Identification of SOX9 Interaction Sites in the Genome of Chondrocytes

Our previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a "master switch" in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies.The ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9.Our results provide a broad understanding of the strategies used by a "master" transcription factor of differentiation in control of the genetic program of chondrocytes

Nephrostomy-free percutaneous nephrolithotripsy: intraoperative hemostasis methods of the percutaneous tract

Review based on the analysis of more than 40 scientific papers published in the Pubmed and Medline databases from 1984 to 2019, dedicated to intraoperative hemostasis of the percutaneous tract and its tightness during nephrostomyfree percutaneous nephrolithotomy (PCNL). The article aimed to summarize scientific data on this issue. We presented information about the history and development of percutaneous surgery in the treatment of urolithiasis. In our review, we have been demonstrated various methods of surgical and intraoperative hemostasis during nephrostomy-free PCNL

Bilateral percutaneous mininephrolithotripsy: simultaneous or staged approach?

Introduction. Patients with bilateral nephrolithiasis are a challenge for the treating physician. Therefore, such patients traditionally are subject to phased surgery to reduce the rate of complications. At the same time, the enhancement of endoscopic technologies and anesthesia makes it possible to perform surgical treatment of bilateral nephrolithiasis simultaneously.Objective. To evaluate the effectiveness and safety of performing simultaneous bilateral percutaneous nephrolithotripsy (PCNL).Materials & methods. The main group comprises 19 patients (avg age 45.0 ± 2.25 years) suffering from bilateral nephrolithiasis (13 men and 6 women). The control group include 20 patients (avg age 45.80 ± 2.29 years) suffering from bilateral nephrolithiasis. Main group patients underwent simultaneous bilateral mini-PCNL, control group patients — staged PCNL within two hospitalisations. The visual analogue scale (VAS) was used to assess the pain severity. Patients noted subjective pain sensations on post-op days 1 and 3. QoL indicators were assessed using the SF-36 general questionnaire, as well as the Russian-language validated version of the WISQoL questionnaire.Results. There were no statistically significant differences between the parameters (the difference between the hemoglobin before and after mini-PCNL was 12 g/l; between the serum creatinine was 18 µmol/l). There was a decrease in total surgery time (121.0 ± 6.1 min) for main group patients compared to (147.0 ± 7.3 min) control group patients (total surgery time during the first and second hospitalisations) and a reduction in hospital stay (4.50 ± 0.23 days) for main group patients compared to control group patients (10.0 ± 0.5 days). Complications observed by us in the two groups were comparable. The valuesobtained on the SF, RE and MH scales in main group patients were higher both on post-op day 1 (67.9 ± 3.39; 56.90 ± 2.85 and 63.80 ± 3.19, respectively) and post-op day 3 (86.80 ± 4.34; 83.70 ± 4.19 and 82.50 ± 4.13, respectively) compared to control group patients during the first and second hospitalizations. Statistically significant differences were also recorded according to the grades "Social functioning" and "Emotional influence" in main group patients (80.90 ± 0.26 and 82.6 ± 0.19, respectively).Conclusion. Simultaneous bilateral mini-PCNL is safe and effective in well-selected patients

Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques

<p>Abstract</p> <p>Background</p> <p>Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components.</p> <p>Objectives</p> <p>To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue.</p> <p>Methods</p> <p>Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified.</p> <p>Results</p> <p>Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (<it>p </it>< 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (<it>p </it>= 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue.</p> <p>Conclusions</p> <p>MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.</p

Selection criteria for minimally invasive endoscopic treatment of urolithiasis depending on stone characteristics

Introduction. According to the guidelines, the stone maximum diameter is one of the main criteria for choosing the method of nephrolithotripsy. When planning an operation, the surgeon focuses not only on the diameter, but also on the renal pelvis anatomy, stone density and number of it, the presence of hydronephrosis, a history of surgery, etc. The maximum diameter is not an exhaustive characteristic that allows you to choose the optimal treatment.Objective. To evaluate the effect of stone volume (compared to its maximum diameter) on the duration of minimally invasive endoscopic nephrolithotripsy.Materials &amp; methods. The study was retrospective. The study included 55 patients (22 women, 33 men), the average age was 47.0 ± 1.9 years. All patients underwent minimally invasive thulium fiber laser nephrolithotripsy for stones up to 20 mm (mean maximum diameter — 13.3 ± 0.6 mm, mean density — 1041.0 ± 48.0 HU). Minimally invasive endoscopic interventions such as retrograde intrarenal surgery (RIRS, n = 30), minipercutaneous and micropercutaneous nephrolithotripsy (miniPNL, n = 16 and microPNL, n = 9, respectively) were performed. Patients with urinary system abnormalities, acute urinary tract infections and patients without stone-free status were excluded from the study. In addition to evaluating standard indicators, the stone volume was calculated in all patients using the formula of a scalene ellipsoid (median volume — 287 [144; 538] mm3). Spearman's rank correlation coefficient (r) with an assessment of the significance level was calculated for the stone maximum diameter and volume for the total sample of patients and for each surgical intervention method separately.Results. The analysis of the total sample of patients reliably revealed a weak correlation (r = 0.39) between the stone maximum diameter and surgery time. And a moderate correlation was found between the stone volume and surgery time (r = 0.53). A similar relationship with the linear distribution was also observed in the analysis in all groups.Conclusion. When choosing minimally invasive laser nephrolithotripsy, it is advisable to focus not only on the stone maximum diameter, density, and localisation, but also on the stone volume, which has a great correlation with the surgery time

Videodensitometric analysis of advanced carotid plaque: correlation with MMP-9 and TIMP-1 expression

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of MMP (TIMP) promote derangement of the extracellular matrix, which is ultimately reflected in plaque images seen on ultrasound. Videodensitometry can identify structural disturbances in plaques.</p> <p>Objectives</p> <p>To establish the correlations between values determined using videodensitometry in B-mode ultrasound images of advanced carotid plaques and the total expression of MMP-9 and TIMP-1 in these removed plaques.</p> <p>Methods</p> <p>Thirty patients underwent ultrasonic tissue characterization of carotid plaques before surgery, using mean gray level (MGL), energy, entropy and homogeneity. Each patient was assigned preoperatively to one of 2 groups: group I, symptomatic patients (n = 16; 12 males; mean age 66.7 ± 6.8 years), and group II, asymptomatic patients (n = 14; 8 males; mean age 67.6 ± 6.81 years). Tissue specimens were analyzed for MMP-9 and TIMP-1 expression. Nine carotid arteries were used as normal tissue controls.</p> <p>Results</p> <p>MMP-9 expression levels were elevated in group II and in normal tissues compared to group I (p < 0.001). TIMP-1 levels were higher in group II than in group I, and significantly higher in normal tissues than in group I (p = 0.039). The MGL was higher in group II compared to group I (p = 0.038). Energy had greater values in group II compared to group I (<it>p </it>= 0.02). There were no differences between patient groups in homogeneity and entropy. Energy positively correlated with MMP-9 and TIMP-1 expression (p = 0.012 and p = 0.031 respectively). Homogeneity positively correlated with MMP-9 and TIMP-1 expression (p = 0.034 and p = 0.047 respectively). There were no correlations between protein expression and MGL or entropy.</p> <p>Conclusions</p> <p>Videodensitometric computer analysis of ultrasound scanning images can be used to identify stable carotid plaques, which have higher total expression levels of MMP-9 and TIMP-1 than unstable plaques.</p

Sox6 Is Necessary for Efficient Erythropoiesis in Adult Mice under Physiological and Anemia-Induced Stress Conditions

BACKGROUND: Definitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions. METHODOLOGY/PRINCIPAL FINDINGS: Sox6fl/flErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6fl/flCaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6fl/flErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1

The Prevalence and Regulation of Antisense Transcripts in Schizosaccharomyces pombe

A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe

p16INK4a Suppression by Glucose Restriction Contributes to Human Cellular Lifespan Extension through SIRT1-Mediated Epigenetic and Genetic Mechanisms

Although caloric restriction (CR) has been shown to increase lifespan in various animal models, the mechanisms underlying this phenomenon have not yet been revealed. We developed an in vitro system to mimic CR by reducing glucose concentration in cell growth medium which excludes metabolic factors and allows assessment of the effects of CR at the cellular and molecular level. We monitored cellular proliferation of normal WI-38, IMR-90 and MRC-5 human lung fibroblasts and found that glucose restriction (GR) can inhibit cellular senescence and significantly extend cellular lifespan compared with cells receiving normal glucose (NG) in the culture medium. Moreover, GR decreased expression of p16INK4a (p16), a well-known senescence-related gene, in all of the tested cell lines. Over-expressed p16 resulted in early replicative senescence in glucose-restricted cells suggesting a crucial role of p16 regulation in GR-induced cellular lifespan extension. The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter. GR resulted in an increased expression of SIRT1, a NAD-dependent histone deacetylase, which has positive correlation with CR-induced longevity. The elevated SIRT1 was accompanied by enhanced activation of the Akt/p70S6K1 signaling pathway in response to GR. Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling. Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems