Micro-Osteoperforations Accelerate Tooth Movement without Exacerbating the Progression of Root Resorption in Rats.

A recent study reported that micro-osteoperforations (MOPs) accelerated tooth movement by activating alveolar bone remodeling. However, very little is known about the relationship between MOPs and external apical root resorption during orthodontic treatment. In this study, in order to investigate the mechanism through which MOPs accelerate tooth movement without exacerbating the progression of root resorption, we measured the volume of the resorbed root, and performed the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method on exposed MOPs during experimental tooth movements in rats. Male Wistar rats (11 weeks old) were divided into three groups: 10 g orthodontic force (optimal force) applied to the maxillary first molar (optimal force: OF group), 50 g orthodontic force application (heavy force: HF group), and 10 g force application plus three small perforations of the cortical plate (OF + MOPs group). On days 1, 4, 7, 10, and 14 after force application, the tooth movement and root volume were investigated by micro-computed tomography. Furthermore, the number of apoptotic cells in the pressured sides of the periodontal ligament (PDL) and surrounding hard tissues were determined by TUNEL staining. The OF + MOPs group exhibited a 1.8-fold increase in tooth movement on days 7, 10, and 14 compared with the OF group. On days 14, the HF group had a higher volume of root loss than the OF and OF + MOPs groups. On the same day, the number of TUNEL-positive cells in the HF group increased at the root (cementum) site whereas that in the OF group increased at the alveolar bone site. Furthermore, the number of TUNEL-positive cells in the OF + MOPs group increased at the alveolar bone site compared with the OF group. These results suggest that MOPs accelerate orthodontic tooth movement without exacerbating the progression of root resorption.

Role of MST2/YAP1 signaling pathway in retinal cells apoptosis and diabetic retinopathy.

Diabetic retinopathy (DR) is a main factor affecting vision of patients, and its pathogenesis is not completely clear. The purpose of our study was to investigate correlations between MST2 and DR progression, and to study the possible mechanism of MST2 and its down pathway in high glucose (HG)-mediated RGC-5 apoptosis. The diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ) 60 mg/kg. HE and TUNEL staining were used to evaluate the pathological changes and apoptosis of retinal cells in rats. Western blot, qRT-PCR and immunohistochemistry showed that levels of MST2 were increased in diabetic group (DM) than control. In addition, the differential expression of MST2 is related to HG-induced apoptosis of RGC-5 cells. CCK-8 and Hoechst 33,342 apoptosis experiments showed that MST2 was required in HG-induced apoptosis of RGC-5 cells. Further research revealed that MST2 regulated the protein expression of YAP1 at the level of phosphorylation in HG-induced apoptosis. Simultaneously, we found that Xmu-mp-1 acts as a MST2 inhibitor to alleviate HG-induced apoptosis. In summary, our study indicates that the MST2/YAP1 signaling pathway plays an important role in DR pathogenesis and RGC-5 apoptosis. This discovery provides new opportunities for future drug development targeting this pathway to prevent DR.

TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries.

Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in a specific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS cell type in the mixed culture and animal models of the CNS diseases and injuries.

Overexpression of KAT8 induces a failure in early embryonic development in mice.

Embryo quality is strongly associated with subsequent embryonic developmental efficiency. However, the detailed function of lysine acetyltransferase 8 (KAT8) during early embryonic development in mice remains elusive. In this study, we reported that KAT8 played a pivotal role in the first cleavage of mouse embryos. Immunostaining results revealed that KAT8 predominantly accumulated in the nucleus throughout the entire embryonic developmental process. Kat8 overexpression (Kat8-OE) was correlated with early developmental potential of embryos to the blastocyst stage. We also found that Kat8-OE embryos showed spindle-assembly defects and chromosomal misalignment, and that Kat8-OE in embryos led to increased levels of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX by affecting the expression of critical genes related to mitochondrial respiratory chain and antioxidation pathways. Subsequently, cellular apoptosis was activated as confirmed by TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) assay. Furthermore, we revealed that KAT8 was related to regulating the acetylation status of H4K16 in mouse embryos, and Kat8-OE induced the hyperacetylation of H4K16, which might be a key factor for the defective spindle/chromosome apparatus. Collectively, our data suggest that KAT8 constitutes an important regulator of spindle assembly and redox homeostasis during early embryonic development in mice.

