Beclin 1, LC3 and P62 Expression in Equine Sarcoids

Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

Selenoprotein T Protects Endothelial Cells against Lipopolysaccharide-Induced Activation and Apoptosis

Sepsis is an exaggerated immune response upon infection with lipopolysaccharide (LPS) as the main causative agent. LPS-induced activation and apoptosis of endothelial cells (EC) can lead to organ dysfunction and finally organ failure. We previously demonstrated that the first twenty amino acids of the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) are sufficient to inhibit EC apoptosis. To identify genes whose regulation by LPS is affected by this N-terminal APEX1 peptide, EC were transduced with an expression vector for the APEX1 peptide or an empty control vector and treated with LPS. Following RNA deep sequencing, genes upregulated in LPS-treated EC expressing the APEX1 peptide were identified bioinformatically. Selected candidates were validated by semi-quantitative real time PCR, a promising one was Selenoprotein T (SELENOT). For functional analyses, an expression vector for SELENOT was generated. To study the effect of SELENOT expression on LPS-induced EC activation and apoptosis, the SELENOT vector was transfected in EC. Immunostaining showed that SELENOT was expressed and localized in the ER. EC transfected with the SELENOT plasmid showed no activation and reduced apoptosis induced by LPS. SELENOT as well as APEX1(1-20) can protect EC against activation and apoptosis and could provide new therapeutic approaches in the treatment of sepsis.

Evaluation of Bacterial Reduction after Root Canal Shaping Using ProTaper Gold and WaveOne Gold Rotary Systems

Introduction The present study evaluated bacterial reduction promoted by the WaveOne system (Dentsply Maillefer, Ballaigues, Switzerland) and ProTaper Gold system (PTG; Dentsply Maillefer) in human extracted central incisors. Methods Sixty-two maxillary central incisors that were infected with Enterococcus faecalis (ATCC 51299) were sterilized with ethylene oxide for 21 days, and then root canal initial bacterial sample was collected with paper points and plated on M-Enterococcus agar. The specimens were randomly divided into two groups according to instrumentation: WaveOne Gold group (n = 30) and PTG group (n = 30). Each group was further subdivided into subgroup A (n = 15) where no activation of the irrigant was performed, and subgroup B (n = 15) where passive ultrasonic activation (PUI) was applied. The other two specimens without contamination were control asepsis. After instrumentation, samples were collected with the use of paper points. The bacterial reduction was calculated using colony-forming unit and quantitative real-time polymerase chain reaction. Data were collected and statistically analyzed. Results All techniques significantly reduced the number of bacteria in the root canal (p < 0.05), in which PTG showed superior bacterial reduction than WaveOne Gold (p > 0.05). The aseptic control group did not show any bacterial growth. PUI showed a significant bacterial reduction with the WaveOne Gold group. Conclusion It can be concluded that the single-file system, WaveOne Gold with the aid of passive ultrasonic irrigation, significantly reduce the bacterial number in the root canal similar to the multifile system, PTG.

Ictal and interictal brain activation in episodic migraine: Neural basis for extent of allodynia

In some patients, migraine attacks are associated with symptoms of allodynia which can be localized (cephalic) or generalized (extracephalic). Using functional neuroimaging and cutaneous thermal stimulation, we aimed to investigate the differences in brain activation of patients with episodic migraine (n = 19) based on their allodynic status defined by changes between ictal and interictal pain tolerance threshold for each subject at the time of imaging. In this prospective imaging study, differences were found in brain activity between the ictal and interictal visits in the brainstem/pons, thalamus, insula, cerebellum and cingulate cortex. Significant differences were also observed in the pattern of activation along the trigeminal pathway to noxious heat stimuli in no allodynia vs. generalized allodynia in the thalamus and the trigeminal nucleus but there were no activation differences in the trigeminal ganglion. The functional magnetic resonance imaging (fMRI) findings provide direct evidence for the view that in migraine patients who are allodynic during the ictal phase of their attacks, the spinal trigeminal nucleus and posterior thalamus become hyper-responsive (sensitized)–to the extent that they mediate cephalic and extracephalic allodynia, respectively. In addition, descending analgesic systems seem as “switched off” in generalized allodynia.

81 Pronuclear formation and SMARCA4 incorporation after intracytoplasmic sperm injection (ICSI) or assisted ICSI in pig zygotes

