Loss of CCDC6 Affects Cell Cycle through Impaired Intra-S-Phase Checkpoint Control

In most cancers harboring Ccdc6 gene rearrangements, like papillary thyroid tumors or myeloproliferative disorders, the product of the normal allele is supposed to be functionally impaired or absent. To address the consequence of the loss of CCDC6 expression, we applied lentiviral shRNA in several cell lines. Loss of CCDC6 resulted in increased cell death with clear shortening of the S phase transition of the cell cycle. Upon exposure to etoposide, the cells lacking CCDC6 did not achieve S-phase accumulation. In the absence of CCDC6 and in the presence of genotoxic stress, like etoposide treatment or UV irradiation, increased accumulation of DNA damage was observed, as indicated by a significant increase of pH2Ax Ser139. 14-3-3σ, a major cell cycle regulator, was down-regulated in CCDC6 lacking cells, regardless of genotoxic stress. Interestingly, in the absence of CCDC6, the well-known genotoxic stress-induced cytoplasmic sequestration of the S-phase checkpoint CDC25C phosphatase did not occur. These observations suggest that CCDC6 plays a key role in cell cycle control, maintenance of genomic stability and cell survival and provide a rational of how disruption of CCDC6 normal function contributes to malignancy

Rare Variant Enrichment analysis Supports

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by aplasia of the female reproductive tract; the syndrome can include renal anomalies, absence or dysgenesis, and skeletal anomalies. While functional models have elucidated several candidate genes, onl

Genome-Wide Association Identifies Nine Common Variants Associated With Fasting Proinsulin Levels and Provides New Insights Into the Pathophysiology of Type 2 Diabetes

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10−8). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10−4), improved β-cell function (P = 1.1 × 10−5), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10−6). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

CCDC6 knock down alters proliferation rate and increases cell death <i>in vitro</i>.

<p>Cells were transduced using lentivirus, expressing two different small hairpins for CCDC6, labeled as sh1 and sh2 and cultured for 48 hours. The same viral vector was applied as control (mock: mock transduced). (<b>A</b>) Western blot analysis using anti-CCDC6 mouse monoclonal antibody demonstrated the efficient knock down of CCDC6 protein expression. Growth curves were performed in triplicates using trypan blue dye exclusion for counting the alive (<b>B</b>) and the dead cells (<b>C</b>). Decreased proliferation rate and increased cell death was observed in the absence of CCDC6. (<b>D</b>) The subG₀/G₁ population, as measured by flow cytometry, is indicative of apoptosis and is significantly increased following CCDC6 knock down. The percentage of survival was calculated for each time point by excluding both early apoptotic and dead cells. (<b>E</b>) Apoptotic cell death was analyzed by Po-PRO and 7-ADD staining. The Po-PRO single-positive cells are early apoptotic while the double positive stained cells for Po-PRO and 7-AAD are late apoptotic and dead cells. All assays were performed in three independent experiments.</p

Deficient S phase checkpoint regulation upon etoposide treatment in the absence of CCDC6.

<p>(<b>A</b>) HCT116 cells were treated with 20 µM etoposide and cells were harvested at predetermined time points for cell cycle analysis. In the absence of CCDC6, no S phase accumulation is observed and the transition to G₂ phase is accelerated. One representative experiment is shown, out of three performed. (<b>B</b>) Concomitant apoptotic cell death was quantified by measuring the subG₀/G₁ DNA content. CCDC6 knock down cells showed higher levels of apoptosis, at earlier time point, in comparison to the control, in response to genotoxic stress upon etoposide treatment. (<b>C</b>) The percentage of cell survival was assessed by gating for PoPRO and 7-AAD negative cells. CCDC6 knock down resulted in lower cell survival upon etoposide induced genotoxic stress. The assays were performed in triplicates.</p

CCDC6 knock down results in altered cellular localization of CDC25C and accelerated G₂/S transition upon etoposide-mediated genotoxic stress.

<p>Control and CCDC6 knock down HCT116 cells were treated with etoposide (20 µM). (<b>A</b>) Cell lysates of HCT116 cells treated with etoposide (20 µM) for 2, 4, 8, 12 and 24 hours and mock control treated with DMSO vehicle were resolved on a SDS-PAGE and probed for 14-3-3σ and CDC25C. 14-3-3σ protein levels were down-regulated in the absence of CCDC6 protein expression and the CDC25C protein level regulation was altered. (<b>B</b>) Cells grown on cover slips were exposed to etoposide for 4, 8, 12, 24 hours, fixed and stained for CDC25C. In mock cells, CDC25C is kept in the cytosol upon etoposide treatment at 8 and 12 hours but is localized in the nucleus in the absence of CCDC6. (<b>C</b>) Cells exposed to etoposide for 12 hours were co-stained for CDC25C and 14-3-3σ. CDC25C is kept in the cytosol upon etoposide treatment and exhibits co-localization with 14-3-3σ (seen in yellow) but enters the nucleus in the absence of CCDC6.</p

Normal Cell cycle progression is altered upon CCDC6 knock down.

<p>Cell cycle analysis was performed using propidium iodide (PI) staining and measuring the DNA content, at the indicated time points, starting 48 hours after transduction. Cell cycle was analyzed with FlowJo software and Jean-Fox algorithm. In all time points both in HCT116 (<b>A</b>), (<b>B</b>) and HeLa (<b>C</b>), (<b>D</b>) the percentage of cells in the S phase is reduced upon knock down of CCDC6 in comparison to the control (mock). One, out of three, representative experiment is shown. (<b>E</b>) HCT116 cells were synchronized by serum starvation for 48 hours followed by restimulation with 5% of FCS. CCDC6 knock down resulted in incomplete arrest at G₁ and not total synchronization, as the control cells. 14 hours after serum stimulation the majority of the control cells are in S phase while CCDC6 knock down cells demonstrated a delay in S phase entering. 4 hours later, control and CCDC6 knock down cells showed the same profile, suggesting shorter duration of S upon CCDC6 knock down. 24 hours later, control cells were cycling normally and CCDC6 knock down cells exhibited a delay in completing G₂ phase and re-entering G₁.</p

Synthesis and biological evaluation of modified purine homo-N-nucleosides containing pyrazole or 2-pyrazoline moiety

9-Substituted (pyrazol-5-yl)methyl- or (2-pyrazolin-5-yl)methyl-9H-purines were synthesized from 9-allyl-6-chloro-9H-purine through the 1,3-dipolar cycloaddition reaction with nitrile imines, prepared in situ from the corresponding hydrazone and NBS/Et3N under MW or from hydrazinoylchloride and Et3N under reflux. The coupling of new 6-chloropurines with amines in H2O under microwaves resulted quantitatively to modified pyrazol-5-yl- or 2-pyrazolin-5-yl adenine homo-N-nucleosides. The new compounds were tested in vitro for their ability to: (i) interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH), (ii) inhibit lipid peroxidation, (iii) inhibit the activity of soybean lipoxygenase, (iv) inhibit in vitro thrombin and for (v) their antiproliferative and cytotoxic activity. Pyrazolines were found to be more potent in vitro. Compound 7a exhibited satisfactory combined antioxidant and anti-lipid peroxidation activity, inhibition of lipoxygenase (89%) and thrombin inhibitory ability, whereas compound 7b exhibited high lipoxygenase inhibitory activity in combination to significant anti-thrombin activity. No compound exhibited a significant cytotoxic activity, while all showed moderate antiproliferative activity