In vivo Gonadoprotective Effects of Myricetin on Cisplatin- Induced Testicular Damage via Suppression of TLR4/NF-kB Inflammation Pathway and Heat-Shock Response / Efectos Gonadoprotectores in vivo de la Miricetina sobre el Daño Testicular Inducido por Cisplatino Mediante la Supresión de la Vía de Inflamación TLR4/NF-kB y la Respuesta al Choque Térmico

SUMMARY: The aim of this study is to reveal the gonadoprotective effects of myricetin (MYC), which has many biological properties, on cisplatin (CP)-induced testicular damage in rats. For this purpose, 40 male Wistar albino rats were divided into 4 groups as Control (group given no treatment), MYC (group given 5 mg/kg/i.p myricetin for 7 days), CP (group given 7 mg/kg/i.p cisplatin at 7th day) and MYC + CP (group given 5 mg/kg/i.p myricetin for 7 days before 7 mg/kg/i.p cisplatin injection). After administrations, testicular tissues of animals were extracted and processed according to tissue processing protocol. Hematoxylin & Eosin staining were performed to evaluate the histopathological changes and Johnsen'sTesticular Biopsy Score (JTBS) was applied and mean seminiferous tubule diameters (MSTD) were measured to compare experimental groups in terms of histopathological changes. Moreover, TLR4, NF-kB, HSP70 and HSP90 expression levels were detected by immunohistochemical staining and the density of immunoreactivity were measured to determine the difference in the expression levels of these factors among groups. Additionally, testicular apoptosis was detected via TUNEL assay. JTBS and MSTD data were significantly lower in CP group compared to other groups and MYC administrations significantly protects testicular tissue against CP-induced damage. Moreover, TLR4, NF-kB, HSP70 and HSP90 expressions and apoptotic cells significantly increased in the CP group (p<0.05). However, MYC administrations exerted a strong gonadoprotective effect on testicular tissue in terms of these parameters in MYC+CP group (p<0.05). According to our results, we suggested that MYC can be considered as a protective agent against cisplatin-induced testicular damage.

El objetivo de este estudio es revelar los efectos gonadoprotectores de la miricetina (MYC), que tiene muchas propiedades biológicas, sobre el daño testicular inducido por cisplatino (CP) en ratas. Para este propósito, se dividieron 40 ratas albinas Wistar macho en 4 grupos: Control (grupo que no recibió tratamiento), MYC (grupo que recibió 5 mg/kg/i.p de miricetina durante 7 días), CP (grupo que recibió 7 mg/kg/i.p de cisplatino al séptimo día) y MYC + CP (grupo que recibió 5 mg/ kg/i.p de miricetina durante 7 días antes de la inyección de 7 mg/ kg/i.p de cisplatino). Después de las administraciones, se extrajeron y procesaron tejidos testiculares de animales según el protocolo de procesamiento de tejidos. Se realizó tinción con hematoxilina y eosina para evaluar los cambios histopatológicos y se aplicó la puntuación de biopsia testicular de Johnsen (JTBS) y se midieron los diámetros medios de los túbulos seminíferos (MSTD) para comparar los grupos experimentales en términos de cambios histopatológicos. Además, los niveles de expresión de TLR4, NF-kB, HSP70 y HSP90 se detectaron mediante tinción inmunohistoquímica y se midió la densidad de inmunorreactividad para determinar la diferencia en los niveles de expresión de estos factores entre los grupos. Además, se detectó apoptosis testicular mediante el ensayo TUNEL. Los datos de JTBS y MSTD fueron significativamente más bajos en el grupo CP en comparación con otros grupos y las administraciones de MYC protegen significativamente el tejido testicular contra el daño inducido por CP. Además, las expresiones de TLR4, NF-kB, HSP70 y HSP90 y las células apoptóticas aumentaron significativamente en el grupo CP (p<0,05). Sin embargo, las administraciones de MYC ejercieron un fuerte efecto gonadoprotector sobre el tejido testicular en términos de estos parámetros en el grupo MYC+CP (p<0,05). Según nuestros resultados, sugerimos que MYC puede considerarse como un agente protector contra el daño testicular inducido por cisplatino.

