D-Loop Mutations as Prognostic Markers in Glioblastoma-A Pilot Study.

Glioblastoma, a highly aggressive brain tumor, poses significant treatment challenges. A deeper investigation into its molecular complexity is essential for the identification of novel prognostic biomarkers and therapeutic strategies, potentially improving patient outcomes in terms of survival and quality of life. While nuclear DNA mutations have been extensively studied, the role of mitochondrial DNA (mtDNA) mutations, specifically in the D-loop region, remains poorly understood. This prospective case-control study aimed to assess the prognostic significance of the mtDNA D-loop m.16126T>C variant in glioblastoma patients. Immunohistochemistry and droplet digital PCR (ddPCR) were employed for mutation analysis, complemented by statistical analyses and a literature review. The study cohort comprised 22 glioblastoma patients (mean age 59.36 ± 14.17, 12 (54.55%) females), and 25 controls (59.48 ± 13.22, 12 (80%) females). The D-loop m.16126T>C variant was observed in four (18%) of the glioblastoma samples and was associated with shorter median survival (9.5 vs. 18 months; p = 0.016, log-rank test). This study underscores the importance of investigating mtDNA, especially D-loop variants, in glioblastoma, suggesting its potential as a prognostic biomarker and, therefore, its possible therapeutic targets, warranting further exploration.

Interleukin-4 during post-exercise recovery negatively correlates with the production of phagocyte-generated oxidants.

Exhaustive run induced a biphasic oxidative response of circulating phagocytes in 16 amateur sportsmen. The first phase involved an increment just after exercise of enhanced whole blood chemiluminescence normalized per phagocyte count, whereas in the second phase a decrement from 1 h post-exercise and ongoing till 24 h. We tested whether plasma Interleukin IL-4, IL-8, IL-10 and Tumor Necrosis Factor α concentrations change in response to exhaustive run and whether there are associations between their levels and delta resting. Moreover, IL-8 and IL-10 significantly increased immediately post-exercise and after 1 h, but later normalized. Tumor necrosis factor α rose by 1.1-times only just after exercise. However, none of these cytokines showed any correlation with the investigated chemiluminescence. Exercise did not alter plasma concentrations of IL-4. However, pre-exercise IL-4 negatively correlated with measured luminescence just after exercise (ρ = -0.54, p < 0.05), and also tended to be negatively associated with decrements of the second phase at 1 h post-exercise ρ = -0.45, p = 0.08. It is suggested that plasma IL-4, by a negative association with blood phagocytes oxidants production, could be involved in the maintenance of proper balance between oxidants and anti-oxidants during strenuous exercise and post-exercise recovery.

Cell-Free DNA: Potential Application in COVID-19 Diagnostics and Management.

WHO has declared COVID-19 as a worldwide, public health emergency. The elderly, pregnant women, and people with associated co-morbidities, including pulmonary disease, heart failure, diabetes, and cancer are the most predisposed population groups to infection. Cell-free DNA is a very commonly applied marker, which is elevated in various pathological conditions. However, it has a much higher sensitivity than standard biochemical markers. cfDNA appears to be an effective marker of COVID-19 complications, and also serves as a marker of certain underlying health conditions and risk factors of severe illness during COVID-19 infection. We aimed to present the possible mechanisms and sources of cfDNA released during moderate and severe infections. Moreover, we attempt to verify how efficiently cfDNA increase could be applied in COVID-19 risk assessment and how it corresponds with epidemiological data.

Current Trends in Cell-Free DNA Applications. Scoping Review of Clinical Trials.

We aimed to summarize the current knowledge about the trends in cfDNA application based on the analysis of clinical trials registered until April 2021. International Clinical Trials Registry Platform (ICTRP) and Clinicaltrials.gov were searched with the keywords: "cf-DNA"; "Circulating DNA"; "Deoxyribonucleic Acid"; and "Cell-Free Deoxyribonucleic Acid". Of 605 clinical trials, we excluded 237 trials, and 368 remaining ones were subject to further analysis. The subject, number of participants, and study design were analyzed. Our scoping review revealed three main trends: oncology (n = 255), non-invasive prenatal diagnostic (n = 48), and organ transplantation (n = 41), and many (n = 22) less common such as sepsis, sport, or autoimmune diseases in 368 clinical trials. Clinical trials are translating theory into clinical care. However, the diagnostic value of cfDNA remains controversial, and diagnostic accuracy still needs to be evaluated. Thus, further studies are necessary until cfDNA turns into a standard in clinical practice.

