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Recent evidence suggests that the fimbriated end of the fallopian tube harbors the precursor cells for many high-grade ovarian cancers, opening the door for development of better screening methods that directly assess the fallopian tube in women at risk for malignancy. Previously we have shown that the karyometric signature is abnormal in the fallopian tube epithelium in women at hereditary risk of ovarian cancer. In this study, we sought to determine whether the karyometric signature in serous tubal intraepithelial carcinoma (STIC) is significantly different from normal, and whether an abnormal karyometric signature can be detected in histologically normal tubal epithelial cells adjacent to STIC lesions. The karyometric signature was measured in epithelial cells from the proximal and fimbriated portion of the fallopian tube in fallopian tube specimens removed from women at: 1) average risk for ovarian cancer undergoing surgery for benign gynecologic indications (n = 37), 2) hereditary risk of ovarian cancer (germline BRCA alterations) undergoing risk-reducing surgery (n = 44), and 3) diagnosed with fimbrial STICs (n = 17). The karyometric signature in tubes with fimbrial STICs differed from that of tubes with benign histology. The degree of karyometric alteration increased with increasing proximity to fimbrial STICs, ranging from moderate in the proximal portion of the tube, to greatest in both normal appearing fimbrial cells near STICs as well as in fimbrial STIC lesions. These data demonstrate an abnormal karyometric signature in STICs that may extend beyond the STIC, potentially providing an opportunity for early detection of fallopian tube neoplasia. • Karyometric assessment reveals a signature that is abnormal in serous tubal intraepithelial carcinoma (STIC). • The abnormal karyometric signature extends to histologically normal fallopian tube epithelial cells beyond the STIC. • An abnormal karyometric signature in the fallopian tube has potential as a target for ovarian cancer screening. [ABSTRACT FROM AUTHOR]

Background: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology.Methods: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp).Results: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43.Conclusion: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation. [ABSTRACT FROM AUTHOR]

Abstract: Histological subtypes of basal cell carcinoma have biological, evolutionary and distinct prognostic behavior. The analysis of characteristics of the nucleus can provide data on their cellular physiology and behavior. The authors of this study evaluated nuclear morphological parameters and textural patterns of chromatin from different subtypes of basal cell carcinoma: nodular (n=37), superficial (n=28) and sclerodermiform (n=28). The parameters were compared between neoplasms' subtypes and with unaffected adjacent basal epithelium. Nuclear area and diameter of sclerodermiform neoplasms were superior to the other subtypes. Chromatin's color intensity and fractal dimension were less intense in superficial subtypes. Nuclear roundness and chromatin's entropy presented lower values in tumors than in normal epithelium. There was significant correlation between morphological and textural variables of normal skin and tumors. Morphometric elements and textural chromatin's homogeneity of basal cell carcinomas may be related to evolutionary, biological and behavior particularities related to each histotype.

We studied the effect of alcohol on the pineal gland of 48 male Wistar rats. Animals were divided into control and experimental groups. The experimental group underwent a previous progressive alcoholisation period with ethanol diluted in water at a concentration of 40%. Animals were sacrificed at 3, 6, 9 and 12 months, and the ultrastructure, karyometric indices, and number of synaptic bodies in the pineal gland were analysed. The results showed progressive morphologic alterations in the ethanol-treated animals, which culminated in fatty degeneration of the pineal parenchyma after 6 months. The karyometric indices decreased in both the central and peripheral areas compared with the control group. Moreover, the seasonal rhythmicity observed in the controls disappeared in the experimental groups, whose number of different populations of synaptic bodies (synaptic ribbons and synaptic spherules) considerably lowered with inversion of their normal seasonal rhythm. These results support that chronic alcoholisation leads to fatty degeneration of the pineal parenchyma, and a considerable alteration in nuclear functional rhythms and synaptic bodies. [ABSTRACT FROM AUTHOR]

Intraductal papillary mucinous neoplasms ( IPMN) are pancreatic lesions with uncertain biologic behavior. This study sought objective, accurate prediction tools, through the use of quantitative histopathological signatures of nuclear images, for classifying lesions as chronic pancreatitis ( CP), IPMN, or pancreatic carcinoma ( PC). Forty-four pancreatic resection patients were retrospectively identified for this study (12 CP; 16 IPMN; 16 PC). Regularized multinomial regression quantitatively classified each specimen as CP, IPMN, or PC in an automated, blinded fashion. Classification certainty was determined by subtracting the smallest classification probability from the largest probability (of the three groups). The certainty function varied from 1.0 (perfectly classified) to 0.0 (random). From each lesion, 180 ± 22 nuclei were imaged. Overall classification accuracy was 89.6% with six unique nuclear features. No CP cases were misclassified, 1/16 IPMN cases were misclassified, and 4/16 PC cases were misclassified. Certainty function was 0.75 ± 0.16 for correctly classified lesions and 0.47 ± 0.10 for incorrectly classified lesions ( P = 0.0005). Uncertainty was identified in four of the five misclassified lesions. Quantitative histopathology provides a robust, novel method to distinguish among CP, IPMN, and PC with a quantitative measure of uncertainty. This may be useful when there is uncertainty in diagnosis. [ABSTRACT FROM AUTHOR]

