Fibroblast Nuclei Research Articles

Abstract 5164: Mechanosensor MYL9 regulates cancer cell malignancy in gastrointestinal tumors

Abstract Although the stiffness of tissues likely is involved in the malignant behavior of tumors, it remains to be clarified which molecules control the nature, how it is involved in the invasiveness of tumors, or whether any marker is available for the prediction of cancer patient prognosis. In the present study, we studied the role of Myl9, a non-muscle-type, myosin light chain by the experiment in vitro, and assessed the usefulness in the stratification of patients in vivo as the precision medicine. Given that Myl9 is involved in the contraction of cell skeleton, cell hardness, and alterations of cell morphology in various tissues, we first examined whether the expression of the Myl9 is associated with the clinical status of tumors by immunohistochemistry. The results of 45 cases with colon cancer and pancreatic cancer indicated that the increased expression of Myl9 is associated significantly with a reduced provability of overall survival and disease-free survival of those cancers. Moreover, we noted the differential expression of Myl9 in epithelial cancer cells and mesenchymal fibroblasts, i.e., the accumulation of Myl9 staining in cell nuclei of fibroblasts. The experiment of 3-dimensional culture with cancer cells and fibroblasts confirmed the results. Furthermore, we investigated whether Myl9 overexpression is involved in the biologic behavior of gastrointestinal cancer cells. The results showed that the rentiviral-mediated overexpression of Myl9 resulted in an increase of cell proliferation and invasion as well as tumorigenicity in mice. The present study indicates that Myl9 protein can play a fundamental role in the malignant behaviors of gastrointestinal cancer cells. Citation Format: Masamitsu Konno, Kiminori Yanagisawa, Katsunori Matsushita, Ayumu Asai, Jun Koseki, Michiya Matsusaki, Shinji Deguchi, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Mechanosensor MYL9 regulates cancer cell malignancy in gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5164.

Notch3 protein expression in skin fibroblasts from CADASIL patients

AimCADASIL is an inherited cerebrovascular disease caused by mutations in the NOTCH3 gene. Notch signaling is involved in a broad spectrum of function, from the cell proliferation to apoptosis. Thus far, because the molecular mechanism underlying the pathological alterations remains unclear and taking into account that fibroblasts contribute to the integrity of the vasculature, our aims was to establish whether fibroblasts, in subjects carrying different NOTCH3 mutations, show abnormalities in the protein expression. MethodsWe performed the investigation on skin fibroblasts in culture obtained from three CADASIL patients and normal subjects. The patients were genetically characterized, and carried a p.R61W, a p.C174T, and p.R103X, mutation respectively. Notch3 expression was first evaluated on fibroblasts by immunofluorescence analysis, then western blot on cellular extract was utilized to validate the immunofluorescence results. ResultsThe Notch3 immunoreactivity was clearly detected along the cellular body and in the cellular nuclei of the control fibroblasts. We observed a marked, statistically significant, reduction of the fluorescence immunoreactivity in the fibroblasts from patient with the classical C174T cysteine mutation and a less pronounced reduction in the other two subject's samples with respect to the normal controls. These data were confirmed by the immunoblot analysis. ConclusionsOur results show that the investigated three NOTCH3 mutations are associated with a reduction of the levels of Notch3 expression in vitro. Because the smooth muscle cells appear to be predominantly involved in this cerebrovascular disease, our result, despite the limitation of the sample size examinated, clearly suggest that also fibroblasts, directly involved in making the vascular basal lamina and in maintaining the vascular integrity, may play an important role in the mechanism responsible for the disease.

Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage.

Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage.

