Dense Chromatin Research Articles

Selective retina therapy and thermal stimulation of the retina: different regenerative properties - implications for AMD therapy

BackgroundSelective Retina Therapy (SRT), a photodisruptive micropulsed laser modality that selectively destroys RPE cells followed by regeneration, and Thermal Stimulation of the Retina (TSR), a stimulative photothermal continuous wave laser modality that leads to an instant sublethal temperature increase in RPE cells, have shown therapeutic effects on Age-related Macular Degeneration (AMD) in mice. We investigate the differences between both laser modalities concerning RPE regeneration.MethodsFor PCR array, 6 eyes of murine AMD models, apolipoprotein E and nuclear factor erythroid-derived 2- like 2 knock out mice respectively, were treated by neuroretina-sparing TSR or SRT. Untreated litter mates were controls. Eyes were enucleated either 1 or 7 days after laser treatment. For morphological analysis, porcine RPE/choroid organ cultures underwent the same laser treatment and were examined by calcein vitality staining 1 h and 1, 3 or 5 days after irradiation.ResultsTSR did not induce the expression of cell-mediators connected to cell death. SRT induced necrosis associated cytokines as well as inflammation 1 but not 7 days after treatment. Morphologically, 1 h after TSR, there was no cell damage. One and 3 days after TSR, dense chromatin and cell destruction of single cells was seen. Five days after TSR, there were signs of migration and proliferation. In contrast, 1 h after SRT a defined necrotic area within the laser spot was seen. This lesion was closed over days by migration and proliferation of adjacent cells.ConclusionsSRT induces RPE cell death, followed by regeneration within a few days. It is accompanied by necrosis induced inflammation, RPE proliferation and migration. TSR does not induce immediate RPE cell death; however, migration and mitosis can be seen a few days after laser irradiation, not accompanied by necrosis-associated inflammation. Both might be a therapeutic option for the treatment of AMD.

Non-cooperative mechanism for bounded and ultrasensitive chromatin remodeling

Chromatin remodeling is an essential form of gene regulation that is involved in a variety of biological processes. We develop a theoretical model that takes advantage of percolation effects at the level of nucleosome interactions, which allows for ultrasensitive chromatin expansion. This model is non-cooperative and readily provides spatial bounds to the expansion region, preventing uncontrolled remodeling events. We explore different chromatin architectures and the ultrasensitivity of the chromatin density as a function of transcription factor concentration. We also compare our model with experimental data involving an inhibitor of nucleosome acetylation. These results suggest a novel mechanism for spatially-bounded chromatin remodeling and they provide means for quantitative comparisons between proposed models of chromatin architecture.

Cytologic features of small cell melanoma.

Small cell melanoma (SCM) is an aggressive variant of malignant melanoma (MM), which has been rarely described in the cytology literature. The aim of this study was to describe the clinical and cytologic features of a series of cases of metastatic SCM with discussion of the differential diagnosis of metastatic SCM diagnosed by fine-needle aspiration (FNA). A retrospective review of cases was performed, identifying two FNA cases and one core biopsy with touch preparation of metastatic SCM. Clinical presentation, cytomorphology features, ancillary tests, and final diagnoses were documented and analyzed. Patients ranged in age from 69 to 85 years-old. Cytomorphologic features included the presence of a monomorphic population of dispersed small round blue cells, with scant cytoplasm, high nuclear to cytoplasmic ratios, dense nuclear chromatin, and inconspicuous nucleoli. Acinar like arrangement (n=2) and nuclear molding (n=1) were also present. All cases showed diffuse positivity for the melanocytic markers SOX10 and Melan A by immunohistochemistry (IHC). Expression of neuroendocrine markers was variable. Diagnosing metastatic SCM at unusual anatomic sites by FNA cytology is a challenging task, especially in patients without known prior history of melanoma. Cytomorphology of SCM is unique, differing from conventional MM in many aspects, including the presence of acinar formations and a lack of typical melanoma features, such as large cells, intracytoplasmic melanin, and macronucleoli. IHC is critical for establishing the diagnosis of SCM.

ENDOBRONCHIAL SCHWANNOMA: VERY RARE TUMOR DISCOVERED ACCIDENTLY DURING FOB FOR LEFT LOWER LOBE COLLAPSE

TOPIC: Lung Cancer TYPE: Global Case Reports INTRODUCTION: In the mediastinum, Schwannomas are fairly common. Endobronchial position is extremely uncommon, with only a few cases reported in the literature. CASE PRESENTATION: An 80-year-old man presented in the emergency department with a 3-day history of fever 39 ◦C, general malaise, dry cough, myalgia, headache &loss of appetite. Chest X-ray on admission (IMAGE 1) revealed partial right lower lobe collapse (RLL). A diagnosis of aspiration pneumonia secondary to dysphagia was established, received AB with chest physiotherapy with gradual improvement of his condition. CKD, former smoker SI 20, AF, daistoic dysfunction. Then the patient started to be deteriorated with decrease conscious level due to metabolic cause where invasive mechanical ventilation was initiated. CXR was done which revealed left lower lobe collapse. IMAGE 2). Fiberoptic bronchoscopy showed Mucous plug obstructing left lower lobe bronchus which relieved by frequent suction.a nodule in the left bronchial tree, at the ridge between left upper lobe bronchus and left lower lobe bronchus. (IMAGE 3) The nodule was about 1 cm diameter, surface was smooth, shiny and vascularized. o Bronchoscopic removal of this nodules with mild bleeding which controlled spontaneously.o Another nodule on right side detected on ridge between right lower lobe bronchus and middle lobe bronchus. (IMAGE 4) Microscopic description:Given microphotographs (IMAGE 5 and IMAGE 6 display histological features of Bronchial schwannoma, comprising of a relatively circumscribed sub bronchial spindle cell neoplasm showing compact hypercellular (Antoni A) and hypocellular (Antoni B) areas. Nuclear palisading around fibrillary process (verocay bodies) is also appreciable.Cytologicaly cells are narrow, elongated and wavy with tapered ends interspersed with collagen fibers. Tumor cells have ill-defined cytoplasm, dense chromatin and mild atypia. No mitotic figures identified.o (IMAGE 7) S100: Immunohistochemical stains show diffuse S100 positivity. Extended panel of immunohistochemical including Cytokeratin, CD117, SMA, CD34 and H-Caldesomon also performed and all turns negative.consultation to thoracic surgery team was done to elect the best way of management as the patient has is not candidate for surgery at time being. DISCUSSION: Despite being a rare neoplasm schwannoma can occur in any area of the tracheobronchial tree(1,2,3,4). Most of symptoms are nonspecific(5) The accurate diagnosis depends upon histopathological criteria including presence of typical Antoni A formation and Verocay bodies in hematoxylin and eosin stains (6) Bronchoscopic treatment has been recently utilized for benign tracheobronchial tumors. It has been shown to be a safe and effective tool.(7,8) CONCLUSIONS: This is a case of endobronchial schwannoma was discovered accidently during FOB for left lower lobe collapse due to mucous plug, diagnosed according to histopathological criteria. REFERENCE #1: Shah H, Garbe L, Nussbaum E, Dumon JF, Chiodera PL, Cavaliere S. Benign tumors of the tracheobronchial tree: endoscopic characteris- tics and role of laser resection. Chest 1995; 107: 1744-51.2. Kasahara K, Fukuoka K, Konishi M, et al. Two cases of endobronchial neurilemmoma and review of the literature in Japan. Intern Med 2003; 42: 1215-8.3. Stack PS, Steckler RM. Tracheal neurilemmoma: case report and re- view of the literature. Head Neck 1990; 12: 436-9.4. Shirakusa T, Takada S, Yamazaki S, et al. Intrabronchial neurilem- moma: review of cases in Japan. Thorac Cardiovasc Surg 1989; 37: 388-90.5. Oxner CR, Shinners MJ, Godin DA, Lyn IT, Brent RB, McFadden PM. Obstructing tracheobronchial schwannoma. J La State Med Soc 2005; 157: 159-61.6.Lee BR, Choi YD, Kim YI, Lim SC, Kwon YS. Endobronchial schwannoma treated by rigid bronchoscopy with argon plasma co- agulation. Tuberc Respir Dis 2012; 73: 174-7. 7. Suzuki H, Sekine Y, Motohashi S, et al. Endobronchial neurogenic tumors treated by transbronchial electrical snaring and Nd-YAG la- ser abrasion: report of three cases. Surg Today 2005; 35: 243-6.8. Kwon YS, Kim H, Koh WJ, et al. Clinical characteristics and efficacy of bronchoscopic intervention for tracheobronchial leiomyoma. Respirology 2008; 13: 908-12. DISCLOSURES: No relevant relationships by Shereef AbdelWahab, source=Web Response No relevant relationships by Usama Abu Elhassan, source=Web Response No relevant relationships by MOHAMED HUSSEIN AHMED, source=Web Response No relevant relationships by MUHAMMAD RIZWAN BASHIR, source=Web Response No relevant relationships by Mohammed Elgazzar, source=Web Response No relevant relationships by Sidra Jahangir, source=Web Response No relevant relationships by Magda Rizk, source=Web Response no disclosure on file for Ayman Shaalan

Effects of Biotin and Folic Acid on Motility, Viability, Morphology, Chromatin Density and Integrity of Cryopreserved and Thawed Sperm in Normozoospermic Men

Effects of Biotin and Folic Acid on Motility, Viability, Morphology, Chromatin Density and Integrity of Cryopreserved and Thawed Sperm in Normozoospermic Men

Efficiency of SpCas9 and AsCpf1 (Cas12a) programmable nucleases at genomic safe harbor loci in HEK293 cells

Rationale: The development of eukaryote genome engineering tools based on CRISPR-Cas programmable bacterial nucleases systems opens wide horizons for gene therapies, human disease cell modeling, as well as investigation into manifestation of disease phenotypes and visualization of cellular processes. The safety and approximation of experiments both at the cellular and organismal levels depend on the accuracy of introducing double-stranded breaks into the target DNA regions. The search for new variants of more accurate CRISPR-Cas nucleases and evaluation of their ability to hydrolyze nucleosome DNA in vivo is considered a critical task for the development of the genome engineering technologies.Aim: To analyze the activity of the programmable nuclease AsCpf1 (Cas12a), with low level of off-target activity, in the human genome loci that are safe for the introduction of transgenic constructs (“safe harbor”) and to compare its efficiency with that of the widely used SpCas9 nuclease in HEK293 cells.Materials and methods: We performed the bioinformatics analysis of the association between target regions with nucleosomes and other proteins in the safe harbor loci AAVS1 and GSH-Ch1 and the transcriptionally inactive gene MYBPC3 (cardiac myosin binding protein 3) based on ATAC-seq data for the HEK293FT cells obtained from the NCBI SRA database. Plasmids encoding SpCas9 and AsCpf1 nucleases and guide RNA to the target regions were constructed and transfected into the HEK293FT cells. Events in the target regions of the HEK293FT cell genome were studied in the sequenograms with the TIDE algorithm.Results: The results of the ATAC-seq experiments for HEK293FT cells have shown that the AAVS1 locus can be referred as open chromatin with a low nucleosome density, while the GSH-Ch1 locus can be attributed to closed chromatin. In HEK293FT cells, the cardiac MYBPC3 gene has intermediate chromatin density. Assessment of the efficiency of introducing breaks into the studied HEK293FT cell chromatin loci by nucleases has shown that SpCas9 is able to cope with chromatin of any nucleosome density, while AsCpf1 can effectively introduce DNA breaks only at loci with open chromatin, such as AAVS1 and MYBPC3. Editing events occur at a very low rate at the GSH-Ch1 locus with a high nucleosome density.Conclusion: We have found low efficiency of the AsCpf1 nuclease in the genomic safe harbor locus GSH-Ch1, which is characterized by a high nucleosome density. When planning an experiment on AsCpf1 nuclease genome editing, the epigenetic chromatin landscape and the nucleosome density should be considered, as well as chromatin opening substances should be used.

Abstract MP12: The Epigenetic Remodeling Protein Brg1 Is Implicated In Vascular Progenitor Cell Contribution To Pathological Vascular Remodeling And Fibrosis

Vascular fibrosis is an irreversible consequence of vascular remodeling that ensues in many cardiovascular diseases. Currently, no treatments are available to target chronic vascular fibrosis. We identified a unique population of multipotent smooth muscle-derived Sca1+ progenitor cells that reside in the vascular adventitia (AdvSca1-SM cells). While AdvSca1-SM cells can differentiate into mature smooth muscle cells (SMCs) and repair the vessel wall, AdvSca1-SM cells also have the potential to differentiate into myofibroblasts and greatly contribute to vascular fibrosis after acute vascular injury. The epigenetic remodeling protein Brg1 is upregulated in AdvSca1-SM cells in the setting of vascular injury, but how Brg1 influences AdvSca1-SM differentiation is unknown. Using in vitro and in vivo approaches, our goal was to define the role of Brg1 in AdvSca1-SM cells. We hypothesized that Brg1 regulates chromatin density around genetic loci associated with fibrosis to preferentially drive AdvSca1-SM cell differentiation towards pathologic myofibroblasts. Further, we hypothesized that pharmacologic inhibition of Brg1 will disrupt AdvSca1-SM differentiation into myofibroblasts and attenuate vascular fibrosis. Mice subjected to carotid ligation and treated with the highly specific Brg1 inhibitor, PFI-3, exhibited decreased vascular fibrosis, reduced neointima, and decreased adventitial expansion of AdvSca1-SM cells as compared to mice that received vehicle control. This in vivo data was complemented by in vitro studies where cultured AdvSca1-SM cells stimulated with TGF-β expressed myofibroblast genes including αSMA and periostin, and co-treatment with PFI-3 blocked TGF-β-induced myofibroblast marker expression at the mRNA and protein level. In conclusion, these results support the hypothesis that Brg1 is a major regulator of AdvSca1-SM myofibroblast differentiation and may represent a novel targetable protein to treat vascular fibrosis.

Tackling Tumour Cell Heterogeneity at the Super-Resolution Level in Human Colorectal Cancer Tissue.

Simple SummaryTumour cell heterogeneity is the most fundamental problem in cancer diagnosis and therapy. Micro-diagnostic technologies able to differentiate the heterogeneous molecular, especially metastatic, potential of single cells or cell clones already within early primary tumours of carcinoma patients would be of utmost importance. Single molecule localisation microscopy (SMLM) has recently allowed the imaging of subcellular features at the nanoscale. However, the technology has mostly been limited to cultured cell lines only. We introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma patient tissue sections, illustrating, as a first example, changes in nuclear chromatin nanostructure and microRNA intracellular distribution within carcinoma cells as opposed to normal cells, chromatin accessibility and microRNAs having been shown to be critical in gene regulation and metastasis. We believe this technology to have an enormous potential for future differential diagnosis between individual cells in the tissue context.Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients.

Positive and Negative Impact of RNA Pol II Transcription on DNA Replication Progression

Conflicts between RNA Polymerase II transcription and DNA replication are associated with increased DNA damage and genome instability. Nevertheless, how exactly transcription can affect replication progression through genes is not known. Performing a genome-wide analysis of transcription and replication together we found that transcription increases the velocity of replication across long transcribed genes, because transcription reduces chromatin density along long genes. Conversely, transcription progression dictates where replication forks pause and stall in short highly transcribed genes. Sites of replication forks stalling/pausing are associated with increased DNA damage levels, representing bonafide transcription-replication collisions sites. Importantly, these sites overlap with breakpoints of rearrangements identified in cancers. Such rearrangements involving transcription-replication collisions sites can be induced in vitro following treatment with ATM and ATR inhibitors. Altogether, our findings highlight two opposing effects of transcription on replication progression, with positive and negative consequences for replication fork progression and genome instability.

A critical role for linker DNA in higher-order folding of chromatin fibers.

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.

Pulmonar Granular Cell Tumor in a Horse

Background: The occurrence of neoplasms in horses is relatively low. Granular cell tumor is a seldom diagnosed neoplasm, usually benign, of mesenchymal origin. Controversies exist regarding its origin, which is possibly from Schwann cells or cells with neuroendocrine differentiation. Despite being one of the main primary neoplasms in the lungs of horses, the number of cases is low in comparison to that of secondary lung tumors. Thus, this study proposes to report the anatomopathological aspects in a horse with granular cell tumor of primary pulmonary origin. Case: An 11-year-old female Quarter Horse breed underwent exploratory right lateral thoracotomy after presenting with chronic respiratory changes. During the operation, tumor masses were found in the right and left caudal pulmonary lobes. Due to the severity of clinical respiratory signs and the extent of the lesions, the animal was subjected to euthanasia and anatomopathological examination. Upon necroscopic examination, a tumor mass was found in the middle third of the left caudal lobe, rounded to flattened, measuring 10.0 × 8.0 cm in height and length, white in color, of firm consistency, smooth and regular surface and rising to the lung surface. When sectioned, the mass showed to be composed of multiple firm and dense circular lobes, separated by a thin layer of connective tissue. The tumor invaded the lumen of nearby segmental and subsegmental bronchi, which were partially or totally obstructed by the mass. In the right lung, multiple similar nodules were observed, accompanied by peritumoral hemorrhage. Histopathological analysis of the new formation revealed a dense cluster of cells that expanded over the lung parenchyma. The neoplastic cells were pleomorphic, moderately cohesive, without defined borders, with abundant cytoplasm, densely eosinophilic and finely granular. Intracytoplasmic granules were well evidenced by periodic acid Schiff staining (PAS). The cell nucleus was rounded to oval, excentric, markedly basophilic and with dense chromatin. There was moderate anisocytosis and mild anisokaryosis, with rare mitotic figures. Immunohistochemical analysis revealed positive staining for anti-vimentin and anti-S100 antibodies, confirming the diagnosis of granular cell tumor.Discussion: Granular cell tumors have no predisposition as to breed, sex or age. However, most of the described cases are reported in female horses aged around 13 years. The advanced age of the diagnosed animals may be related to late definitive diagnosis, since the clinical signs are nonspecific and treated palliatively like other respiratory diseases. Macroscopically, this tumor is more common in the multinodular form and, as observed in this case, it has a greater capacity for infiltration. Histologically, the visualization of large, polygonal cells, with a wide cytoplasm containing eosinophilic granules leads to the diagnosis of granular cell tumor. However, PAS staining and immunohistochemical tests were essential for the diagnostic conclusion in this report, confirming the presence of cytoplasmic granules and the mesenchymal and neuroectodermal origin of this neoplasm, respectively. Thus, considering the low occurrence of pulmonary granular cell tumor, the description of this case contributes to the basis of the knowledge of medical-veterinary professionals about this tumor in its clinical and diagnostic aspects.Keywords: Schwann cells, immunohistochemistry, neoplasia, lung.Descritores: células de Schwann, imuno-histoquímica, neoplasia, pulmão.Título: Tumor de células granulares pulmonar em um equino

Spatial density of open chromatin: an effective metric for the functional characterization of topologically associated domains

Topologically associated domains (TADs) are spatial and functional units of metazoan chromatin structure. Interpretation of the interplay between regulatory factors and chromatin structure within TADs is crucial to understand the spatial and temporal regulation of gene expression. However, a computational metric for the sensitive characterization of TAD regulatory landscape is lacking. Here, we present the spatial density of open chromatin (SDOC) metric as a quantitative measurement of intra-TAD chromatin state and structure. SDOC sensitively reflects epigenetic properties and gene transcriptional activity in TADs. During mouse T-cell development, we found that TADs with decreased SDOC are enriched in repressed developmental genes, and the joint effect of SDOC-decreasing and TAD clustering corresponds to the highest level of gene repression. In addition, we revealed a pervasive preference for TADs with similar SDOC to interact with each other, which may reflect the principle of chromatin organization.

Abstract MP173: Loss of DCHS1 Promotes Mitral Valve Prolapse Through Cytoskeleton Destabilization

Introduction: Mitral valve prolapse (MVP) is a major source of morbidity and mortality and is becoming one of the most common indications for cardiac surgery. Our group has previously reported that MVP is associated with missense mutations in DCHS1 , a gene widely implicated in tissue development and organization. Purpose: We recently performed two-hybrid screens and co-immunoprecipitation assays that define a novel complex between DCHS1, LIX1L, and SEPT9 (DLS). Our current studies investigate the role of this interaction in mitral valve morphogenesis. Methods: Wild-type (WT), DCHS1, and/or LIX1L heterozygous neonate murine heart tissue was isolated for primary culture of cardiac fibroblasts (CFs) and analyzed with immunocytochemistry, collagen, compaction, fibrin pillar to post assays, and actin sedimentation assays. Nuclear morphology and chromatin density were conducted for functional read-outs of cytoskeletal aberrations. Morphologic and functional analyses of murine mitral valves were completed. Results: Loss of actin architecture and collagen compaction capability were seen in CFs deficient of DCHS1 and/or LIX1L. Delivery of a peptide that disrupted the LIX1L-SEPT9 binding domain likewise destroyed actin architecture and reduced ECM compaction. In-vivo epistasis studies support these findings as mitral valve enlargement was exacerbated in DCHS1/LIX1L compound heterozygote mice compared to DCHS1 and LIX1L heterozygotes. Echocardiography of 11-month DCHS1/LIX1L compound heterozygote mice revealed pronounced MVP and increased left atrium diameter relative to DCHS1 and LIX1L heterozygotes. Filamentous SEPTIN9 localized to the actin ultrastructure and was also lost with disturbance of the DLS complex. Nuclei stretch and chromatin compaction were reduced in DCHS1/LIX1L compound heterozygote CFs. Further, histone H4 modifications were observed in DCHS1 KO mouse embryonic fibroblasts that indicate reductions in gene expression. Conclusion: DCHS1, through its interaction with the septin cytoskeleton, must regulate and stabilize actin dynamics in mitral VICs leading to proper valve development. Non-surgical interventions for MVP may be identified through establishing the downstream effects of DCHS1 mutations in our model.

Inhibition of Serine Metabolism Promotes Resistance to Cisplatin in Gastric Cancer.

BackgroundSerine provides important precursors of protein, lipid, and nucleotide synthesis needed for tumor cell growth. Phosphoglycerate dehydrogenase (PHGDH), a key rate-limiting enzyme in the serine de novo synthesis pathway, is highly expressed in many tumor types (including gastric cancer) and negatively correlated with overall survival. Cisplatin is a chemotherapeutic drug commonly used in the treatment of gastric cancer. In this study, we mainly investigated the relationship between serine metabolism and resistance to cisplatin in gastric cancer cells, as well as the regulatory mechanism involved in this process.Materials and MethodsWe determined the effect of different concentrations of serine or a PHGDH inhibitor combined with cisplatin or oxaliplatin on the viability and apoptosis of SGC7901, BGC823, and MGC803 cells via the Cell Counting Kit-8 and Hoechst 33258 staining, respectively. Western blotting was utilized to detect the relative protein expression. Furthermore, we investigated DNA damage through the micrococcal nuclease sensitivity assay detected using agarose gels.ResultsWe found that reduced concentrations of serine or inhibition of PHGDH hindered the toxicity and pro-apoptotic effects of cisplatin on gastric cancer cells. Moreover, the addition of serine could reverse the sensitivity of gastric cancer cells to cisplatin. Moreover, we found that DNA damage was reduced by treatment with PHGDH inhibitor NCT-503 or CBR-5884. Inhibition of serine metabolism induced a decrease in H3K4 tri-methylation, which was reversed by JIB-04 (inhibitor of H3K4 demethylase). The tolerance of gastric cancer cells to cisplatin was relieved by JIB-04. Through micrococcal nuclease experiments, we further found that inhibiting the activity of PHGDH strengthened chromatin tightness.ConclusionInhibition of serine metabolism reduced H3K4 tri-methylation and increased the density of chromatin, which leads to decreased toxicity and pro-apoptotic effect of platinum chemotherapeutic drugs on gastric cancer cells.

Shedding light on the chromatin changes that modulate shade responses.

Perception of vegetation proximity or plant shade informs of potential competition for resources by the neighboring vegetation. As vegetation proximity impacts on both light quantity and quality, perception of this cue by plant photoreceptors reprograms development to result in responses that allow plants to compete with the neighboring vegetation. Developmental reprogramming involves massive and rapid changes in gene expression, with the concerted action of photoreceptors and downstream transcription factors. Changes in gene expression can be modulated by epigenetic processes that alter chromatin compaction, influencing the accessibility and binding of transcription factors to regulatory elements in the DNA. However, little is known about the epigenetic regulation of plant responses to the proximity of other plants. In this manuscript, we review what is known about plant shade effects on chromatin changes at the cytological level, that is, changes in nuclear morphology and high order chromatin density. We address which are the specific histone post-transcriptional modifications that have been associated with changes in shade-regulated gene expression, such as histone acetylation and histone methylation. Furthermore, we explore the possible mechanisms that integrate shade signaling components and chromatin remodelers to settle epigenetic marks at specific loci. This review aims to be a starting point to understand how a specific environmental cue, plant shade, integrates with chromatin dynamics to implement the proper acclimation responses.

Chromatin Compaction Leads to a Preference for Peripheral Heterochromatin

A layer of dense heterochromatin is found at the periphery of the nucleus. Because this peripheral heterochromatin functions as a repressive phase, mechanisms that relocate genes to the periphery play an important role in regulating transcription. Using Monte Carlo simulations, we show that an interaction that attracts euchromatin and heterochromatin equally to the nuclear envelope will still preferentially locate heterochromatin to the nuclear periphery. This observation considerably broadens the class of possible interactions that result in peripheral positioning to include boundary interactions that either weakly attract all chromatin or strongly bind to a randomly chosen 0.05% of nucleosomes. The key distinguishing feature of heterochromatin is its high chromatin density with respect to euchromatin. In our model, this densification is caused by heterochromatin protein 1’s preferential binding to histone H3 tails with a methylated lysine at the ninth residue, a hallmark of heterochromatin. We find that a global rearrangement of chromatin to place heterochromatin at the nuclear periphery can be accomplished by attaching a small subset of loci, even if these loci are uncorrelated with heterochromatin. Hence, factors that densify chromatin determine which genomic regions condense to form peripheral heterochromatin.

Role of nucleosome positioning in 3D chromatin organization and loop formation

We present a physics-based polymer model that can investigate 3D organization of chromatin accounting for DNA elasticity, DNA-bending due to nucleosomes, and 1D organization of nucleosomes along DNA. We find that the packing density of chromatin oscillates between densities corresponding to highly folded and extended configurations as we change the nucleosome organization (length of linker DNA). We compute the looping probability of chromatin and show that the presence of nucleosomes increases the looping probability of the chain compared to that of a bare DNA. We also show that looping probability has a large variability depending on the nature of nucleosome organization and density of linker histones.

Anthocyanins isolated from Oryza Sativa L. protect dermal fibroblasts from hydrogen peroxide-induced cell death

Background: Oxidative stress, cellular toxicity, and inflammation lead to skin damage, which results in premature skin aging. Recently, anthocyanins (ANT) have received much attention as dietary anti-oxidants involved in the prevention of oxidative damage. Materials and Methods: This study investigated the effects of ANT extracted from black rice (Oryza sativa L.) on the survival of rat dermal fibroblasts (RDFs) after oxidative stress-induced cellular damage by hydrogen peroxide (H2O2) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We further investigated the apoptosis-inducing effects of ANT using 4′,6-diamidino-2-phenylindole and Annexin V staining. The effect of ANT extract on autophagy was confirmed by reverse transcription polymerase chain reaction of the autophagy-related microtubule-associated protein 1B light chain 3 (LC3-II) and ffluorescence microscopy of the LC3-II protein. Results: The high-performance liquid chromatography results indicated the presence of cyanidin-3-O-glucoside in both extracts. The study demonstrated that the addition of crude or purified ANT extract before H2O2 treatment increased RDF cell viability. Pretreatment with ANT decreased the number of cells exhibiting dense chromatin fragments and DNA condensation, which are characteristics of apoptotic cell death. ANT decreased the number of late apoptotic/necrotic (Annexin + and propidium iodide (PI) +) cells and early apoptotic (Annexin V + and PI-) cells. Furthermore, ANT inhibited the H2O2-mediated induction of LC3-II gene expression in RDFs. Conclusion: The contribution of autophagy induction to the protective effects of ANT was verified by the observed decrease in the mRNA and protein expression of LC3-II. These results suggest the therapeutic potential of polyphenolic compounds extracted from O. sativa L. in oxidative damage-induced skin aging.

