Chromatin Masses Research Articles

Inhibition of lncEPS by TLR4/NF-κB pathway induces ventilator-induced lung injury by decreasing its binding to and upregulating Hspa5

Whether LincRNA erythroid prosurvival (LncEPS) reduces VILI remains unclear. A GSE200932 microarray was used to screen differentially expressed genes (DEGs). A VILI mouse model was constructed by mechanical ventilation (MV), with or without TAK242 or SN50 pretreatment. Airway transfection with adeno-associated virus (AAV) was used to overexpress lncEPS in alveolar macrophages (AMs). Lung tissues were collected to assess pathological injury and macrophage polarization. NLRP3 inflammasome, TLR4/ NF-κB pathway activation and heat shock protein family A member 5 (Hspa5) in lung tissue and AMs wre evaluated. LncEPS localization and regulatory changes were assessed using in situ hybridization and RT–PCR. Lung tissues after lncEPS overexpression were subjected to transcriptomics. Chromatin isolation and mass spectrometry (MS) were performed to identify proteins interacting with lncEPS. GSE200932 microarray showed that the DEGs were related to NF-κB pathway, Toll-like receptor pathway and NOD-like receptor pathway. TAK242 or SN50 treatment increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation by inhibiting TLR4/NF-κB pathway in VILI. Inhibition of TLR4/NF-κB pathway upregulated lncEPS expression in AMs. Overexpression of lncEPS increased polarization of M2 macrophages and decreased NLRP3 inflammasome activation, eventually alleviating VILI in mice. Mechanistically, lncEPS bound to Hspa5 and downregulated its expression to inhibit inflammatory response.

DeepPRMS: advanced deep learning model to predict protein arginine methylation sites.

Protein methylation is a form of post-translational modifications of protein, which is crucial for various cellular processes, including transcription activity and DNA repair. Correctly predicting protein methylation sites is fundamental for research and drug discovery. Some experimental techniques, such as methyl-specific antibodies, chromatin immune precipitation and mass spectrometry, exist for predicting protein methylation sites, but these techniques are time-consuming and costly. The ability to predict methylation sites using in silico techniques may help researchers identify potential candidate sites for future examination and make it easier to carry out site-specific investigations and downstream characterizations. In this research, we proposed a novel deep learning-based predictor, named DeepPRMS, to identify protein methylation sites in primary sequences. The DeepPRMS utilizes the gated recurrent unit (GRU) and convolutional neural network (CNN) algorithms to extract the sequential and spatial information from the primary sequences. GRU is used to extract sequential information, while CNN is used for spatial information. We combined the latent representation of GRU and CNN models to have a better interaction among them. Based on the independent test data set, DeepPRMS obtained an accuracy of 85.32%, a specificity of 84.94%, Matthew's correlation coefficient of 0.71 and a sensitivity of 85.80%. The results indicate that DeepPRMS can predict protein methylation sites with high accuracy and outperform the state-of-the-art models. The DeepPRMS is expected to effectively guide future research experiments for identifying potential methylated protein sites. The web server is available at http://deepprms.nitsri.ac.in/.

Post-Translational Modifications in Histones and Their Role in Abiotic Stress Tolerance in Plants.

Abiotic stresses profoundly alter plant growth and development, resulting in yield losses. Plants have evolved adaptive mechanisms to combat these challenges, triggering intricate molecular responses to maintain tissue hydration and temperature stability during stress. A pivotal player in this defense is histone modification, governing gene expression in response to diverse environmental cues. Post-translational modifications (PTMs) of histone tails, including acetylation, phosphorylation, methylation, ubiquitination, and sumoylation, regulate transcription, DNA processes, and stress-related traits. This review comprehensively explores the world of PTMs of histones in plants and their vital role in imparting various abiotic stress tolerance in plants. Techniques, like chromatin immune precipitation (ChIP), ChIP-qPCR, mass spectrometry, and Cleavage Under Targets and Tag mentation, have unveiled the dynamic histone modification landscape within plant cells. The significance of PTMs in enhancing the plants' ability to cope with abiotic stresses has also been discussed. Recent advances in PTM research shed light on the molecular basis of stress tolerance in plants. Understanding the intricate proteome complexity due to various proteoforms/protein variants is a challenging task, but emerging single-cell resolution techniques may help to address such challenges. The review provides the future prospects aimed at harnessing the full potential of PTMs for improved plant responses under changing climate change.

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis.

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.

Predicting chemoevasion potential of cancer cells from statistical chromatin packing behavior.

In malignancy, genetic, metabolic, or environmental insults may remodel chromatin structure, potentially increasing the plasticity of aberrant cell state transitions. Observed alterations to the epigenetic landscape of chromatin include gain-of-function mutations of Polycomb repressor EZH2 that block differentiation. Nucleosome remodeling and higher-order chromatin organization may also increase malignancy, with abnormalities ranging from prevalent mutations of ATP-dependent chromatin remodelers to mixing between transcriptionally active and inactive compartments. However, a universal relationship between chromatin structure and the disease state does not yet exist. Here, using the first principles of physics, we demonstrate that chromatin packing, or the statistical distribution of chromatin density in 4D, is a genome-wide regulator of phenotypic plasticity. The nucleus is a highly crowded, heterogeneous environment where DNA, RNA, proteins, and other macromolecules exert excluded volume effects, altering the kinetics and efficiency of transcription reactions. As chromatin is the major crowder in the nucleus, characterizing the chromatin mass density distribution facilitates a more complete understanding of transcription processes in a realistic nuclear environment. Combining high-resolution chromatin scanning transmission electron microscopy (ChromSTEM) and live-cell, label-free Partial Wave Spectroscopy (PWS) along with polymer physics-based analysis, we identified the existence of spatially separable packing domains, around 80 nm in radius. We then employed our understanding of fundamental chromatin packing to build a chromatin-mediated model of transcription that incorporates two pertinent aspects of phenotypic plasticity: transcriptional malleability, the ability of a cell to upregulate certain genes within a critical time frame, and transcriptional heterogeneity, a cell population's range of functional states. Finally, we extended our transcription model to predict how the average chromatin packing behavior of a cell population contributes directly to cancer cell survival upon chemotherapy treatment. Experimental observations confirmed model predictions, underlying the importance of chromatin packing in chemoevasion processes.

Evaluation & comparison of micronuclei in smokers with & without diabetes

Background: Micronuclei are chromatin masses that arise from chromosomal fragments or whole chromosome that lack behind at the anaphase stage of cell division. Their presence in cells means the number of chromosomal aberrations arising during mitosis. Cigarette smoke contains over 4000 chemical carcinogens, with 200 known carcinogens that show significant genotoxic effects in human cells. Chronic high levels of glucose during Diabetes Mellitus are thought to increase oxidative stress and the formation of free radicals that in turn damage cells. Reactive oxygen species chemically attack cellular components altering metabolism, inflammatory mediators, and antioxidant defense mechanisms, overall favoring the pathogenesis of the disease and the persistence of genetic damage. Scoring of micronuclei is considered a DNA damage biomarker for chromosomal damage and instability. Aim: Considering this, we decided to evaluate & compare the frequency of micronuclei in oral epithelial cells in smokers & in diabetics. Materials & Methods: A total number of eligible 150 individuals were included in the study; 50 smokers without diabetes, 50 smokers with diabetes & 50 age and sex matched non smoker healthy individuals group as controls. Oral epithelial cells were taken from buccal smears and subjected to PAP stain. Mean micronuclei were taken from all the subjects. Results: Least number of micronuclei was found in non smoker, non diabetic group & highest numbers were found in smoker, Diabetic group. Conclusion: Micronuclei assay is non-invasive technique that offers a very simple method for obtaining information on status of the epithelial cells, particularly DNA damage, proliferative potential of basal cells and cell death.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. Cerevisiae.

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model.

The establishment of an appropriate costimulatory phenotype is crucial for dendritic cells (DCs) to maintain a homeostatic state with optimal immune surveillance and immunogenic activities. The upregulation of CD80/86 and CD40 is a hallmark costimulatory phenotypic switch of DCs from a steady state to an activated one for T cell activation. However, knowledge of the regulatory mechanisms underlying this process remains limited. In this study, we identified a Zbtb46 homolog from a zebrafish model. Zbtb46 deficiency resulted in upregulated cd80/86 and cd40 expression in kidney marrow-derived DCs (KMDCs) of zebrafish, which was accompanied with a remarkable expansion of CD4+/CD8+ T cells and accumulation of KMDCs in spleen of naive fish. Zbtb46 -/- splenic KMDCs exhibited strong stimulatory activity for CD4+ T cell activation. Chromatin immunoprecipitation-quantitative PCR and mass spectrometry assays showed that Zbtb46 was associated with promoters of cd80/86 and cd40 genes by binding to a 5'-TGACGT-3' motif in resting KMDCs, wherein it helped establish a repressive histone epigenetic modification pattern (H3K4me0/H3K9me3/H3K27me3) by organizing Mdb3/organizing nucleosome remodeling and deacetylase and Hdac3/nuclear receptor corepressor 1 corepressor complexes through the recruitment of Hdac1/2 and Hdac3. On stimulation with infection signs, Zbtb46 disassociated from the promoters via E3 ubiquitin ligase Cullin1/Fbxw11-mediated degradation, and this reaction can be triggered by the TLR9 signaling pathway. Thereafter, cd80/86 and cd40 promoters underwent epigenetic reprogramming from the repressed histone modification pattern to an activated pattern (H3K4me3/H3K9ac/H3K27ac), leading to cd80/86 and cd40 expression and DC activation. These findings revealed the essential role of Zbtb46 in maintaining DC homeostasis by suppressing cd80/86 and cd40 expression through epigenetic mechanisms.

Lipid metabolism-related lncRNA SLC25A21-AS1 promotes the progression of oesophageal squamous cell carcinoma by regulating the NPM1/c-Myc axis and SLC25A21 expression.

BackgroundObesity alters metabolic microenvironment and is thus associated with several tumours. The aim of the present study was to investigate the role, molecular mechanism of action, and potential clinical value of lipid metabolism–related long non‐coding RNA (lncRNA) SLC25A21‐AS1 in oesophageal squamous cell carcinoma (ESCC).MethodsA high‐fat diets (HFDs)‐induced obesity nude mouse model was established, and targeted metabolomics analysis was used to identify critical medium‐long chain fatty acids influencing the growth of ESCC cells. Transcriptomic analysis of public dataset GSE53625 confirmed that lncRNA SLC25A21‐AS1 was a lipid metabolism–related lncRNA. The biological function of lncRNA SLC25A21‐AS1 in ESCC was investigated both in vivo and in vitro. Chromatin immunoprecipitation(ChIP)assay, RNA‐pull down, mass spectrometry, co‐IP, and RNA IP(RIP) were performed to explore the molecular mechanism. Finally, an ESCC cDNA microarray was used to determine the clinical prognostic value of SLC25A21‐AS1 by RT‐qPCR.ResultsPalmitic acid (PA) is an important fatty acid component of HFD and had an inhibitory effect on ESCC cell lines. LncRNA SLC25A21‐AS1 expression was downregulated by PA and associated with the proliferation and migration of ESCC cells in vitro and in vivo. Mechanistically, SLC25A21‐AS1 interacted with nucleophosmin‐1 (NPM1) protein to promote the downstream gene transcription of the c‐Myc in the nucleus. In the cytoplasm, SLC25A21‐AS1 maintained the stability of SLC25A21 mRNA and reduced the intracellular NAD+/NADH ratio by influencing tryptophan catabolism. Finally, we demonstrated that high expression of SLC25A21‐AS1 promoted resistance to cisplatin‐induced apoptosis and was correlated with poor tumour grade and overall survival.ConclusionsHFD/PA has an inhibitory effect on ESCC cells and SLC25A21‐AS1 expression. SLC25A21‐AS1 promotes the proliferation and migration of ESCC cells by regulating the NPM1/c‐Myc axis and SLC25A21 expression. In addition, lncRNA SLC25A21‐AS1 may serve as a favourable prognostic biomarker and a potential therapeutic target for ESCC.

