Capsaicin triggers autophagic cell survival which drives epithelial mesenchymal transition and chemoresistance in bladder cancer cells in an Hedgehog-dependent manner

Bladder cancer (BC) is a common urologic tumor characterized by high risk of recurrence and mortality. Capsaicin (CPS), used as an intravesical drug for overactive bladder, was demonstrated to induce cell death in different cancer cells including BC cells.Here we found that treatment of high-grade BC cells with high dose of CPS triggers autophagy. Infact, the CPS treatment alters the redox homeostasis by inducing production of radicals, mitochondrial depolarization, alterations of ADP/ATP ratio and activation of AMPK pathway stimulating the autophagic process in BC cells. The inhibition of autophagy, by using the specific inhibitor bafilomycin A or Beclin 1 knock-down, enhanced the CPS-induced cell death, demonstrating that CPS-induced autophagy acts as a pro-survival process in BC cells. By using PCR arrays and FACS analysis, we found that the CPS-treated BC cells displayed typical mesenchymal features of the epithelial mesenchymal transition (EMT) as elongated shape and over-expression of vimentin, α5 and β1 integrin subunits, integrin-like kinase and the anti-apoptotic Bcl-2 proteins. Moreover, we demonstrated that CPS treatment stimulates upregulation of Dhh/Ptch2/Zeb2 members of the Hedgehog signaling pathway, increases CD24, VEGFA and TIMP1 and decreases CD44 and ALCAM mRNA expression levels. By PTCH2 knock-down we found that the Hedgehog signaling pathway is involved in the CPS-induced autophagy and EMT phenotype.Finally, we also showed that the CPS-resistant EMT-positive BC cells displayed an increased drug-resistance to the cytotoxic effects of mitomycin C, gemcitabine and doxorubicine drugs commonly used in BC therapy

Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21(Waf/Cip1) dependent manner.

Glioblastoma is associated with a poor overall survival despite new treatment advances. Antiangiogenic strategies targeting VEGF based on tyrosine kinase inhibitors (TKIs) are currently undergoing extensive research for the treatment of glioma. Herein we demonstrated that the TKI axitinib induces DNA damage response (DDR) characterized by γ-H2AX phosphorylation and Chk1 kinase activation leading to G2/M cell cycle arrest and mitotic catastrophe in U87, T98 and U251 glioma cell lines. Moreover, we found that p21(Waf1/Cip1) increased levels correlates with induction of ROS and senescence-associated cell death in U87 and T98 cell lines, which are reverted by N-acetyl cysteine pretreatment. Conversely, U251 cell line showed a resistant phenotype in response to axitinib treatment, as evidenced by cell cycle arrest but no sign of cell death. The combinatorial use of axitinib with other therapies, with the aim of inhibiting multiple signaling pathways involved in tumor growth, can increase the efficiency of this TKI. Thus, we addressed the combined effects of axitinib with no toxic doses of the proteasome inhibitor bortezomib on the growth of U87 and T98 axitinib- sensitive and axitinib-resistant U251 cell lines. Compared to single treatments, combined exposure was more effective in inhibiting cell viability of all glioma cell lines, although with different cell death modalities. The regulation of key DDR and cell cycle proteins, including Chk1, γ-H2AX and p21(Waf1/Cip1) was also studied in glioma cell lines. Collectively, these findings provide new perspectives for the use of axitinib in combination with Bortezomib to overcome the therapy resistance in gliomas

Effect of LPS-induced inflammatory state on some aspects of reproductive function of rabbit does