Analysis of Let-7 Family miRNA in Plasma as Potential Predictive Biomarkers of Diagnosis for Papillary Thyroid Cancer.

The most common histological type of thyroid cancer is papillary thyroid carcinoma (PTC). Radical resection of the thyroid gland is currently the recommended method of treatment. Almost 75% of thyroidectomies performed just for diagnostic purposes are benign. Thus, the confirmation of innovative and more precise noninvasive biomarkers holds promise for the detection of PTC, which may decrease the number of unnecessary thyroid lobectomies. In this work, using the droplet digital PCR (ddPCR) method, we have analyzed the level of five miRNAs (let-7a, let-7c, let-7d, let-7f, and let-7i) in the plasma of patients with PTC and compared them with those of a healthy control group to investigate whether miRNAs also have value in the management of PTC. Levels of four miRNAs, namely let-7a, let-7c, let-7d, and let-7f, were significantly higher in PTC patients than healthy controls. Thus, the analysis of circulating let-7 can be a useful tool and support the currently used methods for PTC diagnosis. However, our observation requires further research on a larger patient group.

Altered levels of circulating nuclear and mitochondrial DNA in patients with Papillary Thyroid Cancer.

Papillary thyroid cancer is the most common thyroid cancer type. However, diagnostics based on fine needle biopsy cannot make a definitive diagnosis in 25% of thyroid nodules. Additionally, approximately 70% to 80% of thyroid lobectomies performed just for diagnostic purposes are benign. Despite this, biopsy still remains the main method of evaluation of thyroid nodules. Cell-free DNA (cf-DNA) measurement could give a new diagnostic opportunities which may reduce the number of unnecessary thyroid procedures. In this study, using a qPCR, we have examined the nuclear cf-DNA and mitochondrial cf-DNA in the plasma of 32 patients. We have found that the level of nuclear cf-DNA is almost 2-fold increased (median 3 089 vs. 1 872, p = 0.022), whereas mitochondrial cf-DNA content was significantly decreased in respect to healthy controls (median 44 992 vs. 92 220, p = 0.010). The ROC curve analysis showed high specificity for nuclear cf-DNA and mitochondrial cf-DNA, which may serve as a useful tool to decrease the number of unneeded surgeries. Our study reports the first epidemiological evidence for lower mitochondrial cf-DNA content in the patient group, what suggests that apart from nuclear cf-DNA also mitochondrial cf-DNA is affected by disease development.

Potential of Liquid Biopsy in Papillary Thyroid Carcinoma in Context of miRNA, BRAF and p53 Mutation.

BACKGROUND: Liquid biopsy is a minimally invasive detection method for molecular biomarkers such as miRNA and cell free DNA in body fluids. Deregulations of miRNA are involved in papillary thyroid carcinoma (PTC), one the most common endocrine malignancy. The most widespread common mutations detected in papillary thyroid cancers are BRAF mutations. Many studies indicate that the BRAF mutation is related to deregulation of miRNA. p53 has an important role in cell cycle control, DNA repair and apoptosis. Moreover, the p53 can regulate the expression of miRNAs and thus participate in thyroid oncogenesis. OBJECTIVE: In this review, we briefly summarize the present state of knowledge about miRNA, BRAF and p53 mutation in the development of PTC and the possibility of using detecting BRAF mutation and miRNA expression in liquid biopsy. RESULTS: The use of the plasma miRNA expression profile in combination with the BRAF mutation analysis in cf-DNA may be a valuable tool in management of PTC. CONCLUSION: Numerous molecular variation characterize recent diagnostic and prognostic markers and therapeutic targets for this type of cancer, which offer unique chances for further research and clinical development of innovative treatment strategies for thyroid cancer.

Eradication of LIG4-deficient glioblastoma cells by the combination of PARP inhibitor and alkylating agent.

