Proximal Region Research Articles

The Control of the Crossover Localization in Allium.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F1 diploid (2n = 2x = 8C + 8F) and F1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

Transcription factors Dmrt1a, Foxl2, and Nr5a1a potentially interact to regulate cyp19a1a transcription in ovarian follicles of ricefield eel (Monopterus albus)

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a, foxl2, and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10–50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100–200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well.

AGE-DEPENDENT CARTILAGE DEGENERATION THROUGH NRF2/ARE MODULATION OF SOX9 EXPRESSION

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE1 did not. A functional ARE2 site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

The Genomic Variation and Differentially Expressed Genes on the 6P Chromosomes in Wheat-Agropyron cristatum Addition Lines 5113 and II-30-5 Confer Different Desirable Traits.

Wild relatives of wheat are essential gene pools for broadening the genetic basis of wheat. Chromosome rearrangements and genomic variation in alien chromosomes are widespread. Knowledge of the genetic variation between alien homologous chromosomes is valuable for discovering and utilizing alien genes. In this study, we found that 5113 and II-30-5, two wheat-A. cristatum 6P addition lines, exhibited considerable differences in heading date, grain number per spike, and grain weight. Genome resequencing and transcriptome analysis revealed significant differences in the 6P chromosomes of the two addition lines, including 143,511 single-nucleotide polymorphisms, 62,103 insertion/deletion polymorphisms, and 757 differentially expressed genes. Intriguingly, genomic variations were mainly distributed in the middle of the chromosome arms and the proximal centromere region. GO and KEGG analyses of the variant genes and differentially expressed genes showed the enrichment of genes involved in the circadian rhythm, carbon metabolism, carbon fixation, and lipid metabolism, suggesting that the differential genes on the 6P chromosome are closely related to the phenotypic differences. For example, the photosynthesis-related genes PsbA, PsbT, and YCF48 were upregulated in II-30-5 compared with 5113. ACS and FabG are related to carbon fixation and fatty acid biosynthesis, respectively, and both carried modification variations and were upregulated in 5113 relative to II-30-5. Therefore, this study provides important guidance for cloning desirable genes from alien homologous chromosomes and for their effective utilization in wheat improvement.

SHAPE DIFFERENCES IN THE SEMITENDINOSUS FOLLOWING TENDON HARVESTING FOR ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION

Following anterior cruciate ligament reconstruction (ACLR) using a semitendinosus (ST) autograft measures such as length, cross-sectional area, and volume may not fully describe the effects of tendon harvest on muscle morphology as these discrete measures cannot characterize three-dimensional muscle shape. This study aimed to determine between-limb ST shape similarity and regional morphology in individuals with a unilateral history of ACLR using a ST graft, and healthy controls.A secondary analysis of magnetic resonance imaging was undertaken from 18 individuals with unilateral history of ST ACLR and 18 healthy controls. ST muscles were manually segmented, and shape similarity were assessed between limbs and groups using Jaccard index (0-1) and Hausdorff distance (mm). ST length (cm), peak cross-sectional area (CSA) (cm2), and volume (cm3) was compared between surgically reconstructed and uninjured contralateral limbs, and between the left and right limbs of control participants with no history of injury. Cohen's d was reported as a measure of effect size.Compared to healthy controls, the ACLR group had significantly (p<0.001, d= −2.33) lower bilateral ST shape similarity. Furthermore, the deviation in muscle shape was significantly (p<0.001, d= 2.12) greater in the ACLR group. Within the ACLR group, maximum Hausdorff distance indicated ST from the ACLR limb deviated (23.1±8.68 mm) from the shape of the healthy contralateral ST, this was observed particularly within the distal region of the muscle. Compared to the uninjured contralateral limb and healthy controls, deficits in peak cross-sectional area and volume in ACLR group were largest in proximal (p<0.001, d= −2.52 to −1.28) and middle (p<0.001, d= −1.81 to −1.04) regions.Findings highlight morphological features in distal ST not identified by traditional discrete morphology measures. ST shape was most different in the distal region of the muscle, despite deficits in CSA and volume being most pronounced in proximal and middle regions. ST shape following ACLR may affect force transmission and distribution within the hamstrings and contribute to persistent deficits in knee flexor and internal rotator strength.