Dual‐Drug Delivery Using Dextran‐Functionalized Nanoparticles Targeting Cardiac Fibroblasts for Cellular Reprogramming

AbstractThe inability of the heart to recover from an ischemic insult leads to the formation of fibrotic scar tissue and heart failure. From the therapeutic strategies under investigation, cardiac regeneration holds the promise of restoring the full functionality of a damaged heart. Taking into consideration the presence of vast numbers of fibroblasts and myofibroblasts in the injured heart, direct fibroblast reprogramming into cardiomyocytes using small drug molecules is an attractive therapeutic option to replenish the lost cardiomyocytes. Here, a spermine‐acetalated dextran‐based functional nanoparticle is developed for pH‐triggered drug delivery of two poorly water soluble small molecules, CHIR99021 and SB431542, both capable of increasing the efficiency of direct reprogramming of fibroblast into cardiomyocytes. Upon functionalization with polyethylene glycol and atrial natriuretic peptide, the biocompatibility of the nanosystem is improved, and the cellular interactions with the cardiac nonmyocytes are specifically augmented. The dual delivery of the compounds is verified in vitro, and the compounds exerted concomitantly anticipate biological effects by stabilizing β‐catenin (CHIR99021) and by preventing translocation of Smad3 to the nucleus of (myo)fibroblasts (SB431542). These observations highlight the potential of this nanoparticle‐based system toward improved drug delivery and efficient direct reprogramming of fibroblasts into cardiomyocyte‐like cells, and thus, potential cardiac regeneration therapy.

Safety Assessment of the Auto Manipulation Device for Acupuncture in Sprague‐Dawley Rats: Preclinical Evaluation of the Prototype

Background The Auto Manipulation Device for Acupuncture (AMDA) is designed for providing stable, quantified effects and higher frequency when doing lifting and thrusting manipulation. The purpose of this study is to investigate the safety of manipulation by AMDA in different frequency and duration in healthy rats. Methods The study was divided into two parts: single intervention and once a day for a week. 12 rats and 15 rats were randomly allocated to different groups: Control (needle insertion only), AMDA (2Hz/10Mins), AMDA (2Hz/20Mins), AMDA (20Hz/10Mins), and AMDA (20Hz/20Mins) for single and repeated interventions. Real-time physiological functions, laboratory data, and the bilateral muscle tissue of acupoint (ST 36) were obtained after the intervention. Results We found neither real-time physiological functions nor laboratory data differences between control group and AMDA groups in both parts. In the muscle tissue samples, the slight damage had been observed in the AMDA group with a frequency of 2 Hz for 20 minutes after once intervention, and the repeated session groups noted more obvious tissue damage with fibrotic change. Although the period was shorter, higher frequency manipulation caused more damage that fibroblast nuclei became more slender and obvious. However, no significant adverse effect was noted such as crippled and molting in the whole process. Conclusion Our study suggested that the safety issue of AMDA operation in rats is feasible because there was no difference between control group and AMDA groups among real-time physiological functions and laboratory data. However, manipulation with higher frequency should be more preserved.

Rapports entre la capsule antérieure de la gléno-humérale et le muscle sous-scapulaire : une étude anatomique et histologique

IntroductionThe purpose of this study was to delineate the anatomic relationship between the anterior articular capsule and the adjacent subscapularis by measuring the dimensions of the anterior articular capsule attachment and the subscapularis foot print on the humerus, as well as investigating the interface between the two structures. Materials and methodsThree shoulder specimens underwent histological analysis; for histological analysis, cross-sections through the subscapularis-capsule complex were harvested at the tendinous and muscular insertion sites. The dimensions of the anterior articular capsule attachment and the subscapularis footprint (including the tendinous and muscular insertions) were measured in thirteen cadaveric shoulder specimens. ResultsHistologically, the articular capsule has thin and loosely arranged collagen fibers with many interspersing fibroblast nuclei, whereas the outer layer of the articular capsule blends into a layer of more loosely spaced and less organized collagen fibers. This interface between the subscapularis and the underlying articular capsule is filled with more loosely spaced and less organized collagen fibers. The macroscopic evaluation showed that the minimum articular capsule width (4.2mm, SD 2.2mm) was located at its initiation 4.9mm (SD, 2.1mm) inferior to the superior margin of the subscapularis; the corresponding subscapularis footprint width measured 10.1mm (SD, 4.9mm). The maximum articular capsule width was 11.1mm (SD, 3.7mm) and was located 5mm distal to the inferior margin of the tendinous footprint. The maximum subscapularis footprint width was 15.8mm (SD, 2.9mm); the corresponding articular capsule attachment measured 5.2mm (SD, 1.8mm). ConclusionsOur results suggest that the anterior articular capsule attachment of the glenohumeral joint complements the footprint of the subscapular is and occupies a larger area of the lesser tubercle and metaphysis of the humerus than previously documented. The histological study confirms the presence of a demarcation between the subscapularis and articular capsule, specifically more significant at the region medial to the tendon insertion and at the muscular insertion of the subscapularis.

