Chemical modification of extracellular matrix by cold atmospheric plasma‐generated reactive species affects chondrogenesis and bone formation

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein‐2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD‐treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd.     

Surveillance and epidemiology of syphilis,gonorrhoea and chlamydia in the non-European Union countries of the World Health Organization European Region, 2015 to 2020

BackgroundEpidemics of sexually transmitted infections (STI) are a major public health challenge in the World Health Organization (WHO) European Region.AimWe aimed to provide an overview of case reporting and other surveillance data for syphilis, gonorrhoea and chlamydia for the non-European Union (EU)/European Economic Area (EEA) countries of the Centre and East part of the WHO European Region as per classification used by the WHO Regional Office for Europe (WHO/Europe) and the European Centre for Disease Prevention and Control.MethodsData were provided by the surveillance agencies of the Member States for the period 2015 to 2019 through the WHO/Europe Communicable Diseases Annual Reporting Form. We analysed reported cases, explored data reported to the WHO Gonococcal Antimicrobial Surveillance Programme (GASP) and performed a review of publications on antimicrobial resistance (AMR) in gonorrhoea in the period 2015 to 2020 using systematic methodology.ResultsFrom 2015 to 2019, in most of the countries with three or more data points, there was a pattern of decrease in reported syphilis, gonorrhoea and chlamydia cases, which is in contrast to the EU/EEA. The number of reported cases per 100,000 population was 0.4–26.5 for syphilis, 0–18.5 for gonorrhoea and 0–43.3 for chlamydia. Four countries reported recent data on AMR in gonorrhoea to GASP, and we identified further publications from Georgia, Russia and Ukraine.ConclusionWe found wide heterogeneity in reported rates of STI. There is a strong need to improve availability and quality of STI surveillance data in the non-EU/EEA countries.     

Implementing quality assurance: the design and operation of codes of practice

As services become more dispersed and increasing emphasis is given to ensuring that they are of good quality, concern has been expressed as to how this can be achieved. Achievement of quality services must not be at the expense of staff: imposition of standards from “above” is likely to reduce morale and motivation and create rigid practices which could reproduce in smaller settings some of the worst features of institutional life. This paper outlines a strategy which requires staff and user participation in the active pursuit of quality and suggests how quality may be looked at in a practical way in services. It suggests that pursuit of quality is an important and major task which must be given real priority by service providers and managers.     

Dendritic cells in cancer immunology

The clinical success of immune checkpoint therapy(ICT)has produced explosive growth in tumor immunology research because ICT was discovered through basic studies of immune regulation.Much of the current translational efforts are aimed at enhancing ICT by identifying therapeutic targets that synergize with CTLA4 or PD1/PD-L1 blockade and are solidly developed on the basis of currently accepted principles.Expanding these principles through continuous basic research may help broaden translational efforts.With this mindset,we focused this review on three threads of basic research directly relating to mechanisms underlying ICT.Specifically,this review covers three aspects of dendritic cell(DC)biology connected with antitumor immune responses but are not specifically oriented toward therapeutic use.First,we review recent advances in the development of the cDC1 subset of DCs,identifying important features distinguishing these cells from other types of DCs.Second,we review the antigen-processing pathway called cross-presentation,which was discovered in the mid-1970s and remains an enigma.This pathway serves an essential in vivo function unique to cDC1s and may be both a physiologic bottleneck and therapeutic target.Finally,we review the longstanding field of helper cells and the related area of DC licensing,in which CD4 T cells influence the strength or quality of CD8 T cell responses.Each topic is connected with ICT in some manner but is also a fundamental aspect of cell-mediated immunity directed toward intracellular pathogens.     

Programmed Cell Death (Apoptosis) and Its Role in the Pathogenesis of Lower Extremity Varicose Veins

p < 0.03). This increased apoptotic activity was not observed in media or intima of either vein group (p < 0.001). No significant difference in immunoreactivity to bcl-2 protein was observed in varicose vein specimens as compared to controls. Varicose vein specimens demonstrated increased nuclear expression of cyclin D1 whereas its cytoplasmic expression was significantly diminished (p≤0.02). These data show that programmed cell death is inhibited in varicose veins. Differential expression of cyclin D1 suggests that it may deregulate cell cycle events, thereby leading to varicosity formation.     

