Strengthening of the Blood Safety System in the National Blood Transfusion Service - Implementation of the European Union IPA Project - at the Institute for Transfusion Medicine of the Republic of Macedonia

The Safety of the Blood Supply in any country is of utmost importance to safeguard patients from serious adverse events of blood transfusion. Implementation of a Quality System in the Blood Transfusion Service, with support of Government and Ministry of Health is a key element to guarantee safe blood. The IPA TAIB 2009 project - Strengthening of the Blood Safety System executed in 2013/14 provided the means to start implementing a Quality System in the Institute for Transfusion Medicine of the Republic of Macedonia. This project aimed to ultimately bring the Blood Transfusion Service to European Union standards, allowing the exchange of blood components and all other types of collaboration with other European Union countries in future. The project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel

RT-PCR for expression of embryonic stem cell specific genes.

<p>OCT4, NANOG and CRIPTO1 in human ESCs during culture (A), during 21 days after induction of hESC differentiation (B), and in adult human dermal fibroblasts cultured in 2% oxygen with FGF2 supplementation (C). Restriction digest of 646 bp OCT4 amplicon from human embryonic stem cells (D), control fibroblasts (E), and iPSCs (F). ApaI restriction digest of OCT4 amplicon in fibroblasts grown in 2% oxygen and FGF2 supplementation (G), and various transformed, multipotent and differentiated cells (H). NCCIT – teratocarcinoma, NTERA2– teratocarcinoma, SHSY – neuroblastoma, SMCs – smooth muscle cells, HUVECs – human umbilical vein endothelial cells, hMSCs – human mesenchymal stem cells. Only the embryonic OCT4A contains ApaI restriction site.</p

Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approach

Background. Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients. Materials and methods. We used a commercially available antibody array that includes 656 antibodies to analyse blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with GBM

Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

<div><p>The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally-regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms.</p></div

Protein expression of stem cell specific genes.

<p>Western blots for OCT4 in human embryonic stem cells (hESC), teratocarcinoma cells and adult human dermal fibroblasts (A, C) or muscle fibroblasts (B) using different antibodies. Complete cell lysates or nuclear and cytoplasmic fractions (N and C, respectively) were separated on 10% SDS PAGE, transferred to PVDF and blotted with different antibodies. Fibroblasts were grown for 7 days under low (2 or 5%) or ambient (19%) oxygen, in the presence or absence of FGF2 (+ and −, respectively).</p

Restriction digest of 646 bp OCT4 amplicon from human embryonic carcinoma cells during 21 days of differentiation in medium containing retinoic acid.

<p>Restriction digest of 646 bp OCT4 amplicon from human embryonic carcinoma cells during 21 days of differentiation in medium containing retinoic acid.</p

Schematic representation of restriction sites in 646-PCR amplicon.

<p>Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. <b>ApaI</b> restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. <b>HinfI</b> digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. <b>BglI</b> digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. <b>XhoI</b> does not digest OCT4-pg4.</p

RT-PCR for expression of embryonic stem cell specific genes.

<p>OCT4, NANOG and CRIPTO1 in human ESCs during culture (A), during 21 days after induction of hESC differentiation (B), and in adult human dermal fibroblasts cultured in 2% oxygen with FGF2 supplementation (C). Restriction digest of 646 bp OCT4 amplicon from human embryonic stem cells (D), control fibroblasts (E), and iPSCs (F). ApaI restriction digest of OCT4 amplicon in fibroblasts grown in 2% oxygen and FGF2 supplementation (G), and various transformed, multipotent and differentiated cells (H). NCCIT – teratocarcinoma, NTERA2– teratocarcinoma, SHSY – neuroblastoma, SMCs – smooth muscle cells, HUVECs – human umbilical vein endothelial cells, hMSCs – human mesenchymal stem cells. Only the embryonic OCT4A contains ApaI restriction site.</p

Immunocytochemistry of OCT4 in human embryonic stem cells (hESCs) and fibroblasts.

<p>Cells were cultured in control (ambient 19% O₂, no FGF2) and 2% oxygen with FGF2 supplementation for 7 days. Cells were stained with three different OCT4 antibodies, as indicated. Red – OCT4, Green – actin; Blue – DNA.</p