Comprehensive transcriptomic analysis of papillary thyroid cancer: potential biomarkers associated with tumor progression.

PURPOSE: Identification of stage-specific prognostic/predictive biomarkers in papillary thyroid carcinoma (PTC) could lead to its more efficient clinical management. The main objective of this study was to characterize the stage-specific deregulation in genes and miRNA expression in PTC to identify potential prognostic biomarkers. METHODS: 495 RNASeq and 499 miRNASeq PTC samples (stage I-IV) as well as, respectively, 56 and 57 normal samples were retrieved from The Cancer Genome Atlas (TCGA). Differential expression analysis was performed using DESeq 2 to identify deregulation of genes and miRNAs between sequential stages. To identify the minority of patients who progress to higher stages, we performed clustering analysis on stage I RNASeq data. An independent PTC RNASeq data set (BioProject accession PRJEB11591) was also used for the validation of the results. RESULTS: LTF and PLA2R1 were identified as two promising biomarkers down-regulated in a subgroup of stage I (both in TCGA and in the validation data set) and in the majority of stage IV of PTC (in TCGA data set). hsa-miR-205, hsa-miR-509-2, hsa-miR-514-1 and hsa-miR-514-2 were also detected as up-regulated miRNAs in both PTC patients with stage I and stage III. Hierarchical clustering of stage I samples showed substantial heterogeneity in the expression pattern of PTC indicating the necessity of categorizing stage I patients based on the expressional alterations of specific biomarkers. CONCLUSION: Stage I PTC patients showed large amount of expressional heterogeneity. Therefore, risk stratification based on the expressional alterations of candidate biomarkers could be an important step toward personalized management of these patients.

CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function.

G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic target. This study aimed to assess the metabolic functionality of GPR55 ligands using CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55 signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10-7-10-4M) stimulated insulin secretion (p < 0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4) beta cells, with 2-2.7-fold (p < 0.001) increase demonstrated in BRIN-BD11 cells (10-4M). The insulinotropic effect of Abn-CBD (42 %), AM251 (30 %) and PEA (53 %) were impaired (p < 0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602 completely abolished (p < 0.001). Gpr55 ablation abolished the release of intracellular Ca2+ upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-CBD and AM251 (1.7-3-fold; p < 0.01) was greatly diminished (p < 0.001) in Gpr55 null cells. Orally administered Abn-CBD and AM251 (0.1 µmol/kgBW) improved GIP (p < 0.05-p < 0.01), GLP-1 (p < 0.05-p < 0.001), glucose tolerance (p < 0.001) and circulating insulin (p < 0.05-p < 0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p < 0.05) and insulin release (p < 0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects of Abn-CBD (p < 0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1 release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a novel approach for the treatment of type-2-diabetes.

Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells.

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

Gene editing in the context of an increasingly complex genome.

The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule -and how these relate to regulation- have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin.In this review, we chart the course of the major advances in the biomedical sciences in the era pre- and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA- RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms.Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.

Post-LASIK exacerbation of granular corneal dystrophy type 2 in members of a chinese family.

PurposeThe post-LASIK exacerbation of corneal dystrophy, otherwise asymptomatic, is almost exclusively associated with the TGFBI gene mutations at codon 124 in exon 4 and codon 555 in exon 12. It is our intention to demonstrate that the pre-operative genetic screening for TGFBI mutations should be mandatory for refractive surgery candidates.Patients and MethodsIn this study, we reviewed the proband's post-LASIK slit-lamp and in vivo confocal microscopy images and genetic testing results, and performed genetic testing on eleven additional members of the family to investigate the penetrance of corneal dystrophy in asymptomatic members who carry the mutation.ResultsThe proband demonstrated a post-LASIK exacerbation of Granular Corneal Dystrophy type 2 (GCD2), identified as a TGFBI R124H mutation. Three of the 11 family members tested positive for the same R124H mutation as the proband.ConclusionThe lesson learned from this case is that the genetic screening of TGFBI mutations must be incorporated into the pre-operative screening procedures to prevent exacerbation and recurrence, which eventually could lead to the need for a corneal transplant.

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

A retrospective audit of the extent and nature of domestic violence cases identified over a three year period in the two district command units of the police service of Northern Ireland.

The work load of forensic medical officers (FMOs) who provide medical cover for the Coleraine and Limavady district command units (DCU) of the police service of Northern Ireland (PSNI) in dealing with domestic violence was investigated over a three year period from 1st April 2003 to 31st March 2006. A total of 128 cases involving domestic violence were identified during this three year period. There was a significant increase from 4% (32/791) in the first year to 6.6% (56/851, p<0.01) in the number of cases of identified domestic violence in the second year which dropped to 4.2% (40/956) in the third year. Victims were identified in 38% of these domestic violence cases with 62% being identified as assailants. It was noted that there was a significant difference in the proportion of male assailants (96.2%) from female assailants (3.8%). Fifty-four percent of victims were noted to have injuries in accordance with the more serious injury categories of assault of actual bodily harm (AOABH) and grievous bodily harm (GBH). Domestic incidents occurred at the home in 91% of cases, with the FMO being the primary contact in 97% of cases. Alcohol was implicated in 56% of all domestic violence cases recorded.

Drug facilitated sexual assault--a review.

This review was undertaken to identify the evolutionary process in the current understanding of allegations of drug facilitated sexual assault (DFSA), so that those who work in this field may gain a better understanding of the complexities involved in such cases. Several definitions of DFSA are provided as well as a list of intoxicating substances which have so far been incriminated in this crime. Perception and alcohol use is addressed, whilst an examination of intoxication and victim outcomes reveals disturbing but important information which needs to be centrally placed within health education campaigns with a degree of urgency. The review identifies the effects of alcohol on sexual behaviour, drinking patterns and specific quantitative research indicating very high alcohol levels in some instances. In practical terms, suggestions are made following Operation Matisse, to address prevention, early detection and easier identification of DFSA cases so that victims' needs are prioritised and appropriately addressed.