ARF6-Dependent Regulation of P2Y Receptor Traffic and Function in Human Platelets

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y1 and P2Y12 purinoceptors. Recently, we demonstrated that P2Y1 and P2Y12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y1 and P2Y12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y1 or P2Y12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function

ADP-Ribosylation Factor 6 Expression and Activation Are Reduced in Myometrium in Complicated Pregnancies

ARF6 (ADP-ribosylation factor 6) small GTP binding protein plays critical roles in actin cytoskeleton rearrangements and membrane trafficking, including internalisation of G protein coupled receptors (GPCR). ARF6 operates by cycling between GDP-bound (inactive) and GTP-bound (active) forms and is a potential regulator of GPCR-mediated uterine activity during pregnancy and labour. ARF6 contains very low intrinsic GTP binding activity and depends on GEFs (guanine nucleotide exchange factors) such as CYTH3 (cytohesin 3) to bind GTP. ARF6 and CYTH3 were originally cloned from human placenta, but there is no information on their expression in other reproductive tissues.The expression of ARF6, ARF1, and CYTH1-4 was investigated by measuring mRNA (using RT-PCR) and protein levels (using immunoblotting) in samples of myometrium obtained from non-pregnant women, and women with normal pregnancies, before or after the spontaneous onset of labour. We also analysed myometrial samples from women with spontaneous preterm labour and from women with complicated pregnancies requiring emergency preterm delivery. The GST)-effector pull down assay was used to study the presence of active ARF6 and ARF1 in all myometrial extracts.ARF6, ARF1 and CYTH3 but not CYTH1, CYTH2 and CYTH4 were expressed in all samples and the levels did not change with pregnancy or labour. However, ARF6 and CYTH3 but not ARF1 levels were significantly reduced in complicated pregnancies. The alterations in the expression of ARF6 and its GEF in human myometrium indicate a potential involvement of this signalling system in modulating the response of myometrial smooth muscle in complicated pregnancies. The levels of ARF6-GTP or ARF1-GTP did not change with pregnancy or labour but ARF6-GTP levels were significantly decreased in women with severe complications of pregnancy.We have demonstrated a functional ARF6 system in human myometrium and a correlation between ARF6 level and activity in uterine and abnormal pregnancy

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Immunohistochemical localization of ARF6 and CYTH3 in human uterine tissue sections.

<p>A. Sections of human placenta and liver show clear ARF6 and CYTH3 immunostaining. Negative controls on the right hand side were obtained with pre-immune serum. B. ARF6 and CYTH3 showing dense intracellular staining in non-pregnant endometrial glands and uniform intracellular staining pattern in both non-pregnant and pregnant myometrial cells. The images were taken at 40× magnification.</p

Expression of ARF1, ARF6 and CYTH3 mRNAs in human myometrium.

<p>A. RT-PCR analysis of the expression of ARF6, ARF1 and CYTH1-4 mRNAs in human placenta and myometrium. Lanes 1, MDA-MB-231 cell cDNA (+ve control), 2, water (-ve control), 3, gene specific plasmid (+ve control), 4, non-pregnant (NP) myometrium cDNA, 5, placental cDNA and 6, -RT NP myometrium cDNA (-ve control). B and C. RT-PCR analysis of ARF6/ARF1 and CYTH 1-4 mRNA expression, respectively, in human myometrium in different conditions. (i). ARF6, ARF1 and CYTH1-4 cDNA fragments were amplified from the myometrium of non-pregnant (NP), spontaneous labour (SL), not-in labour (NIL), preterm-spontaneous labour (NP-SL) and preterm- not in labour (PT-NIL) by PCR using the specific primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037954#pone-0037954-t001" target="_blank">Table 1</a>. The intensity of the bands was quantified by densitometric scanning. The expression levels of ARF6, ARF1 and CYTH3 mRNAs were normalised to the amount of GAPDH in each sample and expressed as percentage relative to the NP expression (shown as 100%). The histogram represents means ± SEM of 4 samples for each condition (ii). *represents P<0.001, One way ANOVA with Tukey’s multiple comparison test.</p

The GST-pulldown analysis of active ARF1 and ARF6 levels in the myometrium.

<p>A. Analysis of the ARF1-GTP and ARF6-GTP levels in human placenta and myometrium. Lanes 1, Placenta and 2, NP myometrium. B. The GST-pulldown analysis of active ARF1 and ARF6 levels in human myometrium of NP, NIL, SL, NP-SL and PT-NIL. The tissue lysates from NP, NIL, SL, PT-NIL and PT-SL were incubated with GST-GGA3 PBD resin to precipitate ARF1-GTP or ARF6-GTP as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037954#s2" target="_blank">materials and method</a> section. The total ARF1 and ARF6 expression in the cell lysates (upper panel) and the activated ARF1 and ARF6 that was pulled down with GST-GGA3 PBD (lower panel) were detected by immunoblotting using an anti-ARF1 rabbit polyclonal and an anti-ARF6 mouse monoclonal antibody respectively (i). The intensity of the bands was quantified by densitometric scanning. The data were expressed as percentage of total ARF1 or ARF6 that is GTP bound. The histogram represents an average of 4 samples for each condition (ii).</p

GST-GGA3 PBD binding to ARF6-GTP.

<p>Myometrium tissue isolated from non-pregnant woman was lysed and the lysate was incubated with 0.1 mM GTPγS or 1 mM GDP for 30 min. The glutathione resin coupled to GST-GGA3 PBD was added to the lysate and continued the incubation for further 2 h. After washing the beads, the protein bound to the beads were analysed by immunoblotting using an anti-ARF6 mouse monoclonal antibody. The lysates that were not incubated with beads were used as input controls in immunoblotting.</p