The clinical application of a new, widely applicable method known as Karyomapping to carry out a total of 55 clinical cases of preimplantation genetic diagnosis (PGD) for single gene disorders is reported. Conventional polymerase chain reaction (PCR) testing was carried out in parallel to the new method for all cases. Clinical application of Karyomapping in this study resulted in three live births and nine clinical pregnancies out of 20 cases with a transfer. All in all, results presented in this study indicate that Karyomapping is a highly efficient, accurate and robust method for PGD of single gene disorders. Karyomapping can offer a more comprehensive assessment of the region of interest than conventional PCR analysis, allowing for more embryos to receive diagnosis (99.6% versus 96.8%), whereas its wide applicability reduces substantially the time that patients have to wait before starting their in vitro fertilization (IVF) cycle. Nonetheless, inclusion of elements of conventional PCR methodology, such as direct mutation detection, may be required in cases in which the gene of interest is in a region with reduced single nucleotide polymorphism (SNP) coverage (e.g. telomeric regions), when offering PGD for consanguineous couples, or in cases where no samples from additional family members are available. [ABSTRACT FROM AUTHOR]

Purpose: Human papillomaviruses (HPV) have been associated with placental inflammation resulting in high-risk preterm birth. The premise for this study was that treatment with anti-inflammatory pentoxifylline would inhibit HPV-mediated placental cell death. The objectives were: (1) to study the effects of HPV-16 exposure on trophoblast cells and (2) to evaluate pentoxifylline in preventing the damaging effects of HPV-16. Methods: Mouse embryos (1-cell) were cultured (G1plus medium, 72 h, 37 °C, 5 % CO2 in air), divided into four groups at blastocyst-stage and incubated to implantation stage. Implanted trophoblasts were exposed to either HPV-16 (group 1), concomitant HPV-16 and 3 mM pentoxifylline (group 2), 3 mM pentoxifylline only (group 3) or control medium (group 4) and further incubated for 24 h. HPV-16 were from SiHa cell lysates. Trophoblast structural integrity was assessed by morphometric analysis while apoptosis and necrosis were detected using dual-stain fluorescence assay. Results: Trophoblast outgrowth was reduced by 90 % ( P < 0.05) in HPV-16 presence (629 ± 265 µ, mean + SEM) when compared with controls (6,456 ± 795 µ). Pentoxifylline attenuated the effects of HPV-16 (4,308 ± 362 µ). Nuclear size of HPV-16 infected trophoblasts was smallest among the groups ( P < 0.005). HPV-16 decreased cell viability and increased necrosis but not apoptosis. Pentoxifylline prevented HPV-16 associated necrosis in trophoblasts. Conclusions: HPV-16 decreased nuclear size and trophoblast outgrowth but these effects were attenuated by the phosphodiesterase inhibitor, pentoxifylline. The action of HPV-16 on trophoblasts was increased cell necrosis suggesting that HPV-16 pathogenesis involved either cAMP inhibition and/or activated TNF pathways. [ABSTRACT FROM AUTHOR]

Summary Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate [ 1–3 ]. Whereas the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property [ 4–7 ], it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development [ 8 ]. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. Whereas previous studies suggested a role for the N/C volume ratio in MBT timing [ 1, 9–13 ], none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. Whereas the DNA-to-cytoplasmic ratio has been implicated in MBT timing [ 1, 9–18 ], our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development. [ABSTRACT FROM AUTHOR]

Summary Chromosomes are folded, spatially organized, and regulated by epigenetic marks. How chromosomal architecture is connected to the epigenome is not well understood. We show that chromosomal architecture of Arabidopsis is tightly linked to the epigenetic state. Furthermore, we show how physical constraints, such as nuclear size, correlate with the folding principles of chromatin. We also describe a nuclear structure, termed KNOT , in which genomic regions of all five Arabidopsis chromosomes interact. These KNOT ENGAGED ELEMENT ( KEE ) regions represent heterochromatic islands within euchromatin. Similar to PIWI-interacting RNA clusters, such as flamenco in Drosophila , KEE s represent preferred landing sites for transposable elements, which may be part of a transposon defense mechanism in the Arabidopsis nucleus. [ABSTRACT FROM AUTHOR]

This research describes the pineal complex histology in juvenile and adult Cichlasoma dimerus, and the effect of different photoperiods on its cell morphology. In both juveniles and adults, the pineal complex of C. dimerus has three components: the pineal organ, consisting of a pineal vesicle ( PV) and a pineal stalk, the parapineal organ and the dorsal sac. Although a strong morphological resemblance exists between the two stages, different synthesis patterns of cone and rod opsins were detected in the two life stages. An effect of the photoperiod length was observed on putative pinealocytes' activity from the PV, measured indirectly through nuclear area morphometry. Individuals exposed to a natural photoperiod ( 14L: 10D) had smaller nuclear areas (mean ± s.e. = 13·82 ± 1·52 µm2) than those exposed to a short photoperiod (8:16) (21·45 ± 2·67 µm2; P < 0·001). Eventually, the nuclear area of pinealocytes could be used as a putative indicator of melatonin synthesis in fishes where it is difficult to obtain plasma samples, e. g. due to its small size or age. This work constitutes one of the few comparative descriptions of the pineal complex of juvenile and adult teleost and suggests potential approaches for the study of melatonin synthesis in fish larvae or small adult fishes. [ABSTRACT FROM AUTHOR]

