The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a ...real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria,
,
,
,
, and
, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent
semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for
was 97.5%, for
96.88%, and for
95.24%. The sensitivity for
and
was 100%. The Spearman correlation factor of two different measurements was 0.976 for
, 0.967 for
, 0.949 for
, 0.966 for
, and 0.917 for
. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.
The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning ...prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.
The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.
Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage.
In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.
Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in ...conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within ±0.5 log₁₀ unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.
Objective:
To report a case of a 12-year-old boy with intellectual disability and attention deficit hyperactivity disorder, who came to surgery for an examination due to a minor bulge on the left ...thumb, which had been growing for the previous month. His mother denied any trauma.
Methods:
After the removal of the clinically ambiguous bulge and a pathohistological confirmation that it was a periungual fibroma, complete patient analysis was performed due to the presence of hypomelanotic macules and a suspected tuberous sclerosis.
Results:
Considering the presence of hypomelanotic macules, as one of the main criteria, possible TS diagnosis was set.
Conclusion:
Early detection of the symptoms of TS enables a timely provision of protocols for further patient monitoring, which affects the patient’s morbidity and mortality.
Background
The spread and invasion of malignant melanoma cells involve degradation and reorganization of the extracellular matrix by the activation of several matrix metalloproteinases (MMPs). This ...study analyzed the expression of MMP‐1, MMP‐2, and MMP‐13 proteins in primary nodular melanoma (NM) and dysplastic nevi (DN) as a significant risk factor for melanoma development. The secondary goal was to analyze the correlation of MMPs protein expression in NM with tumor invasion, BRAF V600 mutation status, and overall survival.
Methods
Immunohistochemistry for MMP‐1, MMP‐2, and MMP‐13 was performed on nodular melanoma (n = 52) and dysplastic nevi (n = 28) on tissue microarray (TMA). BRAF V600 mutation analysis on NM samples was performed by the Sanger sequencing method.
Results
A high level of MMPs expression in NM samples (>30%) compared with DN (<8%) was statistically significant (P < 0.001). BRAF V600 mutations were detected in 15 of 39 (38.5%) NM samples. This study revealed an interesting finding that MMP‐1 and MMP‐13 protein expression in the BRAF V600 mutated melanomas were significantly lower than in the BRAF V600 wild type (P < 0.05).
Conclusion
Cox analysis revealed that Clark categories, Breslow thickness, and MMP‐1 high protein expression are predictive factors for shorter overall survival (P < 0.05).
A case of a 41-year-old woman with a history of nodular melanoma (NM), associated with an indurated dome-shaped blue-black nodule with a diameter of 1.2 cm in the gluteal region, is presented. ...Clinical diagnosis of the lesion, present from birth, was blue nevus. Recently, the nodule has been showing a mild enlargement and thus complete resection was performed. Histological analysis revealed a pigmented lesion with an expansive pattern of extension into the dermis and the subcutaneous adipose tissue. The lesion displayed an alveolar pattern as well as a pigmented dendritic cell pattern. The histology was consistent with cellular blue nevus (CBN); however, the history of NM which was excised one year earlier, as well as the clinical information about the slow growing lesion, included a differential diagnosis of CBN, borderline melanocytic tumor, and malignant blue nevus. Additional immunohistochemical (HMB-45, p16, and Ki-67) and molecular (BRAF V600E mutation) analyses were performed on both lesions: the CBN-like and the previously excised NM. Along with lesion history and histological analyses, p16 staining and BRAF were useful diagnostic tools for confirming the benign nature of CBN in this case.
Mitochondrial DNA (mtDNA) has been used for decades as a predominant tool in population genetics and as a valuable addition to forensic genetic research, owing to its unique maternal inheritance ...pattern that enables the tracing of individuals along the maternal lineage across numerous generations. The dynamic interplay between evolutionary forces, primarily genetic drift, bottlenecks, and the founder effect, can exert significant influence on genetic profiles. Consequently, the Adriatic islands have accumulated a subset of lineages that exhibits remarkable absence or rarity within other European populations. This distinctive genetic composition underscores the islands’ potential as a significant resource in phylogenetic research, with implications reaching beyond regional boundaries to contribute to a global understanding. In the initial attempt to expand the mitochondrial forensic database of the Croatian population with haplotypes from small isolated communities, we sequenced mitogenomes of rare haplogroups from different Croatian island and mainland populations using next-generation sequencing (NGS). In the next step and based on the obtained results, we refined the global phylogeny of haplogroup N1a, HV2, and X by analyzing rare haplotypes, which are absent from the current phylogenetic tree. The trees were based on 16 novel and 52 previously published samples, revealing completely novel branches in the X and HV2 haplogroups and a new European cluster in the ancestral N1a variant, previously believed to be an exclusively African–Asian haplogroup. The research emphasizes the importance of investigating geographically isolated populations and their unique characteristics within a global context.
Malignant melanoma (MM) is known to avoid the host’s immune response. Studies on in vitro melanoma cell lines link the microphthalmia-associated transcription factor (MITF) with the regulation of the ...PD-L1 expression. It seems that MITF affects the activation of the gene responsible for PD-L1 protein expression. Several proteins, including Bcl-2 and Cyclin D1, play major roles in malignant melanoma cell cycle regulation and survival. Our study aims to assess the relationship between MITF, Bcl-2, and cyclin D1 protein expression and the expression of the PD-L1 molecule. Additionally, we examined the association of BRAF mutation, MITF, and CCND1 gene amplification with PD-L1 protein expression. We performed immunohistochemical staining on fifty-two tumour samples from patients diagnosed with nodular melanoma (NM). BRAF V600 mutation, MITF, and CCND1 gene amplification analyses were analyzed by the Sanger sequencing and QRT-PCR methods, respectively. Statistical analyses confirmed the significant inverse correlation between cyclin D1 and PD-L1 expression (p = 0.001) and correlation between PD-L1 and MITF protein expression (p = 0.023). We found a statistically significant inverse correlation between the present MITF gene amplification and PD-L1 (p = 0.007) and MITF protein expression (p = 3.4 ×10–6), respectively. Our study, performed on clinical NM materials, supports the in vitro study findings providing a rationale for the potential MITF-dependent regulation of PD-L1 expression in malignant melanoma.
The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same ...cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).
Considering that nodular melanoma (NM) has the potential to show an early distant metastasis, there is an urgent need for the discovery and evaluation of new diagnostic and prognostic biomarkers. We ...aimed to investigate the protein expression of membrane and nuclear epidermal growth factor receptor (EGFR), cyclin D1, and the corresponding gene status in NM samples and correlate the results obtained with clinicopathological parameters and overall survival of patients. Immunohistochemical and fluorescence in-situ hybridization analyses were carried out on tissue microarrays constructed from 110 NM samples, 30 compound nevi, and 38 dysplastic nevi. NM samples showed 24% strong cyclin D1 and 37% strong Ki67 protein expression compared with 3 and 0% strong cyclin D1 and Ki67 expression in the control group. Membrane EGFR expression was detected in 50% of NM cases, whereas EGFR gene amplification was detected in only 4% of NM cases. Multiple NM samples presented simultaneous membrane and nuclear EGFR expression. We found a negative correlation between tumor thickness and membrane EGFR expression. It was also observed that membrane EGFR 3+ NM samples presented ulceration significantly more often than membrane EGFR-negative (0) NM samples. In univariate analysis, carried out on 44 patients with follow-up data, both nuclear and membrane EGFR overexpression showed a correlation with a shorter overall survival. Nuclear EGFR (++, +++) showed 3.06 and membrane EGFR (2+, 3+) showed 2.76 higher risk of mortality compared with patients with low and negative nuclear and membrane EGFR expression (P<0.05).
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