A novel tRNA-derived fragment AS-tDR-007333 promotes the malignancy of NSCLC via the HSPB1/MED29 and ELK4/MED29 axes

BackgroundTransfer RNA-derived fragments (tRFs) are a new class of small non-coding RNAs. Recent studies suggest that tRFs participate in some pathological processes. However, the biological functions and mechanisms of tRFs in non-small cell lung cancer (NSCLC) are largely unknown.MethodsDifferentially expressed tRFs were identified by tRF and tiRNA sequencing using 9 pairs of pre- and post-operation plasma from patients with NSCLC. Quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to determine the levels of tRF in tissues, plasma, and cells. Gain- and loss-of-function experiments were implemented to investigate the oncogenic effects of tRF on NSCLC cells in vitro and in vivo. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pulldown, mass spectrum, RNA immunoprecipitation (RIP), Western blot, co-immunoprecipitation (Co-IP) assays, and rescue experiments were performed to explore the regulatory mechanisms of tRF in NSCLC.ResultsAS-tDR-007333 was an uncharacterized tRF and significantly up-regulated in NSCLC tissues, plasma, and cells. Clinically, AS-tDR-007333 overexpression could distinguish NSCLC patients from healthy controls and associated with poorer prognosis of NSCLC patients. Functionally, overexpression of AS-tDR-007333 enhanced proliferation and migration of NSCLC cells, whereas knockdown of AS-tDR-007333 resulted in opposite effects. Mechanistically, AS-tDR-007333 promoted the malignancy of NSCLC cells by activating MED29 through two distinct mechanisms. First, AS-tDR-007333 bound to and interacted with HSPB1, which activated MED29 expression by enhancing H3K4me1 and H3K27ac in MED29 promoter. Second, AS-tDR-007333 stimulated the expression of transcription factor ELK4, which bound to MED29 promoter and increased its transcription. Therapeutically, inhibition of AS-tDR-007333 suppressed NSCLC cell growth in vivo.ConclusionsOur study identifies a new oncogenic tRF and uncovers a novel mechanism that AS-tDR-007333 promotes NSCLC malignancy through the HSPB1-MED29 and ELK4-MED29 axes. AS-tDR-007333 is a potential diagnostic or prognostic marker and therapeutic target for NSCLC.

Hemagglutination in gill capillaries of sheepshead, Archosargus probatocephalus (Perciformes: Sparidae), infected by a myxosporidean.

During a survey Myxozoa, four specimens of the sheepshead (18 ± 1.5 cm and 59 ± 2.5 g) (Archosargus probatocephalus) were collected in the Ipioquinha river (Maceió/AL). Transmission electron microscopy observations revealed erythrocyte agglutinations in gill capillaries located near spherical cysts containing myxospores of the genus Henneguya. This hemagglutination partially or totally obstructed the gill capillaries. Erythrocytes occurred in close adherence to each other, with a closed intercellular space. A few lysed erythrocytes were observed among agglutinated cells. The reduced lumen of the capillaries was partially filled with amorphous dense homogenous material adhering to the erythrocytes. In addition, heterogeneous masses of irregular lower electron density were observed in the reduced channel of the capillary. The agglutinated erythrocytes appeared dense and homogenous, lacking cytoplasmic organelles. The nuclei had the appearance of normal condensed chromatin masses, generally without visible nucleoli. This occurrence of hemagglutination only in the capillaries located in close proximity to the developing myxozoan cysts suggests that parasite development may be a factor triggering erythrocyte agglutination. This is supported by previous experimental studies that showed a probable correlation between parasitic infections and hemagglutination. Nonetheless, further studies are necessary in order to better understand the physicochemical processes involved in this phenomenon.

Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells.

Simple SummaryHuman glioblastoma (GBM) cells are particularly resistant to nuclear fragmentation upon cytotoxic insult. To date, nuclear disassembly is the biological point-of-no-return of the apoptotic process. The injured cell that does not go beyond this point goes into a failed apoptosis and has potential for recovery. Nuclear disassembly is governed by caspases and, ultimately, by DFF40/CAD endonuclease. GBM cells express low levels of DFF40/CAD protein. Our aim was to assess whether this endonuclease could be activated to facilitate nuclear fragmentation in GBM cells. We revealed that GBM cells can activate their nuclear pool of DFF40/CAD in a caspase-dependent manner when treated with gossypol. Gossypol enabled DFF40/CAD assembly into high-order structures, facilitating nuclear dismantling. The identification of such compounds, pushing cells toward the point-of-no-return of apoptosis, will provide new tools to hamper the recovery of injured cells, slowing down tumor progression.Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.

Subcellular patterns of SPE-6 localization reveal unexpected complexities in Caenorhabditis elegans sperm activation and sperm function.

To acquire and maintain directed cell motility, Caenorhabditis elegans sperm must undergo extensive, regulated cellular remodeling, in the absence of new transcription or translation. To regulate sperm function, nematode sperm employ large numbers of protein kinases and phosphatases, including SPE-6, a member of C. elegans’ highly expanded casein kinase 1 superfamily. SPE-6 functions during multiple steps of spermatogenesis, including functioning as a “brake” to prevent premature sperm activation in the absence of normal extracellular signals. Here, we describe the subcellular localization patterns of SPE-6 during wild-type C. elegans sperm development and in various sperm activation mutants. While other members of the sperm activation pathway associate with the plasma membrane or localize to the sperm’s membranous organelles, SPE-6 surrounds the chromatin mass of unactivated sperm. During sperm activation by either of two semiautonomous signaling pathways, SPE-6 redistributes to the front, central region of the sperm’s pseudopod. When disrupted by reduction-of-function alleles, SPE-6 protein is either diminished in a temperature-sensitive manner (hc187) or is mislocalized in a stage-specific manner (hc163). During the multistep process of sperm activation, SPE-6 is released from its perinuclear location after the spike stage in a process that does not require the fusion of membranous organelles with the plasma membrane. After activation, spermatozoa exhibit variable proportions of perinuclear and pseudopod-localized SPE-6, depending on their location within the female reproductive tract. These findings provide new insights regarding SPE-6’s role in sperm activation and suggest that extracellular signals during sperm migration may further modulate SPE-6 localization and function.

Formation of Non-Nucleoplasmic Proteasome Foci during the Late Stage of Hyperosmotic Stress.

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.

Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis.

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.

HISTOPATHOLOGICAL GRADING OF ORAL SQUAMOUS CELL CARCINOMA BY MICRONUCLEI ASSAY, A POTENTIAL BIOMARKER IN ORAL EXFOLIATED CELLS

Background: Micronucleus is a microscopically visible round or oval cytoplasmic chromatin mass in the extra nuclear vicinity, originated from aberrant mitosis, which consists of eccentric chromosomes that have failed to reach spindle poles during mitosis and are used as biomarkers for assessment of DNA damage. Micronuclei in exfoliated oral epithelial cells have been shown in some studies to correlate with severity of this genotoxic damage. This severity can be measured in terms of grading of the lesions. Aim: To correlate frequency of micronuclei (MN) in oral exfoliated cells in clinically diagnosed cases of oral squamous cell carcinoma (OSCC) followed by a histopathological grading. Materials and Methods : The study subjects consisted of clinically diagnosed cases of OSCC. Healthy subjects without any tobacco consumption habits formed the control group.The cytosmears from both groups were stained with rapid Papanicolaou stain. MN were identified according to the criteria given by Tolbert et al. Results: The frequency of MN was three to four times higher in patients with OSCC as compared to patients in the control group and the difference was found to be highly significant. In 75% cases, the cytological grade as determined by the frequency of micronuclei correlated with the histopathological grade and this observation was statistically significant. Conclusions : The MN is potentially an excellent candidate to serve as such a biomarker for prediction of the grade of OSCC.

Evidence for anaphase pulling forces during C. elegans meiosis.

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

Correlación entre recuento de micronúcleos y hábito tabáquico: Un estudio hospitalario

Background: Micronucleus is a microscopically visible cyto-plasmic chromatin mass in the extranuclear vicinity, originating from aberrant mitosis, which consists of eccentric chromosomes that have failed to reach spindle poles during mitosis. The present study was designed to evaluate and compare cytogenetic changes in the buccal mucosa of smokers and non-smokers based on the occurrence of micronuclei. The study aimed to determine the correlation between the micronuclei count and the frequency and duration of smoking habit. Materials and Methods: Two groups (smokers and non-smokers) of 34 individuals each were examined. Cytological buccal smears were taken from participants using a moistened wooden spatula and stained with standard Papanicolaou stain. Presence of micronuclei was assessed at 40X magnification using a light microscope and a count per 500 cells was determined. The results of the study were analyzed statistically using Mann-Whitney U test, Spearman's rank correlation coefficient and Student t-test. Result: Smears from smokers showed a significant increase in the total number of micronuclei per 500 cell count compared to non-smokers. There was a strong positive correlation between the occurrence of micronuclei and the frequency and duration of smoking. A age-related increase in older age groups was also observed. Conclusion: The study reveals a strong positive correlation between the occurrence of micronuclei and the frequency and duration of smoking. This observation is vital in the utilization of the micronuclei detection in smears as a prognostic, educational and interventional tool in the management of patients with smoking habits.

Chromatin-mediated regulators of meiotic recombination revealed by proteomics of a recombination hotspot

BackgroundMeiotic recombination hotspots control the frequency and distribution of Spo11 (Rec12)-initiated recombination in the genome. Recombination occurs within and is regulated in part by chromatin structure, but relatively few of the many chromatin remodeling factors and histone posttranslational modifications (PTMs) have been interrogated for a role in the process.ResultsWe developed a chromatin affinity purification and mass spectrometry-based approach to identify proteins and histone PTMs that regulate recombination hotspots. Small (4.2 kbp) minichromosomes (MiniCs) bearing the fission yeast ade6-M26 hotspot or a basal recombination control were purified approximately 100,000-fold under native conditions from meiosis; then, associated proteins and histone PTMs were identified by mass spectrometry. Proteins and PTMs enriched at the hotspot included known regulators (Atf1, Pcr1, Mst2, Snf22, H3K14ac), validating the approach. The abundance of individual histones varied dynamically during meiotic progression in hotspot versus basal control MiniCs, as did a subset of 34 different histone PTMs, implicating these as potential regulators. Measurements of basal and hotspot recombination in null mutants confirmed that additional, hotspot-enriched proteins are bona fide regulators of hotspot activation within the genome. These chromatin-mediated regulators include histone H2A-H2B and H3-H4 chaperones (Nap1, Hip1/Hir1), subunits of the Ino80 complex (Arp5, Arp8), a DNA helicase/E3 ubiquitin ligase (Rrp2), components of a Swi2/Snf2 family remodeling complex (Swr1, Swc2), and a nucleosome evictor (Fft3/Fun30).ConclusionsOverall, our findings indicate that a remarkably diverse collection of chromatin remodeling factors and histone PTMs participate in designating where meiotic recombination occurs in the genome, and they provide new insight into molecular mechanisms of the process.

A parthenogenetic quasi-program causes teratoma-like tumors during aging in wild-type C. elegans

A long-standing belief is that aging (senescence) is the result of stochastic damage accumulation. Alternatively, senescent pathology may also result from late-life, wild-type gene action (i.e., antagonistic pleiotropy, as argued by Williams) leading to non-adaptive run-on of developmental programs (or quasi-programs) (as suggested more recently by Blagosklonny). In this study, we use existing and new data to show how uterine tumors, a prominent form of senescent pathology in the nematode Caenorhabditis elegans, likely result from quasi-programs. Such tumors develop from unfertilized oocytes which enter the uterus and become hypertrophic and replete with endoreduplicated chromatin masses. Tumor formation begins with ovulation of unfertilized oocytes immediately after exhaustion of sperm stocks. We show that the timing of this transition between program and quasi-program (i.e., the onset of senescence), and the onset of tumor formation, depends upon the timing of sperm depletion. We identify homology between uterine tumors and mammalian ovarian teratomas, which both develop from oocytes that fail to mature after meiosis I. In teratomas, futile activation of developmental programs leads to the formation of differentiated structures within the tumor. We report that older uterine tumors express markers of later embryogenesis, consistent with teratoma-like activation of developmental programs. We also present evidence of coupling of distal gonad atrophy to oocyte hypertrophy. This study shows how the Williams Blagosklonny model can provide a mechanistic explanation of this component of C. elegans aging. It also suggests etiological similarity between teratoma and some forms of senescent pathology, insofar as both are caused by quasi-programs.