Complex effects of flavopiridol on the expression of primary response genes

<p>Abstract</p> <p>Background</p> <p>The Positive Transcription Elongation Factor b (P-TEFb) is a complex of Cyclin Dependent Kinase 9 (CDK9) with either cyclins T1, T2 or K. The complex phosphorylates the C-Terminal Domain of RNA polymerase II (RNAPII) and negative elongation factors, stimulating productive elongation by RNAPII, which is paused after initiation. P-TEFb is recruited downstream of the promoters of many genes, including primary response genes, upon certain stimuli. Flavopiridol (FVP) is a potent pharmacological inhibitor of CDK9 and has been used extensively in cells as a means to inhibit CDK9 activity. Inhibition of P-TEFb complexes has potential therapeutic applications.</p> <p>Results</p> <p>It has been shown that Lipopolysaccharide (LPS) stimulates the recruitment of P-TEFb to Primary Response Genes (PRGs) and proposed that P-TEFb activity is required for their expression, as the CDK9 inhibitor DRB prevents localization of RNAPII in the body of these genes. We have previously determined the effects of FVP in global gene expression in a variety of cells and surprisingly observed that FVP results in potent upregulation of a number of PRGs in treatments lasting 4-24 h. Because inhibition of CDK9 activity is being evaluated in pre-clinical and clinical studies for the treatment of several pathologies, it is important to fully understand the short and long term effects of its inhibition. To this end, we determined the immediate and long-term effect of FVP in the expression of several PRGs. In exponentially growing normal human fibroblasts, the expression of several PRGs including FOS, JUNB, EGR1 and GADD45B, was rapidly and potently downregulated before they were upregulated following FVP treatment. In serum starved cells re-stimulated with serum, FVP also inhibited the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 were upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition than ensues transcription of certain PRGs.</p> <p>Conclusions</p> <p>We have shown that certain PRGs are transcribed in the presence of FVP in a manner that might be independent of CDK9, suggesting a possible alternative mechanism for their transcription when P-TEFb kinase activity is pharmacologically inhibited. These results also show that the sensitivity to FVP is quite variable, even among PRGs.</p

C-reactive protein level and obesity as cardiovascular risk factors in polycystic ovary syndrome

Objective: To investigate the role of C-reactive protein(CRP) level elevation and obesity for the increased cardiovasculardisease risk in polycystic ovary syndrome(PCOS).Methods: A hundred and nine patients with PCOS and 30age matched healthy volunteers with regular menstrualcycle are involved in the study. PCOS group is furthersubdivided into three subgroups according to the bodymass index (BMI). Subgroups included 54 with BMI<25,22 with BMI 25-30, and 33 with BMI>30. Blood samplesfor glucose, insulin, uric acid, and CRP were collected inthe morning after overnight fasting (12 hours). Homeostasismodel assessment-insulin resistance (HOMA-IR)was calculated. Results: Fasting blood glucose, insulin,and HOMA-IR was significantly higher in PCOS group(p=0.02, p=0.01 and p=0.02). CRP level was higher insubgroup with BMI>30. High CRP level in PCOS wasfound to be independent from BMI (p<0.001). HOMA-IRand insulin level was higher in the subgroup with BMI>30.When compared with the control group high insulin levelwas the only to be statistically significant in obese PCOSpatients (p=0.005). HOMA-IR was higher in PCOS subgroupwith BMI>30 when compared with controls and thePCOS subgroup with BMI<25 (p<0.001, p= 0.003).Conclusion: Obesity, hyperinsulinemia, and high CRPlevels are seemed to be related and potentiating eachother in PCOS. Struggling with obesity is one of the mostimportant issues for preventive medicine.Key words: PCOS, CRP, obesity, cardiovascular ris

Metabolic dysfu nctio n-As socia ted fatty liver disease and fibrosis status in patients with Type 2 Diabetes Treated at Internal medicine clinics: Türkiye DAHUDER awareness of fatty liver disease (TR-DAFLD) study