Age-Related Changes in the Expression of Lamin B Receptors in Human Dermal Fibroblasts

The goal of this research was to study the content of lamin B receptors in human dermal fibroblasts during different age periods (from a 20-week pregnancy to the age of 85) in correlation with their proliferative activity. Lamin B receptors and proliferating cell nuclear antigen (PCNA) were identified in skin sections by indirect immunohistochemistry. Our results have shown that an increase in the share of fibroblasts with positive staining for the lamin B receptors and an increase in the expression level of lamin B receptors in fibroblast nuclei are observed during the period from age zero to 20 years of age as compared with other age periods. The total fibroblast count, including positively PCNA-stained dermal fibroblasts, decreased most significantly from the age of 20. The increase in the content of lamin B receptors in dermal fibroblasts, which is observed from birth until the age of 20, can be considered one of the triggering moments leading to the agedependent decrease in counts of human dermal fibroblasts.

The relationship of the anterior articular capsule to the adjacent subscapularis: An anatomic and histological study

IntroductionThe purpose of this study was to delineate the anatomic relationship between the anterior articular capsule and the adjacent subscapularis by measuring the dimensions of the anterior articular capsule attachment and the subscapularis footprint on the humerus, as well as investigating the interface between the two structures. Materials and methodsThree shoulder specimens underwent histological analysis; for histological analysis, cross-sections through the subscapularis-capsule complex were harvested at the tendinous and muscular insertion sites. The dimensions of the anterior articular capsule attachment and the subscapularis footprint (including the tendinous and muscular insertions) were measured in thirteen cadaveric shoulder specimens. ResultsHistologically, the articular capsule has thin and loosely arranged collagen fibers with many interspersing fibroblast nuclei, whereas the outer layer of the articular capsule blends into a layer of more loosely spaced and less organized collagen fibers. This interface between the subscapularis and the underlying articular capsule is filled with more loosely spaced and less organized collagen fibers. The macroscopic evaluation showed that the minimum articular capsule width (4.2mm, SD 2.2mm) was located at its initiation 4.9mm (SD, 2.1mm) inferior to the superior margin of the subscapularis; the corresponding subscapularis footprint width measured 10.1mm (SD, 4.9mm). The maximum articular capsule width was11.1 mm (SD, 3.7mm) and was located 5mm distal to the inferior margin of the tendinous footprint. The maximum subscapularis footprint width was 15.8mm (SD, 2.9mm); the corresponding articular capsule attachment measured 5.2mm (SD, 1.8mm). ConclusionsOur results suggest that the anterior articular capsule attachment of the glenohumeral joint complements the footprint of the subscapularis and occupies a larger area of the lesser tubercle and metaphysis of the humerus than previously documented. The histological study confirms the presence of a demarcation between the subscapularis and articular capsule, specifically more significant at the region medial to the tendon insertion and at the muscular insertion of the subscapularis.

Colloidal synthesis of biocompatible iron disulphide nanocrystals

The aim of this research was to synthesis biocompatible iron disulphide nanocrystals at different reaction temperatures using the colloidal synthesis methodology. Synthesis was conducted at the 220–240 °C range of reaction temperatures at intervals of 5 °C in an inert argon atmosphere. The toxicity of iron disulphide nanocrystals was evaluated in vitro using mouse fibroblast cell line. Two complementary assays were conducted: the first to evaluate cell viability of the fibroblast via an MTT assay and the second to determine the preservation of fibroblast nuclei integrity through DAPI staining, which labels nuclear DNA in fluorescence microscopes. Through TEM and HRTEM, we observed a cubic morphology of pyrite iron disulphide nanocrystals ranging in sizes 25–50 nm (225 °C), 50–70 nm (230 °C) and >70 nm (235 °C). Through X-ray diffraction, we observed a mixture of pyrite and pyrrohotite in the samples synthesized at 225 °C and 240 °C, showing the best photocatalytic activity at 80% and 65%, respectively, for the degradation of methylene blue after 120 minutes. In all experimental groups, iron disulphide nanocrystals were biocompatible, i.e. no statistically significant differences were observed between experimental groups as shown in a one-way ANOVA and Tukey’s test. Based on all of these results, we recommend non-cytotoxic semiconductor iron sulphide nanocrystals for biomedical applications.

Spatial organization of fibroblast and spermatocyte nuclei with different B-chromosome content in Korean field mouse, Apodemus peninsulae (Rodentia, Muridae).

Korean field mouse (Apodemus peninsulae) shows a wide variation in the number of B chromosomes composed of constitutive heterochromatin. For this reason, it provides a good model to study the influence of the number of centromeres and amount of heterochromatin on spatial organization of interphase nuclei. We analyzed the three-dimensional organization of fibroblast and spermatocyte nuclei of the field mice carrying a different number of B chromosomes using laser scanning microscopy and 3D fluorescence in situ hybridization. We detected a co-localization of the B chromosomes with constitutive heterochromatin of the chromosomes of the basic set. We showed a non-random distribution of B chromosomes in the spermatocyte nuclei. Unpaired B chromosomes showed a tendency to occur in the compartment formed by the unpaired part of the XY bivalent.

Effect of plasma rich in growth factors on the early phase of healing of surgically severed Achilles tendon in sheep: histological study

ABSTRACTTendon injuries are the most frequent musculoskeletal problems, constituting 30–50% of all sport-related lesions. Efficient handling of early stage tendon injury and healing can accelerate tendon repair as well as improve the quality of newly formed tendons. Plasma rich in growth factors (PRGF) is an autologous biological therapy that has been proposed to treat tendon injuries. To elucidate the effect of this treatment on the early stage of tendon healing, 14 sheep were used to perform the present study. The right Achilles tendon was surgically severed and repaired with sutures. Seven animals were treated with PRGF and the other seven with saline solution after surgical tendon reconstruction. Tendons were ultrasound-guide infiltrated 1 week after the induced injury. Two weeks later, the sheep were euthanized and histochemical and immunohistochemical analyses were performed. The tendons with PRGF showed reduced infiltration of inflammatory cells compared to those treated with saline solution. Analysis of the blood vessels, morphometric data of fibroblast nuclei, and collagen fibres in the extracellular matrix did not show statistical differences between groups. These findings suggest the important role that PRGF therapy plays in the modulation of inflammatory response in Achilles tendon injuries, leading to acceleration of the tendon healing process, shortening the convalescence period.

Epigenetic Modifications to H3K9 in Renal Tubulointerstitial Cells after Unilateral Ureteric Obstruction and TGF-β1 Stimulation.

