Raman microspectroscopy: toward a better distinction and profiling of different populations of dental stem cells

Aim To characterize stem cells originating from different dental tissues (apical papilla [SCAP], dental follicle [DFSC], and pulp [DPSC]) and test the capacity of Raman microspectroscopy to distinguish between the three dental stem cell types. Methods SCAR DFSC, and DPSC cultures were generated from three immature wisdom teeth originating from three patients. Cell stemness was confirmed by inducing neuro-, osteo-, chondro-, and adipo-differentiaton and by mesenchymal marker expression analysis by flow-cytometry and real-time polymerase chain reaction. Cellular components were then evaluated by Raman microspectroscopy. Results We found differences between SCAP, DFSC, and DPSC Raman spectra. The ratio between proteins and nucleic acids (748/770), a parameter for discriminating more differentiated from less differentiated cells, showed significant differences between the three cell types. All cells also displayed a fingerprint region in the 600-700 cm(-1) range, and characteristic lipid peaks at positions 1440 cm(-1) and 1650 cm(-1). Conclusion Although different dental stem cells exhibited similar Raman spectra, the method enabled us to make subtle distinction between them

Marked epithelial to mesenchymal transition in surgical margins of oral cancer-an in vitro study

Epithelial to mesenchymal transition (EMT) is a feature of several types of human cancer, including oral squamous cell carcinoma (OSCC). In the present study, tumor and margin cell cultures obtained from patients with OSCC were used to determine the expression patterns of certain EMT-associated markers, including vimentin, alpha -smooth muscle actin, SLUG and SNAIL. In addition, other EMT-associated features, including clonal, proliferative and migratory potential were compared between the two cell types. Cell cultures were generated from tumor and margin tissue samples from 6 patients and cultured up to the fifth passage. EMT marker expression was assessed by reverse transcription-quantitative PCR. Cell proliferation, colony formation and scratch wound healing assays were conducted to characterize the two cell types in terms of proliferation rates, clonality and motility. All of the studied markers were expressed in tumor and margin cells. Although no significant differences were noted with regard to the aforementioned markers, their expression tended to be higher in margin cultures than in tumor cultures. The expressions of the EMT markers were also higher in the fifth passage compared with those noted at the first with a few exceptions. The rates of proliferation and cell migration were decreased during passages, while the number of colonies was increased in both types of cell culture. Tumor and margin cells indicated certain similarities with regard to EMT transition characteristics

Large-scale recent expansion of European patrilineages shown by population resequencing

The proportion of Europeans descending from Neolithic farmers similar to 10 thousand years ago (KYA) or Palaeolithic hunter-gatherers has been much debated. The male-specific region of the Ychromosome (MSY) has been widely applied to this question, but unbiased estimates of diversity and time depth have been lacking. Here we show that European patrilineages underwent a recent continent-wide expansion. Resequencing of 3.7Mb of MSY DNA in 334 males, comprising 17 European and Middle Eastern populations, defines a phylogeny containing 5,996 single-nucleotide polymorphisms. Dating indicates that three major lineages (I1, R1a and R1b), accounting for 64% of our sample, have very recent coalescent times, ranging between 3.5 and 7.3 KYA. A continuous swathe of 13/17 populations share similar histories featuring a demographic expansion starting similar to 2.1-4.2 KYA. Our results are compatible with ancient MSY DNA data, and contrast with data on mitochondrial DNA, indicating a widespread male-specific phenomenon that focuses interest on the social structure of Bronze Age Europe.Peer reviewe

Association between Periodontopathogens and CRP Levels in Patients with Periodontitis in Serbia