Effetto dell\u2019infiammazione indotta con LPS su alcuni aspetti della funzione riproduttiva nelle coniglie. Scopo della ricerca \ue8 stato quello di studiare un modello d\u2019induzione sperimentale di infiammazione con lipopolisaccaridi (LPS) microbici nella coniglia fattrice e l'eventuale effetto sulla risalita degli spermatozoi. Due gruppi di 6 coniglie fattrici sono state inoculate per via intra-peritoneale rispettivamente con LPS di E. coli 0127:B8 (100 \ub5g/kg peso vivo), o con soluzione fisiologica (controllo). Sono stati rilevati per 72 ore temperatura rettale e il numero dei leucociti; dopo inseminazione artificiale \ue8 stata valutata la risalita degli spermatozoi nel tratto riproduttivo femminile e la situazione ovarica. L\u2019infiammazione sperimentale ha indotto un rilevante incremento della temperatura rettale e sostanziali modifiche a livello di leucociti che sono comunque scomparse entro 72 ore. Anche il numero di spermatozoi risaliti \ue8 stato significativamente pi\uf9 basso a livello di corna uterine e addirittura nullo a livello dell\u2019ovidutto. In conclusione si pu\uf2 affermare che \ue8 possibile costruire un modello d\u2019induzione dello stato infiammatorio nella coniglia mediante inoculazione intra-peritoneale di 100 \ub5g LPS/kg di peso vivo.The efficiency of the cycled production system in rabbit farms is greatly conditioned by the fertility rate of does. Nulliparous does generally exhibit high fertility rate (Castellini et al., 1998), whereas the reproductive performances of multiparous does goes down. One reason of this reduction is related to the use of intensive reproductive rhythms which implies an overlapping between lactation and insemination which often produces a severe energy deficit. As in other mammals, lactation shows a strong hormonal antagonism with the reproductive activity. An other cause of hypo-fertility depends on the sanitary condition of does. Genital tract inflammation and/or infection is one of the major causes of infertility (Gram et al., 2002) and often determined by incorrect practices of artificial insemination (AI). It has been demonstrated that uterine infection negatively affects fertility (Facchin et al., 1999) and prolongs the life span of corpora lutea (Boiti et al., 1999) due to uterine leukocytes infiltration, reduced prostaglandins synthesis and increased spermatozoa reabsorption. Lipopolysaccarides (LPS), constituents of the Gram-negative germ wall, are potent stimulators of prostaglandins synthesis and are widely used to simulate inflammation in several district and organs. The aim of the paper was to verify the effect of an LPS-induced inflammatory state on some aspects of reproductive function of non-lactating rabbit does

MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protei

Food restriction during pregnancy in rabbits: effects on hormones and metabolites involved in energy homeostasis and metabolic programming

This study examined the effects of food restriction during rabbit pregnancy on hormones and metabolites involved in energy homeostasis and metabolic programming. Pregnant does were assigned to four groups: the control group was fed a standard ration while the others received a restricted amount of food (30% restriction) during early (0-9 days), mid (9-18 days), and late (19-28 days) pregnancy. The pregnancy induced a coordinated range of adaptations to fulfil energy requirements of both mother and foetus, such as hyperleptinaemia and hyperinsulinaemia, reduced insulin sensitivity, increased cortisol and non-esterified fatty acid. Food restriction altered leptin, insulin, T3, non-esterified fatty acids and glucose concentrations depending on the gestational phase in which it was applied. Collectively, present data confirm that the endocrinology of pregnancy and the adaptive responses to energy deficit make the rabbit an ideal model for studying nutritional-related disorders and foetal programming of metabolic disease. (C) 2014 Elsevier Ltd. All rights reserved

The Use of Premixed Calcium Silicate Bioceramic Sealer with Warm Carrier-Based Technique: A 2-Year Study for Patients Treated in a Master Program

Background: Recently several calcium silicate flowable sealers have been introduced as endodontic materials for the root canal. This clinical study tested the use of a new premixed calcium silicate bioceramic sealer in association with the Thermafil warm carrier-based technique (TF). Epoxy-resin-based sealer with the warm carrier-based technique was the control group. Methodology: Healthy consecutive patients (n = 85) requiring 94 root canal treatments were enrolled in this study and assigned to one filling group (Ceraseal-TF n = 47, AH Plus-TF n = 47) in accordance with operator training and best clinical practice. Periapical X-rays were taken preoperatively, after root canal filling and after 6, 12 and 24 months. Two evaluators blindly assessed the periapical index (PAI) and sealer extrusion in the groups (k = 0.90). Healing rate and survival rate were also evaluated. Chi-square tests was used to analyze significant differences between the groups. Multilevel analysis was performed to evaluate the factors associated with healing status. Results: A total of 89 root canal treatments in 82 patients were analyzed at the end-line (24 months). The total drop-out was 3.6% (3 patients; 5 teeth). A total of 91.1% of healed teeth (PAI 1-2) was observed in Ceraseal-TF, with 88.6% in AH Plus-TF. No significant difference was observed on healing outcome and survival among the two filling groups (p > 0.05). Apical extrusion of the sealers occurred in 17 cases (19.0%). Of these, 6 occurred in Ceraseal-TF (13.3%) and 11 in AH Plus-TF (25.0%). Three Ceraseal extrusions were radiographically undetectable after 24 months. All the AH Plus extrusions did not change during the evaluation time. Conclusions: The combined use of the carrier-based technique and premixed CaSi-based bioceramic sealer showed clinical results comparable with carrier-based technique and epoxy-resin-based sealer. The radiographical disappearance of apically extruded Ceraseal is a possible event in the first 24 months