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Highlights: [•] Mechanisms of nuclear size and shape regulation. [•] Cell cycle events that influence nuclear morphology. [•] Nuclear morphology, chromatin organization, and gene expression. [•] Nuclear morphology in cancer. [ABSTRACT FROM AUTHOR]

Abstract: The gerbil is a rodent considered a good model for studies of prostatic morphophysiology under different experimental conditions. Studies involving castration and steroidal blockers of aged gerbils showed that the glandular epithelium persists after long-term therapy, preventing the organ atrophy. Thus, the objective of this study was to evaluate the phenotypic characteristics and behavior of prostatic epithelial cells that remained after different periods of hormone ablation in aged gerbils. The identification of elements that influenced the survival of this cell type was performed by morphometric, nuclear phenotypes, ultrastructural and immune histochemical analysis. The most significant responses to treatment, by analyzing morphometric features, were observed during the first three time points (day 1, day 3, and day 7), after which there appeared to be an adjustment of the gland to the hormone ablation. All treatments led to changes in the state of chromatin condensation, DNA methylation pattern and phenotypic changes indicated cell senescence. Additionally, an increase in the basal cells seemed to guarantee self-renewal properties to the epithelium. These data indicate that changes occur at many levels, including gene expression and nuclear architecture in the epithelial cells, when aging and steroidal blockade are associated. These aspects are important when considering castration-resistant prostate cancer, a malignant tumor posing difficult therapeutic intervention. [Copyright &y& Elsevier]

Objective: Presurgical, window of opportunity trials have been proposed as a model to assess the activity of preventive and therapeutic interventions in a cost-effective manner in prostate cancer (CaP). The aim of the study was to explore karyometry as a method for monitoring the efficacy of intervention with preventive agents in patients with CaP Materials and methods: The material used in this investigation was from the 2F study, i.e., an Italian prospective randomized phase lib presurgical study of finasteride vs. low-dose flutamide vs. placebo in men with CaP. Image analysis was performed in 16 cases treated with finasteride, 24 with flutamide, and 20 with placebo. For all these cases, CaP and normal looking secretory epithelium were present in the pretreatment biopsies as well as the post-treatment ex-vivo biopsies obtained from the radical prostatectomy specimens. Results: To establish a direction of nuclear change from normal to malignancy, i.e., the so-called line of progression, a discriminant function was derived with the normal looking epithelium in the pretreatment biopsies as one endpoint, and the CaP in the pretreatment biopsies as the other. The discriminant function was then applied to the post-treatment groups. The increase in relative nuclear area was the dominant feature. In the placebo group, 15 out of 20 CaP (75%) cases had a higher discriminant function score at the end of study, with a significant increase of the mean score by 90%. The flutamide treated CaP cases had increased discriminant function scores in 19 out of 24 cases (79%) and an increase of the mean score by 43%; the 5 cases with lower scores involved only minor reductions. In contrast, the finasteride treated CaP cases had increased discriminant function scores for 8 out of 16 cases (50%), but the increase in the mean score was by only 8%. Conclusion: This exploratory study establishes that karyometric monitoring can track the results of subtle nuclear changes induced by preventive interventions in men with CaP, thus allowing assessment of agent activity in a cost-effective manner. [ABSTRACT FROM AUTHOR]

Abstract: Telomeres are crucial for the maintenance of genome stability through “capping” of chromosome ends to prevent their recognition as double-strand breaks, thus avoiding end-to-end fusions or illegitimate recombination [1–3]. Similar to other genomic regions, telomeres participate to the nuclear architecture while being highly mobile. The interaction of telomeres with nuclear domains or compartments greatly differs not only between organisms but also between cells within the same organism. It is also expected that biological processes like replication, repair or telomere elongation impact the distribution of chromosome extremities within the nucleus, as they probably do with other regions of the genome. Pathological processes such as cancer induce profound changes in the nuclear architecture, which also affects telomere dynamics and spatial organization. Here we will expose our present knowledge on the relationship between telomeres and nuclear architecture and on how this relationship is affected by normal or abnormal telomere metabolisms. [Copyright &y& Elsevier]