Cancer cells often accumulate spontaneous and treatment-induced DNA damage i.e. potentially lethal DNA double strand breaks (DSBs). Targeting DSB repair mechanisms with specific inhibitors could potentially sensitize cancer cells to the toxic effect of DSBs. Current treatment for glioblastoma includes tumor resection followed by radiotherapy and/or temozolomide (TMZ) - an alkylating agent inducing DNA damage. We hypothesize that combination of PARP inhibitor (PARPi) with TMZ in glioblastoma cells displaying downregulation of DSB repair genes could trigger synthetic lethality. In our study, we observed that PARP inhibitor (BMN673) was able to specifically sensitize DNA ligase 4 (LIG4)-deprived glioblastoma cells to TMZ while normal astrocytes were not affected. LIG4 downregulation resulting in low effectiveness of DNA-PK-mediated non-homologous end-joining (D-NHEJ), which in combination with BMN673 and TMZ resulted in accumulation of lethal DSBs and specific eradication of glioblastoma cells. Restoration of the LIG4 expression caused loss of sensitivity to BMN673+TMZ. In conclusion, PARP inhibitor combined with DNA damage inducing agents can be utilized in patients with glioblastoma displaying defects in D-NHEJ.

Daily Smoking and Subjective Health Complaints in Adolescence.

INTRODUCTION: Using data from the Health Behaviour in School-aged Children survey, this study used a repeated cross-sectional design to examine associations between daily smoking, gender, and self-reported health complaints in five cohorts of adolescents over a 16-year period. METHODS: Data were from nationally representative cohorts of 15-year-old youth in Norway in 1993/1994, 1997/1998, 2001/2002, 2005/2006, and 2009/2010 (n total = 7761). Dependent variables were psychological, somatic, and total health complaints. A mixed GLM model examined main and interaction effects of smoking (daily, intermittent, nonsmoking), year, and gender in predicting complaints. Time periods were segmented to compare trends across smoking groups in specific periods. RESULTS: Prevalence of daily smoking declined from 15.5% (1993/1994) to 6.0% (2009/2010). All health complaint scores were significantly higher for smokers and for girls (vs. boys). Smoking status by year interactions were significant for all complaint variables during the period of sharpest decline of daily smoking prevalence (2001/2002-2005/2006), with daily smokers experiencing increases in health complaints while intermittent and nonsmokers did not. Smoking status by gender interactions were significant for all health complaint variables, indicating that the main effect for gender (females higher) was even stronger among smokers compared with nonsmokers. Using year as unit of analysis, the size of mean differences between daily smokers and intermittent/nonsmokers in total complaints was significantly negatively correlated with daily smoking prevalence (-.963, n = 5, p < .01). CONCLUSIONS: As prevalence of daily smoking declined, daily smokers reported higher levels of complaints, suggesting increasing health problems within this group. Girls who smoke daily had particularly elevated levels of complaints. IMPLICATIONS: This study indicates that the relationship between daily smoking and concurrent health symptomatology in adolescents is changing over time, with higher levels of health complaints reported as overall smoking prevalence declines. To our knowledge, this finding has not previously been reported. If youth are smoking to cope with distress, pain, or other health concerns, tobacco control objectives will be increasingly difficult to achieve with adolescents. Levels of health complaints are particularly high among girls who are daily smokers. The findings suggest that restrictive measures and persuasive communications may not be sufficient tobacco prevention strategies for adolescent populations. Young smokers may need counseling and support.

The Role of miRNA in Papillary Thyroid Cancer in the Context of miRNA Let-7 Family.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy. RET/PTC rearrangement is the most common genetic modification identified in this category of cancer, increasing proliferation and dedifferentiation by the activation of the RET/PTC-RAS-BRAF-MAPK-ERK signaling pathway. Recently, let-7 miRNA was found to reduce RAS levels, acting as a tumor suppressor gene. Circulating miRNA profiles of the let-7 family may be used as novel noninvasive diagnostic, prognostic, treatment and surveillance markers for PTC.

Spontaneous in vitro senescence of glioma cells confirmed by an antibody against IDH1R132H.