Background/Aims: This awareness study aimed to determine the ultrasound (US) examination rates in relation to US-confirmed metabolic dysfunction-associated fatty liver disease (MAFLD) diagnosis in internal medicine outpatients with type 2 diabetes (T2D) across Turkiye. Materials and Methods: A total of 6283 T2D patients were included in this multicenter retrospective cohort study conducted at 17 internal medicine clinics across Turkiye. The presence and indications for US performed within the last 3 years were recorded along with US-confirmed MAFLD rates, laboratory findings on the day of US, and referral rates. Fibrosis-4 (FIB-4) index was calculated to estimate the risk of advanced liver fibrosis (FIB-4 index ≥ 1.3). Results: Overall, 1731 (27.6%) of 6283 patients had US examination, which revealed MAFLD diagnosis in 69.9% of cases. In addition, 24.4% of patients with US-confirmed MAFLD were at risk of advanced fibrosis (FIB-4 index ≥ 1.3), and the referral rate was 15.5%. Conclusion: In conclusion, our findings emphasize an insufficient MAFLD awareness among clinicians and the likelihood of most of T2D patients to be at risk of living with an unknown status regarding their MAFLD and advanced fibrosis ris

Discovery, characterization and mechanism of RNA and CDNA-mediated DNA double-strand break repair

A double-strand break (DSB) is one of the most deleterious DNA lesions and its repair is crucial for genome stability. Even if a single DSB is not repaired precisely, this could cause mutations, chromosomal rearrangements, cell death, and apoptosis. The safest mechanism to repair a DSB is homologous recombination (HR). HR requires an identical or nearly identical DNA template, such as a sister chromatid or a homologous chromosome to retrieve the missing genetic information and accomplish error-free repair. In special cases, HR can occur between RNA molecules, such as RNA molecules in RNA viruses. However, very little is known about RNA-DNA HR. Previously, it was demonstrated that synthetic RNA-containing molecules can serve as templates for repairing defective or broken homologous chromosomal DNA in yeast, human and bacterial cells, but it remained unclear whether cellular RNA transcripts can recombine with genomic DNA. Here, we investigated whether yeast cells can use transcript RNA as a template to repair a chromosomal DSB either directly or indirectly, if the RNA is converted first into a DNA copy, cDNA. We developed a system to detect HR between chromosomal DNA and transcript RNA in budding yeast, Saccharomyces cerevisiae. We focused on repair of a chromosomal DSB occurring either in a homologous but remote locus (trans) or in the same transcript-generating locus (cis) in yeast. We proved that transcript RNA can repair a DSB indirectly, via cDNA. Moreover, we found that cDNA repair is much more frequent in the trans than in the cis system. Interestingly, in the absence of Ribonuclease H1 and H2 (RNases H1 and H2), we could detect DSB repair even in conditions that strongly inhibit cDNA formation, suggesting direct DSB repair by transcript RNA. In contrast to DSB repair by cDNA, the direct DSB repair by transcript RNA is more efficient in the cis than in the trans system, despite the higher abundance of the transcript in the trans system. These results suggest that the vicinity of the transcript RNA to the break site in the cis system may facilitate DSB repair. DSB repair by transcript RNA in cis is promoted by the HR protein Rad52 but not Rad51, in agreement with the demonstration that the yeast and human Rad52 proteins efficiently catalyze annealing of RNA to a DSB-like DNA end in vitro. We also showed that yeast cells expressing hypomorphic mutants of RNase H2, which correspond to the human RNase H2 mutants that are associated with the neuroimmunological disease, Aicardi Goutieres (AGS) syndrome, have increased frequency of DSB repair by cDNA, significantly higher than in wild-type RNase H2 cells. In addition, we showed that in contrast to DSB repair by single strand DNA (ssDNA) oligonucleotides (oligos), RNA templated DSB repair is not dependent on factors that are major players in DNA end resection. This result could be explained by a mechanism in which transcript RNA repairs a DSB in conditions of limited end resection via an inverse strand exchange reaction. Our study provides proof and initial characterization of a new mechanism of DNA repair and HR mediated by RNA in yeast, and unravels novel aspects in the complex relationship between RNA and DNA in genome stability.Ph.D

Böbrek yetmezlikli hastalarda FLAIR imajlarda subaraknoid aralık hiperintensitesi: Tanısal bir tuzak

Subaraknoid aralık SAA hiperintensitesi, manyetik rezonans görüntülemenin MRG FLAIR sekansında ayırıcı tanısı dikkatlice yapılması gereken önemli bir bulgudur. FLAIR sekansında SAA hiperintensitesinin subaraknoid kanama SAK , leptomeningeal karsinomatozis, menenjit gibi ciddi tedavi gerektiren birçok nedeni vardır. Kontrast madde, vasküler pulsasyonlar, beyin omurilik sıvısı BOS pulsasyonları ve hareket artefaktları sadece tuzak görüntülerdir, bu nedenle de tedavi gerektirmezler. Böbrek yetmezlikli hastalar için birçok komorbidite sebebi bulunmaktadır. Bunlardan bir tanesi serebrovasküler olaylardır. Nörolojik semptomlara sahip hastalarda MRG tanı için en iyi tercihtir. Fakat hastaya beyin MR tetkiki öncesinde kontrastlı başka bir MRG incelemesi uygulan- mışsa gadolinyumun SAA’da gecikmiş klirensi ve persistansı ortaya çıkar. Sonuçta bu durum ciddi patolojileri taklit edebilir. Bu olgu sunumunda daha önce kontrastlı boyun MRG uygulanan böbrek yetmezlikli olgunun SAA hiperintensitesine ait ilginç görüntülerini sunmayı amaçladı

Hyperintensity in the subarachnoid space on FLAIR MRI in patients with renal insufficiency: A diagnostic pitfall

Subaraknoid aralık (SAA) hiperintensitesi, manyetik rezonans görüntülemenin (MRG) FLAIR sekansında ayırıcı tanısı dikkatlice yapılması gereken önemli bir bulgudur. FLAIR sekansında SAA hiperintensitesinin subaraknoid kanama (SAK), leptomeningeal karsinomatozis, menenjit gibi ciddi tedavi gerektiren birçok nedeni vardır. Kontrast madde, vasküler pulsasyonlar, beyin omurilik sıvısı (BOS) pulsasyonları ve hareket artefaktları sadece tuzak görüntülerdir, bu nedenle de tedavi gerektirmezler. Böbrek yetmezlikli hastalar için birçok komorbidite sebebi bulunmaktadır. Bunlardan bir tanesi serebrovasküler olaylardır. Nörolojik semptomlara sahip hastalarda MRG tanı için en iyi tercihtir. Fakat hastaya beyin MR tetkiki öncesinde kontrastlı başka bir MRG incelemesi uygulan- mışsa gadolinyumun SAA'da gecikmiş klirensi ve persistansı ortaya çıkar. Sonuçta bu durum ciddi patolojileri taklit edebilir. Bu olgu sunumunda daha önce kontrastlı boyun MRG uygulanan böbrek yetmezlikli olgunun SAA hiperintensitesine ait ilginç görüntülerini sunmayı amaçladık.Subarachnoid space (SAS) hyperintensity in fluid attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) is an important finding that differantial diagnosis must be done carefully. FLAIR hyperintensity has lots of reasons: subarachnoid hemor- rhage (SAH), leptomemingeal carcinomatosis, meningitis, etc. which are required serious treatment. Some of the reasons such as contrast media, vascular-cerebrospinal fluid (CSF) pulsation, motion artifact are only pitfall, so no treatment is necessary for them. Patients with renal failure have comorbidities. One of them is cerebrovascular events and if patient has neurologic symptoms, MRI is the best choice for diagnosis. But if patient was applied gadolinium enhanced MRI previously, it will be confusing. Because delayed clearance and persistance of gadolinium on SAS may mimick the serious pathologies mentioned above. This case report highlights the interesting appearance of SAS in a patient with renal insufficiency who underwent previous contrast-enhanced neck MRI