NOX4–TIM23 interaction regulates NOX4 mitochondrial import and metabolic reprogramming

Pulmonary fibrosis is a progressive lung disease characterized by macrophage activation. Asbestos-induced expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (NOX4) in lung macrophages mediates fibrotic progression by the generation of mitochondrial reactive oxygen species (ROS), modulating mitochondrial biogenesis, and promoting apoptosis resistance; however, the mechanism(s) by which NOX4 localizes to mitochondria during fibrosis is not known. Here, we show that NOX4 localized to the mitochondrial matrix following asbestos exposure in lung macrophages via direct interaction with TIM23. TIM23 and NOX4 interaction was found in lung macrophages from human subjects with asbestosis, while it was absent in mice harboring a conditional deletion of NOX4 in lung macrophages. This interaction was localized to the proximal transmembrane region of NOX4. Mechanistically, TIM23 augmented NOX4-induced mitochondrial ROS and metabolic reprogramming to oxidative phosphorylation. Silencing TIM23 decreased mitochondrial ROS and oxidative phosphorylation. These observations highlight the important role of the mitochondrial translocase TIM23 interaction with NOX4. Moreover, this interaction is required for mitochondrial redox signaling and metabolic reprogramming in lung macrophages.

Abstract 6619: Virtual staining enabled combined morphological and spatial transcriptomic analysis of individual malignant B cells and local tumor microenvironments

Abstract B-cell lymphomas, commonly classified by histological presentation and B-cell lineage, are in fact heterogeneous diseases with respect to gene expression and tumor microenvironment. Certain lymphomas, such as Hodgkin and T-cell rich B-cell lymphoma, may contain fewer than 1% malignant cells in a largely inflammatory background. These rare malignant cells have unique interactions with local tumor microenvironments (TME), in some cases driving distinct gene expression patterns for individual B-cells. This presents a challenging fundamental biology landscape, which can create uncertainty in directing patient care or developing new therapies. The lack of tools allowing subtyping of malignant B-cells while simultaneously preserving the context of local TMEs and providing deep spatial transcriptomic sequencing further complicates analysis. Here we have developed an integrated, single-slide workflow of virtual H&E staining using the inherent tissue autofluorescence (PictorLabs, Los Angeles, CA) of formalin-fixed, paraffin embedded lymphoma tissue sections on the GeoMx® Digital Spatial Profiler (Nanostring, Seattle, WA) combined with spatial expression analysis of 1,800 genes using the Cancer Transcriptome Atlas (CTA) panel. The H&E virtual staining allows for the assessment of B-cell lymphoma subtypes, identification and phenotyping of malignant B-cells and the immediately proximal (within 30µm) TME regions by a certified pathologist. These regions of interest (ROI) are spatially profiled for transcriptional expression determined by NGS sequencing of the RNA target specific DNA oligo tags. We benchmarked our system by analyzing four lymphoma cases: Nodular sclerosis classical Hodgkin, Nodular lymphocyte-predominant Hodgkin, T-cell/histiocyte rich large B-cell, and Diffuse large B-cell lymphomas. Using virtual H&E staining on the GeoMx Digital Spatial Profiler platform, a pathologist correctly identified the diagnosis and subtype based on the morphology. Regions of inflammatory B- and T-cells could be distinguished from lymphoma cells on virtual staining, allowing subset segregation for digital transcriptional expression profiling with the GeoMx CTA panel. Additionally, background TME more distant to B-cell lymphoma as well as regions of high T-cell infiltration were selected for profiling. Analysis of the ROIs revealed distinct transcriptional profiles of malignant B-cells (including cell-of-origin signature), the signaling contributions of local TMEs (versus non-adjacent regions) and confirmed that the expression patterns are further influenced by the presence of T-cell infiltration. Citation Format: Michael E. Kallen, Alyssa Rosenbloom, Zihang Fang, Raymond Kozikowski, Yair Rivenson, Kevin de Haan, Serge Alexanian, Mark Conner, Daniel Newhouse, Felicia New, Yan Liang, Kyla Teplitz, Joachim Schmid, Jaemyeong Jung. Virtual staining enabled combined morphological and spatial transcriptomic analysis of individual malignant B cells and local tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6619.

Abstract 6778: Spatial analysis of genomic signatures on colorectal cancer pathogenesis using the GeoMx® Digital Spatial Profiler

Abstract Background: The development of colorectal cancer (CRC) is a complex process, involving multiple sequential molecular aberrations that contribute to disease progression. Understanding the immunopathogenesis of CRC particularly within the spatial context of tumor microenvironment (TME) may elucidate the genomic and biological changes to improve patient prognosis for adjuvant therapies and identifying potential drug targets. However, this can be challenging, as genomic signatures from sub-cellular populations and varying levels of immune cell infiltration in TME may be lost in bulk sequencing. In this study, we used the NanoString GeoMx® Digital Spatial Profiler (DSP) to spatially select epithelial, proximal stromal and distal stromal regions of TMEs on a total of 10 FFPE samples (5 colon cancer patients and 5 healthy donor samples) and profiled up to 1,800 genes included in the Cancer Transcriptome Atlas (CTA) panel. Methods: Selection of regions of interest (ROI) were guided by pathology assessment of H&E images and fluorescent markers (CD45, PanCK, Syto13). Tumor and stroma (proximal and distal) regions were profiled through geometric ROI selection in PanCK+ (epithelial) and CD45+ (stromal/immune) enriched areas respectively, followed by collection of indexed oligonucleotides and sequencing on NextSeq 550 Illumina instrument. Differential expression and pathways enrichment analyses was performed using R BioConductor package DESeq2 and DSP GeoMx analysis suite. A subset of samples were tested for bulk RNA expression using PanCancer IO360 panel on NanoString nCounter Flex system and data analyzed using nSolver software 3.0 and TIS algorithm App. Results: The CTA panel is designed to profile the global immune response and all aspects of tumor microenvironment biology. From differential gene expression analysis of epithelial and distal/proximal stromal ROIs, we have identified biomarkers such as SOX9, IRAK2 and NLRP3 that are specifically upregulated in CRC samples compared to the healthy cohort. Comprehensive pathways analysis revealed an over activation of NF-kB signaling, extracellular matrix organization and interferon signaling pathways in the CRC cohort. Suppression of the NF-kB signaling pathway is a potential therapeutic approach in the treatment of colon cancer. Conclusion: With this spatial study using GeoMx CTA panel, we have highlighted key genes and pathways involved in CRC in order to provide a much-needed understanding of the underlying mechanisms as well as immune signatures in the development of colon cancer. Citation Format: Xiang Li, Qinqin Zha, Brigitte Lovell, Jane Reed, Anna Juncker-Jensen, Tricia Peters, Lakshmi Chandramohan. Spatial analysis of genomic signatures on colorectal cancer pathogenesis using the GeoMx® Digital Spatial Profiler. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6778.

Defining the fine structure of promoter activity on a genome-wide scale with CISSECTOR.

Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as survey of regulatory elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel. Here we show how a generalized linear model (GLM) can be used to transform genome-scale SuRE data into a high-resolution genomic track that quantifies the contribution of local sequence to promoter activity. This coefficient track helps identify regulatory elements and can be used to predict promoter activity of any sub-region in the genome. It thus allows in silico dissection of any promoter in the human genome to be performed. We developed a web application, available at cissector.nki.nl, that lets researchers easily perform this analysis as a starting point for their research into any promoter of interest.

Abstract 1494: PBRM1-deficient PBAF complexes target de novo genomic loci to activate NF-κB pathway in clear cell renal cell carcinoma

Abstract PBRM1 is an accessory subunit of the PBAF subclass of the SWI/SNF chromatin remodeler and the inactivation of PBRM1 is the second most frequent mutational event in kidney tumorigenesis. However, the impact of PBRM1 loss on chromatin remodeling, especially pertaining to kidney tumorigenesis, has not been examined previously. Here we show that in VHL-deficient ccRCC tumors, PBRM1 deficiency results in altered PBAF complexes that retain the association between SMARCA4 and ARID2 but disengage BRD7 from SMARCA4. The PBRM1-deficient PBAF complexes redistribute from promoter proximal regions to distal enhancer regions containing motifs of pro-tumorigenic factors including NF-κB. Subsequently, PBRM1-deficient cells display heightened NF-κB activity across multiple cell models. The ATPase function of SMARCA4 maintains chromatin occupancy of both pre-existing and newly acquired RELA specific to PBRM1 loss, and activates downstream target gene expression. Proteasome inhibitor bortezomib reverses NF-κB activation by reducing RELA occupancy and delays growth of PBRM1-deficient tumors. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumorigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes. Citation Format: Xiaosai Yao, Jing Han Hong, Amrita Nargund, Michelle Shu Wen Ng, Hong Lee Heng, Zhimei Li, Peiyong Guan, Masahiro Sugiura, Pek Lim Chu, Loo Chien Wang, Xiaofen Ye, Robert Yauch, Kenneth Tou-En Chang, Radoslaw M. Sobota, Patrick Tan, Bin Tean Teh. PBRM1-deficient PBAF complexes target de novo genomic loci to activate NF-κB pathway in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1494.

Associations between lower extremity muscle fat fraction and motor performance in myotonic dystrophy type 2: A pilot study.

Although muscle structure measures from magnetic resonance imaging (MRI) have been used to assess disease severity in muscular dystrophies, little is known about how these measures are affected in myotonic dystrophy type 2 (DM2). We aim to characterize lower extremity muscle fat fraction (MFF) as a potential biomarker of disease severity, and evaluate its relationship with motor performance in DM2. 3-Tesla MRIs were obtained from nine patients with DM2 and six controls using a T1W-Dixon protocol. To calculate MFF, muscle volumes were segmented from proximal, middle, and distal regions of the thigh and calf. Associations between MFF and motor performance were calculated using Spearman's correlations (ρ). Mean age of DM2 participants was 62 ± 11 y (89% female), and mean symptom duration was 20 ± 12 y. Compared to controls, the DM2 group had significantly higher MFF in the thigh and the calf segments (p-value = .002). The highest MFF at the thigh in DM2 was located in the posterior compartment (39.7 ± 12.9%) and at the calf was the lateral compartment (31.5 ± 8.7%). In the DM2 group, we found a strong correlation between the posterior thigh MFF and the 6-min walk test (ρ = -.90, p-value = .001). The lateral calf MFF was also strongly correlated with the step test (ρ = -0.82, p-value = .006). Our pilot data suggest a potential correlation between lower extremity MFF and some motor performance tests in DM2. Longitudinal studies with larger sample sizes are required to validate MFF as a marker of disease severity in DM2.

Temporally and regionally distinct morphogenetic processes govern zebrafish caudal fin blood vessel network expansion.

Blood vessels form elaborate networks that depend on tissue-specific signalling pathways and anatomical structures to guide their growth. However, it is not clear which morphogenetic principles organize the stepwise assembly of the vasculature. We therefore performed a longitudinal analysis of zebrafish caudal fin vascular assembly, revealing the existence of temporally and spatially distinct morphogenetic processes. Initially, vein-derived endothelial cells (ECs) generated arteries in a reiterative process requiring vascular endothelial growth factor (Vegf), Notch and cxcr4a signalling. Subsequently, veins produced veins in more proximal fin regions, transforming pre-existing artery-vein loops into a three-vessel pattern consisting of an artery and two veins. A distinct set of vascular plexuses formed at the base of the fin. They differed in their diameter, flow magnitude and marker gene expression. At later stages, intussusceptive angiogenesis occurred from veins in distal fin regions. In proximal fin regions, we observed new vein sprouts crossing the inter-ray tissue through sprouting angiogenesis. Together, our results reveal a surprising diversity among the mechanisms generating the mature fin vasculature and suggest that these might be driven by separate local cues.

MP79-11 A SHAM-CONTROLLED RANDOMIZED TRIAL OF LOW INTENSITY SHOCKWAVE THERAPY FOR ERECTILE DYSFUNCTION

You have accessJournal of UrologyCME1 Apr 2023MP79-11 A SHAM-CONTROLLED RANDOMIZED TRIAL OF LOW INTENSITY SHOCKWAVE THERAPY FOR ERECTILE DYSFUNCTION Irwin Goldstein, Sue W. Goldstein, and Noel N. Kim Irwin GoldsteinIrwin Goldstein More articles by this author , Sue W. GoldsteinSue W. Goldstein More articles by this author , and Noel N. KimNoel N. Kim More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003356.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We performed a sham-controlled, randomized prospective trial in men with ED using an electrohydraulic shockwave device FDA cleared for connective tissue activation and improved blood flow. METHODS: This single-blind study was performed in men with ED naïve to acoustic wave and shockwave therapy. Patients were randomized to treatment and assigned to active low intensity shockwave therapy (LiSWT) (4 Hz, 0.12 mJ/mm2) or sham treatment, 2:1. Arm 1 consisted of 3 treatments of 5000 shocks every 3 weeks. Arm 2 consisted of 5000, 3000, and 3000 shocks during weeks 1, 2, and 3, respectively, followed by an identical cycle of treatment 3 weeks later. Doppler ultrasound and grayscale imaging with a 15.4 MHz probe were performed under pharmacologic erection at weeks 20 and 32. Subjects completing sham treatment were unblinded and crossed over to the opposite arm for active treatment. Post-treatment end diastolic velocity (EDV) and peak systolic velocity (PSV) were measured, and visual grading scores were used to assess extent of hypoechoic regions in the corpora cavernosa. Data were analyzed by 2-way repeated measures ANOVA with Geisser-Greenhouse correction. Pairwise comparisons were performed to baseline used Dunnett’s multiple comparison test. Missing data were imputed by “last observation carried forward”. RESULTS: Although powered for 60, recruitment was limited due to COVID and 36 subjects (22 active, 14 sham) were randomized. Sham treatments showed no significant changes in outcome measures. The number of subjects with improved visual grading scores in the proximal region was consistently higher in active LiSWT vs sham (Arm 1=88.9% vs. 11.1%; Arm 2=40.0% vs. 20.0%, respectively) with statistical significance in Arm 1 at weeks 20 (p=0.005) and 32 (p=0.001). Sham subjects rolled over to active LiSWT also had improved grayscale ratings (Arm 1=33.3% vs. 11.1%; Arm 2=40.0% vs. 20.0%). After LiSWT, greater numbers of patients had higher PSV, lower EDV, or no worsening of blood flow parameters relative to baseline. Decrease in EDV was statistically significant in active treatment Arm 2 at Week 32 (p=0.003). Mean IIEF-EF scores were nominally higher in subjects in active treatment who had improved visual grading scores vs those with no improvement. Adverse events were transient. CONCLUSIONS: Flaccid penile LiSWT appears to be safe and efficacious for treating ED based on statistically significant changes from baseline between sham and active treatments in primary outcome measures. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1145 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Irwin Goldstein More articles by this author Sue W. Goldstein More articles by this author Noel N. Kim More articles by this author Expand All Advertisement PDF downloadLoading ...

