Changes In Nuclear Size Research Articles

The Role of β-Dystroglycan in Nuclear Dynamics.

Dystroglycan is a ubiquitously expressed heterodimeric cell-surface laminin receptor with roles in cell adhesion, signalling, and membrane stabilisation. More recently, the transmembrane β-subunit of dystroglycan has been shown to localise to both the nuclear envelope and the nucleoplasm. This has led to the hypothesis that dystroglycan may have a structural role at the nuclear envelope analogous to its role at the plasma membrane. The biochemical fraction of myoblast cells clearly supports the presence of dystroglycan in the nucleus. Deletion of the dystroglycan protein by disruption of the DAG1 locus using CRISPR/Cas9 leads to changes in nuclear size but not overall morphology; moreover, the Young's modulus of dystroglycan-deleted nuclei, as determined by atomic force microscopy, is unaltered. Dystroglycan-disrupted myoblasts are also no more susceptible to nuclear stresses including chemical and mechanical, than normal myoblasts. Re-expression of dystroglycan in DAG1-disrupted myoblasts restores nuclear size without affecting other nuclear parameters.

Abstract A024: Ploidy as a predictive biomarker for gemcitabine sensitivity in triple-negative breast cancers

Abstract The polyploid giant cancer cell (PGCC) state is a common response of cancer cells to various stressors including chemotherapy, irradiation, hypoxia and viral infection. Upon stress, PGCCs adopt an endoreplication in which the genome replicates, mitosis is omitted, and cells grow in size, causing drug resistance. How accessible endoreplication is to a cell, therefore, directly translates to its resistance to therapy. In this study, we hypothesized that endoreplication and PGCC state are more accessible to cancer cells that already have a higher ploidy content prior to therapy. To test this hypothesis, we developed a comprehensive three-tier framework consisting of i) computational, ii) in-vitro and iii) in silico components. First, we designed a set of ordinary differential equations (ODEs) to mathematically model how cells enter and exit the PGCC state in response to a given stressor. We engineered how this in silico approach interacts with in vitro experiments into a broadly applicable software solution called CLONEID. The software uses computer vision to monitor phenotypic changes in cell and nuclear size from standard bright-field microscopy and classify cells into PGCC and non-PGCC states. We used CLONEID to test various therapeutic agents for their ability to select for a stable near-tetraploid (4N) population in a set of near-diploid (2N) cell lines. Through spontaneous cell fusions, we also obtained tetraploid breast cancer cells matched with their parental lines. Altogether, this framework enabled us to i) monitor the PGCC state experimentally in ii) two sets of matched isogenic cell lines with 2N and 4N DNA content to iii) model the successful entry and exit rates to and from the endoreplication state, respectively. As the first application of this framework, we tested the ability of our 2N and 4N TNBC lines (SUM159 and MDA-MB-231) to access the PGCC state upon treatment with 18 chemotherapy agents. Among those drugs, we observed that only gemcitabine caused continued cell growth without cell division in both tetraploid SUM159 and MDA-MB-231 cells whereas near-diploid parental lines were hypersensitive to the treatment. Consequently, tetraploid cancer cells continued to safely grow in the presence of gemcitabine. Furthermore, these PGCCs re-entered the proliferative cell cycle and grew in cell number when treatment is terminated. Gemcitabine-based chemotherapy is a standard treatment for patients with TNBC although its efficacy is limited mainly due to drug resistance. We expect our findings and three-component framework strategy to help stratify the TNBC patient population by their response to gemcitabine. In addition, our mathematical modelling approach has the promising potential to inform personalized dose optimization and to effectively decrease administered gemcitabine dose for a subset of patients, which would alleviate severe therapy-associated side effects and co-morbidities. Citation Format: Vural Tagal, Jackson Cole, Daria Miroshnychenko, Andriy Marusyk, Noemi Andor. Ploidy as a predictive biomarker for gemcitabine sensitivity in triple-negative breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A024.