Pigs are considered an important experimental model for their biological similarities to humans, including their potential as organ donors in xenotransplantation. Unfortunately, in this species conventional invitro fertilization results in high polyspermic rates. ICSI avoids polyspermy and ICSI-mediated gene edition could be a powerful technique to produce genetically modified pigs. However, ICSI is not yet efficient in pigs. Moreover, the ATP-dependent chromatin remodeller, SMARCA4, translocates to the pronuclei soon after fertilization and its mislocalization or reduction leads to poor embryo development. The aim of this study was to assess whether assisted activation or the use of the piezo drill (PD) during ICSI improves pronuclear (PN) formation rates and to analyse SMARCA4 intensity levels in pronuclei. First, cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured invitro for 44h. Matured and denuded oocytes were subjected to (1) ICSI (n=47), (2) ICSI assisted by PD (ICSIp, n=21), (3) ICSI assisted by electrical activation (ICSIe, n=39), and (4) electrical activation as an haploid parthenogenetic control (HAP, n=21). Presumptive zygotes were fixed for 20min in 4% formaldehyde solution 18h after injection or activation and incubated with SMARCA4 antibody (1:100) and Alexa Fluor (1:1000) as a secondary antibody. Then, the zygotes were classified according to the presence of PN in 2 PN (2-PN), 1 PN with the presence of a semi-condensed or condensed sperm (1-PN), and semi-condensed or condensed sperm with no evidence of PN (no activation). Zygotes that exhibited a different pattern were included in the “other” category. A region of interest was drawn around each PN and the average pixel intensity of SMARCA4 was determined with ImageJ image processing software. Data were analysed by Fisher’s exact test and Kruskal–Wallis test using GraphPad software (GraphPad Inc.). Differences were considered significant at P&lt;0.05. We found no significant differences in 2-PN formation rates among groups after ICSI (ICSI n=16, 34.04%; ICSIe n=10, 25.64%; ICSIp n=6, 28.57%). As expected, the majority of the HAP zygotes exhibited 1 PN (n=14, 66.67%). In contrast, in most of the zygotes of all experimental groups, SMARCA4 was found to be localised in both PN, being absent in polar bodies, metaphase plate, or condensed sperm. Interestingly, out of the total 2-PN porcine ICSI zygotes of all experimental groups (n=25), 7 zygotes (28%) showed clear asymmetric intensity levels between PN. The rest of the ICSI zygotes (n=18, 72%) showed a similar SMARCA4 intensity level between PN. In conclusion, our results suggest that neither the use of piezo drill or electrical activation improves PN formation or SMARCA4 pattern. It remains to be determined whether the asymmetric levels of SMARCA4 between PN observed in some zygotes could be associated with a lower embryo developmental competence.

Acute exposure to hyperosmotic conditions reduces sperm activation by urine in the yellowtail tetra Astyanax altiparanae, a freshwater teleost fish

In freshwater fish with external fertilization, sperm sampling can be contaminated with urine, which triggers motility and gives rise to decreased fertilization success. The maintenance of freshwater fish in hyperosmotic conditions may reduce urine production and improve sperm quality. Thus, the aim of this work was to verify if acute exposure to various NaCl concentrations improves sperm quality in the yellowtail tetra Astyanax altiparanae. Spermiation was induced using a single dose of carp pituitary gland (5 mg kg-1) and the males were maintained at various NaCl concentrations: NaCl 0.00% (control), NaCl 0.45% (hypoosmotic), NaCl 0.9% (isosmotic) and NaCl 1.0% (hyperosmotic) for 6 h at 26 °C. Sperm was collected and verified for activation by urine and motility traits. At 0.00%, 0.45%, and 0.90%, the sperm was motile just after sampling, indicating activation by urine. Surprisingly, at hyperosmotic conditions, no activation was observed. Other sperm and motility parameters did not show any statistical differences, including sperm viability (P = 0.7083), concentration (P = 0.9030), total motility (P = 0.6149), VCL (curvilinear velocity; P = 0.1216), VAP (average path velocity; P = 0.1231) and VSL (straight-line velocity; P = 0.1340). Our results indicate that acute maintenance at hyperosmotic conditions eliminates sperm activation by urine and maintains sperm quality. Such a new procedure is interesting for both basic and applied sciences, including reproductive practice in fish.

CircRNA circ-0110102 Function as Anti-oncogenic Gene in Hepatocellular Carcinoma through Modulating miR-580-5p/CCL2 Pathway

Background Circular RNAs (circRNAs) have been shown to have critical regulatory roles in tumor biology, whereas their contributions in hepatocellular carcinoma (HCC) still remains enigmatic. The purpose of this study was to investigate the molecular mechanisms involved in hsa_circ_0110102 in the occurrence and development of HCC. Results hsa_circ_0110102 was significantly down-regulated in HCC cell lines and tissues, low hsa_circ_0110102 expression levels were associated with poor prognosis. Knockdown hsa_circ_0110102 significantly inhibited cell proliferation, migration and invasion. In addition, the interaction between hsa_circ_0110102 and miR-580-5p was predicted and verified by luciferase assay and RNA pull-down, indicating that hsa_circ_0110102 function as sponge of miR-580-5p. Moreover, miR-580-5p which could directly bind to the 3’-UTR of CCL2 and induce its expression, then active the COX-2/PGE2 pathway in macrophage via FoxO1 in p38 MAPK dependent manner. Furthermore, the Δ256 mutant of FoxO1 showed no activation effect. These results concluded that hsa_circ_0110102 act as a sponge for miR-580-5p and decreased CCL2 secretion in HCC cells, then inhibits pro-inflammatory cytokine release from activated macrophage by regulating the COX-2/PGE2 pathway. Conclusions These results indicating that hsa_circ_0110102 serves as a potential prognostic predictor or therapeutic target for HCC.