Background and aims. Recent epidemiological studies have shown that individuals with periodontitis have a significantly higher risk of developing coronary heart disease, which might be attributed to the complex microbiota in the dental plaque. Periodontopathogens have been reported as risk factors for cardiovascular disease. This study evaluated association of chronic periodontitis and periodontopathogens with CRP in systemically healthy Serbian adults. Materials and methods. Serum C-reactive protein levels were measured in 24 patients with moderate periodontitis, 26 patients with severe periodontitis, and 25 periodontally healthy subjects. Periodontal health indicators included gingival bleeding on probing and periodontal disease status. Patients with moderate periodontitis had low attachment loss and pocket depths of <4 mm. Patients with severe periodontitis had high AL and pocket depth of >5 mm. The control group with healthy gingiva had gingival sulcus of <2 mm and no attachment loss. Presence of periodontopathogens in subgingival plaque samples was analyzed by polymerase chain reaction. Results. The periodontal parameters and CRP levels were significantly higher in the patients with periodontitis. Patients who had both severe and moderate periodontitis had higher mean CRP levels. The percentage of subjects with elevated CRP leves of >5 mol/L was greater in the higher clinical AL group compared to the group with less attachment loss. Presence of periodontopathogens was also associated with elevated CRP levels and poor periodontal status. Conclusions. PD and subgingival periodontopathogens are associated with increased CRP levels. These findings suggest that periodontal infection may contribute to systemic inflammatory burden in otherwise healthy individuals

Methylation of tumour suppressor genes in benign and malignant salivary gland tumours: a systematic review and meta-analysis

The aim of the present systematic review was to critically analyse the relationship between tumour suppressor genes (TSGs) promoter methylation, a potent mechanism of gene silencing, and the development of salivary gland tumours, as well as the possible effect on clinical/histological characteristics. Review protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) database (registration ID CRD42020218511). A comprehensive search of Web of Science, Scopus, PubMed, and Cochrane Central Register of Controlled Trials was performed utilizing relevant key terms, supplemented by a search of grey literature. Newcastle-Ottawa Quality Assessment Scale (NOQAS) was used for the quality assessment of included studies. Sixteen cross-sectional and 12 case-control studies were included in the review, predominantly dealing with methylation in TSGs related to DNA repair, cell cycle, and cell growth regulation and differentiation. Quantitative synthesis could be performed on P16 (inhibitor of cyclin-dependent kinase 4a), RASSF1A (Ras association domain family 1 isoform A) and MGMT (O6-methylguanine DNA methyltransferase) genes only. It showed that P16 and RASSF1A genes were more frequently methylated in salivary gland tumours compared to controls (P = .0002 and P < .0001, respectively), while no significant difference was observed for MGMT. Additionally, P16 did not appear to be related to malignant transformation of pleomorphic adenomas (P = .330). In conclusion, TSG methylation is involved in salivary gland tumour pathogenesis and several genes might play a considerable role. Further studies are needed for a better understanding of complex epigenetic deregulation during salivary gland tumour development and progression

Analysis of microsatellite markers D18S70 and d20S116 in DNA isolated from dentin: Use in forensic medicine

Introduction. Short tandem repeats and more specifically microsatellites represent a powerful tool in forensic medicine. In the past years, they have been extensively used in human identification and paternity testing. Objective The aim of the present study was to analyze two microsatellite markers in the Serbian population, i.e. to determine the number of alleles and the relevant forensic parameters. Methods. DNA was isolated from teeth samples using standard proteinase K digestion and phenol/chloroform alcohol extraction. PCR products were analyzed on polyacrilamide gels and visualized by AgNO3 staining. Forensic parameters were calculated using the Cervus software. Results. The loci D18S70 and D20S116 were analyzed on a sample of 70 unrelated, healthy adult individuals from Serbia. The number of alleles was determined and Hardy Weinberg equilibrium was confirmed for both loci. D18S70 and D20S116 demonstrated 6 and 8 alleles, respectively. The power of discrimination (PD) and the power of exclusion (PE) for the tested STR loci, D18S70 and D20S116 were 0.92 (PD), 0.41 (PE) and 0.95 (PD), 0.480 (PE), respectively. Conclusion. According to the presented data, D18S70 and D20S116 are most informative markers. Based on allelic frequencies and statistical parameters for forensic testing, it may be suggested that these two microsatellites represent useful markers for individual identification and parentage analysis in the Serbian population.Uvod. Genetičke analize hipervarijabilnih genskih lokusa iz grupe mikrosatelita izolovanih iz različitog biološkog materijala ljudskog porekla nalaze svoju praktičnu primenu u forenzičkoj praksi u svrhu identifikacije, utvrđivanja spornog roditeljstva i srodničkih odnosa. Cilj rada. Cilj rada je bio da se ispita učestalost alela mikrosatelitskih genskih lokusa D18S70 i D20S116 i utvrdi da li se u našoj populaciji nalaze u Hardi-Vajnbergovoj (Hardy-Weinberg) ravnoteži. Takođe su ispitivani forenzički parametri za navedene genetičke markere: moć diskriminacije (PD), moć isključenja (PE), marker informativnosti lokusa, značajan marker očinstva i verovatnoća poklapanja genotipova. Metode rada. U istraživanju je kao biološki materijal ljudskog porekla korišćeno 70 ekstrahovanih zuba. Izolacija DNK iz čvrstog zubnog tkiva obavljana je organskom metodom, nakon čega je utvrđen protokol za PCR amplifikaciju ispitivanih genskih lokusa. Proizvodi PCR amplifikacije analizirani su vertikalnom elektroforezom na dvanaestoprocentnom poliakrilamidnom gelu bojenom etidijum-bromidom, posle čega su analizirani genotipovi. Statistička obrada navedenih forenzičkih parametara vršena je primenom programa Cervus. Rezultati. Učestalosti pojedinih alela i genotipova za analizirane genske lokuse statistički su obrađene i utvrđeno je da se nalaze u Hardi-Vajnbergovoj ravnoteži. Marker D18S70 imao je šest alela, a D20S116 osam alela. PD i PE za D18S70 bili su 0,92, odnosno 0,41, a za D20S116 0,95, odnosno 0,48. Zaključak. STR lokusi D18S70 i D20S116 su se pokazali vrlo informativnim markerima. S obzirom na broj i učestalost alela, kao i vrednosti ključnih forenzičkih parametara, ovi mikrosateliti su pogodni za upotrebu u forenzičke svrhe u našoj populaciji, odnosno moguće ih je primeniti u otkrivanju i analizi spornog roditeljstva

Estimation of total bacteria by real-time PCR in patients with periodontal disease

Introduction Periodontal diseases are associated with the presence of elevated levels of bacteria within the gingival crevice. Objective The aim of this study was to evaluate a total amount of bacteria in subgingival plaque samples in patients with a periodontal disease. Methods A quantitative evaluation of total bacteria amount using quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 20 samples of patients with ulceronecrotic periodontitis and on 10 samples of healthy subjects. The estimation of total bacterial amount was based on gene copy number for 16S rRNA that was determined by comparing to Ct values / gene copy number of the standard curve. Results A statistically significant difference between average gene copy number of total bacteria in periodontal patients (2.55×107) and healthy control (2.37×106) was found (p=0.01). Also, a trend of higher numbers of the gene copy in deeper periodontal lesions (>7 mm) was confirmed by a positive value of coefficient of correlation (r=0.073). Conclusion The quantitative estimation of total bacteria based on gene copy number could be an important additional tool in diagnosing periodontitis.Uvod Parodontopatija se povezuje sa postojanjem povećanog broja bakterija u parodontalnom džepu. Cilj rada Cilj rada je bila kvantifikacija ukupnih bakterija u uzorcima subgingivalnog dentalnog plaka kod osoba obolelih od parodontopatije. Metode rada U 20 uzoraka subgingivalnog plaka ispitanika sa ulceronekroznom parodontopatijom i 10 uzoraka osoba sa zdravim parodoncijumom izvršena je kvantifikacija ukupnog broja bakterija korišćenjem metode qRT- PCR. Kvantifikacija bakterija je zasnovana na određivanju ukupnog broja genskih kopija za rRNK poređenjem sa CT vrednošću standarda. Rezultati Ustanovljena je statistički značajna razlika u prosečnom broju genskih kopija ukupnih bakterija između ispitanika sa parodontopatijom (2,55×107) i ispitanika kontrolne grupe (2,37×106), (p=0,01). Takođe, trend porasta broja genskih kopija ukupnih bakterija s povećanjem dubine parodontalnog džepa (>7 mm) potvrđen je pozitivnom vrednošću koeficijenta korelacije (p=0,073). Zaključak Procena ukupnog broja bakterija na osnovu broja genskih kopija za 16S rRNK može biti važan dodatni parametar u dijagnostikovanju parodontopatije