Abstract LB175: Ploidy as a predictive biomarker for gemcitabine sensitivity in triple-negative breast cancers

Abstract Polyploid giant cancer (PGCC) state is a common response of cancer cells to various stressors including chemotherapy, irradiation, hypoxia and viral infection. Upon stress, PGCCs adopt an endoreplication in which the genome replicates, mitosis is omitted, and cells grow in size, leading to resistance. How accessible endoreplication is to a cell, therefore, directly translates to its resistance to therapy. In this study, we hypothesized that endoreplication and PGCC state are more plausible to cancer cells that already have a higher ploidy content prior to therapy. To test this hypothesis, we developed a comprehensive three-tier framework consisting of i) computational, ii) in vitro and iii) in silico components. We designed a set of ordinary differential equations (ODEs) to model how cells enter and exit the PGCC state in response to a given stressor. We engineered how this in silico approach interacts with in vitro experiments into a broadly applicable software solution called CLONEID. The software uses computer vision to monitor phenotypic changes in cell and nuclear size from standard bright-field microscopy and classify cells into PGCC and non-PGCC states. We used CLONEID to test various therapeutic agents for their ability to select for a stable near-tetraploid population in a set of near-diploid cell lines. Through spontaneous cell fusions, we also obtained tetraploid breast cancer cells matched with their parental lines. Altogether, this framework, now, enables us i) to monitor the PGCC state experimentally ii) in matched cell lines with differential DNA content iii) to model the successful entrance and exit rates to and from the endoreplication state, respectively. As the first application of this framework, we tested the ability of our matched triple-negative breast cancer (TNBC) lines (SUM-159 and MDA-MB-231) to access the PGCC state upon treatment with 18 commonly-used chemotherapy agents. We observed that gemcitabine caused continued cell growth without cell division in both tetraploid SUM-159 and MDA-MB-231 cells whereas near-diploid parental cells were hypersensitive to the treatment. Consequently, tetraploid cancer cells continued to safely grow in the presence of gemcitabine. Furthermore, these PGCCs re-entered the proliferative cell cycle and grew in cell number when treatment is terminated. Gemcitabine (GEM)-based chemotherapy is a standard treatment for patients with TNBC although its efficacy is limited mainly due to drug resistance. Moreover, such poor response rate is coupled with severe side effects that frequently leads to the failure of vital organs, serious secondary diseases, and co-morbidities. Our findings suggest that ploidy is a predictive biomarker for gemcitabine sensitivity. Thus, we expect our findings and three-component framework strategy to help stratify the TNBC patient population by their response to gemcitabine. In addition, our mathematical modelling approach has the promising potential to inform personalized dose optimization and to effectively decrease administered gemcitabine dose for a subset of patients, which would alleviate therapy-associated side effects and lethalities. Citation Format: Vural Tagal, Jackson P. Cole, Daria Miroshnychenko, Andriy Marusyk, Noemi Andor. Ploidy as a predictive biomarker for gemcitabine sensitivity in triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB175.

Region-specific heterogeneity in neuronal nuclear morphology in young, aged and in Alzheimer's disease mouse brains.

Neurons in the mammalian brain exhibit enormous structural and functional diversity across different brain regions. Compared to our understanding of the morphological diversity of neurons, very little is known about the heterogeneity of neuronal nuclear morphology and how nuclear size changes in aging and diseased brains. Here, we report that the neuronal cell nucleus displays differences in area, perimeter, and circularity across different anatomical regions in the mouse brain. The pyramidal neurons of the hippocampal CA3 region exhibited the largest area whereas the striatal neuronal nuclei were the smallest. These nuclear size parameters also exhibited dichotomous changes with age across brain regions-while the neocortical and striatal neurons showed a decrease in nuclear area and perimeter, the CA3 neurons showed an increase with age. The nucleus of parvalbumin- and calbindin-positive interneurons had comparable morphological features but exhibited differences between brain regions. In the context of activity-dependent transcription in response to a novel environment, there was a decrease in nuclear size and circularity in c-Fos expressing neurons in the somatosensory cortex and hippocampal CA1 and CA3. In an APP/PS1 mutant mouse model of Alzheimer's disease (AD), the neuronal nuclear morphology varies with plaque size and with increasing distance from the plaque. The neuronal nuclear morphology in the immediate vicinity of the plaque was independent of the plaque size and the morphology tends to change away from the plaque. These changes in the neuronal nuclear size and shape at different ages and in AD may be attributed to changes in transcriptional activity. This study provides a detailed report on the differences that exist between neurons in nuclear morphology and can serve as a basis for future studies.