The purpose of this study was to evaluate the effects of zinc, during lactation, on the junctional epithelium and inserted gum of the first upper molar of rats. The study used one-day old male rats, divided into two groups: those whose mother had been treated with 300 mg zinc chloride (ZnC12) in the drinker water (treated group), and those whose mothers did not receive ZnCl2 (control group). After 21 days, the rat pups were sacrificed. Using karyometrical techniques, the greater (D) and smaller (d) nuclear diameters of the different layers of the junctional and inserted gum epithelia were determined, and the mean geometric diameter, D/d ratio, perimeter, area, volume, volume/area ratio, eccentricity, shape coefficient, and the contour index were estimated. The 100-point Merz grid was used with the purpose of evaluating the citoplasmatic and celular volume, the nucleus/citoplasm relationship, number density, outer surface/ basal layer ratio, the thickness of epithelial layers, and the surface density. The results were submitted to statistical analysis using the Wilcoxon-Mann-Whitney test. The nuclei of the studied structures were significantly smaller, and the stereological results demonstrated that there were smaller cells, hence meaning a greater number of cells per mm³ of tissue, in the treated group. Zinc caused changes on the studied epitheliums, according to morphometric and stereological evaluations. [ABSTRACT FROM AUTHOR]

Study Type - Aetiology (case control) Level of Evidence 3b What's known on the subject? and What does the study add? Penile rehabilitation is still controversial regarding good results. Our study shows a non-invasive treatment option to recovery after cavernous nervous damage. The assessment of changes in the intracavernous pressure and karyometry demonstrates the protective effect of annexin-A1 in an animal model of cavernous nerve injury. We found that annexin-A1 effectively preserved erectile function, evidently through significantly protecting the corpus cavernosum tissue against fibrosis. OBJECTIVE To evaluate the protective effect of annexin-A1 against irreversible damage to cavernous tissue after cavernous nerve injury., PATIENTS AND METHODS Thirty Sprague-Dawley male rats were divided into 3 groups; sham-operated rats ( n= 10), bilateral cavernous nerve injury treated intravenously with 100 µg/kg annexin-A1 ( n= 10), and a crush group of rats submitted to bilateral cavernous nerve injury and vehicle ( n= 10). Groups were compared in respect to intracavernous pressure and karyometric parameters., RESULTS After annexin-A1 treatment, the maximum changes in intracavernous pressure responses were significantly higher in the annexin-A1 group compared to the vehicle-only group on the 7th postoperative day (p-value <0.05). Hematoxylin-eosin staining showed that the percentage of cavernosal smooth muscle was higher in the annexin-A1 group. Karyometry showed that the nuclear volume was greater in the annexin-A1 group, as was the major/minor smooth muscle cell diameter ratio compared to the vehicle-only group on the 7th postoperative day (p-value <0.05)., CONCLUSION This is the first report that, by assessing changes in the intracavernous pressure and karyometry, demonstrates the protective effect of annexin-A1 in an animal model of cavernous nerve injury. We found that annexin-A1 effectively preserved erectile function, evidently through significantly protecting the corpus cavernosum tissue against fibrosis. [ABSTRACT FROM AUTHOR]

Feulgen image analysis densitometry (FIA) and image cytometry were used to study the relationship between the DNA content (pgDNA nucleus−1) and nuclear area (μm2) in blood smears of evolutionary tetraploid (4n) sterlet ( Acipenser ruthenus) and stellate sturgeon ( A. stellatus); evolutionary octaploid (8n) Siberian sturgeon ( A. baerii) and Russian sturgeon ( A. gueldenstaedtii); hexaploid (6n) and decaploid (10n) fish found within A. baerii stock; and A. baerii and A. gueldenstaedtii exhibiting dodecaploidy (12n). Standards used for FIA were blood smears of chicken ( Gallus gallus domesticus; 2.5 pgDNA nucleus−1) and diploid and induced triploid tench, Tinca tinca (2.04 and 3.1 pgDNA nucleus−1, respectively). All ploidy levels were first verified by means of flow cytometry. Species of the same ploidy level, however differing in their DNA content, exhibited a similar mean erythrocyte nuclear area, as could be demonstrated on A. ruthenus and A. stellatus (19.27 and 19.79 μm2, respectively) with a respective mean DNA content of 3.72 and 4.68 pgDNA nucleus−1 and the same relationship was found for evolutionary octaploid (8n) A. baerii and A. gueldenstaedtii (29.87 and 30.09 μm2, respectively) with respective mean DNA content 8.29 and 7.87 pgDNA nucleus−1. The 0.19-0.32 pgDNA increments in DNA content of erythrocytes thus had no effect on their nuclear area. With increasing ploidy level, the DNA concentration (pgDNA per μm2 of erythrocyte nuclear area) was found not to increase linearly. The DNA in erythrocyte nuclei appeared to be more and more densely packed with an increase of the ploidy level ( r = 0.98; R2 = 0.95). [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding the methyl-CpG-binding protein MECP2 are the major cause of Rett syndrome, an autism spectrum disorder mainly affecting young females. MeCP2 is an abundant chromatin-associated protein, but how and when its absence begins to alter brain function is still far from clear. Using a stem cell-based system allowing the synchronous differentiation of neuronal progenitors, we found that in the absence of MeCP2, the size of neuronal nuclei fails to increase at normal rates during differentiation. This is accompanied by a marked decrease in the rate of ribonucleotide incorporation, indicating an early role of MeCP2 in regulating total gene transcription, not restricted to selected mRNAs. We also found that the levels of brain-derived neurotrophic factor (BDNF) were decreased in mutant neurons, while those of the presynaptic protein synaptophysin increased at similar rates in wild-type and mutant neurons. By contrast, nuclear size, transcription rates, and BDNF levels remained unchanged in astrocytes lacking MeCP2. Re-expressing MeCP2 in mutant neurons rescued the nuclear size phenotype as well as BDNF levels. These results reveal a new role of MeCP2 in regulating overall RNA synthesis in neurons during the course of their maturation, in line with recent findings indicating a reduced nucleolar size in neurons of the developing brain of mice lacking Mecp2. S TEM C ells 2012;30:2128-2139 [ABSTRACT FROM AUTHOR]

