Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties.

Background: Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics. Methods: BM donors (age: 48-85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed. Results: The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs. Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.

The searching for agents for Alzheimer's disease treatment via the system of self-consistent models.

Robust quantitative structure-activity relationships (QSARs) for hBACE-1 inhibitors (pIC50) for a large database (n = 1706) are established. New statistical criteria of the predictive potential of models are suggested and tested. These criteria are the index of ideality of correlation (IIC) and the correlation intensity index (CII). The system of self-consistent models is a new approach to validate the predictive potential of QSAR-models. The statistical quality of models obtained using the CORAL software (http://www.insilico.eu/coral) for the validation sets is characterized by the average determination coefficient R2v= 0.923, and RMSE = 0.345. Three new promising molecular structures which can become inhibitors hBACE-1 are suggested.

The SC-35 Splicing Factor Interacts with RNA Pol II and A-Type Lamin Depletion Weakens This Interaction.

The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with ß-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are already established in the cytoplasm of the late telophase and at the stage of early cytokinesis. In addition, we observed the occurrence of splicing factors in the nuclear blebs and micronuclei, which are also sites of both transcription and splicing. This conclusion supports the fact that splicing proceeds transcriptionally. According to our data, this process is A-type lamin-dependent. Lamin depletion also reduces the interaction between SC35 and ß-catenin in mitotic cells.

The sequence of amino acids as the basis for the model of biological activity of peptides.

The algorithm of building up a model for the biological activity of peptides as a mathematical function of a sequence of amino acids is suggested. The general scheme is the following: The total set of available data is distributed into the active training set, passive training set, calibration set, and validation set. The training (both active and passive) and calibration sets are a system of generation of a model of biological activity where each amino acid obtains special correlation weight. The numerical data on the correlation weights calculated by the Monte Carlo method using the CORAL software (http://www.insilico.eu/coral). The target function aimed to give the best result for the calibration set (not for the training set). The final checkup of the model is carried out with data on the validation set (peptides, which are not visible during the creation of the model). Described computational experiments confirm the ability of the approach to be a tool for the design of predictive models for the biological activity of peptides (expressed by pIC50).

Alternative isoforms of KDM2A and KDM2B lysine demethylases negatively regulate canonical Wnt signaling.

A precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they still have the ability to bind to CpG islands in promoters and to interact with their protein partners via their other functional domains. We have observed that KDM2A-SF and KDM2B-SF bind to the promoters of axin 2 and cyclin D1, two canonical Wnt signaling target genes, and repress their activity. Moreover, KDM2A-SF and KDM2B-SF are both able to strongly repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to negatively affect the transcription of canonical Wnt signaling target genes by binding to their promoters and by interacting with TCF7L1 and other co-repressors.

Hypocalcaemic cardiomyopathy: a description of two cases and a literature review.

Hypocalcaemic cardiomyopathy is a rare form of dilated cardiomyopathy. The authors here present two cases in which symptomatic dilated cardiomyopathy was the result of severe hypocalcaemia. First, we report about a 26-year-old woman with primary hypoparathyroidism and then about a 74-year-old man with secondary hypoparathyroidism following a thyroidectomy. In both cases, the left ventricular systolic function improved after calcium supplementation. In the first case, a lack of compliance led to a repeated decrease of both serum calcium level and left ventricular systolic function. The authors also present a comprehensive summary of all cases of hypocalcaemic dilated cardiomyopathy that have been described in literature to date. The mean age of the affected patients was 48.3 years, of which 62% were female patients. The most common causes of hypocalcaemic cardiomyopathy are primary hypoparathyroidism (50%) and post-thyroidectomy hypoparathyroidism (26%). In the post-thyroidectomy subgroup, the median time for the development of hypocalcaemic cardiomyopathy is 10 years (range: 1.5 months to 36 years). Hypocalcaemic cardiomyopathy leads to heart failure with reduced ejection fraction in 87% of patients. Generally, the most common complications of hypoparathyroidism and/or hypocalcaemia are cerebral calcifications, cognitive deficit, and cataracts. Once calcium supplementation is administered, the disease has a good prognosis and, in most individuals, a significant improvement (21%) or even normalization (74%) of the left ventricular systolic function occurs.

The study of the index of ideality of correlation as a new criterion of predictive potential of QSPR/QSAR-models.