Nuclear size rectification: A potential new therapeutic approach to reduce metastasis in cancer.

Research on metastasis has recently regained considerable interest with the hope that single cell technologies might reveal the most critical changes that support tumor spread. However, it is possible that part of the answer has been visible through the microscope for close to 200 years. Changes in nuclear size characteristically occur in many cancer types when the cells metastasize. This was initially discarded as contributing to the metastatic spread because, depending on tumor types, both increases and decreases in nuclear size could correlate with increased metastasis. However, recent work on nuclear mechanics and the connectivity between chromatin, the nucleoskeleton, and the cytoskeleton indicate that changes in this connectivity can have profound impacts on cell mobility and invasiveness. Critically, a recent study found that reversing tumor type-dependent nuclear size changes correlated with reduced cell migration and invasion. Accordingly, it seems appropriate to now revisit possible contributory roles of nuclear size changes to metastasis.

Loops, topologically associating domains, compartments, and territories are elastic and robust to dramatic nuclear volume swelling

Layers of genome organization are becoming increasingly better characterized, but less is known about how these structures respond to perturbation or shape changes. Low-salt swelling of isolated chromatin fibers or nuclei has been used for decades to investigate the structural properties of chromatin. But, visible changes in chromatin appearance have not been linked to known building blocks of genome structure or features along the genome sequence. We combine low-salt swelling of isolated nuclei with genome-wide chromosome conformation capture (Hi-C) and imaging approaches to probe the effects of chromatin extension genome-wide. Photoconverted patterns on nuclei during expansion and contraction indicate that global genome structure is preserved after dramatic nuclear volume swelling, suggesting a highly elastic chromosome topology. Hi-C experiments before, during, and after nuclear swelling show changes in average contact probabilities at short length scales, reflecting the extension of the local chromatin fiber. But, surprisingly, during this large increase in nuclear volume, there is a striking maintenance of loops, TADs, active and inactive compartments, and chromosome territories. Subtle differences after expansion are observed, suggesting that the local chromatin state, protein interactions, and location in the nucleus can affect how strongly a given structure is maintained under stress. From these observations, we propose that genome topology is robust to extension of the chromatin fiber and isotropic shape change, and that this elasticity may be beneficial in physiological circumstances of changes in nuclear size and volume.

Chemical Interrogation of Nuclear Size Identifies Compounds with Cancer Cell Line-Specific Effects on Migration and Invasion.

Background: Lower survival rates for many cancer types correlate with changes in nuclear size/scaling in a tumor-type/tissue-specific manner. Hypothesizing that such changes might confer an advantage to tumor cells, we aimed at the identification of commercially available compounds to guide further mechanistic studies. We therefore screened for Food and Drug Administration (FDA)/European Medicines Agency (EMA)-approved compounds that reverse the direction of characteristic tumor nuclear size changes in PC3, HCT116, and H1299 cell lines reflecting, respectively, prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. Results: We found distinct, largely nonoverlapping sets of compounds that rectify nuclear size changes for each tumor cell line. Several classes of compounds including, e.g., serotonin uptake inhibitors, cyclo-oxygenase inhibitors, β-adrenergic receptor agonists, and Na+/K+ ATPase inhibitors, displayed coherent nuclear size phenotypes focused on a particular cell line or across cell lines and treatment conditions. Several compounds from classes far afield from current chemotherapy regimens were also identified. Seven nuclear size-rectifying compounds selected for further investigation all inhibited cell migration and/or invasion. Conclusions: Our study provides (a) proof of concept that nuclear size might be a valuable target to reduce cell migration/invasion in cancer treatment and (b) the most thorough collection of tool compounds to date reversing nuclear size changes specific to individual cancer-type cell lines. Although these compounds still need to be tested in primary cancer cells, the cell line-specific nuclear size and migration/invasion responses to particular drug classes suggest that cancer type-specific nuclear size rectifiers may help reduce metastatic spread.

Nuclear Transport Factor 2 (NTF2) suppresses WM983B metastatic melanoma by modifying cell migration, metastasis, and gene expression

While changes in nuclear structure and organization are frequently observed in cancer cells, relatively little is known about how nuclear architecture impacts cancer progression and pathology. To begin to address this question, we studied Nuclear Transport Factor 2 (NTF2) because its levels decrease during melanoma progression. We show that increasing NTF2 expression in WM983B metastatic melanoma cells reduces cell proliferation and motility while increasing apoptosis. We also demonstrate that increasing NTF2 expression in these cells significantly inhibits metastasis and prolongs survival of mice. NTF2 levels affect the expression and nuclear positioning of a number of genes associated with cell proliferation and migration, and increasing NTF2 expression leads to changes in nuclear size, nuclear lamin A levels, and chromatin organization. Thus, ectopic expression of NTF2 in WM983B metastatic melanoma abrogates phenotypes associated with advanced stage cancer both in vitro and in vivo, concomitantly altering nuclear and chromatin structure and generating a gene expression profile with characteristics of primary melanoma. We propose that NTF2 is a melanoma tumor suppressor and could be a novel therapeutic target to improve health outcomes of melanoma patients.

Prognostic value of proliferation, PD-L1 and nuclear size in patients with superior sulcus tumours treated with chemoradiotherapy and surgery

AimsThe aim of this study was to determine the relationship between proliferative activity, PD-L1 status and nuclear size changes after preoperative chemoradiotherapy (CRT) and the clinical outcome in patients with...

The Transcription Factor Sox2 Is Required to Maintain the Cell Type-Specific Properties and Innervation of Type II Vestibular Hair Cells in Adult Mice.

The sense of balance relies on vestibular hair cells, which detect head motions. Mammals have two types of vestibular hair cell, I and II, with unique morphological, molecular, and physiological properties. Furthermore, each hair cell type signals to a unique form of afferent nerve terminal. Little is known about the mechanisms in mature animals that maintain the specific features of each hair cell type or its postsynaptic innervation. We found that deletion of the transcription factor Sox2 from Type II hair cells in adult mice of both sexes caused many cells in utricles to acquire features unique to Type I hair cells and to lose Type II-specific features. This cellular transdifferentiation, which included changes in nuclear size, chromatin condensation, soma and stereocilium morphology, and marker expression, resulted in a significantly higher proportion of Type I-like hair cells in all epithelial zones. Furthermore, Sox2 deletion from Type II hair cells triggered non-cell autonomous changes in vestibular afferent neurons; they retracted bouton terminals (normally present on only Type II cells) from transdifferentiating hair cells and replaced them with a calyx terminal (normally present on only Type I cells). These changes were accompanied by significant expansion of the utricle's central zone, called the striola. Our study presents the first example of a transcription factor required to maintain the type-specific hair cell phenotype in adult inner ears. Furthermore, we demonstrate that a single genetic change in Type II hair cells is sufficient to alter the morphology of their postsynaptic partners, the vestibular afferent neurons.SIGNIFICANCE STATEMENT The sense of balance relies on two types of sensory cells in the inner ear, Type I and Type II hair cells. These two cell types have unique properties. Furthermore, their postsynaptic partners, the vestibular afferent neurons, have differently shaped terminals on Type I versus Type II hair cells. We show that the transcription factor Sox2 is required to maintain the cell-specific features of Type II hair cells and their postsynaptic terminals in adult mice. This is the first evidence of a molecule that maintains the phenotypes of hair cells and, non-cell autonomously, their postsynaptic partners in mature animals.

Accumulative Deformation Characteristics and Microstructure of Saturated Soft Clay under Cross-River Subway Loading.

The cross-river subway in the Hangzhou Bay area often passes through deep, thick, soft soil at the bottom of the river. At the same time, overlying erosion, siltation, and changes in water levels adversely affect the deformation of the subway, thereby causing hidden dangers to its safe operation. Using two-way dynamic triaxial testing, the effects of cyclic loading of the cross-river subway on the soft clay foundation were investigated for the first time, using simulation methodology as the prime objective of the present study. A strain development curve for the soft clay was obtained as a result. Considering the effects of effective confining pressure (p′) and radial cyclic stress ratio (τr), an explicit model of accumulative strain on soft clay under cyclic loading of the cross-river subway was established. The results showed that the accumulative axial strain (εd) was closely related to p′ and τr. Under certain conditions, as p′ and τr increased, the εd produced by the soil tended to decrease. Furthermore, through non-destructive testing based on nuclear magnetic resonance (NMR), pore distribution and pore size changes in soft clay during cyclic loading were analyzed. It was observed that under the action of the cross-river loading, the large internal soft clay pores were transformed into small pores, which manifested as a significant decrease in the number of large pores and an increase in the proportion of small pores. Lastly, the macroscopic dynamic soil characteristics observed during triaxial testing closely correlated with the microscopic pore size of the soil obtained in the NMR test, which indicated that using pore distribution and pore size changes to describe microscopic changes was a valid method.

Synthesis of Novel Structural Hybrids between Aza-Heterocycles and Azelaic Acid Moiety with a Specific Activity on Osteosarcoma Cells.

Nine compounds bearing pyridinyl (or piperidinyl, benzimidazolyl, benzotriazolyl) groups bound to an azelayl moiety through an amide bond were synthesized. The structural analogy with some histone deacetylase inhibitors inspired their syntheses, seeking new selective histone deacetylase inhibitors (HDACi). The azelayl moiety recalls part of 9-hydroxystearic acid, a cellular lipid showing antiproliferative activity toward cancer cells with HDAC as a molecular target. Azelayl derivatives bound to a benzothiazolyl moiety further proved to be active as HDACi. The novel compounds were tested on a panel of both normal and tumor cell lines. Non-specific induction of cytotoxicity was observed in the normal cell line, while three of them induced a biological effect only on the osteosarcoma (U2OS) cell line. One of them induced a change in nuclear shape and size. Cell-cycle alterations are associated with post-transcriptional modification of both H2/H3 and H4 histones. In line with recent studies, revealing unexpected HDAC7 function in osteoclasts, molecular docking studies on the active molecules predicted their proneness to interact with HDAC7. By reducing side effects associated with the action of the first-generation inhibitors, the herein reported compounds, thus, sound promising as selective HDACi.

Evaluation of the relation between Lens Opacities Classification System III grading and nuclear size by direct measurement.

CONTEXT:Although relation between Lens Opacities Classification System III (LOCS III) and nuclear density is established, no data are available about nuclear size at different LOCS III grades.AIMS:The aim of this study is to evaluate the relation between LOCS III grading of nuclear opacity obtained preoperatively and the size of the nucleus obtained during cataract surgery.SETTINGS AND DESIGN:This was a prospective observational study carried out in a hospital attached to medical college.MATERIALS AND METHODS:Patients who underwent manual small-incision cataract surgery or extra-large temporal tunnel cataract extraction and gave consent were included in this study. Institutional Ethics Committee clearance was taken for the study. Preoperative LOCS III grading was obtained at slit-lamp biomicroscope. Ocular dimensions were obtained by preoperative immersion biometry. The thickness and diameter of the nucleus obtained by extraction were measured up to 10 μ accuracy. Data were analyzed for the change in nuclear thickness, nuclear diameter, age, lens thickness, and anterior chamber depth in relation to the LOCS III grade of the nucleus.STATISTICAL ANALYSIS USED:Statistical analysis used in this study was one-way ANOVA, mean, and range.RESULTS:There was a significant increase (P < 0.05) in nuclear thickness, nuclear diameter, and age with increasing LOCS III grade of the nucleus. The change in nuclear size was linear between Grades 1 and 4. The nuclear size did not increase between Grades 4 and 5. It increased steeply from Grade 5 to Grade 6.9.CONCLUSION:LOCS III grading of the nucleus can be utilized for determining the nuclear thickness and diameter preoperatively. These data can be helpful in adjusting machine parameters during phacoemulsification.

Cell adhesion and culture medium dependent changes in the high frequency mechanical vibration induced proliferation, osteogenesis, and intracellular organization of human adipose stem cells

High frequency (HF) mechanical vibration appears beneficial for in vitro osteogenesis of mesenchymal stem cells (MSCs). However, the current mechanobiological understanding of the method remains insufficient. We designed high-throughput stimulators to apply horizontal or vertical high magnitude HF (HMHF; 2.5 Gpeak, 100 Hz) vibration on human adipose stem cells (hASCs). We analyzed proliferation, alkaline phosphatase (ALP) activity, mineralization, and effects on the actin cytoskeleton and nuclei using immunocytochemical stainings. Proliferation was studied on a standard tissue culture plastic (sTCP) surface and on an adhesion supporting tissue culture plastic (asTCP) surface in basal (BM) and osteogenic (OM) culture medium conditions. We discovered that the improved cell adhesion was a prerequisite for vibration induced changes in the proliferation of hASCs. Similarly, the adhesion supporting surface enabled us to observe vibration initiated ALP activity and mineralization changes in OM condition. The horizontal vibration increased ALP activity, while vertical stimulation reduced ALP activity. However, mineralization was not enhanced by the HMHF vibration. We performed image-based analysis of actin and nuclei to obtain novel data of the intracellular-level responses to HF vibration in BM and OM conditions. Our quantitative results suggest that actin organizations were culture medium and stimulation direction dependent. Both stimulation directions decreased OM induced changes in nuclear size and elongation. Consequently, our findings of the nuclear deformations provide supportive evidence for the involvement of the nuclei in the mechanocoupling of HF vibration. Taken together, the results of this study enhanced the knowledge of the intracellular mechanisms of HF vibration induced osteogenesis of MSCs.

Cell migration through three-dimensional confining pores: speed accelerations by deformation and recoil of the nucleus.

Directional cell migration in dense three-dimensional (3D) environments critically depends upon shape adaptation and is impeded depending on the size and rigidity of the nucleus. Accordingly, the nucleus is primarily understood as a physical obstacle; however, its pro-migratory functions by stepwise deformation and reshaping remain unclear. Using atomic force spectroscopy, time-lapse fluorescence microscopy and shape change analysis tools, we determined the nuclear size, deformability, morphology and shape change of HT1080 fibrosarcoma cells expressing the Fucci cell cycle indicator or being pre-treated with chromatin-decondensating agent TSA. We show oscillating peak accelerations during migration through 3D collagen matrices and microdevices that occur during shape reversion of deformed nuclei (recoil), and increase with confinement. During G1 cell-cycle phase, nucleus stiffness was increased and yielded further increased speed fluctuations together with sustained cell migration rates in confinement when compared to interphase populations or to periods of intrinsic nuclear softening in the S/G2 cell-cycle phase. Likewise, nuclear softening by pharmacological chromatin decondensation or after lamin A/C depletion reduced peak oscillations in confinement. In conclusion, deformation and recoil of the stiff nucleus contributes to saltatory locomotion in dense tissues.This article is part of a discussion meeting issue ‘Forces in cancer: interdisciplinary approaches in tumour mechanobiology’.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/β-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas.

The intracellular accommodation of arbuscular mycorrhizal (AM) fungi is a paradigmatic feature of this plant symbiosis that depends on the activation of a dedicated signaling pathway and the extensive reprogramming of host cells, including striking changes in nuclear size and transcriptional activity. By combining targeted sampling of early root colonization sites, detailed confocal imaging, flow cytometry and gene expression analyses, we demonstrate that local, recursive events of endoreduplication are triggered in the Medicago truncatula root cortex during AM colonization. AM colonization induces an increase in ploidy levels and the activation of endocycle specific markers. This response anticipates the progression of fungal colonization and is limited to arbusculated and neighboring cells in the cortical tissue. Furthermore, endoreduplication is not induced in M.truncatula mutants for symbiotic signaling pathway genes. On this basis, we propose endoreduplication as part of the host cell prepenetration responses that anticipate AM fungal accommodation in the root cortex.

Nuclear size changes caused by local motion of cell boundaries unfold the nuclear lamina and dilate chromatin and intranuclear bodies.

The mechanisms by which mammalian nuclear shape and size are established in cells, and become abnormal in disease states are not understood. Here, we tracked motile cells that underwent systematic changes in cell morphology as they moved from 1-D to 2-D micro-patterned adhesive domains. Motion of the cell boundaries during cell motility caused a dynamic and systematic change in nuclear volume. Short time scales (∼1 h) distinguished the dilation of the nucleus from the familiar increase that occurs during the cell cycle. Nuclear volume was systematically different between cells cultured in 3-D, 2-D and 1-D environments. Dilation of the nuclear volume was accompanied by dilation of chromatin, a decrease in the number of folds in the nuclear lamina, and an increase in nucleolar volume. Treatment of 2-D cells with non-muscle myosin-II inhibitors decreased cell volume, and proportionately caused a decrease in nuclear volume. These data suggest that nuclear size changes during cell migration may potentially impact gene expression through the modulation of intranuclear structure.

Persistent Erectile Dysfunction after Discontinuation of 5-Alpha Reductase Inhibitor Therapy in Rats Depending on the Duration of Treatment.

PurposeThe current study is aimed to assess whether a longer duration of 5α-reductase inhibitor (5α-RI) exposure was associated with higher rate of permanent erectile dysfunction (ED) in a rat model.Materials and MethodsMale Sprague-Dawley rats (n=76) were assigned to five groups: (i) normal control group; (ii) dutasteride (0.5 mg/rat/d) for 4-weeks group; (iii) dutasteride for 4-weeks plus 2-weeks of resting group; (iv) dutasteride for 8-weeks group; and (v) dutasteride for 8-weeks plus 2-weeks of resting group. In vivo erectile responses to electrical stimulation, and changes of fibrotic factors and smooth muscle/collagen contents in the corpus cavernosum were evaluated in each group.ResultsDutasteride administration for 4 and 8 weeks significantly decreased erectile parameters compared with the control group. Reduced erectile responses were recovered during 2 weeks of drug-free time in the 4-week treatment group, but were not in the 8-week group. Protein levels of fibrosis-related factors transforming growth factor (TGF)-β1, TGF-β2, and p-Smad/Smad (Smad 2/3) in the corpus cavernosum showed no significant change after 4 weeks of dutasteride oral administration, but were enhanced after 8 weeks. Dutasteride markedly decreased smooth muscle content and increased collagen after 4 and 8 weeks of use, but no nuclear size changes; however, neither group showed significant improvement in the smooth muscle to collagen ratio after the rest period.ConclusionsOur study showed that recovery from ED depended on the duration of medication, and administration of dutasteride for more than 8-weeks in rats could result in irreversible ED even after discontinuation of medication.

Lung-Specific Extracellular Superoxide Dismutase Improves Cognition of Adult Mice Exposed to Neonatal Hyperoxia.

Lung and brain development is often altered in infants born preterm and exposed to excess oxygen, and this can lead to impaired lung function and neurocognitive abilities later in life. Oxygen-derived reactive oxygen species and the ensuing inflammatory response are believed to be an underlying cause of disease because over-expression of some anti-oxidant enzymes is protective in animal models. For example, neurodevelopment is preserved in mice that ubiquitously express human extracellular superoxide dismutase (EC-SOD) under control of an actin promoter. Similarly, oxygen-dependent changes in lung development are attenuated in transgenic SftpcEC−SOD mice that over-express EC-SOD in pulmonary alveolar epithelial type II cells. But whether anti-oxidants targeted to the lung provide protection to other organs, such as the brain is not known. Here, we use transgenic SftpcEC−SOD mice to investigate whether lung-specific expression of EC-SOD also preserves neurodevelopment following exposure to neonatal hyperoxia. Wild type and SftpcEC−SOD transgenic mice were exposed to room air or 100% oxygen between postnatal days 0–4. At 8 weeks of age, we investigated neurocognitive function as defined by novel object recognition, pathologic changes in hippocampal neurons, and microglial cell activation. Neonatal hyperoxia impaired novel object recognition memory in adult female but not male mice. Behavioral deficits were associated with microglial activation, CA1 neuron nuclear contraction, and fiber sprouting within the hilus of the dentate gyrus (DG). Over-expression of EC-SOD in the lung preserved novel object recognition and reduced the observed changes in neuronal nuclear size and myelin basic protein fiber density. It had no effect on the extent of microglial activation in the hippocampus. These findings demonstrate pulmonary expression of EC-SOD preserves short-term memory in adult female mice exposed to neonatal hyperoxia, thus suggesting anti-oxidants designed to alleviate oxygen-induced lung disease such as in preterm infants may also be neuroprotective.