Background: Marginal zone lymphomas are indolent B-cell lymphomas associated with autoimmunity and chronic inflammation. The two most frequent variants are mucosa associated lymphoid tissues marginal zone lymphomas and splenic marginal zone lymphomas. The aim of the study was to determine if it is possible to classify splenic and gastric lymphomas according to karyometric features. Methods: The material consisted of 16 splenic and 14 gastric lymphomas. The measurements were done with the AnalySIS image analysis system. In each case at least 100 nuclei were selected, and 19 different geometric parameters were measured. Results: On statistical analysis, the nuclei of splenic and gastric lymphomas showed differences in most parameters, but significant overlap of the values was present. Neural networks were trained and used for classification of the data. By this method, the nuclei were properly classified with a sensitivity of 0.75 and specificity of 0.71. In addition, in all the cases the majority of the nuclei were properly classified, thus allowing correct classification of all the cases into 'splenic' or 'gastric'. Conclusion: These results support the view that mucosa-associated lymphoid tissue lymphomas and splenic marginal-zone lymphomas are separate entities. [ABSTRACT FROM AUTHOR]

Abstract: Attrition due to safety reasons remains a serious problem for the pharmaceutical industry. This has prompted efforts to develop early predictive in vitro screens that can assist in selecting compounds with a more desirable safety profile early on in the drug discovery process. Here we examined the relationship between physicochemical properties, such as partition coefficient (clogP), topological polar surface area (TPSA), acid dissociation constant (pK a), and in vitro mechanistic endpoints generated using a high content imaging approach. We demonstrate in our initial analysis that compounds with clogP>2 and pK a >5.5 flagged more endpoints than compounds with clogP⩽2 and pK a ⩽5.5. In contrast, TPSA did not stand on its own in predicting cytotoxicity. When this knowledge was applied to eight different mechanistic cytotoxicity endpoints (cell loss, apoptosis, ER stress, DNA fragmentation, mitochondrial potential, nuclear size, neutral lipids/steatosis and lysosomal mass), we found that compounds with such properties preferentially flagged in the lysosomal endpoint. We also saw a slight enrichment of such compounds in the endpoints cell loss, DNA fragmentation and nuclear size. We demonstrate that lysosomal compound accumulation is a potential contributor to cell death and possibly organ toxicity. [Copyright &y& Elsevier]

Abstract: Objectives: Treatment for atypical endometrial hyperplasia (AEH) is based on pathologic diagnosis. About 40% of AEH is found to be carcinoma at surgery. This study''s objective is to derive an objective characterization of nuclei from cases diagnosed as AEH or superficially invasive endometrial cancer (SIEC). Methods: Cases from GOG study 167A were classified by a central pathology committee as AEH (n=39) or SIEC (n=39). High resolution digitized images of cell nuclei were recorded. Features of the nuclear chromatin pattern were computed. Classification rules were derived by discriminant analysis. Results: Nuclei from cases of AEH and SIEC occupy the same range on a progression curve for endometrial lesions. Cases of AEH and SIEC both comprise nuclei of two phenotypes: hyperplastic characteristics and premalignant/neoplastic characteristics. The principal difference between AEH and SIEC is the percentage of premalignant/neoplastic nuclei. When this percentage approaches 50–60% superficial invasion is likely. SIEC may develop already from lesions at the low end of the progression curve. Conclusions: AEH comprises cases which may constitute a low risk group involving <40% of AEH cases. These cases hold a percentage of <20% of nuclei of a preneoplastic phenotype. AEH cases from the central and high end of progression have >40% of nuclei of preneoplastic phenotype. Nuclei of the preneoplastic phenotype in AEH lesions are almost indistinguishable from nuclei in SIEC, where this percentage exceeds 60%. The percentage of nuclei of the preneoplastic phenotype in AEH esions might serve as criterion for assessment of risk for the development of invasive disease. [Copyright &y& Elsevier]

Background and Aims: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. Methods: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. Results: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. Conclusions: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity. [ABSTRACT FROM AUTHOR]