Acetylcholinesterase (AChE) inhibitors, dihydrofolate reductase inhibitors (DHFR), Toxicity in Tetrahymena pyriformis (TP), Acute Toxicity in fathead minnow (TFat), Water solubility (WS), and Acute Aquatic Toxicity in Daphnia magna (DM) are examined as endpoints to establish quantitative structure - property/activity relationships (QSPRs/QSARs). The Index of Ideality of Correlation (IIC) is a measure of predictive potential. The IIC has been studied in a few recent works. The comparison of models for the six endpoints above confirms that the index can be a useful tool for building up and validation of QSPR/QSAR models. All examined endpoints are important from an ecologic point of view. The diversity of examined endpoints confirms that the IIC is real criterion of the predictive potential of a model.

Rifampicin Nanoformulation Enhances Treatment of Tuberculosis in Zebrafish.

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of alveolar macrophages. These cells avidly take up nanoparticles, even without the use of specific targeting ligands, making the use of nanotherapeutics ideal for the treatment of such infections. Methoxy poly(ethylene oxide)- block-poly(ε-caprolactone) nanoparticles of several different polymer blocks' molecular weights and sizes (20-110 nm) were developed and critically compared as carriers for rifampicin, a cornerstone in tuberculosis therapy. The polymeric nanoparticles' uptake, consequent organelle targeting and intracellular degradation were shown to be highly dependent on the nanoparticles' physicochemical properties (the cell uptake half-lives 2.4-21 min, the degradation half-lives 51.6 min-ca. 20 h after the internalization). We show that the nanoparticles are efficiently taken up by macrophages and are able to effectively neutralize the persisting bacilli. Finally, we demonstrate, using a zebrafish model of tuberculosis, that the nanoparticles are well tolerated, have a curative effect, and are significantly more efficient compared to a free form of rifampicin. Hence, these findings demonstrate that this system shows great promise, both in vitro and in vivo, for the treatment of tuberculosis.

Endosomal vacuoles of the prepupal salivary glands of Drosophila play an essential role in the metabolic reallocation of iron.

In the recent past, we demonstrated that a great deal is going on in the salivary glands of Drosophila in the interval after they release their glycoprotein-rich secretory glue during pupariation. The early-to-mid prepupal salivary glands undergo extensive endocytosis with widespread vacuolation of the cytoplasm followed by massive apocrine secretion. Here, we describe additional novel properties of these endosomes. The use of vital pH-sensitive probes provided confirmatory evidence that these endosomes have acidic contents and that there are two types of endocytosis seen in the prepupal glands. The salivary glands simultaneously generate mildly acidic, small, basally-derived endosomes and strongly acidic, large and apical endosomes. Staining of the large vacuoles with vital acidic probes is possible only after there is ambipolar fusion of both basal and apical endosomes, since only basally-derived endosomes can bring fluorescent probes into the vesicular system. We obtained multiple lines of evidence that the small basally-derived endosomes are chiefly involved in the uptake of dietary Fe3+ iron. The fusion of basal endosomes with the larger and strongly acidic apical endosomes appears to facilitate optimal conditions for ferrireductase activity inside the vacuoles to release metabolic Fe2+ iron. While iron was not detectable directly due to limited staining sensitivity, we found increasing fluorescence of the glutathione-sensitive probe CellTracker Blue CMAC in large vacuoles, which appeared to depend on the amount of iron released by ferrireductase. Moreover, heterologous fluorescently-labeled mammalian iron-bound transferrin is actively taken up, providing direct evidence for active iron uptake by basal endocytosis. In addition, we serendipitously found that small (basal) endosomes were uniquely recognized by PNA lectin, whereas large (apical) vacuoles bound DBA lectin.

KDM2A/B lysine demethylases and their alternative isoforms in development and disease.

Aberrant levels of histone modifications lead to chromatin malfunctioning and consequently to various developmental defects and human diseases. Therefore, the proteins bearing the ability to modify histones have been extensively studied and the molecular mechanisms of their action are now fairly well understood. However, little attention has been paid to naturally occurring alternative isoforms of chromatin modifying proteins and to their biological roles. In this review, we focus on mammalian KDM2A and KDM2B, the only two lysine demethylases whose genes have been described to produce also an alternative isoform lacking the N-terminal demethylase domain. These short KDM2A/B-SF isoforms arise through alternative promoter usage and seem to play important roles in development and disease. We hypothesise about the biological significance of these alternative isoforms, which might represent a more common evolutionarily conserved regulatory mechanism.

Depletion of A-type lamins and Lap2α reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2α protein is responsible for compactness of irradiated chromatin.

We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.

Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks.

The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance.

Interaction of spin-labeled HPMA-based nanoparticles with human blood plasma proteins - the introduction of protein-corona-free polymer nanomedicine.

In this paper, we revised the current understanding of the protein corona that is created on the surface of nanoparticles in blood plasma after an intravenous injection. We have focused on nanoparticles that have a proven therapeutic outcome. These nanoparticles are based on two types of biocompatible amphiphilic copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA): a block copolymer, poly(ε-caprolactone) (PCL)-b-poly(HPMA), and a statistical HPMA copolymer bearing cholesterol moieties, which have been tested both in vitro and in vivo. We studied the interaction of nanoparticles with blood plasma and selected blood plasma proteins by electron paramagnetic resonance (EPR), isothermal titration calorimetry, dynamic light scattering, and cryo-transmission electron microscopy. The copolymers were labeled with TEMPO radicals at the end of hydrophobic PCL or along the hydrophilic HPMA chains to monitor changes in polymer chain dynamics caused by protein adsorption. By EPR and other methods, we were able to probe specific interactions between nanoparticles and blood proteins, specifically low- and high-density lipoproteins, immunoglobulin G, human serum albumin (HSA), and human plasma. It was found that individual proteins and plasma have very low binding affinity to nanoparticles. We observed no hard corona around HPMA-based nanoparticles; with the exception of HSA the proteins showed no detectable binding to the nanoparticles. Our study confirms that a classical "hard corona-soft corona" paradigm is not valid for all types of nanoparticles and each system has a unique protein corona that is determined by the nature of the NP material.

Poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) Nanoparticles: Synthesis and Characterization, Enzymatic and Cellular Degradation, Micellar Solubilization of Paclitaxel, and in Vitro and in Vivo Evaluation.

Polyester-based nanostructures are widely studied as drug-delivery systems due to their biocompatibility and biodegradability. They are already used in the clinic. In this work, we describe a new and simple biodegradable and biocompatible system as the Food and Drug Administration approved polyesters (poly-ε-caprolactone, polylactic acid, and poly(lactic- co-glycolic acid)) for the delivery of the anticancer drug paclitaxel (PTX) as a model drug. A hydrophobic polyester, poly(propylene succinate) (PPS), was prepared from a nontoxic alcohol (propylene glycol) and monomer from the Krebs's cycle (succinic acid) in two steps via esterification and melt polycondensation. Furthermore, their amphiphilic block copolyester, poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) (mPEO- b-PPS), was prepared by three steps via esterification followed by melt polycondensation and the addition of mPEO to the PPS macromolecules. Analysis of the in vitro cellular behavior of the prepared nanoparticle carriers (NPs) (enzymatic degradation, uptake, localization, and fluorescence resonance energy-transfer pair degradation studies) was performed by fluorescence studies. PTX was loaded to the NPs of variable sizes (30, 70, and 150 nm), and their in vitro release was evaluated in different cell models and compared with commercial PTX formulations. The mPEO- b-PPS copolymer analysis displays glass transition temperature < body temperature < melting temperature, lower toxicity (including the toxicity of their degradation products), drug solubilization efficacy, stability against spontaneous hydrolysis during transport in bloodstream, and simultaneous enzymatic degradability after uptake into the cells. The detailed cytotoxicity in vitro and in vivo tumor efficacy studies have shown the superior efficacy of the NPs compared with PTX and PTX commercial formulations.

Discontinuous transcription.

Numerous studies based on new single-cell and single-gene techniques show that individual genes can be transcribed in short bursts or pulses accompanied by changes in pulsing frequencies. Since so many examples of such discontinuous or fluctuating transcription have been found from prokaryotes to mammals, it now seems to be a common mode of gene expression. In this review we discuss the occurrence of the transcriptional fluctuations, the techniques used for their detection, their putative causes, kinetic characteristics, and probable physiological significance.

A demethylation deficient isoform of the lysine demethylase KDM2A interacts with pericentromeric heterochromatin in an HP1a-dependent manner.

Histone modifications have a profound impact on the chromatin structure and gene expression and their correct establishment and recognition is essential for correct cell functioning. Malfunction of histone modifying proteins is associated with developmental defects and diseases and detailed characterization of these proteins is therefore very important. The lysine specific demethylase KDM2A is a CpG island binding protein that has been studied predominantly for its ability to regulate CpG island-associated gene promoters by demethylating their H3K36me2. However, very little attention has been paid to the alternative KDM2A isoform that lacks the N-terminal demethylation domain, KDM2A-SF. Here we characterized KDM2A-SF more in detail and we found that, unlike the canonical full length KDM2A-LF isoform, KDM2A-SF forms distinct nuclear heterochromatic bodies in an HP1a dependent manner. Our chromatin immunoprecipitation experiments further showed that KDM2A binds to transcriptionally silent pericentromeric regions that exhibit high levels of H3K36me2. H3K36me2 is the substrate of the KDM2A demethylation activity and the high levels of this histone modification in the KDM2A-bound pericentromeric regions imply that these regions are occupied by the demethylation deficient KDM2A-SF isoform.

Prevalence and Risk Factors of Osteoporosis in Postmenopausal Women with Type 2 Diabetes Mellitus.

OBJECTIVE: Patients with type 2 diabetes (T2DM) are at increased risk of fractures. The aim of this study is to analyze the prevalence and risk factors of osteoporosis and osteoporosis related fractures in postmenopausal women with T2DM. METHODS: A total of 112 postmenopausal women with T2DM and 171 control nondiabetic women received a standardized questionnaire on osteoporosis risk factors, and were evaluated for bone mineral density (BMD, by using a dual energy X-ray absorptiometry), biochemical markers of bone and glucose metabolism, soluble receptor for advanced glycation end products (sRAGE) and its gene polymorphisms (rs1800625 or rs2070600). RESULTS: In T2DM patients the prevalence of osteoporosis was 25% and low trauma vertebral (Vfx) and non-vertebral fractures were found in 8% and 19% women, respectively. When compared between subjects with and without fractures, there were no significant differences in BMD at any site between the groups, except for distal radius, which was significantly lower in T2DM women with Vfx (p<0.05 vs.non-fractured without osteoporosis). We found no associations between bone and glucose metabolism variables, sRAGE and BMD. No significant differences were observed in sRAGE levels according to their rs1800625, rs 2070600 genotype or fracture prevalence. Serum osteocalcin was significantly lower in T2DM women (p<0.01 vs. controls) and in T2DM women with Vfx (p<0.05) vs. non-fractured without osteoporosis. T2DM women with low daily walking activity (< 2 h daily) had significantly higher serum sclerostin levels (p<0.05 vs. those who were walking > 2 h daily). CONCLUSION: Diabetes-specific parameters as well as RAGE polymorphisms did not associate with BMD or fractures in T2DM postmenopausal women. Lower levels of osteocalcin, namely in those with Vfx and higher sclerostin levels in those with low daily walking activity suggest lower bone remodeling and/or decreased bone quality in T2DM.

CORAL Software: Analysis of Impacts of Pharmaceutical Agents Upon Metabolism via the Optimal Descriptors.

BACKGROUNDS: The CORAL software has been developed as a tool to build up quantitative structure- activity relationships (QSAR) for various endpoints. OBJECTIVE: The task of the present work was to estimate and to compare QSAR models for biochemical activity of various therapeutic agents, which are built up by the CORAL software. METHOD: The Monte Carlo technique gives possibility to build up predictive model of an endpoint by means of selection of so-called correlation weights of various molecular features extracted from simplified molecular input-line entry system (SMILES). Descriptors calculated with these weights are basis for building up correlations &quot;structure - endpoint&quot;. RESULTS: Optimal descriptors, which are aimed to predict values of endpoints with apparent influence upon metabolism are crytically compared in aspect of their robustness and heuristic potential. Arguments which are confirming the necessity of reformulation of basics of QSARs are listed: (i) each QSAR model is stochastic experiment. The result of this experiment is defined by distribution into the training set and validation set; (ii) predictive potential of a model should be checked up with a group of different splits; and (iii) only model stochastically stable for a group of splits can be estimated as a reliable tool for the prediction. Examples of the improvement of the models previously suggested are demonstrated. CONCLUSION: The current version of the CORAL software remains a convenient tool to build up predictive models. The Monte Carlo technique involved for the software confirms the principle "QSAR is a random event" is important paradigm for the QSPR/QSAR analyses.

PCNA is recruited to irradiated chromatin in late S-phase and is most pronounced in G2 phase of the cell cycle.

DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.