Evaluation of Sperm DNA Fragmentation Using Two Different Methods: TUNEL via Fluorescence Microscopy, and Flow Cytometry.

Background and Objectives: Sperm DNA fragmentation refers to any break in one or both of the strands of DNA in the head of a sperm. The most widely used methodologies for assessing sperm DNA fragmentation are the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis assay (SCGE-comet), and the terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. The aim of this study was to compare the efficiency and sensitivity of the analysis of sperm DNA fragmentation using TUNEL via fluorescence microscopy, and flow cytometry. Materials and Methods: Semen samples were collected and analyzed for standard characteristics using light microscopy, and for sperm DNA fragmentation using both TUNEL via fluorescence microscopy, and flow cytometry. Results: There were no significant differences in the values of the sperm DNA fragmentation index (DFI) obtained when the analysis was performed using TUNEL or flow cytometry (p = 0.543). Spearman's correlation analysis revealed a significant negative correlation between sperm motility (%) and sperm DNA fragmentation (p < 0.01), as well as between sperm concentration and sperm DNA fragmentation (p < 0.05). The Mann-Whitney U test showed no significant difference in the DFI among couples with repeated implantation failure (RIF) and miscarriages (p = 0.352). Conclusions: Both methods (TUNEL via fluorescence microscopy, and flow cytometry) have a high efficiency and sensitivity in accurately detecting sperm DNA fragmentation, and can be effectively used to assess male fertility.

TUNEL Staining in Sections of Paraffin-Enabled Planarians.

Planarians are a model animal for the study of regeneration and homeostasis. Understanding how planarians control their cellular balance is key to the knowledge of their plasticity. Both apoptotic and mitotic rates can be quantified in "whole mount" planarians. Apoptosis is usually analyzed through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), a technique that detects cell death by identifying DNA breaks. In this chapter we detail a protocol to analyze apoptotic cells in paraffin sections of planarians, which enables a more accurate cellular visualization and quantification than in "whole mount."

In Vivo Electroporation Improves Retinal Delivery of Intravitreally Injected Exosomes.

Purpose: Mesenchymal stem cell (MSC)-derived exosomes are promising therapeutic agents and natural nanoscale delivery platforms for treating degenerative retinal diseases. This study investigated the effect of electroporation on the retinal delivery of intravitreally administered MSC-derived exosomes in a murine model. Methods: Exosomes isolated from adipose tissue-derived MSCs were stained with ExoGlow exosome-specific dye and administered to the right eyes of 40 Sprague-Dawley rats. Electroporation was performed in 20 rats immediately after intravitreal injection (electroporation group); 5 square pulses of 40 V/cm for 50 ms each with 950-ms intervals were administered. The remaining 20 rats were assigned to the no-electroporation group. The eyeballs were harvested 24 h later for evaluation. The total number of fluorescent particles per hyperfield was counted from the retinal flat mounts to quantify the retinal delivery of exosomes. Tissue damage after electroporation was evaluated using retinal histological sections and a terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling (TUNEL) assay. Results: A significantly higher number of fluorescent particles per hyperfield were observed in the retinal flat mounts of the electroporation group compared with that in the no-electroporation group (599.0 ± 307.5 vs. 376.9 ± 175.4; P = 0.013). Retinal histological sections and TUNEL assays showed no signs of tissue damage after electroporation. Conclusions: In vivo electroporation can improve the retinal delivery of intravitreally injected exosomes.

Avoidance of the use of tryptophan in buried chromosomal proteins as a mechanism for reducing photo/oxidative damage to genomes.

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery.

Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

C9orf131 and C10orf120 are not essential for male fertility in humans or mice.

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131-/- and C10orf120-/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131-/-, and C10orf120-/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Occurrence of late-apoptotic symptoms in porcine preimplantation embryos upon exposure of oocytes to perfluoroalkyl substances (PFASs) under in vitro meiotic maturation.

The objectives of this study were to evaluate the effect of perfluoroalkyl substances on early embryonic development and apoptosis in blastocysts using a porcine in vitro model. Porcine oocytes (N = 855) collected from abattoir ovaries were subjected to perfluorooctane sulfonic acid (PFOS) (0.1 µg/ml) and perfluorohexane sulfonic acid (PFHxS) (40 µg/ml) during in vitro maturation (IVM) for 45 h. The gametes were then fertilized and cultured in vitro, and developmental parameters were recorded. After 6 days of culture, resulting blastocysts (N = 146) were stained using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and imaged as stacks using confocal laser scanning microscopy. Proportion of apoptotic cells as well as total numbers of nuclei in each blastocyst were analyzed using objective image analysis. The experiment was run in 9 replicates, always with a control present. Effects on developmental parameters were analyzed using logistic regression, and effects on apoptosis and total numbers of nuclei were analyzed using linear regression. Higher cell count was associated with lower proportion of apoptotic cells, i.e., larger blastocysts contained less apoptotic cells. Upon PFAS exposure during IVM, PFHxS tended to result in higher blastocyst rates on day 5 post fertilization (p = 0.07) and on day 6 post fertilization (p = 0.05) as well as in higher apoptosis rates in blastocysts (p = 0.06). PFHxS resulted in higher total cell counts in blastocysts (p = 0.002). No effects attributable to the concentration of PFOS used here was seen. These findings add to the evidence that some perfluoroalkyl substances may affect female reproduction. More studies are needed to better understand potential implications for continued development as well as for human health.

Valproic acid induced selective apoptosis of ocular fibrous tunic in mice fetuses.

BACKGROUND: Valproic acid (VPA), a prescribed drug commonly used for various neurological perturbations, has been implicated in teratogenic inflictions on developing fetuses during pregnancy. The purpose of this research was to delineate the gross morphological and histological effects of VPA in the developing eye tunics and lens. METHODS: A time-dependent administration of 500 mg/kg VPA to BALB/c groups of female mice was coordinated during organogenesis (gestational days 7, 8, and 9) and compared to controls that received normal saline. Seized fetuses were checked for macroscopic eye anomalies, histological malformations with Azan trichrome staining, and levels of apoptotic activity with the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. RESULTS: Histochemical analysis showed that VPA-treated groups exhibited collagen deficiency (2.5-50% decrease in aniline blue intensity) and a marked increase in TUNEL-positive cells (p < .05) in corneal stroma and sclera/choroid layers while less was detected in retina and lens, when compared to controls. CONCLUSIONS: Since the evaluation of the inner structures did not manifest major differences, we conclude that VPA teratogenic influence display eclectic toxicity, as seen by increased apoptosis to eye layers with high degree fibrous context, particularly the outer tunics.

Thyroid hormone-induced cell death in sea urchin metamorphic development.