Abstract: Background: The aim to find a morphological biomarker of disturbed activity of the lateral amygdaloid nucleus in depression was approached by a karyometric analysis of projection neurons. Methods: The study was performed on paraffin-embedded brains from 19 depressed patients from both the major depressive disorder (MDD) and the bipolar disorder (BD) diagnostic groups, including 10 suicides, and 24 matched controls. The karyometric parameters of the lateral amygdaloid nucleus (La) projection neurons bilaterally were evaluated by the argyrophilic nucleolar organiser region (AgNOR) silver staining method. Results: An increased AgNOR number was found in the right La in suicides compared to controls. The intra-group comparisons between the hemispheres suggest a disturbed amygdaloid lateralisation in depressed patients. The effects were independent from psychotropic medication. There was a strong positive correlation between the nuclear area in La projection neurons and prefrontal limbic areas pyramidal neurons in the right hemisphere specific for suicide and MDD. Limitations: A major limitation of this study is the relatively small number of cases. A further limitation is given by the lack of data on drug exposure across the entire lifespan. Conclusion: The results suggest that depressed patients from both the MDD and BD diagnostic groups exhibit an increased activity of the La output neurons specific for suicidal patients. The distinctness of the diagnostic groups of mood disorders was accentuated in the correlation analysis. This putative hyperactivity was specific for the right hemisphere and psychotropic medication most likely did not counteract it. [ABSTRACT FROM AUTHOR]

The cyclamate, a sweetner substance derived from N-cyclo-hexyl-sulfamic acid, is largely utilized as a non-caloric artificial edulcorant in foods and beverages as well as in the pharmaceutical industry. The objective of this study was to evaluate karyometric and stereological alterations in the rat fetal pancreas resulting from the intraperitoneal administration of sodium cyclamate. The exocrine pancreas of ten fetuses of rats were evaluated, five treated and five controls chosen at random, in which five rats that received from the 10th to 14th days of pregnancy an intraperitoneal daily injection of sodium cyclamate at 60 mg/Kg of body weight during 5 days. At the 20th day of gestation, the animals were removed and weighed, as were their placentas; the length of the umbilical cords also were measured. After the laboratory processing, semi-seriated 6mm cuts stained with haematoxyline and eosine were performed. In seven karyometric parameters (major, minor, and medium diameters, volume, area, perimeter, and volume-area ratio), the increase was statistically significant in the treated group when compared with control group. Stereological parameters showed in the treated group a significant increase in the cellular volume and a significant reduction in the numerical cellular density. These results showed that the sodium cyclamate in pregnant rats led to retardation of fetal development and hypertrophy in the exocrine pancreas of the rat fetuses. [ABSTRACT FROM AUTHOR]

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (−)- cubebin (CUB) and (−)-hinokinin (HNK), was evaluated in the chronic phase of Chagas’ disease by quantifying the enzyme β-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas’ disease. [ABSTRACT FROM AUTHOR]

Abstract: Background: The external globus pallidus (EGP) is thought to play the most important integrating and conveying role in the striatopallidal system involved in the transfer from motivation to action. The aim to find a morphological biomarker of disturbed EGP activity in depression was approached by the karyometric analysis of large projecting neurons. Methods: The study was performed on paraffin-embedded brains from 19 depressed patients from both the major depressive disorder (MDD) and the bipolar disorder (BD) diagnostic groups encompassing 10 suicides and from 24 controls. The karyometric parameters of EGP neurons bilaterally were evaluated by argyrophilic nucleolar organiser (AgNOR) silver staining method. Results: A significantly decreased AgNOR area was found in the left EGP neurons in depressed patients compared to controls. The distinctness of the diagnostic groups and suicidal vs non-suicidal patients was not shown in the statistical comparisons. The AgNOR parameter which was decreased correlated positively with the mean dose of benzodiazepines in non-suicidal patients. Limitations: A major limitation of this study is the relatively small number of cases. A further limitation is given by the lack of data on drug exposure across the whole lifespan of patients. Conclusion: The results suggest disturbed, most likely decreased, activity of the left EGP projecting neurons in depressed patients, a disturbed activity that should hypothetically be counteracted by the applied pharmacotherapy in non-suicidal patients. [Copyright &y& Elsevier]

Abstract: Background: Morphometric studies based on the measurement of cardiocyte nuclei have focused on progressive hypertrophy rather than shape, which is a deciding factor for the diagnosis of hypertrophy in myocardial diseases. The aim of this research was to demonstrate how the digital morphology of cardiocyte nuclei change correlated with the type of myocardial pathology. Materials and Methods: The study groups encompassed 7 hearts with dilated cardiomyopathy (DCM) and 8 hearts with ischemic heart disease (IHD) which were explanted. A comparative group consisting of myocardial hypertrophy was contrasted with a control group of donor heart fragments. Cardiocyte nuclei were evaluated morphometrically on histologic slides. We calculated the nuclear area, length, breadth, perimeter, roundness, elongation, fullness factors, and nuclear chromatin mean gray level. The results were subjected to discriminant analysis. Results: All karyometric measurements analyzed by backward discriminant analysis showed only 2 powerful factors: nuclear breadth and chromatin mean gray level. The Mahalanobis distance showed the proximity of control and hypertrophy groups, whereas differences between IHD and DCM were nonsignificant. Conclusion: The lack of karyometric differences between IHD and DCM suggested a common morphologic response for long-lasting progressive injury. The main morphologic differences were dependent on nuclear chromatin activity/stainability and nuclear breadth, suggesting darker and thinned nuclei in normal and adaptative stages and irregular brighter nuclei in cardiomyopathies. [Copyright &y& Elsevier]

Abstract: In this study, we compared the effect of KNK437 (N-formyl-3, 4-methylenedioxy-benzylidene-γ-butyrolactam), a benzylidene lactam compound, on heat shock and chemical stressor-induced hsp30 gene expression in Xenopus laevis A6 kidney epithelial cells. Previously, KNK437 was shown to inhibit HSE-HSF1 binding activity and heat-induced hsp gene expression. In the present study, Northern and Western blot analysis revealed that pretreatment of A6 cells with KNK437 inhibited hsp30 mRNA and HSP30 and HSP70 protein accumulation induced by chemical stressors including sodium arsenite, cadmium chloride and herbimycin A. In A6 cells subjected to sodium arsenite, cadmium chloride, herbimycin A or a 33 °C heat shock treatment, immunocytochemistry and confocal microscopy revealed that HSP30 accumulated primarily in the cytoplasm. However, incubation of A6 cells at 35 °C resulted in enhanced HSP30 accumulation in the nucleus. Pre-treatment with 100 μM KNK437 completely inhibited HSP30 accumulation in A6 cells heat shocked at 33 or 35 °C as well as cells treated with 10 μM sodium arsenite, 100 μM cadmium chloride or 1 μg/mL herbimycin A. These results show that KNK437 is effective at inhibiting both heat shock- and chemical stress-induced hsp gene expression in amphibian cells. [Copyright &y& Elsevier]

Abstract: Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to several morphological and cell biological properties, and evaluated the survival of in vitro produced, morphologically classified, blastocysts following non-surgical transfer. In vitro and in vivo produced blastocysts were allocated to two groups (classes A and B) on the basis of morphological characteristics. The quality of their actin cytoskeleton, their total cell number, their ability to re-expand after cytochalasin-B treatment and the occurrence of numerical chromosome aberrations were studied and compared. In vivo produced blastocysts were used as a control. Our results indicate that the ability of blastocysts to re-expand after cytochalasin-B-induced actin depolymerization was positively correlated with the morphology of the blastocyst, and associated with the quality of the actin cytoskeleton. Chromosome analysis revealed that mosaicism is inherent to the in vitro production of porcine embryos, but also that in vivo produced blastocysts contained some non-diploid cells. In non-surgical embryo transfer experiments more recipients receiving class A blastocysts were pregnant on Day 20 than those receiving class B blastocysts. One recipient gave birth to six piglets from class A in vitro produced blastocysts, providing a verification of the enhanced viability of blastocysts that were scored as ‘good’ on the basis of their morphology. [Copyright &y& Elsevier]

Abstract: Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes. [Copyright &y& Elsevier]

The objective of this work was to evaluate karyometrically the alterations caused by different strains of Trypanosoma cruzi in the mouse placenta. Pregnant mice, 60-day oíd, were intraperitoneally inoculated with 2 × 105 bloodstream trypomastigotes of the Colombian, Y, Bolivia or RC strain of T cruzi. There were observed clear differences in the karyometric alterations of the trophoblast giant cells and in the spongiotrophoblast cells. The results demónstrate that the Colombian and RC strains cause alterations both in the trophoblast giant cells and in the spongiotrophoblast cells, whereas the Y and Bolivia strains provoke alterations only in the trophoblast giant cells. It is possible concluding that each strain has its own characteristics and that, in spite of the similar type of transmission, it show differential nuances in the placental pathogenic process. [ABSTRACT FROM AUTHOR]

Abstract: Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis. [Copyright &y& Elsevier]

Abstract: Background: Digital nuclear morphometric analysis can capture subtle differences along neoplastic progression. Studies showed different profiles from normal to cancer lesions. Our goal is to utilize this method as biomarker in chemoprevention trials. Methods: Postmenopausal women were randomized to oral (CEE) or transdermal (E2) estrogen replacement therapy (ERT) in association with fenretinide or placebo. Ultrasound-guided fine needle aspiration (FNA) was performed at baseline and after 12 months in a subset of subjects. Results: Ten samples were analyzed by karyometry. E2 compared with CEE increased nuclear area (p=0.01). A similar pattern was observed for other DNA content and chromatin texture features. Fenretinide vs. placebo, increased nuclear area and shape while decreased slope, peak and entropy. Conclusion: Preliminary results indicate that nuclear morphometry is feasible on FNA samples. ERT and fenretinide induced significant karyometric changes. These results support further investigation of this procedure as surrogate biomarker in chemoprevention trial. [Copyright &y& Elsevier]