BACKGROUND: We have recently suggested that glioblastoma cells become spontaneously senescent in cell culture conditions. The antibody specific against IDH1(R132H) offers the perfect opportunity to verify this hypothesis. MATERIALS AND METHODS: We analyzed the features of senescence in 8 glioma cell cultures showing the IDH1(R132H) mutation based on combination of immunocytochemistry, enzymo-cytochemistry, BrdU incorporation assay and real-time microscopic observation. RESULTS: We report that glioma cells showing the IDH1(R132H) mutation become rapidly and spontaneously senescent in vitro. Senescence was observed in both classical and novel serum-free cell culture conditions. Importantly, the senescent IDH1(R132H)-positive cells showed the expression of stemness marker (SOX2). CONCLUSION: In vitro senescence appeared to be the main reason of the difficulties in any kind culturing of glioma cells. 3D cell cultures prolonged the survival and in vitro proliferation of neoplastic IDH1(R132H)-positive cells, however, did not enhance the stabilization efficiency. Senescence of glioma cells is spontaneously triggered in vitro, which offers the opportunity of potential new therapeutic strategies based on this phenomenon.

Reduced expression of ELAVL4 in male meningioma patients.

Meningioma is a frequently occurring tumor of the central nervous system. Among many genetic alternations, the loss of the short arm of chromosome 1 is the second most frequent chromosomal abnormality observed in these tumors. Here, we focused on the previously described and well-established minimal deletion regions of chromosome 1. In accordance with the Knudson suppressor theory, we designed an analysis of putative suppressor genes localized in the described minimal deletion regions. The purpose was to determine the molecular background of the gender-specific occurrence of meningiomas. A total of 149 samples were examined for loss of heterozygosity (LOH). In addition, 57 tumor samples were analyzed using real-time polymerase chain reaction. We examined the association between the expression of selected genes and patient age, gender, tumor grade and presence of 1p loss. Furthermore, we performed an analysis of the most stable internal control for real-time analysis in meningiomas. LOH analysis revealed gender-specific discrepancies in the frequency of 1p aberrations. Moreover, statistical correlation between the gene expression level and gender was significant for the ELAVL4 gene as we found it to be lower in males than in females. We conclude that meningiomas present different features depending on patient gender. We suggest that ELAVL4 can be involved in the pathogenesis of meningiomas in male patients.

Cancer survivors' responses to daily stressors: implications for quality of life.

OBJECTIVE: This study examined cancer survivors' experience of and responses to challenges and stressors associated with everyday living. The impact of daily stressors on quality of life concerns and cortisol patterns was also investigated. METHOD: Participants included 111 cancer survivors who participated in a national telephone diary study of daily experiences (National Study of Daily Experiences). Their responses were compared with those of 111 sociodemographically matched participants with no cancer history using a multilevel modeling approach. Over an 8-day period, participants completed a daily inventory of the occurrence and impact of stressful events, affect, and physical symptoms. Salivary cortisol was sampled four times per day, and indices of awakening response (cortisol awakening response), diurnal slope, and overall output (area under the curve) were examined. RESULTS: Cancer survivors experienced similar numbers and types of stressful events as the comparison group. Although appraisals were largely comparable, cancer survivors showed a modest tendency to perceive stressors as more severe and disruptive, particularly those involving interpersonal tensions. The occurrence of stressors was associated with increased negative affect, decreased positive affect, and increased physical symptoms, but little change in cortisol. Relative to the comparison group, cancer survivors showed less pronounced changes in positive affect and cortisol output when stressors occurred, but a greater increase in negative affect in response to interpersonal conflicts. CONCLUSION: Findings indicate that cancer survivors show a resilient ability to respond to day-to-day stressors and challenges. However, daily stressors can have a significant impact on survivors' mood and physical symptoms and therefore may be an important intervention target.

Amplification of the PDGFRA, KIT and KDR genes in glioblastoma: a population-based study.