Feline Prostatic Carcinoma

Background: Prostatic cancer is a rare condition in cats but should be included as a differential diagnosis whenever middle-aged cats present lower urinary tract signs, such as dysuria and hematuria. Abdominal ultrasound can indicate the disease, but fine-needle aspiration cytology and histopathology are necessary to establish the neoplastic origin and the therapeutic plan. Because of the limited data, no standard-of-care treatment or prognostic information exists in cats with prostate carcinoma. This report describes the clinical signs, diagnosis and surgical approach in a case of prostatic carcinoma in a cat. Case: A 6-year-old, intact male, domestic short-haired cat was presented with a 3-day history of dysuria, hematuria, inappetence, constipation, lethargy and prostration. On physical examination, the cat was in a very poor general condition and abdominal palpation revealed firm mass located caudally to the urinary bladder. The urinary bladder presented high repletion degree, while the large bowel was distended and presented soft faecal content. Blood count, serum biochemistry and urinalysis showed neutrophilic leukocytosis, hypoalbuminemia and high serum creatinine level, and severe hematuria, respectively. Abdominal ultrasound showed a mass located in the prostatic area with hypoechogenic and slightly heterogeneous parenchyma, measuring 3.3 x 3.0 cm. Echo-guided trans-abdominal fine-needle aspiration of the prostate was performed. Microscopically, the cells were round with basophilic cytoplasm, and had round to ovoid nuclei, dense chromatin and prominent nucleoli. Some cells were binucleated and mild anisocytosis and marked anisokaryosis were documented. These findings were compatible with malignant prostatic neoplasia. After initial clinical stabilization, the patient underwent an exploratory laparotomy for tumor resection. Cystotomy followed by pubic osteotomy was performed to access the tumor. Urethral anastomosis was necessary due to adherence of the neoplasm to adjacent structures. However, the patient died during the immediate postoperative period. No complications related to surgery were observed at necropsy. Tissue specimens were collected and stained by hematoxylin and eosin. Prostatic carcinoma was confirmed by immunohistochemistry tests using streptavidin-biotin-peroxidase complex method, and primary antibodies against vimentin, cytokeratin AE1/AE3 and 7. Discussion: Prostatic neoplams are rare in cats and clinical signs are suggestive of lower urinary tract disease, such as dysuria and/or hematuria, associated with tenesmus. Abdominal or rectal palpation seems to be essential to detect the prostatic enlargement. Ultrasound imaging was more sensitive than radiography, since it brings more information about structure, size, form and prostatic internal architecture. The patient’s serum creatinine value above the normal range for cats indicated partial urethral obstruction, due to neoplastic concentric growth. Echo-guided trans-abdominal fine needle aspiration cytology was an effective method to confirm neoplastic etiology. The advanced stage of the disease and poor clinical condition probably contributed to death at the immediate postoperative period, even though the surgical technique seemed to be appropriate as demonstrated at necropsy. Also, the immunohistochemistry tests allowed to confirm the diagnosis and excluded the main differential diagnoses, such as urothelial carcinoma and prostatic sarcomatoid carcinoma.

Spring Model - Chromatin Modeling Tool Based on OpenMM.

Chromatin structure modeling is a rapidly developing field. Parallel to the enormous growth of available experimental data, there is a growing need of building and visualizing 3D structures of nuclei, chromosomes, chromatin domains, and single loops associated with particular gene loci. Here, we present a tool for chromatin domain modeling; it is available as a webservice and standalone python script. Our tool is based on molecular mechanics and utilizes the OpenMM engine for model generation. In this method the user provides contacts between chromatin regions and obtains a 3D structure that satisfies them. Additional parameters allow for the control of fibre stiffness, initial structure adjustments and simulation resolution, there are also options for structure refinement and modeling in a spherical container. The user may provide contacts in the form of bead indices, or insert interactions in genome coordinates sourced from BEDPE files. After the simulation is complete, the user is able to download the structure in the Protein Data Bank (PDB) format for further analysis. We dedicate this tool to all who are interested in chromatin structures. It is suitable for quick visualization of datasets, studying the impact of structural variants (SVs), inspecting the effects of adding and removing particular contacts, and measuring features such as maximum distances between sites (e.g.promoter-enhancer), or local chromatin density.