Mechanisms of the growth inhibitory effects of the isoflavonoid biochanin A on LNCaP cells and xenografts

BACKGROUND: Isoflavones inhibit the growth of some types of tumor cells, including prostate adenocarcinoma. This study used LNCaP cells and xenografts to investigate the mechanisms of the antiproliferative effects of biochanin A, a major isoflavone present in red clover but not soy-derived products. METHODS: LNCaP cells were exposed to varying doses of biochanin A to evaluate viability, DNA synthesis, and DNA fragmentation (TUNEL) analysis. Regulation of gene expression was determined by using Western immunoblotting and cDNA microarrays. Anti-tumorigenic effects were evaluated by using athymic mice with LNCaP flank tumors. RESULTS: Biochanin A induced a dose-dependent inhibition of proliferation and [(3)H]thymidine incorporation that correlated with increased DNA fragmentation, indicative of apoptosis. Western blot analyses of cell cycle regulatory proteins revealed that biochanin A significantly decreased expression of cyclin B and p21, whereas flow cytometry showed that cells were accumulating in the G(0)/G(1) phase. cDNA microarray analyses identified 29 down-regulated genes with six reduced below assay detection limits. Eleven genes were up-regulated, including 9 that were undetectable in controls. In mice with LNCaP xenografts, biochanin A significantly reduced tumor size and incidence. CONCLUSION: These results indicate that biochanin A inhibits prostate cancer cell growth through induction of cell cycle arrest and apoptosis. Biochanin A-regulated genes suggest multiple pathways of action. Biochanin A inhibits the incidence and growth of LNCaP xenograft tumors in athymic mice.     

Targeted Next-generation Sequencing of Advanced Prostate Cancer Identifies Potential Therapeutic Targets and Disease Heterogeneity

Background

Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue.

Objective

To demonstrate the feasibility of a novel next-generation sequencing (NGS)–based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa.

Design, setting, and participants

FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression.

Intervention

At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform.

Outcome measurements and statistical analysis

A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations.

Results and limitations

We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected.

Conclusions

This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.     

The effect of oxygen impurities on the stability and structural properties of vacancy-ordered and -disordered ZrCx

Theoretical calculations predict several long-range ordered sub-stoichiometric zirconium carbide phases to be stable at low temperature, rather than a random (disordered solution) distribution of vacancies. However, experimental synthesis of vacancy-ordered phases is extremely challenging and not all predicted phases have been experimentally observed. It has been hypothesised that the inevitable oxygen contamination in experimental samples may affect the vacancy ordering. In this work, the stability and structural properties of the vacancy-ordered and vacancy-disordered phases are investigated as a function of oxygen defect concentration using first-principles calculations. The observed trends are explained in terms of changes to the local bonding in the presence of varying amounts of oxygen and vacancies. It is found that the relative stability of the ordered phases (compared to the disordered phase at the same composition) decreases as oxygen concentration increases, and some vacancy-ordered phases are destabilised by the level of oxygen impurities found in experimental samples. This suggests that oxygen contamination is a contributing factor to the challenge of synthesising ordered zirconium carbides, and gives insight that may assist fabrication in the future. The volume of all ZrC_x (x ≤ 1) phases was found to decrease with increasing oxygen concentration, which can be attributed to the different ionocovalent nature of the C–Zr and O–Zr bonds. The volume of the vacancy-ordered phases within the expected oxygen solubility limit is greater than the disordered phase of the same composition, which is explained in terms of the relative bond strengths surrounding different vacancy distributions.

First-principles calculations are used to explore vacancy ordering in zirconium carbide at various stoichiometries as affected by oxygen impurities. Atomic bonding and electronic charge distribution are linked to stability and volume trends as a function of O concentration.