however, it showed defects in cell-cycle regulation and apoptosis. Both mutant and Trp53-null mouse embryonic fibroblasts (MEFs) were readily transformed by oncogenes, and the corresponding mice were prone to tumours. We conclude that the determining pathway for Trp53 tumour-suppressor function in mice requires the transactivation domain.

Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.

Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.

The evolution of diverse biological responses to DNA damage: insights from yeast and p53.

The cellular response to ionizing radiation provides a conceptual framework for understanding how a yeast checkpoint system, designed to make binary decisions between arrest and cycling, evolved in a way as to allow reversible arrest, senescence or apoptosis in mammals. We propose that the diversity of responses to ionizing radiation in mammalian cells is possible because of the addition of a new regulatory control module involving the tumour-suppressor gene p53. We review the complex mechanisms controlling p53 activity and discuss how the p53 regulatory module enables cells to grow, arrest or die by integrating DNA damage checkpoint signals with the response to normal mitogenic signalling and the aberrant signalling engendered by oncogene activation.

Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase.

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine-pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.

Recombinase-mediated gene activation and site-specific integration in mammalian cells.

A binary system for gene activation and site-specific integration, based on the conditional recombination of transfected sequences mediated by the FLP recombinase from yeast, was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequence to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporter. By the reverse reaction, integration of transfected DNA was targeted to a specific chromosomal site. The results suggest that FLP could be used to mosaically activate or inactivate transgenes for analysis of vertebrate development, and to efficiently integrate transfected DNA at predetermined chromosomal locations.

Genetic dissection of a mammalian replicator in the human beta-globin locus.

The timing and localization of DNA replication initiation in mammalian cells are heritable traits, but it is not known whether initiation requires specific DNA sequences. A site-specific recombination strategy was used to show that DNA sequences previously identified as replication initiation sites could initiate replication when transferred to new chromosomal locations. An 8-kilobase DNA sequence encompassing the origin of DNA replication in the human beta-globin locus initiated replication in the simian genome. Specific deletions within the globin origin did not initiate replication in these chromosomal sites. These data suggest that initiation of DNA replication in mammalian cells requires specific sequence information and extend the replicon hypothesis to higher eukaryotes.

Participation of the human beta-globin locus control region in initiation of DNA replication.

The human beta-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human beta-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3' to 5' direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.

[Obligatory vaccination reporting in Saxony-Anhalt. Possibilities and limitations of establishing a computerized vaccination registry]. / Impfmeldepflicht in Sachsen-Anhalt. Möglichkeiten und Grenzen der Entwicklung eines elektronischen Impfregisters.

Vaccination registries are databases intended to assess and manage complete vaccination data of as many individuals as possible in a population under survey. The task of these registries is to identify low vaccination rates on the individual and population level, to enable systems of reminding individuals, to focus vaccination campaigns and to maximize overall vaccination coverage. Saxony-Anhalt is the only federal state of Germany to have a law that prescribes the reporting of vaccinations. Vaccinations of children up to the age of 7 are reported to the regional public health services. However, as the law provides no regulations as to how the data should be registered and processed, the development of a vaccination registry depends entirely on the initiative and cooperation of the "players in vaccination". The key players in vaccination in Saxony-Anhalt have recently created a Vaccination-Committee, which set out to develop the theoretical standards and a software prototype for the establishment of a computerized vaccination registry. Recent developments in the public health reporting system of Saxony-Anhalt (which strives to modernize its computerized assessment of child and adolescent health) are now opening the possibility to integrate the vaccination registry into the commercially available child health software.

Mouse bites dogma: how mouse models are changing our views of how P53 is regulated in vivo.