Analysis of antimicrobial effect of MTAD solution in infected canal system using PCR technique

Introduction. Clinically acceptable antiseptic should possess organolithic-mineralolithic properties and antimicrobial efficacy, and should be non-toxic. Objective. The aim of the paper was to assess the presence of genomes of the most common microorganisms (Porphyromonas gingivalis, Agregatibacter actinomycetemcomitans, Tanerella forsythensis, Prevotella intermedia, Treponema denticola and Enterococcus faecalis) in infected tooth root canals before and after rinsing with solution of doxycycline, citric acid and detergent Tween-80 (MTAD) in patients with clinically diagnosed primary apex periodontitis. Methods. The content of primarily infected canals before and after using the MTAD solution was used as a biological material in which the presence of microorganisms DNA was proved. For the detection of bacterial genome the multiplex PCR technique was applied. Results. The percentage of positive samples before canal treatment was 100%. In infected root canals E. faecalis was most dominant (37%). In a relatively high percentage we detected P. intermedia (25%), A. actinomycetemcomitans (20%), T. denticola (17%), T. forsythensis (15%) and P. gingivalis (10%). After rinsing the canal system using MTAD solution, there was a statistically significant decrease in E. faecalis (12%), P. intermedia (0%), T. forsythensis (0%) and P. gingivalis (0%). The presence of other bacteria was also diminished but not statistically significantly. Conclusion. With the application of multiplex PCR technique which provided a simultaneous amplification of various genomic sequences, using several pairs of primers, the most dominant in infected root canals were E. faecalis. P. intermedia, A. actinomycetemcomitans, T. denticola, T. forsythensis and P. gingivalis. After mechanic treatment and irrigation of root canals with MTAD solution, P. intermedia, P. gingivalis and T. forsythensis were not found. The presence of E. faecalis, A. actinomycetemcomitans and T. denticola was diminished, however, not statistically significantly.Uvod. Klinički prihvatljiv intrakanalni antiseptik mora imati organolitičko-mineralolitičko dejstvo i antibakterijsku efikasnost i ne sme biti toksičan. Cilj rada. Cilj rada je bio da se ispita postojanje genoma najčešćih mikroorganizama (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Treponema denticola i Enterococcus faecalis) u inficiranim kanalima korena zuba pre i posle irigacije rastvorom doksiciklina, limunske kiseline i deterdženta Tween­80 (MTAD) kod osoba s klinički dijagnostikovanim primarnim apeksnim parodontitisom. Metode rada. Kao biološki materijal u kojem je dokazivano prisustvo DNK mikroorganizama korišćen je sadržaj iz primarno inficiranih kanala pre i posle irigacije rastvorom MTAD. Za otkrivanje bakterijskog genoma primenjena je multipleksna tehnika PCR. Rezultati. Procenat pozitivnih uzoraka pre obrade kanala bio je 100%. U inficiranim kanalima korena zuba najčešći je bio E. faecalis (37%). U relativno visokom procentu otkriveni su i: P. intermedia (25%), A. actinomycetemcomitans (20%), T. denticola (17%), T. forsythensis (15%) i P. gingivalis (10%). Posle irigacije kanalnog sistema rastvorom MTAD utvrđeno je statistički značajno smanjenje učestalosti E. faecalis (12%), P. intermedia (0%), T. forsythensis (0%) i P. gingivalis (0%). Učestalost ostalih bakterija takođe se smanjila, ali ne statistički značajno. Zaključak. Primenom multipleksne PCR tehnike, koja omogućava istovremenu amplifikaciju genskih sekvenci uz korišćenje dva para prajmera, u inficiranim kanalima korena zuba najčešće je utvrđen E. faecalis. U relativno visokom procentu otkrivene su P. intermedia, A. actinomycetem­comitans, T. denticola, T. forsythensis i P. gingivalis. Posle hemomehaničke obrade i irigacije kanala rastvorom MTAD nisu zabeležene P. intermedia, P. gingivalis i T. forsythensis, dok je učestalost E. faecalis, A. actinomycetemcomitans i T. denticola smanjena, ali ne statistički značajno