Summary: Sedentary endoparasitic nematodes are root parasites that interact with their hosts in a remarkable way. These obligate biotrophic pathogens establish an intimate relationship with their host plants, inducing the redifferentiation of root cells into specialized feeding cells. The successful establishment of feeding cells is essential for nematode development. Root-knot nematodes, of the genus Meloidogyne, have evolved strategies enabling them to induce feeding cell formation in thousands of plant species, probably by manipulating fundamental elements of plant cell development. Feeding cells enlarge and are converted into multinucleate giant cells through synchronous nuclear divisions without cell division. Fully differentiated giant cells may contain more than a hundred polyploid nuclei that may have undergone extensive endoreduplication. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of the typical root gall. Giant cell formation requires extensive changes to gene expression. The induction of feeding cells remains poorly understood, but it is thought that effectors secreted by the nematode play a key role in parasitism, with potential direct effects on recipient host cells. In this review, we focus on the most recent investigations of the molecular basis of the plant–root-knot nematode interaction. Recently, microarray technology has been used to study the plant response to Meloidogyne spp. infection. Such a genome-wide expression profiling provides a global view of transcriptional changes, especially for genes involved in cell wall, transport processes and plant defense responses during giant cell formation. The identification of nematode-responsive plant genes constitutes a major step toward understanding how root-knot nematodes dramatically alter root development to induce and maintain giant cells. The characterization of nematode secretions as parasitism effectors and the development of RNAi technology should improve our understanding of the molecular events and regulatory mechanisms involved in plant parasitism. Finally, Meloidogyne genome sequences should provide further insight into plant–root-knot nematode interactions. [Copyright &y& Elsevier]

Abstract: Automatic centromere identification and polarity assignment are two key factors in the automatic karyotyping of human chromosomes. A multi-stage rule-based computer scheme has been investigated to automatically detect centomeres and determine polarities for both abnormal and normal metaphase chromosomes. The scheme first implements a modified thinning algorithm to identify the medial axis of a chromosome and extracts three feature profiles. Based on a set of pre-optimized classification rules, the scheme adaptively identifies the centromere and then assigns corresponding polarity. An image dataset of 2287 chromosomes acquired from 24 abnormal and 26 normal Giemsa metaphase cells is utilized to optimize and test the scheme. The overall accuracy is 91.4% for centromere identification and 97.4% for polarity assignment. The experimental results demonstrate that our scheme can be successfully applied to diverse chromosomes, which include those severely bent and abnormal chromosomes extracted from cancer cells. [Copyright &y& Elsevier]

The present work studied the adverse effects of maternal exposure of rats to alcohol during lactation, on the development of their offspring. Histometric evaluation by karyometry and of the palatine epithelium of the sucking was performed. Two groups of animals, one coming from mothers exposed to drinking water containing 20% ethanol during the total lactation period and the other of controls coming from mothers receiving only alcohol-free drinking water during this period. On the 21 first day of lactation the young of each group were aleatorily selected and following anesthesia, their heads severed; after histological treatment, serial 6 μm sections on the frontal plane at the molar level, stained with hematoxilin and eosin, were obtained. The experimental results produced, suggest that suckling from ethanol-treated mothers, show a significant decrease of body weight, significant alterations of karyometric and stereological measurements that determined hypotrophy of palatine epithelial cells and toxicity of ethanol at the palatine epithelium. [ABSTRACT FROM AUTHOR]

The objective of this study was the evaluation of aspartame effects on morphometric alterations of the glomerulus, proximal and distal convoluted tubules and collecting ducts of the rat fetal kidney during organogenesis. Fifteen pregnant rats averaging 24 g body weight, were divided into 3 groups (n=5 each) of controls, rats treated with aspartame exposed to room temperature and rats treated with aspartame heated to 40°C. Animals were given 14mg/kg aspartame by the intragastric route on the 9th, 10th and 11th days of pregnancy. Karyometric and stereological techniques estimated morphological changes. A significant decrease of fetal body weight was observed in the group given aspartame kept at room temperature, compared to controls. Karyometry permitted the estimation of the significant nuclear variations observed in the cells of the glomerulus, proximal and distal convoluted tubules and collecting ducts of aspartame-treated rat fetuses. Stereolgical parameters showed statistically significantly increased cell volume and decreased numerical cell density in fetal kidneys of rats treated with aspartame heated to 40° compared to controls. These results indicate that the use of aspartame leads to alterations in all renal structures studied, suggesting this product's nephrotoxicity. [ABSTRACT FROM AUTHOR]

Ofloxacin presents an ample spectrum of antimicrobial action,,including combating Mycobacterium leprae, and is currently employed as a substitute when the use of rifampicin is impossible. The objective of this work was to study alterations in testicular cell nuclei of suckling rats, whose dams were submitted to oral application of ofloxacin, and respective control groups. The method utilized was morphometry by the karyometric technique. The main structures observed in histological preparations of the testicles were interstitial cells, spermatogonias, and sustentacular cells. 10 Wistar rats were utilized, four treated and five controls, in the period of the first 25 days of life, whose dams received ofloxacin 12 mg/ Kg of body weight / day orally, before being killed on the 25th day after birth. The karyometric study of interstitial cells and spermatogonias revealed that there were no changes in the form of their nuclei (p> 0.05). Since sustentacular cell nuclei presented increased major diameter, minor diameter, mean geometric diameter, volume, area, volume/area ratio and perimeter, as well as an augmented and statistically different eccentricity (p <0.05) in suckling pups whose dams were administered ofloxacin, the nuclei presented larger size and more elongated form. It was concluded that the sustentacular cells were more sensitive to the ofloxacin effect at the administered dose. [ABSTRACT FROM AUTHOR]