P53 is a transcription factor that can cause cells to be eliminated by apoptosis or senescent-like arrest upon its activation by irreparable genetic damage, excessively expressed oncogenes, or a broad spectrum of other stresses. As P53 executes life and death decisions, its activity must be stringently regulated, which implies that it is not likely to be controlled by a simple regulatory mechanism involving a binary on-off switch. This brief review will summarize a subset of the new information presented at the 10th P53 workshop in Dunedin, New Zealand in November 2004 as well as very recent publications that provide new insights into the molecular regulators of P53. Data emerging from mouse models provide a fundamentally different view of how P53 is regulated than suggested by more traditional in vitro approaches. The differences between cell culture and mouse models demonstrate the importance of preserving stoichiometric relationships between P53 and its various regulators to obtain an accurate view of the relevant molecular mechanisms that control P53 activity.

Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.

BACKGROUND: The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells. RESULTS: The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells. CONCLUSIONS: The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.

ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage.

BACKGROUND: Embryonic stem (ES) cells can contribute precursors to all adult cell lineages. Consequently, damage to ES cell genomes may cause serious developmental malfunctions. In somatic cells, cell-cycle checkpoints limit DNA damage by preventing DNA replication under conditions that may produce chromosomal aberrations. The tumor suppressor p53 is involved in such checkpoint controls and is also required to avoid a high rate of embryonic malformations. We characterized the cell-cycle and DNA-damage responses of ES cells to elucidate the mechanisms that prevent accumulation or transmission of damaged genomes during development. RESULTS: ES cells derived from wild-type mice did not undergo cell-cycle arrest in response to DNA damage or nucleotide depletion, although they synthesized abundant quantities of p53. The p53 protein in ES cells was cytoplasmic and translocated inefficiently to the nucleus upon nucleotide depletion. Expression of high levels of active p53 from an adenovirus vector could not trigger cell cycle arrest. Instead, ES cells that sustained DNA damage underwent p53-independent apoptosis. The antimetabolite-induced p53-dependent arrest response was restored in ES cells upon differentiation. CONCLUSIONS: Cell-cycle regulatory pathways in early embryos differ significantly from those in differentiated somatic cells. In undifferentiated ES cells, p53 checkpoint pathways are compromised by factors that affect the nuclear localization of p53 and by the loss of downstream factors that are necessary to induce cell-cycle arrest. A p53-independent programmed cell death pathway is effectively employed to prevent cells with damaged genomes from contributing to the developing organism. The p53-mediated checkpoint controls become important when differentiation occurs.

c-Myc, genome instability, and tumorigenesis: the devil is in the details.

The c-myc oncogene acts as a pluripotent modulator of transcription during normal cell growth and proliferation. Deregulated c-myc activity in cancer can lead to excessive activation of its downstream pathways, and may also stimulate changes in gene expression and cellular signaling that are not observed under non-pathological conditions. Under certain conditions, aberrant c-myc activity is associated with the appearance of DNA damage-associated markers and karyotypic abnormalities. In this chapter, we discuss mechanisms by which c-myc may be directly or indirectly associated with the induction of genomic instability. The degree to which c-myc-induced genomic instability influences the initiation or progression of cancer is likely to depend on other factors, which are discussed herein.

Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells.

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.

Formation of an inverted duplication can be an initial step in gene amplification.

We have developed a gene transfer approach to facilitate the identification and isolation of chromosomal regions which are prone to high-frequency gene amplification. Such regions are identified by assaying for transformants which show high-frequency resistance to PALA and/or methotrexate by amplification of a vector containing the genes which encode the enzyme targets of these antiproliferative agents. We identified 2 of 47 transformants which displayed high-frequency amplification of the transfected genes, and in this report we describe the analysis of one of them (L46). Molecular analysis of the integration site in transformant L46 revealed that the donated genes were at the center of an inverted duplication which spanned more than 70 kilobase pairs and consisted largely of host DNA. The data suggest that integration of the transfected sequences generates a submicroscopic molecule containing the inverted duplication and at least 750 kilobases of additional sequences. The donated sequences and the host sequences were readily amplified and lost in exponentially growing cultures in the absence of drug selection, which suggests that the extrachromosomal elements are acentric. In contrast to the instability of this region following gene insertion, the preinsertion site was maintained at single copy level under growth conditions which produced copy number heterogeneity in L46. The implications of our results for mechanisms of genetic instability and mammalian gene amplification are discussed.