The aim of this study was to establish the frequency of amplification of tyrosine kinase receptor genes PDGFRA, KIT and KDR (VEGFR2) at 4q12 in glioblastomas at a population level, and to assess whether such alterations have any clinical impact. Screening of 390 glioblastomas from a population-based study by differential PCR revealed amplification of the PDGFRA, KIT and KDR genes in 33 (8.5%), 17 (4.4%) and 13 (3.3%) glioblastomas, respectively. None of these alterations was prognostic for overall survival. Patients with glioblastoma showing KIT amplification were significantly younger than those with glioblastoma showing no amplification (51.7 ± 21.7 years vs. 59.3 ± 13.1 years; P=0.0231). Twelve glioblastomas showed concurrent amplification of the PDGFRA, KIT and KDR genes, whereas 18 glioblastomas showed PDGFRA amplification only. A significant inverse association was observed between KIT amplification and EGFR amplification (P=0.0260), whereas a borderline positive association was found between KIT amplification and TP53 mutation (P=0.0579). No significant difference was observed in the frequency of amplification of these genes in primary and secondary glioblastomas or in glioblastomas with and without IDH1 mutations, suggesting that amplification of PDGFRA, KIT and KDR may be implicated in the pathogenesis of a small fraction of both subtypes of glioblastoma.

Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies.

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor-EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor-EGFRvIII.

Molecular classification of low-grade diffuse gliomas.

The current World Health Organization classification recognizes three histological types of grade II low-grade diffuse glioma (diffuse astrocytoma, oligoastrocytoma, and oligodendroglioma). However, the diagnostic criteria, in particular for oligoastrocytoma, are highly subjective. The aim of our study was to establish genetic profiles for diffuse gliomas and to estimate their predictive impact. In this study, we screened 360 World Health Organization grade II gliomas for mutations in the IDH1, IDH2, and TP53 genes and for 1p/19q loss and correlated these with clinical outcome. Most tumors (86%) were characterized genetically by TP53 mutation plus IDH1/2 mutation (32%), 1p/19q loss plus IDH1/2 mutation (37%), or IDH1/2 mutation only (17%). TP53 mutations only or 1p/19q loss only was rare (2 and 3%, respectively). The median survival of patients with TP53 mutation ± IDH1/2 mutation was significantly shorter than that of patients with 1p/19q loss ± IDH1/2 mutation (51.8 months vs. 58.7 months, respectively; P = 0.0037). Multivariate analysis with adjustment for age and treatment confirmed these results (P = 0.0087) and also revealed that TP53 mutation is a significant prognostic marker for shorter survival (P = 0.0005) and 1p/19q loss for longer survival (P = 0.0002), while IDH1/2 mutations are not prognostic (P = 0.8737). The molecular classification on the basis of IDH1/2 mutation, TP53 mutation, and 1p/19q loss has power similar to histological classification and avoids the ambiguity inherent to the diagnosis of oligoastrocytoma.

cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer.

BACKGROUND: Recently published data showed discrepancies between P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. METHODS: To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. RESULTS: We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. CONCLUSION: In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells.

Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of betaIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

Smoking is associated with worse mood on stressful days: results from a national diary study.

BACKGROUND: Many smokers report smoking because it helps them modulate their negative affect (NA). The stress induction model of smoking suggests, however, that smoking causes stress and concomitant NA. Empirical support for the stress induction model has primarily derived from retrospective reports and experimental manipulations with non-representative samples of smokers. Moreover, prior studies have typically not considered contextual factors (e.g., daily stressors) that may impact the smoking-NA relationship. PURPOSE: The aim of this study was to assess the stress induction model of smoking using a prospective design in a nationally representative sample of smokers while simultaneously examining the impact of daily stressors on the relationship between smoking and NA. We hypothesized that smoking and NA would be positively related, and this relationship would be intensified by exposure to daily stressors. METHODS: A national sample of middle-aged smokers (N = 256) were called on eight consecutive evenings to assess stressor exposure and intensity. Participants also reported on their daily NA and indicated the number of cigarettes they smoked. Analyses were conducted using hierarchical linear modeling to determine the relationship between daily smoking, NA, and stress. RESULTS: Smoking more than usual was associated with increased NA on days when respondents were exposed to any stressors. Smoking more than usual had no effect on NA on days when no stressors were encountered. Moreover, the moderating effect of stressor exposure remained significant even after controlling for the number and intensity of daily stressors reported. CONCLUSIONS: While smokers report that smoking alleviates their NA, our study suggests that the exact opposite may occur, particularly on stressful days. When smokers smoke more than usual on days when the encounter stress, they are likely to feel emotionally worse off.