Introduction: Epigenetic regulation of fibrogenesis through post-translational histone modifications (marks) may be a key determinant of progression in renal disease. In this study, we examined the distribution and acquisition of histone 3 Lysine 9 (H3K9) marks after injury and stimulation with the pro-fibrotic cytokine TGF-β1. Our focus was on their presence in activated fibroblasts (myofibroblasts) and epithelial cells (epithelial-mesenchymal transition).Methods and Results: Immunofluorescent microscopy was used to examine global H3K9 acetylation (H3K9Ac) and tri-methylation (H3K9Me3) after unilateral ureteric obstruction (UUO) in mice. Confocal, super resolution microscopy and flow cytometry were used to determine the in vitro effect of TGF-β1 on structural arrangement of these marks, and their relationship with α-smooth muscle actin (αSMA) expression, a marker of myofibroblasts and early EMT. The number of individual histone marks was increased 10 days after UUO (p < 0.05 vs. control), with both marks clearly seen in various cell types including proximal tubules and myofibroblasts. Sub-nuclear microscopy in primary rat renal fibroblasts and a proximal tubule cell line (NRK-52e) showed that H3K9Ac was co-localized with phosphorylated-Ser2 RNA polymerase II (pRNAPol II), while H3K9Me3 was not, consistent with permissive and repressive effects on gene expression respectively. In both cell types H3K9Ac was diffusely distributed throughout the nucleus, while H3K9Me3 was found in compartments resembling the nucleolus, and in the case of the fibroblast, also juxtapositioned with the nuclear membrane. TGF-β1 had no effect on H3K9Ac marks in either cell, but resulted in a redistribution of H3K9Me3 within the fibroblast nucleus. This was unrelated to any change in mitogenesis, but was associated with increased αSMA expression.Conclusion: These findings highlight why it is important to consider the epigenetics of each cell individually, because whilst no overall enrichment occurred, renal myofibroblast differentiation was accompanied by distinct changes in histone mark arrangements.

Alleviation of Ultraviolet B-Induced Photodamage by Coffea arabica Extract in Human Skin Fibroblasts and Hairless Mouse Skin.

Coffea arabica extract (CAE) containing 48.3 ± 0.4 mg/g of chlorogenic acid and a trace amount of caffeic acid was found to alleviate photoaging activity in human skin fibroblasts. In this study, polyphenol-rich CAE was investigated for its antioxidant and antiinflammatory properties, as well as for its capability to alleviate ultraviolet B (UVB)-induced photodamage in BALB/c hairless mice. The results indicated that 500 μg/mL of CAE exhibited a reducing power of 94.7%, ferrous ion chelating activity of 46.4%, and hydroxyl radical scavenging activity of 20.3%. The CAE dose dependently reduced UVB-induced reactive oxygen species (ROS) generation in fibroblasts. Furthermore, CAE inhibited the UVB-induced expression of cyclooxygenase-2 and p-inhibitor κB, and the translocation of nuclear factor-kappa B (NF-κB) to the nucleus of fibroblasts. In addition, CAE alleviated UVB-induced photoaging and photodamage in BALB/c hairless mice by restoring the collagen content and reduced UVB-induced epidermal hyperplasia. CAE also inhibited UVB-induced NF-κB, interleukin-6, and matrix metalloproteinase-1 expression in the hairless mouse skin. The results indicated that CAE exhibits antiphotodamage activity by inhibiting UV-induced oxidative stress and inflammation. Therefore, CAE is a candidate for use in antioxidant, antiinflammatory, and antiphotodamage products.

Intracellular Angiotensin-II Interacts With Nuclear Angiotensin Receptors in Cardiac Fibroblasts and Regulates RNA Synthesis, Cell Proliferation, and Collagen Secretion.

BackgroundCardiac fibroblasts play important functional and pathophysiological roles. Intracellular (“intracrine”) angiotensin‐II (Ang‐II) signaling regulates intercellular communication, excitability, and gene expression in cardiomyocytes; however, the existence and role of intracrine Ang‐II signaling in cardiac fibroblasts is unstudied. Here, we evaluated the localization of Ang‐II receptors on atrial fibroblast nuclei and associated intracrine effects of potential functional significance.Methods and ResultsImmunoblots of subcellular protein‐fractions from isolated canine atrial fibroblasts indicated the presence of nuclear Ang‐II type 1 receptors (AT1Rs) and Ang‐II type 2 receptors (AT2Rs). Fluorescein isothiocyanate–Ang‐II binding displaceable by AT1R‐ and AT2R‐blockers was present on isolated fibroblast nuclei. G‐protein subunits, including Gαq/11, Gαi/3, and Gβ, were observed in purified fibroblast nuclear fractions by immunoblotting and intact‐fibroblast nuclei by confocal immunocytofluorescence microscopy. Nuclear AT1Rs and AT2Rs regulated de novo RNA synthesis ([α32P]UTP incorporation) via IP3R‐ and NO‐dependent pathways, respectively. In intact cultured fibroblasts, intracellular Ang‐II release by photolysis of a membrane‐permeable caged Ang‐II analog led to IP3R‐dependent nucleoplasmic Ca2+‐liberation, with IP3R3 being the predominant nuclear isoform. Intracellular Ang‐II regulated fibroblast proliferation ([3H]thymidine incorporation), collagen‐1A1 mRNA‐expression, and collagen secretion. Intracellular Ang‐II and nuclear AT1R protein levels were significantly increased in a heart failure model in which atrial fibrosis underlies atrial fibrillation.ConclusionsFibroblast nuclei possess AT1R and AT2R binding sites that are coupled to intranuclear Ca2+‐mobilization and NO liberation, respectively. Intracellular Ang‐II signaling regulates fibroblast proliferation, collagen gene expression, and collagen secretion. Heart failure upregulates Ang‐II intracrine signaling‐components in atrial fibroblasts. These results show for the first time that nuclear angiotensin‐II receptor activation and intracrine Ang‐II signaling control fibroblast function and may have pathophysiological significance.

Activatable bispecific liposomes bearing fibroblast activation protein directed single chain fragment/Trastuzumab deliver encapsulated cargo into the nuclei of tumor cells and the tumor microenvironment simultaneously.

This work demonstrates the design of activatable bispecific liposomes aimed to target HER2, a poor prognosis tumor marker in many tumor types, and fibroblast activation protein (FAP), a universal tumor marker overexpressed on tumor fibroblasts and pericytes of almost all solid tumors. Encapsulating liposomes with a quenched concentration of a NIRF dye which only fluoresced after cellular degradation and activation enabled reliable visualization of the destination of the cargo in cells and animal studies. Conjugating single chain antibody fragments directed to FAP, together with Trastuzumab, a humanized monoclonal antibody for HER2 resulted in the activatable bispecific liposomes. In animal models of xenografted human breast tumors, the remarkable ability of the bispecific probes to simultaneously deliver the encapsulated dye into the nuclei of target tumor cells and tumor fibroblasts could be demonstrated. Hence, the bispecific probes represent model tools with high significance to address tumor heterogeneity and manage Trastuzumab resistance in the future.

P16INK4A enhances the transcriptional and the apoptotic functions of p53 through DNA-dependent interaction.

p16INK4A and p53 are two important tumor suppressor proteins that play essential roles during cell proliferation and aging through regulating the expression of several genes. Here, we report that p16INK4A and p53 co-regulate a plethora of transcripts. Furthermore, both proteins colocalize in the nucleus of human primary skin fibroblasts and breast luminal cells, and form a heteromer whose level increases in response to genotoxic stress as well as aging of human fibroblasts and various mouse organs. CDK4 is also present in this heteromeric complex, which is formed only in the presence of DNA both in vitro using pure recombinant proteins and in vivo. We have also shown that p16INK4A enhances the binding efficiency of p53 to its cognate sequence presents in the CDKN1A promoter in vitro, and both proteins are present at the promoters of CDKN1A and BAX in vivo. Importantly, the fourth ankyrin repeat of p16INK4A and the C-terminal domain of p53 were necessary for the physical association between these two proteins. The physiologic importance of this association was revealed by the inability of cancer-associated p16INK4A mutants to interact with p53 and to transactivate the expression of its major targets CDKN1A and BAX in the p16-defective U2OS cells expressing either wild-type or mutated p16INK4A . Furthermore, the association between p16INK4A and p53 was capital for their nuclear colocalization, the X-ray-dependent induction of p21 and Bax proteins as well as the induction of apoptosis in various types of cells. Together, these results show DNA-dependent physical interaction between p16INK4A and p53.

Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways

SummaryUnderstanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

Lamin A and lamin-associated polypeptide 2 (LAP-2) in human skin fibroblasts in the process of aging

Mutations of lamin genes lead to diseases, one of which is progeria. This disease is caused by violation of splicing of lamin A gene and accumulation of farnesylated prelamin A (progerin) in the nucleus. LAP-2 is an important factor that regulates and stabilizes the nucleoplasmic pool of lamin A. However, roles of lamin A and LAP-2 in renewal regulation of the population of fibroblasts in the human dermis in different age periods still have not been investigated. The purpose of this investigation was to define expression of lamin A and LAP-2 in human dermal fibroblasts at different ages. Lamin A and LAP-2 were detected in skin sections by indirect immunohistochemistry. It was found that the number of fibroblasts containing lamin A gradually decreased from 90.4 to 76.9% from 20 weeks of pregnancy to 85 years. It was revealed that 32% of dermal fibroblasts were positively stained for LAP-2 in the period from 20 weeks of pregnancy to 20 years, 37.8% in the period of 21–40 years, and 49–51% in the period of 41–85 years. Lamin A expression levels in fibroblast nuclei were essentially constant from 20 weeks of pregnancy to 85 years, while LAP-2 content in fibroblast nuclei was reducing from 0 to 20 years as compared with the embryonic period and was gradually increasing after 21 year. The total number of fibroblasts and PCNA-positive fibroblasts in the dermis decreased with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of pregnancy to 20 years. The results suggest involvement of lamin A and LAP-2 in the age-related decrease in the number of fibroblasts in the human dermis.

Following DNA Compaction During the Cell Cycle by X-ray Nanodiffraction.

X-ray imaging of intact biological cells is emerging as a complementary method to visible light or electron microscopy. Owing to the high penetration depth and small wavelength of X-rays, it is possible to resolve subcellular structures at a resolution of a few nanometers. Here, we apply scanning X-ray nanodiffraction in combination with time-lapse bright-field microscopy to nuclei of 3T3 fibroblasts and thus relate the observed structures to specific phases in the cell division cycle. We scan the sample at a step size of 250 nm and analyze the individual diffraction patterns according to a generalized Porod's law. Thus, we obtain information on the aggregation state of the nuclear DNA at a real space resolution on the order of the step size and in parallel structural information on the order of few nanometers. We are able to distinguish nucleoli, heterochromatin, and euchromatin in the nuclei and follow the compaction and decompaction during the cell division cycle.

Lamin B1 and lamin B2 in human skin in the process of aging

The aim of this work was to study B type lamins in human skin at different ages. Lamins B1 and B2 were detected in sections of the skin by indirect immunohistochemistry. There were 62,3 % of dermal fibroblasts with positive staining for lamin B1 at the period from 20 to 40 weeks of gestation. From birth to 40 years, 41-42 % of fibroblasts containing lamin B1 were found in the dermis. In age interval from 41 to 85 years, 57-60 % of dermal fibroblasts had a positive staining for lamin B1. The number of fibroblasts containing lamin B2 was gradually decreased from 80,6 to 68,6 % from 20 weeks of gestation to 85 years old. Expression of lamin B1 in the nuclei of fibroblasts was reduced from birth to 40 years old. Content of lamin B2 in the nuclei of fibroblasts was almost constant from 20 weeks of gestation to 85 years old. Number of fibroblasts in dermis was diminished with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of gestation to 20 years old. Results allow to suggest the participation of lamin B1 in triggering age-dependent decrease in the number of fibroblasts in the dermis in humans.