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Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin-6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c-Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf-null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53-null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM-induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53-independent manner, and we show that p21-null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6-mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti-inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage. ; L.M. was recipient of an FPU contract from the Spanish Ministry of Education (MECD). Work in the laboratory of M.S. was funded by the CNIO and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (RETOS project), the European Research Council (ERC Advanced Grant), the European Union (RISK-IR project), and the Botin Foundation and Banco Santander (Santander Universities Global Division). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Sí

Xiaolan He,1,2,* Peng Liu,1,2,* Xiao Zhang,1,2,* Zhenhua Jiang,1,2 Nan Gu,1,2 Qun Wang,1,2 Yan Lu1,2 1Department of Pain Medicine; 2Department of Anesthesiology & Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, People's Republic of China*These authors contributed equally to this workCorrespondence: Yan Lu; Qun WangDepartment of Pain Medicine, Xijing Hospital, Fourth Military Medical University, 127 West Changle Road, Xi'an, 710032, People's Republic of ChinaTel +86 134 8815 6067; +86 1399129 2113Fax +86 29 8477 1262Email 13488156067@163.com; wqfmmu77@163.comPurpose: Spinal glycinergic neurons function as critical elements of a spinal gate for pain and itch. We have recently documented that spinal PKCγ+ neurons receive the feedforward inhibitory input driven by Aβ primary afferent. The glycinergic neurons control the excitability of PKCγ+ neurons and therefore gate mechanical allodynia. However, a dynamic or electrophysiological analysis of the synaptic drive on spinal glycinergic interneurons from primary afferent fibers is largely absent. The present study was aimed to analyze the synaptic dynamics between spinal glycinergic interneurons and primary afferents using a genetic labeled animal model.Materials and Methods: The GlyT2-P2A-iCre mice were constructed by the CRISPR/Cas9 technology. The GlyT2-iCre-tdTomato mice were then generated by crossing the GlyT2-P2A-iCre mice with fluorescent reporter mice. Patch-clamp whole-cell recordings were used to analyze the dynamic synaptic inputs to glycinergic neurons in GlyT2-iCre-tdTomato mice. The distribution of GlyT2-tdTomato neurons in the spinal dorsal horn was examined by the immunohistochemistry method. The firing pattern and morphological features of GlyT2-tdTomato neurons were also examined by electrophysiological recordings and intracellular injection of biocitin.Results: The GlyT2-P2A-iCre and GlyT2-tdTomato mice were successfully constructed. GlyT2-tdTomato fluorescence was colocalized extensively with immunoreactivity of glycine, GlyT2 and Pax2 in somata, confirming the selective expression of the transgene in glycinergic neurons. GlyT2-tdTomato neurons were mainly distributed in spinal lamina IIi through IV. The firing pattern and morphological properties of GlyT2-tdTomato neurons met the features of tonic central or islet type of spinal inhibitory interneurons. The majority (72.1%) of the recorded GlyT2-tdTomato neurons received primary inputs from Aβ fibers.Conclusion: The present study indicated that spinal GlyT2-positive glycinergic neurons mainly received primary afferent Aβ fiber inputs; the GlyT2-P2A-iCre and GlyT2-tdTomato mice provided a useful animal model to further investigate the function of the GlyT2+-PKCγ+ feedforward inhibitory circuit in both physiological and pathological conditions.Keywords: glycinergic neurons, spinal cord, GlyT2, Aβ fiber, feed-forward inhibitory circuitry

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2018 ; Includes bibliographical references (leaves. 81-88). ; Text in English; Abstract: Turkish and English. ; DNA methylation is epigenetic events commonly occurs in mammalian genome starting from formation of embryo to the end of life. Especially, hypermethylation in tumor suppressor genes, corresponds the cancer growth and detection of DNA methylation in these genes crucial for the diagnosis of cancer. Water soluble polythiophenes are frequently used for the detection of biomolecules through the optoelectronic properties. In this study, detection of DNA methylation of multiple tumor suppressor p16INK4A gene via polythiophene based optical sensor was achieved. Newly designed, synthesized and characterized poly(1-(3-((4-methylthiophen-3-yl) oxy) propyl)-1,4diazabicyclo [2.2.2] octan-1-ium bromide) was used during characterization of DNA sequences and detection of DNA methylation. The target sequence position is +137 to +156 in p16INK4A gene which have three potential CG dinucleotide to be methylated. Detection of DNA methylation based on sodium bisulfite treatment, complementary sequence of unmethylated ssDNA and the conformational change of water soluble polythiophene. In our fluorometric analysis, unmethylated sequence/complementary successfully hybridized and dsDNA Io/I ratio is under the 1.40 while the methylated sequence/complementary hybridization failed due to different base content and remain as ssDNA and, Io/I ratio is higher than 1,60. The novelty of work is detection mechanism is PCR and FRET free with a range of 300 ng to 700 ng sample requirement. Characterization of homopurines, homopyrimidines, methylated and unmethylated sequence with cationic polythiophenes also accomplished. PolyG (10), polyG (20) and polyA (10) yielded a no signal in UV-VIS region while the polyA (20) yielded a 100 nm red shift. Furthermore, PolyC (10), PolyC (20), PolyT (10), PolyT (20) yielded three vibrionic peaks at 505 nm, 545 nm and 595 nm with different intensities and unique isosbestic points. All 10 bases long homopyrimidine and homopurine have a unique quencher character with cationic polythiophene. Lastly, conformational change of polythiophene investigated with computational methods and heptamer used as a model. ; DNA metilasyonu, embriyonun oluşmasından başlayarak yaşamın sonuna kadar kadar memeli genomunda yaygın olarak görülen epigenetik olaylardır. Özellikle tümör basklıyacı genlerde oluşan hipermetilasyonlar kanser büyümesine neden olurlar ve bu genlerdeki DNA metilasyon tespiti kanser teşhisi için kritiktir. Suda çözünen tiyofenler optoelektronik özelliklerinden dolayı biyomoleküllerin tayininde sıklıkla kullanılmaktadır. Bu çalışmada, politiyofen tabanlı optic sensor ile çoklu tümör baskılayıcı p16INK4A genindeki DNA metilasyonu tayini başarılı bir şekilde yapıldı. Dizayn edilen, sentezlenen ve karakterizasyonu yapılan poly(1-(3-((4-metiltiyofen-3-il) oksi) propil)1,4-diazabisiklo [2.2.2] oktan-1-yum bromür) DNA sekanslarının karakterizasyonunda ve DNA metilasyon tayininde kullanıldı. p16INK4A genindeki üç tane potansiyel metillenebilir CG dinükleotidi bulunan hedef sekansın pozisyonu +137 ile +156 arasındadır. DNA metilasyonun tayininin temeli, sodium bisulfit işlemine, metillenmemiş sekansın tamamlayıcı sekansına ve politiyofenin konformasyonal değişimine dayanmaktadır. Yapılan florometrik analizler sonucunda, metillenmemiş sekans/tamamlayıcı sekans ile başarılı bir şekilde hibridize oldu ve çift sarmal DNA Io/I oranı 1.40 değerinin altında bulundu. Metilli sekans/tamamlayıcı sekansın hibridizasyonu farklı baz içeriği yüzünden başarısız oldu ve tek sarmal DNA olarak kaldı ve Io/I oranı 1.60 değerinin üzerinde bulundu. Bu çalışmanın yenilikçi tarafı, PCR ve FRET bağımsız tayin metodu olup 300 ng ile 700 ng aralığında örnek miktarın tayin için yeterli olmasıdır. Aynı zamanda katyonik tiyofen ile homopürin, homopirimidin, metilli sekans ve metilsiz sekansıların karakterizasyonları başarı ile gerçekleştirildi. PolyG (10), polyG (20) and polyA (10) için UV-VIS bölgesinde sinyal değişimi gözlemlenmezken polyA (20) 100 nm lik bir kırmızı kaymaya neden oldu. Buna ek olarak, PolyC (10), PolyC (20), PolyT (10), PolyT (20) için farkı şiddete ve eşsiz isosbestik noktasına sahip olan 505 nm, 545 nm ve 595 nm de üç sinyal gözlemlendi. Politiyofenin konformasyonal değişimi hesaplamalı metodlarla araştırılarak yedimer model olarak seçilmiştir.

"Background: Human papillomavirus (HPV) E6 and E7 oncoproteins are vital for developing HPV induced penile carcinoma. The viral oncoproteins play a central role in oncogenesis by interacting with several cellular regulatory proteins, such as p16INK4a and p53. Many studies suggest that these proteins showed clinical utility in predicting nodal disease, cancer specific survival, overall survival and even tumor grade. Understanding the molecular mechanism involved in the carcinogenesis of penile cancer could offer biomarkers for disease progression, treatment response and potential targeted therapies; (2) Methods: This paper is a prospective study on a group of 100 patients who underwent prostate surgery during 2013 and 2014 in the Urology Clinic of "Carol Davila" Central Military Emergency University Hospital Bucharest. They were tested for HPV by PCR and IHC (p16) methods; (3) Results: 11 cases (22%) of HPV were found in the cluster of patients tested. PCR and P16 were the HPV diagnostic tests used. In order to determine the consistency of the 2 tests, the Cohen's kappa coefficient was used at a p level < 0.05. The PCR method had a sensitivity of 81.8% and a specificity of 94.9%. The P16 method had a sensitivity of 63.6% and a specificity of 89.7%."

Abstract
Some properties of Salmonella-infected cells overlap with immunogenic cell death. In this study, we demonstrated that intracellular infection of melanoma with Salmonella typhimurium induced high immunogenicity in melanoma cells, leading to antitumor effects with melanoma-antigen-specific T-cell responses. Murine B16F10 melanoma cells were infected with tdTomato-expressing attenuated S. typhimurium (VNP20009; VNP-tdT), triggering massive cell vacuolization. VNP-tdT-infected B16F10 cells were phagocytosed efficiently, which induced the activation of antigen-presenting cells with CD86 expression in vitro. Subcutaneous coimplantation of uninfected and VNP-tdT-infected B16F10 cells into C57BL/6 mice significantly suppressed tumor growth compared with the implantation of uninfected B16F10 cells alone. Inoculation of mice with VNP-tdT-infected B16F10 cells elicited the proliferation of melanoma-antigen (gp100)-specific T cells, and it protected the mice from the second tumor challenge of uninfected B16F10 cells. These results suggest that Salmonella-infected tumor cells acquire effective adjuvanticity, leading to ideal antitumor immune responses.

AbstractThe development of dysplastic changes in oral epithelial lesions is a potential long‐term complication after hematopoietic stem cell transplantation (HSCT). This may be related to mechanisms including radiation and chemotherapy regimens, chronic graft‐versus‐host disease (cGVHD), inflammation, and prolonged immunosuppression. The current case describes a 54‐year‐old male with multiple myeloma treated by autologous and allogenic HSCT followed by development of cGVHD (mouth, skin and the eyes) with the complaint of diffuse white lesions on the buccal mucosa, tongue, and palate. A biopsy performed with histopathological analysis revealed moderate to severe epithelial dysplasia with hyperkeratosis, positive for p16INK4A as a surrogate marker for human papillomavirus (HPV). Our finding suggests a possible association of oral dysplasia and HPV in patients after receiving allogenic HSCT with the necessity of more clinical follow‐ups for those patients that may be at a higher risk for the development of oral dysplasia associated with HPV.

If a specific biological mechanism could be determined by which a carcinogen increases lung cancer risk, how might this knowledge be used to improve risk assessment? To explore this issue, we assume (perhaps incorrectly) that arsenic in cigarette smoke increases lung cancer risk by hypermethylating the promoter region of gene p16INK4a, leading to a more rapid entry of altered (initiated) cells into a clonal expansion phase. The potential impact on lung cancer of removing arsenic is then quantified using a three‐stage version of a multistage clonal expansion (MSCE) model. This refines the usual two‐stage clonal expansion (TSCE) model of carcinogenesis by resolving its intermediate or "initiated" cell compartment into two subcompartments, representing experimentally observed "patch" and "field" cells. This refinement allows p16 methylation effects to be represented as speeding transitions of cells from the patch state to the clonally expanding field state. Given these assumptions, removing arsenic might greatly reduce the number of nonsmall cell lung cancer cells (NSCLCs) produced in smokers, by up to two‐thirds, depending on the fraction (between 0 and 1) of the smoking‐induced increase in the patch‐to‐field transition rate prevented if arsenic were removed. At present, this fraction is unknown (and could be as low as zero), but the possibility that it could be high (close to 1) cannot be ruled out without further data.

Psoriasis is a common inflammatory skin disease involving a cross-talk between epidermal and immune cells. The role of specific epidermal stem cell populations, including hair follicle stem cells (HF-SCs) in psoriasis is not well defined. Here, we show reduced expression of c-JUN and JUNB in bulge HF-SCs in patients with scalp psoriasis. Using lineage tracing in mouse models of skin inflammation with inducible deletion of c-Jun and JunB, we found that mutant bulge HF-SCs initiate epidermal hyperplasia and skin inflammation. Mechanistically, thymic stromal lymphopoietin (TSLP) was identified in mutant cells as a paracrine factor stimulating proliferation of neighboring non-mutant epidermal cells, while mutant inter-follicular epidermal (IFE) cells are lost over time. Blocking TSLP in psoriasis-like mice reduced skin inflammation and decreased epidermal proliferation, VEGFα expression, and STAT5 activation. These findings unravel distinct roles of HF-SCs and IFE cells in inflammatory skin disease and provide novel mechanistic insights into epidermal cell interactions in inflammation. ; We thank Drs. M. Serrano and M. Perez-Moreno for the Gt(ROSA)26Sortrn4(ACTB-tdTomato,-EGFP)Luo/J and K15-Cre-PGR mouse lines. We are very grateful to Drs. M. Perez-Moreno, F. Real, O. Uluckan, L. Bakiri and the laboratory members of the Sibilia and Wagner groups for critical reading of the manuscript and valuable suggestions. We thank V. Bermeo, G. Medrano, S. Leceta, O. Grana, and M. Perez for their technical help and IT support. We acknowledge R. Paus laboratory members for the shipment of hair follicle samples. N.G.L. received funding from the People programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no 608765. A.I is funded by the Institute of Health Carlos III (PI16/01430). The Wagner laboratory was funded by a grant from the Spanish Ministry of Economy and competitiveness (SAF2015-70857RE, cofounded by the European Regional Development Fund) and is ...

El pdf del artículo es la versión post-print.-- et al. ; In mouse epidermal carcinogenesis, the latest stage of malignant progression involves the transition from squamous cell carcinoma to a highly aggressive type of tumor with spindle morphology. In this work, we have isolated a minor epithelial cell subpopulation (CarC-R) contained in the highly malignant spindle carcinoma cell line CarC. CarC-R exhibited a drastic reduction in tumorigenicity when compared with CarC, but CarC-R-induced tumors were mainly sarcomatoid, although they subsequently reverted to the epithelial phenotype when tumor explants were recultured in vitro. Several single-cell clones with either stable epithelial or fibroblastic phenotypes were isolated from an explanted CarC-R tumor (CarC-RT). All these cell lines contained the same specific point mutation in H-Ras codon 61, but while CarC spindle cells had lost the normal H-Ras allele, it was retained in CarC-R- and CarC-RT-derived cell lines. Furthermore, CarC cells have inactivated p16INK4a and p19INK4a/ARF transcription, while CarC-R and CarC-RT clones expressed p19 mRNA and protein but not p16. Altogether, these results suggest that CarC-R represents a precursor stage to CarC in malignant progression. Spectral karyotyping analysis revealed that CarC-R was highly aneuploid and contained many chromosomal abnormalities. In contrast, CarC had a diploid or tetraploid modal chromosome number and contained a specific T(14;15) translocation in all of the analysed metaphases. The T(14;15) translocation was present in only a minority (1.9%) of CarC-R cells, but it was widely spread in CarC-RT and its derived cell clones, regardless of their epithelial or fibroblastic phenotype, indicating that T(14;15) segregates with malignancy. © 2005 Nature Publishing Group All rights reserved. ; This study was supported by grants from the 'Ministerio de Educacióo n y Ciencia' of Spain (SAF2004-04902 to MQ, and BOS2002-00232 to JLB), 'Fondo de Investigación Sanitaria' (Red de Centros de Cáncer, RTICCC, CO3/10) to MQ, and by the European Union (Cost Action B19, 'Molecular Cytogenetics of Solid Tumours') to JCC. ; Peer Reviewed