Thyroid hormones (THs) are important regulators of development, metabolism and homeostasis in metazoans. Specifically, they have been shown to regulate the metamorphic transitions of vertebrates and invertebrates alike. Indirectly developing sea urchin larvae accelerate the formation of juvenile structures in response to thyroxine (T4) treatment, while reducing their larval arm length. The mechanisms underlying larval arm reduction are unknown and we hypothesized that programmed cell death (PCD) is linked to this process. To test this hypothesis, we measured larval arm retraction in response to different THs (T4, T3, rT3, Tetrac) and assessed cell death in larvae using three different methods (TUNEL, YO-PRO-1 and caspase-3 activity) in the sea urchin Strongylocentrotus purpuratus. We also compared the extent of PCD in response to TH treatment before and after the invagination of the larval ectoderm, which marks the initiation of juvenile development in larval sea urchin species. We found that T4 treatment results in the strongest reduction of larval arms but detected a significant increase of PCD in response to T4, T3 and Tetrac in post-ingression but not pre-ingression larvae. As post-ingression larvae have initiated metamorphic development and therefore allocate resources to both larval and the juvenile structures, these results provide evidence that THs regulate larval development differentially via PCD. PCD in combination with cell proliferation likely has a key function in sea urchin development.

Frontiers of the Lower Palaeolithic expansion in Europe: Tunel Wielki Cave (Poland).

Peopling of Central Europe by Middle Pleistocene hominids is highly debatable, mainly due to the relatively harsh climatic and environmental conditions that require cultural and anatomical adjustments. At least several archaeological sites certify human occupation in the region dated back to MIS 13-11, but they represent open-air settlements. Based on the new fieldwork conducted in Tunel Wielki Cave, we can date the human occupation traces in the cave to MIS 14-12. Bipolar-on-anvil knapping technique prevails in the lithic assemblage, made exclusively in flint. The obtained results have given ground for studying the frontiers of human oikumene and the required cultural adaptive abilities.

In vivo fluorescent TUNEL detection of single stranded DNA gaps and breaks induced by dnaB helicase mutants in Escherichia coli.

The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress. If decoupled, vulnerable ssDNA would persist, likely leading to double strand breaks (DSBs) or requiring replication restart mechanisms downstream of a stall. In either case, free 3'-OH strands would exist, resulting from ssDNA gaps in the leading strand or complete DSBs. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) can enzymatically label ssDNA ends with bromo-deoxy uridine triphosphate (BrdU) to detect free 3'-OH DNA ends in the E. coli genome. Labeled DNA ends can be detected and quantified using fluorescence microscopy or flow cytometry. This methodology is useful in applications where in situ investigation of DNA damage and repair are of interest, including effects from enzyme mutations or deletions and exposure to various environmental conditions.

Transcriptional activity and splicing factors are preserved during physiological apoptosis.

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Aripiprazole sensitizes head and neck cancer cells to ionizing radiation by enhancing the production of reactive oxygen species.

Drug repositioning is an alternative process for drug development in cancer. Specifically, it is a strategy for the discovery of new antitumor drugs by screening previously approved clinical drugs. On the basis of this strategy, aripiprazole, an antipsychotic drug, was found to have anticancer activity. In this study, we investigated the radiosensitizing effects of aripiprazole on head and neck cancer cells at sublethal doses of ionizing radiation (IR) in vitro and in vivo. Treatment with aripiprazole suppressed the growth of head and neck cancer cells in a concentration-dependent manner, as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Intriguingly, aripiprazole significantly enhanced the sensitivity of these cells to the IC50 dose of IR. The combination of aripiprazole with IR synergistically increased annexin and propidium iodide double-positive and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cell populations, and induced cleaved poly(ADP-ribose) polymerase and caspase-3 expression, indicating the induction of apoptosis in these cells. Aripiprazole and IR-induced apoptosis were accompanied by an increase in reactive oxygen species and was almost completely suppressed by the addition of the antioxidant, N-acetylcysteine. Finally, aripiprazole greatly sensitized xenograft tumors to IR at doses that did not affect tumor growth. Taken together, these results suggest that aripiprazole could be considered a potent radiosensitizer for head and neck cancer.

Toxicity of Amphotericin B in Rabbit Corneal Epithelial Cells Stored in Optisol™-GS: Corneal Epithelial Cell Morphology and Migration.

PURPOSE: To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability. METHODS: Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6-8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured. RESULTS: At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group. CONCLUSIONS: The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery.