Proteomic analysis of tyrosine phosphorylation during human liver transplantation

BACKGROUND: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. RESULTS: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH(2)/SH(3 )adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. CONCLUSION: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes

The Effect of Distance Learning via SMS on Knowledge & Satisfaction of Pregnant Women

Introduction: Nowadays, cell phone has proved to be a useful device for education and access to health services particularly in developing countries. The present study aimed to determine the effect of distance learning program via SMS during pregnancy on the knowledge and satisfaction of pregnant women. Methods: This experimental study was conducted in Isfahan city during 2012-13. The sample included 300 eligible pregnant women who were selected through convenience method and assigned randomly to two groups. The control group received routine prenatal trainings while the experimental group received those trainings plus a daily pregnancy age-appropriate educational message. The knowledge of the participants was assessed at the beginning and the end of trainings using a researcher-made questionnaire, and the satisfaction of the experimental group was also assessed by a researcher-made questionnaire. Data were analyzed using chi-square tests, independent t-test, paired t-test and ANOVA. Results: The mean and standard deviation of pre-test scores were 8.62±3.33 and 8.69±4.09 in experimental and control group respectively, indicating no statistically significant difference (p=0.86). However, comparison of post-test mean scores in the experimental group (11.68±3.80) and the control group (7.56±3.53) showed a significant difference (P<0.001). Evaluation of the experimental group’s satisfaction with SMS method showed that 100 percent of participants were satisfied with the sequence and the number of the messages and 98.1 percent of them expressed satisfaction with message contents. Overall, 98.7 percent of participants were satisfied with this method. Conclusion: The results of the study showed that the use of SMS in presenting pregnancy trainings could lead to increased knowledge and satisfaction of pregnant women. Therefore, planning and implementation of this method along with pregnancy trainings are recommended

Immunogenicity and safety of RAZI recombinant spike protein vaccine (RCP) as a booster dose after priming with BBIBP-CorV: a parallel two groups, randomized, double blind trial

Abstract Background The immunity induced by primary vaccination is effective against COVID-19; however, booster vaccines are needed to maintain vaccine-induced immunity and improve protection against emerging variants. Heterologous boosting is believed to result in more robust immune responses. This study investigated the safety and immunogenicity of the Razi Cov Pars vaccine (RCP) as a heterologous booster dose in people primed with Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV). Methods We conducted a randomized, double-blind, active-controlled trial in adults aged 18 and over primarily vaccinated with BBIBP-CorV, an inactivated SARS-CoV-2 vaccine. Eligible participants were randomly assigned (1:1) to receive a booster dose of RCP or BBIBP-CorV vaccines. The primary outcome was neutralizing antibody activity measured by a conventional virus neutralization test (cVNT). The secondary efficacy outcomes included specific IgG antibodies against SARS-CoV-2 spike (S1 and receptor-binding domain, RBD) antigens and cell-mediated immunity. We measured humoral antibody responses at 2 weeks (in all participants) and 3 and 6 months (a subgroup of 101 participants) after the booster dose injection. The secondary safety outcomes were solicited and unsolicited immediate, local, and systemic adverse reactions. Results We recruited 483 eligible participants between December 7, 2021, and January 13, 2022. The mean age was 51.9 years, and 68.1% were men. Neutralizing antibody titers increased about 3 (geometric mean fold increase, GMFI = 2.77, 95% CI 2.26–3.39) and 21 (GMFI = 21.51, 95% CI 16.35–28.32) times compared to the baseline in the BBIBP-CorV and the RCP vaccine groups. Geometric mean ratios (GMR) and 95% CI for serum neutralizing antibody titers for RCP compared with BBIBP-CorV on days 14, 90, and 180 were 6.81 (5.32–8.72), 1.77 (1.15–2.72), and 2.37 (1.62–3.47) respectively. We observed a similar pattern for specific antibody responses against S1 and RBD. We detected a rise in gamma interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukin 2 (IL-2) following stimulation with S antigen, particularly in the RCP group, and the flow cytometry examination showed an increase in the percentage of CD3 + /CD8 + lymphocytes. RCP and BBIBP-CorV had similar safety profiles; we identified no vaccine-related or unrelated deaths. Conclusions BBIBP-CorV and RCP vaccines as booster doses are safe and provide a strong immune response that is more robust when the RCP vaccine is used. Heterologous vaccines are preferred as booster doses. Trial registration This study was registered with the Iranian Registry of Clinical Trial at www.irct.ir , IRCT20201214049709N4. Registered 29 November 2021

ARFGAP1 localizes to lipid droplets as revealed by immuno-gold transmission electron microscopy.

<p>Thin frozen sections of HepG2 cells fixed 4 hours after addition of 0.5 mM oleate were incubated individually with antibodies to either PLIN2 (white arrowheads) or ARFGAP1 (black arrowheads) followed by gold-conjugated secondary antibodies to reveal endogenous proteins at the ultra structural level. Bars  = 100 nm.</p

Cyclic AMP reverses lipid droplet association of ARFGAP1.

<p>McA-RH7777 cells were incubated with 0.5 mM oleate for 4 hours after which cyclic AMP (cAMP) was added to a final concentration of 37.5µg/ml. The cells were simultaneously stained for endogenous ARFGAP1 (red) and neutral lipid with Bodipy 493/503 (green). Bars  = 5µm.</p

Lipid droplet localization of ARFGAP1 is distinct from other endogenous Golgi and ER markers.

<p>Indirect immunofluorescence of COPB1 in HepG2 cells, ARF1 in McA-RH7777 cells, TMED7 and CANX in HepG2 cells before (4 right panels) and 4 hours after addition of 0.5 mM oleate. Enlarged regions of interest are shown in the 4 left panels. Bars  = 7µm.</p

ARFGAP1 relocates from the Golgi apparatus to lipid droplets upon addition of oleate.

<p>HepG2 cells were fixed and processed for indirect immunofluorescence before (-OA) and 4 hours after addition of 0.5 mM oleate and stained to reveal endogenous ARFGAP1 (green) and lipid droplets (red) using a rabbit polyclonal to ARFGAP1 and Bodipy 558/568 C₁₂, respectively. Bars  = 5µm.</p

Quantification of ARFGAP1 associated with lipid droplets

<p>. Endogenous ARFGAP1 as revealed by indirect immunofluorescence was quantified before (-OA) and 4 hours after addition of oleate (+OA) in HepG2 and McA-RH7777 cells expressed as % cells with ARFGAP1 staining (as revealed through indirect immunofluorescence) or % Bodipy-stained droplets associated with ARFGAP1 per cell. On the right, total area of Bodipy-stained lipid droplets per µm² per cell.</p