Remote Ischemic Preconditioning (RIPC) Modifies the Plasma Proteome in Children Undergoing Repair of Tetralogy of Fallot: A Randomized Controlled Trial

Background

Remote ischemic preconditioning (RIPC) has been applied in paediatric cardiac surgery. We have demonstrated that RIPC induces a proteomic response in plasma of healthy volunteers. We tested the hypothesis that RIPC modifies the proteomic response in children undergoing Tetralogy of Fallot (TOF) repair.

Methods and Results

Children (n=40) were randomized to RIPC and control groups. Blood was sampled at baseline, after cardiopulmonary bypass (CPB) and 6, 12 and 24h post-CPB. Plasma was analysed by liquid chromatography mass spectrometry (LC-MS) in an untargeted approach. Peptides demonstrating differential expression (p<0.01) were subjected to tandem LC-MS/MS and protein identification. Corresponding proteins were identified using the NCBI protein database. There was no difference in age (7.3±3.5vs6.8±3.6 months)(p=0.89), weight (7.7±1.8vs7.5±1.9 kg)(p=0.71), CPB time (104±7vs94±7 min)(p=0.98) or aortic cross-clamp time (83±22vs75±20 min)(p=0.36). No peptides were differentially expressed at baseline or immediately after CPB. There were 48 peptides with higher expression in the RIPC group 6h post-CPB. This was no longer evident at 12 or 24h, with one peptide down-regulated in the RIPC group. The proteins identified were: inter-alpha globulin inhibitor (42.0±11.8 vs 820.8±181.1, p=0.006), fibrinogen preproprotein (59.3±11.2 vs 1192.6±278.3, p=0.007), complement-C3 precursor (391.2±160.9 vs 5385.1±689.4, p=0.0005), complement C4B (151.5±17.8 vs 4587.8±799.2, p=0.003), apolipoprotein B100 (53.4±8.3 vs 1364.5±278.2, p=0.005) and urinary proteinase inhibitor (358.6±74.9 vs 5758.1±1343.1, p=0.009). These proteins are involved in metabolism, haemostasis, immunity and inflammation.

Conclusions

We provided the first comprehensive analysis of RIPC-induced proteomic changes in children undergoing surgery. The proteomic changes peak 6h post-CPB and return to baseline within 24h of surgery.

Trial Registration

ACTR.org.au ACTRN12610000496011     

Expression and promoter methylation status of hMLH1, MGMT,APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.     

Shrimps of an ancient Balkan lake: Bionomy and conservation

In order to have a comprehensive evaluation and classification of the natural biota of Lake Pamvotis, the present study aims at investigating shrimps’ bionomic traits. Information on shrimps’ habitat preferences, abundances, and syntopic species in relation to the physicochemical profile of the lake’s water are investigated for the first time. The study was carried out on a bi-monthly base, at six littoral sites of the lake. Samples’ study from different habitats and seasons revealed that the freshwater shrimp Atyaephyra thyamisensis was the most abundant species, accounting for 44.76% of the total taxa catch, while the grass shrimp Palaemonetes antennarius was less abundant (7.54%). Syntopic fish species in the littoral zone of Lake Pamvotis such as Economidichthys pygmaeus, Gambusia holbrooki, Knipowitschia caucasica and Rutillus panosi showed interannual differences with abundances of 24.12%, 19.13%, 4.26% and 0.20%, respectively. Correspondence analysis revealed clear patterns between species and stations. A. thyamisensis was predominant in shallow, well oxygenated water bodies rich with aquatic vegetation, but it was absent from deeper habitats. P. antennarius was found mainly in lentic water bodies, rocky substratum and deeper habitats. Taking into account the high ecological importance of the freshwater shrimps in ecosystems’ energy flow, ecological and biological data of lake’s shrimps are discussed and presented thoroughly. Threats and conservation measures for both shrimp species are debated also in detail.     

Non-β-cell progenitors of β-cells in pregnant mice

Pregnancy is a normal physiological condition in which the maternal β-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of β-cells to analyse the origin of new β-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, β-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HP AP). First, we conclude that the lineage tracing system was highly specific for β-cells. Secondly, we scored the proportion of the β-cells marked with HP AP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labeling index in pregnant animal pancreata, compared to nonpregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labeling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-β-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only β-cell duplication, but also the activation of a non-β-cell progenitor population. Further, there was no transdifferentiation of β-cells to other cell types in a two and half month period following labeling, including the period of pregnancy.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

Proteomics studies of childhood pilocytic astrocytoma

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.     

Multiple loci are associated with white blood cell phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.