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

MP42-19 BILAYERED AUTOLOGOUS ADIPOSE-DERIVED MESENCHYMAL CELL-GELATIN SHEETS RECONSTRUCT URETERAL TISSUES IN RABBITS

You have accessJournal of UrologyCME1 Apr 2023MP42-19 BILAYERED AUTOLOGOUS ADIPOSE-DERIVED MESENCHYMAL CELL-GELATIN SHEETS RECONSTRUCT URETERAL TISSUES IN RABBITS Noriyuki Ogawa, Tetsuya Imamura, Tomonori Minagawa, Teruyuki Ogawa, and Osamu Ishizuka Noriyuki OgawaNoriyuki Ogawa More articles by this author , Tetsuya ImamuraTetsuya Imamura More articles by this author , Tomonori MinagawaTomonori Minagawa More articles by this author , Teruyuki OgawaTeruyuki Ogawa More articles by this author , and Osamu IshizukaOsamu Ishizuka More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003280.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Treatments of ureteral defects or strictures due to disease or trauma require reconstruction by urological surgery. However, those surgeries have some limitations, such as reoperation, or depending on ureteral length. Tissue engineering techniques would provide novel approaches to avoid the limitations. In this study, we fabricated bilayered autologous adipose-derived mesenchymal cell (AMC)-gelatin sheets and transplanted them into rabbits to replace surgically excised ureteral segments. METHODS: AMCs harvested from abdominal adipose tissues of female New Zealand White rabbits were cultured on collagen-coated dishes and labeled with a red fluorescent dye, PKH26, for later identification. Monolayers of AMCs that were cultured on temperature-responsive dishes were harvested by a gelatin hydrogel sheet. Two AMC-gelatin sheets were then overlaid together with the cell sides juxtaposed, forming a bilayered AMC-gelatin sheet. Following each partial ureterectomy of approximately 1 cm, the bilayered autologous AMC-gelatin sheet was transplanted, joining the proximal and distal ends of the remaining the ureter (experimental group; n=9). Control animals were conducted with a bilayered acellular-gelatin sheet (control group; n=9). RESULTS: At 4 and 8 weeks after transplantation, the proximal regions of ureters of control groups exhibited flexures and dilations compared with experimental groups. Both the control and experimental reconstructed ureteral walls had smooth muscle layers; however, those in the experimental reconstructed ureteral walls were significantly thicker and better organized than those in the control groups. In addition, the lumina of the 8-week reconstructed ureteral tissues in experimental groups did not show histological strictures as seen in the control ureters. Some AMCs differentiated into smooth muscle marker-positive cells. The experimental ureteral walls contained smooth muscle cells derived from the PKH26-labeled AMCs and others that were derived through migration and differentiation of cells from the remaining proximal and distal ends of the original ureter. CONCLUSIONS: These results indicated that the bilayered AMC-gelatin sheets could replace and/or reconstruct ureter tissues. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e575 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Noriyuki Ogawa More articles by this author Tetsuya Imamura More articles by this author Tomonori Minagawa More articles by this author Teruyuki Ogawa More articles by this author Osamu Ishizuka More articles by this author Expand All Advertisement PDF downloadLoading ...

Endoscopic retrograde cholangiopancreatography and endoscopic decompression in the malignant obstruction of the extrahepatic biliary tracts – a retrospective analysis

Background. Endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic biliary decompression are of the highest priority among minimally invasive procedures for the correction of the malignant obstruction of the extrahepatic biliary tracts. For this purpose, nasobiliary drainage and plastic or metal stents are used. However, it is not always possible to perform the endoscopic drainage of biliary ducts when there is a malignant obstruction. Purpose – to assess the immediate results of the use of the ERCP and endoscopic biliary decompression in malignant obstruction of the extrahepatic biliary tracts depending on the degree of obstruction. Materials and methods. A retrospective analysis of 106 cases of the malignant obstruction of the extrahepatic biliary tracts was performed. Out of 106 patients, 43 (40,6%) were males, and 63 (59,4%) were females. The age range of the patients was from 42 to 90 (68,4±11,1) years. Obstruction at the level of the major duodenal papilla was in 18 (17%) patients; at the level of the distal third of the common bile duct – in 66 (62,3%) patients; at the level of the middle third of the common bile duct – in 11 (10,4%) patients; at the level of the proximal third of the common bile duct – in 11 (10,4%) patients. Results. Out of 106 patients we managed to perform the ERCP in 94 (88,7%), and in 79 (84%) of them endoscopic drainage was successful. The complications after the ERCP were observed in 9 (8,5%) patients. In the malignant obstruction of the extrahepatic biliary tracts at the level of the major duodenal papilla we managed to perform the ERCP and restore the bile outflow in 17 out of 18 patients. In the malignant obstruction of the common bile duct, we managed to perform the ERCP in 77 (87,5%) out of 88 patients. In the obstruction at the level of the distal third, the endoscopic drainage was successful in 50 (87,7%) out of 57 patients who had undergone the ERCP. In the obstruction of the middle and proximal thirds of the common bile duct, the endoscopic drainage was successful in 12 (60%) out of 20 patients in which we managed to perform the ERCP. In the tumoral obstruction of the common bile duct, decompression at the level of the distal third was more successful (p = 0,027). Taking into consideration the obstruction of the major duodenal papilla, endoscopic biliary decompression was much more effective in the obstruction at the level of the major duodenal papilla and the distal third of the common bile duct compared to the obstruction of more proximal regions of the common bile duct (p = 0,002). In the tumoral obstruction of the extrahepatic biliary tracts at the level of the middle and proximal thirds of the common bile duct, the development of acute pancreatitis was characteristic of this process (p = 0,027). Conclusions. The performed retrospective analysis of the use of the ERCP in the malignant obstruction of the extrahepatic biliary tracts showed higher effectiveness of the endoscopic decompression in the obstruction of the distal regions of the extrahepatic biliary tracts compared with the obstruction at the level of the middle and proximal thirds of the common bile duct. Acute pancreatitis as a complication of the ERCP and endoscopic surgeries was significantly more often observed in the obstruction at the level of the middle and proximal thirds of the common bile duct.

Adipose Tissue Caveolin-1 Upregulation in Obesity Involves TNF-α/NF-κB Mediated Signaling

Obesity is characterized by chronic low-grade inflammation. Obese people have higher levels of caveolin-1 (CAV1), a structural and functional protein present in adipose tissues (ATs). We aimed to define the inflammatory mediators that influence CAV1 gene regulation and the associated mechanisms in obesity. Using subcutaneous AT from 27 (7 lean and 20 obese) normoglycemic individuals, in vitro human adipocyte models, and in vivo mice models, we found elevated CAV1 expression in obese AT and a positive correlation between the gene expression of CAV1, tumor necrosis factor-alpha (TNF-α), and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). CAV1 gene expression was associated with proinflammatory cytokines and chemokines and their cognate receptors (r ≥ 0.447, p ≤ 0.030), but not with anti-inflammatory markers. CAV1 expression was correlated with CD163, indicating a prospective role for CAV1 in the adipose inflammatory microenvironment. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of CAV1 mRNA/proteins, which were elevated by administering exogenous TNF-α. Mechanistically, TNF-α induces CAV1 gene transcription by mediating NF-κB binding to its two regulatory elements located in the CAV1 proximal regulatory region. The interplay between CAV1 and the TNF-α signaling pathway is intriguing and has potential as a target for therapeutic interventions in obesity and metabolic syndromes.

Magnetic Resonance Imaging Measurements of the Proximal Palmar Cortex of the Third Metacarpal Bone and the Suspensory Ligament in Non-Lame Endurance Horses before and after Six Months of Training.

Proximal metacarpal injury is common in endurance horses, yet exercise-induced changes in this region have not been described. This study aimed to document objective exercise-induced changes in the proximal palmar cortex of the third metacarpal bone (PcMcIII) and the suspensory ligament (SL). Low-field magnetic resonance (MR) images of both proximal metacarpal regions were obtained from six novice and six experienced horses, before and after six months of endurance training. Measurements were acquired in T1-weighted transverse MR images at four levels and included the thickness of the PcMcIII, the mediolateral width, and the dorsopalmar depth of the entire SL and its lobes. We used t-tests or their nonparametric equivalents to compare the measurements from the two examinations and both novice and experienced horses. The medial aspect of PcMcIII was significantly thicker in experienced horses than in novice horses at 2 and 3 cm distal to the carpometacarpal joint. This likely reflects the cumulative effect of long-term exercise and possibly age. The PcMcIII was significantly thicker medially than laterally. There was no significant difference between pre- and post-season measurements. Six months of endurance training were not sufficient to induce changes in the thickness of PcMcIII or the SL that are detectable in low-field MR images.

Hereditary multiple exostosis: a case report and brief review regarding the risk of malignancy

Osteocartilaginous exostoses is the most common benign bone tumor of childhood whose predominant growth on cartilage with potential impact on bone development and architecture, with potential risk of fractures and installation of malignant neoplasms. We present a case of an 11 -year -old female child, admitted to the unit complained of pain in the proximal region of the left ulna and distal third of the ipsilateral tibia with evolution of one year and suspected hereditary multiple exostosis whose histopathological result was positive for osteochondroma. The conduct for this case consisted of surgical extraction of the bone mass and outpatient follow -up for the case of relevant angular deviation of limbs, fractures or emergence of neoplasia suspected of malignancy.

Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization.

Synthetic genome evolution provides a dynamic approach for systematically and straightforwardly exploring evolutionary processes. Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detect over 260 000 rearrangement events after the SCRaMbLEing of a yeast strain harboring 5.5 synthetic yeast chromosomes (synII, synIII, synV, circular synVI, synIXR and synX). Remarkably, we find that the rearrangement events exhibit a specific landscape of frequency. We further reveal that the landscape is shaped by the combined effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. The enormous numbers of rearrangements mediated by SCRaMbLE provide a driving force to potentiate directed genome evolution, and the investigation of the rearrangement landscape offers mechanistic insights into the dynamics of genome evolution.