Molecular Regulation of SATB1 in Regulatory T-cells

In this study we have identified SATB1, a nuclear protein that recruits chromatin-remodeling factors and regulates numerous genes, as a novel effector molecule in Treg cells. Our interest in SATB1 resulted from a genome wide expression profile of Treg cells and conventional T-cells (Tconv cells). SATB1 was a prominent candidate gene that constantly repressed in Treg cells and highly expressed in Tconv cells. The dominant repression of SATB1 expression in Treg cells could be confirmed at mRNA, protein, and single cell level under resting and different stimulation conditions in humans and mice. In contrast, SATB1 is expressed at high levels in Tconv cells and is further enhanced following physiological stimulation. The inverse expression pattern of FOXP3, the main transcription factor in shaping and maintaining Treg cell identity, in relation to SATB1 led us to hypothesize its active involvement in regulation of SATB1. On the one hand, induction of FOXP3 was associated with inhibition of SATB1. This could be demonstrated by induction of FOXP3 in naïve CD4+ T-cells converted to induced Treg cells (iTreg) or in CD4+ T-cells ectopically overexpressing FOXP3 after lentiviral transduction. On the other hand, using different genetic approaches loss of FOXP3 expression in Treg cells results in relieving the FOXP3-mediated repression and leads to an upregulation of SATB1. Furthermore, confocal microscopy on lymphocytes form scurfy and normal mice interestingly showed mutually excluding staining patterns. While the SATB1 signal is low in normal FOXP3-expressing thymocytes, it is high in thymocytes expressing a mutated non-functional FOXP3 from scurfy animals. FOXP3 as a transcription factor has been linked to direct binding to DNA, thereby regulating gene expression. To investigate whether FOXP3 can directly bind to the SATB1 genomic locus FOXP3-ChIP tiling arrays were performed. The analysis of tiling array data provided us with several putative FOXP3 binding sites in the promoter and intronic regions of the SATB1 locus which were confirmed by ChIP qRT-PCR. Furthermore, we were able to demonstrate high specificity of the binding and determine the binding coefficients of FOXP3 to several motifs in the SATB1 locus by filter retention assays. To assess whether this binding has functional relevance, we performed reporter assays and showed that FOXP3 reduces lucifierase activity for several binding regions clearly supporting that FOXP3 regulates SATB1 transcription by direct binding to the genomic locus. Interstingly, we showed that FOXP3 also controls SATB1 gene expression indirectly at post-transcriptional level via miRNAs. Indeed we identified several FOXP3 dependent miRNA that have been linked to post-transcriptional regulation of gene expression. FOXP3-ChIP tiling arrays showed FOXP3 peaks within these miRNAs loci. Furthermore, silencing of FOXP3 reversed this enrichment, whereas over-expression of FOXP3 induced their expression. Binding of FOXP3-dependent miRNAs to the 3´UTR of SATB1 in reporter assays confirms the suppressive effect of these miRNAs on SATB1 expression. An additional level of regulation of gene expression is exerted by epigenetic modifactions of the respective genomic locus. Epigenetic changes control the accessibility of a genomic locus by permissive or inhibitory histone modifications as well as methylation of CpG islands. Although, we did not observe differences in the methylation pattern of the CpG islands at the SATB1 locus between Treg cells and Tconv cells, we observed more permissive and less repressive histone marks at the SATB1 genomic locus in Tconv cells and the opposite in Treg cells which is in line with the expression data and aforementioned described regulatory mechanism of SATB1 expression in Treg cells. Besides the molecular mechanism regulating SATB1 expression in Treg cells, we further delineated the functional consequences of induction of SATB1 in Treg cells. Lentiviral over-expression of SATB1 in human and murine Treg cells resulted in the edition of gene expression and function of Treg cells. The striking observation was the abrogation of the capacity of Treg cells to suppress the proliferation of responder cells in vitro, in addition to the production of proinflammatory cytokines like IL-4 and IFN-γ. These findings suggested that Treg cells acquire an effector phenotype a finding which is further corroborated on a genome wide level. Gene expression profiles of SATB1 overexpressing Treg cells showed that many proinflammatory genes have been switched on upon induction of SATB1 expression in Treg cells which promotes skewing of regulatory towards effector programs. To further prove the antagonistic effect of SATB1 on the regulatory function of Treg cells in vivo, we adoptively transferred Treg cells overexpressing SATB1 with naïve CD4+ cells into RAG2-/- mice. In this experimental setting Treg cells failed to suppress inflammation in vivo and subsequently the mice developed colitis. In conclusion, SATB1 is an important effector molecule whose expression is tightly regulated in Treg cells. SATB1 upregulation in Treg cells results in aquisition of proinflammatory properties and attenuated suppressive function in vitro and in vivo. Therefore, FOXP3-mediated repression of SATB1 expression in Treg cells seems to be an important regulatory circuit crucial to maintain suppressive function of these cells

Effect of Active and Passive Smoking on Retinal Nerve Fibre Layer and Ganglion Cell Complex

Aim. To evaluate the possible structural and functional changes in the retinal nerve fibre layer (RNFL) and the ganglion cell complex (GCC) of chronic smokers and compare them with those of passive healthy smokers using spectral domain optical coherence tomography (SD-OCT) and pattern electroretinogram (PERG). Materials and Methods. We include 80 active chronic smokers and 80 age- and sex-matched healthy passive smokers. After a full ophthalmological examination, SD-OCT and PERG were tested for all participants. Urinary levels of cotinine and creatinine with subsequent calculation of the cotinine creatinine ratio (CCR). Results. Inferior and superior quadrants of RNFL were thinner in group I, but nasal and temporal quadrants did not show significant difference between the groups. There were no significant differences of GCC values between the two groups. There was no significant difference of PERG-P50 amplitude and latency; however, PERG-N95 showed significant difference between the two groups. Multiple regression analyses demonstrated that the number of cigarettes/day, urinary cotinine, and PERG-N95 amplitude are the most important determinants for both superior and inferior RNFL thicknesses. Conclusion. RNFL thickness decreases in chronic, healthy, heavy cigarette smokers, and this thinning is related to the number of cigarettes/day, urinary cotinine, and PERG-N95 latency and amplitude

Evaluation of Rezum therapy as a minimally invasive modality for management of Benign Prostatic Hyperplasia: A prospective observational study

Objective: To evaluate safety and efficacy of Rezum therapy as a minimally invasive modality for management of benign prostatic hyperplasia in patients with prostate volume 80cc. Methods: Between June 2020 and February 2023, A total of 98 patients diagnosed with BPH and managed by Rezum were included in this study. Patients were divided based on their prostate volume of either less than 80 cc or greater than 80 cc. We evaluated several parameters related to their condition, including prostate volume, post-voiding residual (PVR) before and after surgery, number of treatments received, maximum urine flow rate (Qmax) before and after surgery and mean follow- up periods. Results: The mean age was 68 years (SD 11.2). The median prostatic volume was 62 cc (IQR 41, 17). A maximum of 9 treatments were administered. Six months was determined to be the average post-operative follow-up period (IQR: 3.5-7.2). The mean preoperative total PSA was 2.7 (IQR 1, 2), preoperative mean PVR was 79.8 cm3, preoperative mean Qmax was 8.2 ml/s (IQR 4.7-10.5), and median post-operative days until catheter removal was four days (IQR 3,1). Post-operative PVR was 24.7 cm3 (IQR 18.2, 29.4) and the mean post-operative Qmax was 18.3 ml/s (SD 6.3). Qmax levels significantly increased, by an average of 8.2 ml/s (SD 7.13) (p < 0.001). Similarly, a decrease of average PVR of 97.28 cm3 (SD 95.85) (p < 0.001) was detected, which is a substantial reduction. Between prostates less 80cc and those over 80cc, there were no appreciable differences in Qmax or PVR (p-values: 0.435 and 0.431, respectively). Conclusions: From our study, we conclude that Rezum water vapor thermal therapy, as a minimally invasive modality, is an effective and safe surgical option for management of benign prostatic hyperplasia of men with moderate to severe lower urinary tract symptoms (LUTS). This procedure has been shown to be effective in patients with varying larger prostate volumes

Safety and efficacy of percutaneous nephrolithotripsy in comorbid patients: A 3 years prospective observational study

Purpose: To report the result of percutaneous nephrolithotripsy (PCNL) via standard nephrostomy tract in a single training institution. The perioperative complications in relation to the comorbid state are particularly assessed. Patients and methods: A prospective interventional study between January 2019 to November 2022, included 210 patients scheduled for PCNL. The average age was 40.3 ± 11.8 years (range 18- 67 years). Patients were categorized into two groups. The first group comprised 146 cases (69 .5%) with no associated co-morbidities while the second group 64 (30.5%) had co-morbidities such as obesity in 4 cases (1.9%), hypertension (HTN) in 24 cases (11.4%) cases, diabetes mellitus (DM) in 17 (8.1%) cases, history of recurrent stone surgery in 11 (5.2%) cases and more than one in 8 cases (3.8%). Co-morbidities, stone burden, location of stone, time of surgery, stay in the hospital, further operations, and negative events were among the reported data. Complications and the stone-free rate were the main outcome indicators. Results: Intraoperative complications were reported in 40 (18.8%) patients (18 group 1 and 22 group 2) during PCNL. Bleeding occurred in 22 (10.5%) patients (9 group 1 and 13 group 2), blood transfusions were needed in 4 (1.9%) (2 group 1 and 2 group 2), extravasation was observed in 11 patients (5.2%) (6 group 1 and 5 group 2) and cardiac arrhythmia in 3 (1.4%) (1 group 1 and 2 group 2) patients. Postoperative complications occurred in 61 patients (29%) (24 group 1 and 37 group 2) in the form of fever in 10 patients (4.8 %) (3 group 1 and 7 group 2) and prolonged leakage in 50 patients (23.8%) (21 group 1 and 29 group 2). One patient of group 2 died from postoperative sepsis. Extravasation and postoperative leakage were higher in diabetic patients than in non-diabetics. Stonefree rate was 60.5% (127 of 210). Clinically significant residual fragments (CSRFs) found in 70 cases (33.3%) (33 group 1 and 37 group 2). In 13 cases (6.2%) (5 group 1 and 8 group 2), clinically insignificant residual fragments (CIRFs) were found. In 8 (3 group 1 and 5 group 2) of the 13 cases, spontaneous stone passage was observed within 4-6 weeks of surgery. Residual stones in three cases (1 group 1 and 2 group 2) were asymptomatic and 4 mm or less, whereas stones increased in two cases of group 2. Among all factors studied, stone burden was significantly correlated to both intraoperative and postoperative complications. The occurrence of postoperative fever increased with large stone burden. Conclusions: PCNL is a therapeutic modality that is effective, feasible, and safe for a wide range of patients with concurrent medical issues. A steep curve is required to reduce intraoperative and postoperative complications

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.Marc Beyer... Timothy Sadlon...Simon C Barry... et al

Detailed Genetic Analysis for Identifying QTLs Associated with Drought Tolerance at Seed Germination and Seedling Stages in Barley

Drought induces several challenges for plant development, growth, and production. These challenges become more severe, in particular, in arid and semiarid countries like Egypt. In terms of production, barley ranks fourth after wheat, maize, and rice. Seed germination and seedling stages are critical stages for plant establishment and growth. In the current study, 60 diverse barley genotypes were tested for drought tolerance using two different treatments: control (0-PEG) and drought (20%-PEG). Twenty-two traits were estimated for seed germination and seedling parameters. All traits were reduced under drought stress, and a significant variation was found among genotypes under control and stress conditions. The broad-sense heritability estimates were very high under both control and drought for all traits. It ranged from 0.63 to 0.97 under the control condition and from 0.89 to 0.97 under drought, respectively. These high heritabilities suggested that genetic improvement of drought tolerance in barley at both stages is feasible. The principal component analysis revealed that root-related parameters account for the largest portion of phenotypic variation in this collection. The single-marker analysis (SMA) resulted in 71 quantitative trait loci (QTLs) distributed across the seven chromosomes of barley. Thirty-three QTLs were detected for root-length-related traits. Many hotspots of QTLs were detected for various traits. Interestingly, some markers controlled many traits in a pleiotropic manner; thus, they can be used to control multiple traits at a time. Some QTLs were constitutive, i.e., they are mapped under control and drought, and targeting these QTLs makes the selection for drought tolerance a single-step process. The results of gene annotation analysis revealed very potential candidate genes that can be targeted to select for drought tolerance

Natural Variation Uncovers Candidate Genes for Barley Spikelet Number and Grain Yield under Drought Stress

Drought stress can occur at any growth stage and can affect crop productivity, which can result in large yield losses all over the world. In this respect, understanding the genetic architecture of agronomic traits under drought stress is essential for increasing crop yield potential and harvest. Barley is considered the most abiotic stress-tolerant cereal, particularly with respect to drought. In the present study, worldwide spring barley accessions were exposed to drought stress beginning from the early reproductive stage with 35% field capacity under field conditions. Drought stress had significantly reduced the agronomic and yield-related traits such as spike length, awn length, spikelet per spike, grains per spike and thousand kernel weight. To unravel the genetic factors underlying drought tolerance at the early reproductive stage, genome-wide association scan (GWAS) was performed using 121 spring barley accessions and a 9K single nucleotide polymorphisms (SNPs) chip. A total number of 101 significant SNPs, distributed over all seven barley chromosomes, were found to be highly associated with the studied traits, of which five genomic regions were associated with candidate genes at chromosomes 2 and 3. On chromosome 2H, the region between 6469300693-647258342 bp includes two candidate drought-specific genes (HORVU2Hr1G091030 and HORVU2Hr1G091170), which are highly associated with spikelet and final grain number per spike under drought stress conditions. Interestingly, the gene expression profile shows that the candidate genes were highly expressed in spikelet, grain, spike and leaf organs, demonstrating their pivotal role in drought tolerance. To the best of our knowledge, we reported the first detailed study that used GWAS with bioinformatic analyses to define the causative alleles and putative candidate genes underlying grain yield-related traits under field drought conditions in diverse barley germplasm. The identified alleles and candidate genes represent valuable resources for future functional characterization towards the enhancement of barley cultivars for drought tolerance

Genetic basis of drought tolerance during seed germination in barley.

Drought is one of the harshest abiotic stresses hindering seed germination, plant growth, and crop productivity. A high rate and uniformity of germination under stressful conditions are vital for crop establishment and growth; thus, for productivity. A better understanding of the genetic architecture of seed germination under drought stress is a prerequisite for further increasing yield potential. Barley is considered one of the most abiotic stresses-tolerant cereals. Elucidating the drought tolerance of barley during seed germination would indeed pave the way towards improving the performance of all cereals. However, we still know relatively little about the genetic control of drought tolerance during the seed germination phase. In our study, 218 worldwide spring barley accessions were subjected to PEG-induced drought during seed germination. Induced drought stress "20% PEG" significantly reduced the seed germination parameters and seedling related traits. A genome-wide association scan (GWAS) was used to identify genomic regions associated with our trait of interest. In total, 338 single nucleotide polymorphisms (SNPs) were found to be associated with several traits distributed across seven barley chromosomes, of which 26 genomic regions were associated with candidate genes. The current study found some of the quantitative trait loci (QTL) that have previously been reported to be linked to seed germination-related traits under drought conditions, as well as some new associations. Noteworthy, the identified QTL colocalized with a number of genes (within interval ±0.5 Mbp) that are exclusively distributed on chromosomes 1H, 2H, and 5H. The annotation of these genes in barley shows their roles in drought tolerance through encoding different transcription factors. The function of the identified genes during seed germination was also confirmed by the annotation of their counterparts in Arabidopsis. The current analyses show the power of the GWAS both for identifying putative candidate genes and for improving plant adaptive traits in barley

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes