A deep hybrid learning pipeline for accurate diagnosis of ovarian cancer based on nuclear morphology.

Nuclear morphological features are potent determining factors for clinical diagnostic approaches adopted by pathologists to analyze the malignant potential of cancer cells. Considering the structural alteration of the nucleus in cancer cells, various groups have developed machine learning techniques based on variation in nuclear morphometric information like nuclear shape, size, nucleus-cytoplasm ratio and various non-parametric methods like deep learning have also been tested for analyzing immunohistochemistry images of tissue samples for diagnosing various cancers. We aim to correlate the morphometric features of the nucleus along with the distribution of nuclear lamin proteins with classical machine learning to differentiate between normal and ovarian cancer tissues. It has already been elucidated that in ovarian cancer, the extent of alteration in nuclear shape and morphology can modulate genetic changes and thus can be utilized to predict the outcome of low to a high form of serous carcinoma. In this work, we have performed exhaustive imaging of ovarian cancer versus normal tissue and developed a dual pipeline architecture that combines the matrices of morphometric parameters with deep learning techniques of auto feature extraction from pre-processed images. This novel Deep Hybrid Learning model, though derived from classical machine learning algorithms and standard CNN, showed a training and validation AUC score of 0.99 whereas the test AUC score turned out to be 1.00. The improved feature engineering enabled us to differentiate between cancerous and non-cancerous samples successfully from this pilot study.

DNA content contributes to nuclear size control in Xenopus laevis.

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.

Control of cellular organization and its coordination with the cell cycle.

Cells organize themselves to maintain proper shape, structure, and size during growth and division for their cellular functions. However, how these cellular organizations coordinate with the cell cycle is not well understood. This review focuses on cell morphogenesis and size of the membrane-bound nucleus in the fission yeast Schizosaccharomyces pombe. Growth polarity, an important factor for cell morphogenesis, in rod-shaped fission yeast is restricted to the cell tips and dynamically changes depending on the cell cycle stage. Furthermore, nuclear size in fission yeast is proportional to the cell size, resulting in a constant ratio between nuclear volume and cellular volume (N/C ratio). This review summarizes the signaling pathway(s) involved in growth polarity control and key factors involved in N/C ratio control and provides their roles in coordination between cell organization and the cell cycle.

Constitutive expression of a fluorescent protein reports the size of live human cells.

Cell size is important for cell physiology because it sets the geometric scale of organelles and biosynthesis. A number of methods exist to measure different aspects of cell size, but each has significant drawbacks. Here, we present an alternative method to measure the size of single human cells using a nuclear localized fluorescent protein expressed from a constitutive promoter. We validate this method by comparing it to several established cell size measurement strategies, including flow cytometry optical scatter, total protein dyes, and quantitative phase microscopy. We directly compare our fluorescent protein measurement with the commonly used measurement of nuclear volume and show that our measurements are more robust and less dependent on image segmentation. We apply our method to examine how cell size impacts the cell division cycle and reaffirm that there is a negative correlation between size at cell birth and G1 duration. Importantly, combining our size reporter with fluorescent labeling of a different protein in a different color channel allows measurement of concentration dynamics using simple wide-field fluorescence imaging. Thus, we expect our method will be of use to researchers interested in how dynamically changing protein concentrations control cell fates.

Unravelling nuclear size control.

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin α in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

Proliferation of Cells with Severe Nuclear Deformation on a Micropillar Array.

Cellular responses on a topographic surface are fundamental topics about interfaces and biology. Herein, a poly(lactide- co-glycolide) (PLGA) micropillar array was prepared and found to trigger significant self-deformation of cell nuclei. The time-dependent cell viability and thus cell proliferation was investigated. Despite significant nuclear deformation, all of the examined cell types (Hela, HepG2, MC3T3-E1, and NIH3T3) could survive and proliferate on the micropillar array yet exhibited different proliferation abilities. Compared to the corresponding groups on the smooth surface, the cell proliferation abilities on the micropillar array were decreased for Hela and MC3T3-E1 cells and did not change significantly for HepG2 and NIH3T3 cells. We also found that whether the proliferation ability changed was related to whether the nuclear sizes decreased in the micropillar array, and thus the size deformation of cell nuclei should, besides shape deformation, be taken into consideration in studies of cells on topological surfaces.

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration.

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.

PKC-mediated phosphorylation of nuclear lamins at a single serine residue regulates interphase nuclear size in Xenopus and mammalian cells.

How nuclear size is regulated is a fundamental cell-biological question with relevance to cancers, which often exhibit enlarged nuclei. We previously reported that conventional protein kinase C (cPKC) contributes to nuclear size reductions that occur during early Xenopus development. Here we report that PKC-mediated phosphorylation of lamin B3 (LB3) contributes to this mechanism of nuclear size regulation. By mapping PKC phosphorylation sites on LB3 and testing the effects of phosphomutants in Xenopus laevis embryos, we identify the novel site S267 as being an important determinant of nuclear size. Furthermore, FRAP studies demonstrate that phosphorylation at this site increases lamina dynamics, providing a mechanistic explanation for how PKC activity influences nuclear size. We subsequently map this X. laevis LB3 phosphorylation site to a conserved site in mammalian lamin A (LA), S268. Manipulating PKC activity in cultured mammalian cells alters nuclear size, as does expression of LA-S268 phosphomutants. Taken together, these data demonstrate that PKC-mediated lamin phosphorylation is a conserved mechanism of nuclear size regulation.

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed-ended and open-ended learning.

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience-dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open-ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin-positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed-ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin-positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed-ended and open-ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975-994, 2017.

Use of Xenopus cell-free extracts to study size regulation of subcellular structures.

Striking size variations are prominent throughout biology, at the organismal, cellular, and subcellular levels. Important fundamental questions concern organelle size regulation and how organelle size is regulated relative to cell size, also known as scaling. Uncovering mechanisms of organelle size regulation will inform the functional significance of size as well as the implications of misregulated size, for instance in the case of nuclear enlargement in cancer. Xenopus egg and embryo extracts are powerful cell-free systems that have been utilized extensively for mechanistic and functional studies of various organelles and subcellular structures. The open biochemical nature of the extract permits facile manipulation of its composition, and in recent years extract approaches have illuminated mechanisms of organelle size regulation. This review largely focuses on in vitro Xenopus studies that have identified regulators of nuclear and spindle size. We also discuss potential relationships between size scaling of the nucleus and spindle, size regulation of other subcellular structures, and extract experiments that have clarified developmental timing mechanisms. We conclude by offering some future prospects, notably the integration of Xenopus extract with microfluidic technology.

Uniform Contraction-Expansion Description of Relative Centromere and Telomere Motion.

Internal organization and dynamics of the eukaryotic nucleus have been at the front of biophysical research in recent years. It is believed that both dynamics and location of chromatin segments are crucial for genetic regulation. Here we study the relative motion between centromeres and telomeres at various distances and at times relevant for genetic activity. Using live-imaging fluorescent microscopy coupled to stochastic analysis of relative trajectories, we find that the interlocus motion is distance-dependent with a varying fractional memory. In addition to short-range constraining, we also observe long-range anisotropic-enhanced parallel diffusion, which contradicts the expectation for classic viscoelastic systems. This motion is linked to uniform expansion and contraction of chromatin in the nucleus, and leads us to define and measure a new (to our knowledge) uniform contraction-expansion diffusion coefficient that enriches the contemporary picture of nuclear behavior. Finally, differences between loci types suggest that different sites along the genome experience distinctive coupling to the nucleoplasm environment at all scales.

Utility of nuclear morphometry in effusion cytology.

BACKGROUND: The cytological analysis of serous effusions is a common investigation and yields important diagnostic information. However, the distinction of reactive mesothelial cells from malignant cells can sometimes be difficult for the cytopathologist. Hence cost-effective ancillary methods are essential to enhance the accuracy of cytological diagnosis. The aim of this study was to examine the utility of nuclear morphometry in differentiating reactive mesothelial cells from malignant cells in effusion smears. MATERIALS AND METHODS: Sixty effusion smears consisting of 30 effusions cytologically classified as malignant (adenocarcinomas) and 30 benign effusions showing reactive mesothelial cells were included in the study. ImageJ was used to measure the nuclear area, perimeter, maximal feret diameter, minimal feret diameter and the circularity. A total of ten representative cells were studied in each case. RESULTS: Significant differences were found between benign and malignant effusions for the nuclear area, perimeter, maximal feret diameter and minimal feret diameter. No significant difference was found for circularity, a shape descriptor. Receiver operating characteristic (ROC) curve analysis revealed that nuclear area, perimeter, maximal feret diameter, and minimal feret diameter are helpful in discriminating benign and malignant effusions. CONCLUSIONS: Computerised nuclear morphometry is a helpful ancillary technique to distinguish benign and malignant effusions. ImageJ is an excellent cost effective tool with potential diagnostic utility in effusion cytology.

Accelerated telomere reduction and hepatocyte senescence in tolerated human liver allografts.

BACKGROUND: In living donor liver transplantation, the biological organ age of the donated allograft is unknown in young patients who receive grafts from older donors. Few studies have focused on the effects of aging on allografts in the state of tolerance. The purpose of this study was to assess the biological organ age of liver grafts. METHODS: In 20 tolerated allografts over a 10-year post-transplant follow-up period, the relative telomere lengths were measured by multiplex quantitative polymerase chain reaction, and hepatocyte nuclear size and cell cycle phase markers were determined by immunohistochemistry. The results were compared with the same measurements that had been obtained prior to transplantation in the recipients' pre-implantation donor livers. Tolerance was defined strictly as a condition in which the allograft functioned normally and showed normal histology without any histological signs of rejection, fibrosis or inflammation in the absence of immunosuppression. RESULTS: First, telomere length correlated with chronological donor age (n=41). Accelerated telomere reduction was seen in tolerated grafts compared with the predicted telomere length of each allograft calculated from the regression line of donor livers. Tolerated grafts were associated with higher hepatocyte p21 expression and greater nuclear area than in the donor livers prior to transplantation. CONCLUSIONS: These findings suggest that allografts age more rapidly than in the normal population, and that grafts may reach the limit of proliferative capacity even in the state of tolerance.

A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype.

Tribbles related protein 3 (TRB3) pseudokinase plays a crucial role in cell proliferation, migration and morphogenesis during development. In our recent study, an introduction of human TRB3 gene into mouse mammary tumor cells caused an increase of proliferation of tumor cells and their nuclear size. In the current study, to examine whether this gene causes de novo morphological changes in a specific organ site we have developed a novel variation of the transgenic mouse model that conditionally expresses human TRB3 (hTRB3) gene using Cre-recombinase (Cre)/loxP recombination system. By injecting hTRB3 transgene construct into pronuclei of mouse embryo, we eventually obtained four hTRB3 mice. The gene expression was controlled by infection of adenovirus-expressing Cre via the tail vein of hTRB3 mouse. In Cre-mediated hTRB3 mouse, expression of the hTRB3 protein was detected in the cytoplasm of hepatocytes in the liver. Expression of this protein was also seen in lymphocytes in the spleen, glomerular endothelial cells, and epithelial cells of collecting duct of the kidney. In hepatocytes of the hTRB3 mouse, nuclear size was significantly greater than that of the wild type mouse, indicating that hTRB3 can play a role at least in part in hepatic morphogenesis. The present animal model may provide a system for evaluation of de novo morphological changes induced by a specific transgene in a specific organ site.

Comparison of morphology of bulbar conjunctival cells assessed by impression cytology versus scrape and smear methods.

PURPOSE: To assess the morphology of superficial bulbar conjunctival cells collected by impression cytology or by scraping. METHODS: Freshly procured sheep eyes from a slaughterhouse were washed and then conjunctival impression cytology (CIC) samples taken from the nasal aspect of the bulbar conjunctiva on a Biopore Millicell filter unit, or cells scraped from the same location and smeared onto glass slides. After alcohol fixation, all samples were stained with Giemsa. Cell images were obtained by light microscopy at 200× magnification, and subjected to morphometry. RESULTS: Cells obtained by scrape/smear processing appeared larger and less densely staining than cells procured by CIC. This appearance was confirmed by morphometry with measures of cell longest dimension LONG being 27.7 ± 1.3 µm and 21.7 ± 1.5 µm, respectively, while cell AREAC measures averaged 543 ± 42 µm(2) and 333 ± 51 µm(2); these differences were significant (p < 0.001). Similarly, cell nucleus length (NUCLONG) and nucleus area (AREANUC) were also larger in smeared cells (p < 0.001). Various calculations of nucleo-cytoplasmic ratios as based on dimensions or area were only slightly different when comparing cells obtained by the two methods, indicating that the glass slide-smeared cells simply spread out more than cells on the CIC filters. CONCLUSIONS: Differences in cell and nucleus size can result from use of different processing methods. Therefore, in making comparisons between sets of conjunctival cells, it is important that cell collection and processing methods be considered.

Scaling the primate lateral geniculate nucleus: niche and neurodevelopment in the regulation of magnocellular and parvocellular cell number and nucleus volume.

New stereological assessments of lateral geniculate nucleus (LGN) neuron numbers and volumes in five New World primates (Cebus apella, Saguinus midas niger, Alouatta caraya, Aotus azarae, and Callicebus moloch) and compiled LGN volumes for an additional 26 mammals were analyzed for a better understanding of visual system evolution. Both the magnocellular (M)- and the parvocellular (P)-cell populations scale allometrically with brain volume in primates, P cells with a significantly higher slope such that, for every increase in M neuron number, P neuron numbers more than double (ln scale; y = 0.89x + 2.42R(2) = 0.664). In diurnal primates, the ratio of P to M cells was slightly but significantly higher than in nocturnal primates. For all mammals, including primates, LGN volume was unrelated to nocturnal or diurnal niche but showed marked differences in slope and intercept depending on taxonomic group. The allometric scaling of M and P cells can be related to the order of neurogenesis, with late-generated P cells increasing with positive allometry compared with the earlier-generated M cells. This developmental regularity links relative foveal representation to relative isocortex enlargement, which is also generated late. The small increase in the P/M cell ratio in diurnal primates may result from increased developmental neuron loss in the M-cell population as it competes for limited termination zones in primary visual cortex.

Volumen nuclear de hepatocitos de hígados sometidos a un factor estimulante humoral en ratones isogénicos linea IOR / Nuclear volume of hepatocytes in livers subjected to a humoral stimulating factor in isogenic rats, IOR line

Se estudió el comportamiento del volumen nuclear de los hepatocitos en 14 muestras del lóbulo medio de hígados de ratones isogénicos IOR que fueron sometidos a tres dosis de citosol de hígado regenerativo al inicio del experimento. Las muestras fueron tomadas durante ocho días con frecuencia de doce horas. El corte se dividió en zonas proximal, medial y distal al hilio. Se aplico el método morfométrico para la determinación del volumen nuclear de los hepatocitos a partir de su diámetro mayor y menor. Los resultados mostraron que los valores más altos del volumen nuclear esta en la zona proximal al hilio, aunque en todas las zonas existen diferencias significativas entre los valores obtenidos en la primera mitad de tiempo del experimento respecto a la parte final, con marcada orientación a decrecer lo que puede ser debido a la presencia de factores humorales estimulantes en el citosol regenerativo

We studied the behavior of the nuclear volume of hepatocytes in 14 samples of the middle lobe of isogenic rats liver IOR that were subjected to three doses of regenerating liver cytosol at the beginning of the experiment. Samples were taken during eight days with a frequency of twelve hours. The incision was divided into the proximal, medial and distal to the ileum zones. The Morphometric method was applied to determine the nuclear volume of hepatocytes from its major and minor diameter. The results showed that the highest values of the nuclear volume are located in the proximal zone to the ileum, although in all zones there are significant differences between the values obtained in the first half of the experiment in relation to the final part, with marked orientation to decrease what can be due to the presence of stimulant humoral factors in the regenerative cytosol

Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes.

The quest to understand how the mechanical and geometrical environment of cells impacts their behavior and fate has been a major force driving the recent development of new technologies in cell biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap between the classical and simple cell culture plate and the biological reality of actual tissue. In tissues, cells have their physical space constrained by neighboring cells and the extracellular matrix. Here, we propose a simple and versatile device to precisely and dynamically control this confinement parameter in cultured cells. We show that there is a precise threshold deformation above which the nuclear lamina breaks and reconstructs, whereas nuclear volume changes. We also show that different nuclear deformations correlate with the expression of specific sets of genes, including nuclear factors and classical mechanotransduction pathways. This versatile device thus enables the precise control of cell and nuclear deformation by confinement and the correlative study of the associated molecular events.

Cell-size-dependent control of organelle sizes during development.

During development, cells differentiate into diverse cell types with different sizes. The size of intracellular organelles often correlates with the size of the cell, which may be important for cell homeostasis. The nucleus is a well-known example of an organelle whose size correlates with cell size. However, the mechanical basis of the correlation is unknown. The lengths of the mitotic spindle and contractile ring are emerging as model system to investigate the cell-size-dependent control mechanisms of organelle size. Mechanistic models are proposed for the cell-size-dependent control of these organelles. Understanding the cell-size dependency of organelle sizes is expected to impact not only on the morphogenesis of the individual organelle, but also on cell homeostasis, cell cycle progression, and cell differentiation.

DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer.

OBJECTIVE: The present study was undertaken to analyze the impact of epigenetic alterations with a main focus on nuclear area, aneuploidy, hyperploidy, and proliferation in 70 ovarian cancer specimens. METHODS: Morphometric changes and somatic chromosomal ploidy status were assessed by Feulgen spectrophotometry. DNA-hypomethylation of LINE1 repeats was analyzed by means of MethyLight PCR, and methylation levels of satellite 2 (Sat2) and satellite alpha (Satα) DNA sequences in chromosome 1 were measured by Southern blot analysis. These parameters were analyzed with regard to correlations as well as to recurrence and survival. RESULTS: We identified a significant association between LINE1 DNA-hypomethylation and patient age (p=0.029). Furthermore, LINE1 DNA-hypomethylation was positively correlated with the nuclear area (r=0.47; p<0.001) and the proliferation index (r=0.36; p<0.001). Univariate survival analysis showed that the nuclear area and LINE1 DNA-hypomethylation were prognostic factors for overall (p=0.015 and =0.006, respectively) and progression-free survival (p=0.020 and p=0.001 respectively), the percentage of aneuploidy only for overall survival (p=0.031). Subgroup survival analyses revealed that the prognostic value of these factors is strictly confined to mucinous cancers. In serous cancers no prognostic value could be pointed out for any analyzed parameter. Multivariate analysis of the entire cohort showed that the percentage of hyperploidy was an independent prognostic parameter for overall survival (p=0.003) and LINE1 DNA-hypomethylation for progression-free survival (p=0.03). In mucinous cancers nuclear area and LINE1 DNA-hypomethylation were found to be independent predictors of progression-free and overall survival. CONCLUSIONS: In this study we identified the correlations between early cancer-associated genome DNA-hypomethylation, nuclear morphometric changes, somatic chromosomal ploidy status and the proliferation index. Prognostic relevance of nuclear area and LINE1 DNA-hypomethylation was revealed exclusively in mucinous ovarian cancers.