Ovulation induction in rabbit does submitted to artificial insemination by adding lecirelin to the semen dose: preliminary results

The aim of this study was to analyze the effect of intravaginal administration of lecirelin on ovulation induction in rabbit does. Eighty pluriparous does (Martini genetic strain) were submitted to AI using a seminal dose of 0.5 ml containing about 10 million sperms. To stimulate ovulation, 4 homogeneous groups were submitted to different treatments: Control Group: 0.2 ml intramuscular administration of lecirelin (Dalmarelin, Fatro\uae); 0.2 Group: 0.2 ml intravaginal administration of lecirelin; 0.6 Group: 0.6 ml intravaginal administration of lecirelin; 2.0 Group: 2 ml intravaginal administration of lecirelin. In groups receiving an intra-vaginal administration, 25 \ub5g/ml Dalmarelin was diluted in the seminal dose using benzilic alcohol (20 mg/ml) as excipient. Blood samples were collected from all females, to determine LH prior (-60, -30 and 0 minutes) and (30, 60, 90, 120 and 180 minutes) after AI, and progesterone once a week for 4 weeks. After 7 days from AI, 10 does per group were euthanized in order to analyze the ovarian status. The does of control group showed a high LH peak after 30 minutes from AI; whereas intra-vaginal administration of 0.2 and 0.6 ml determined a lower increase of LH blood concentration after 2 hours. The highest dose did not produce any LH or progesterone increase. The ovary status showed a higher number of corpora lutea in Control group (P<0.05), followed by 0.2 and 0.6 ones, whereas embryos were recorded only in Control and 0.2 groups. The unsuccessful of the other experimental groups could be ascribed to the negative effect of benzilic alcohol on seminal characteristic. Only 30% of 0.2 group does were pregnant and the prolificacy was 8 kits/doe. Compared to the control group, the progesterone concentration in pregnant does showed lower value in 0.2 group. In conclusion, in spite of the obtained results, it will be necessary to test different Dalmarelin formulations (lower volume, different excipient) to recommend the minimal dose to inject intravaginally for inducing ovulation

Technological and Enzymatic Characterization of Autochthonous Lactic Acid Bacteria Isolated from Viili Natural Starters

Viili, a Finnish ropy fermented milk, is traditionally manufactured through spontaneous fermentation, by mesophilic lactic acid bacteria and yeast-like fungi, or back-slopping. This study evaluated four natural viili starters as sources of lactic acid bacteria for dairy production. Back-slopping activation of the studied viili samples was monitored through pH and titratable acidity measurements and enumeration of mesophilic lactic acid bacteria. Sixty lactic acid bacteria isolates were collected, molecularly identified, and assayed for acidification performance, enzymatic activities, production of exopolysaccharides (EPSs), presence of the histidine decarboxylase (hdcA) gene of Gram-positive bacteria, and production of bacteriocins. A neat predominance of Lactococcus lactis emerged among the isolates, followed by Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus lactis, and Lactococcus cremoris. Most isolates exhibited proteolytic activity, whereas only a few enterococci showed lipase activity. Five isolates identified as L. cremoris, L. lactis, and E. faecalis showed a good acidification performance. Most of the isolates tested positive for leucine arylamidase, whereas only one E. durans and two L. lactis isolates were positive for valine arylamidase. A few isolates also showed a positive reaction for beta-galactosidase and alpha- and beta-glucosidase. None of the isolates produced EPSs or bacteriocins. The hdcA gene was detected in five isolates identified as L. lactis and E. faecium. A few L. cremoris and L. lactis isolates for potential use as starter or adjunct cultures for dairy processing were finally identified

Single strain high-depth ngs reveals high rdna (Its-lsu) variability in the four prevalent pathogenic species of the genus candida

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra-and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput highdepth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology