Molecular biology techniques for testing hepatitis B virus, hepatitis C virus and human immunodeficiency virus in 6-donation pools regardless of serological test results and alanine aminotransferase values

Wstęp: W jednostkach polskiej służby krwi badania przeglądowe technikami biologii molekularnej (NAT) w kierunku zakażeń HCV, HIV i HBV są wykonywane w pojedynczych donacjach lub w pulach po 6 donacji. Celem tego typu badań jest identyfikacja zakażonych dawców, gdy nieobecne są markery serologiczne - w szczególności we wczesnym okresie zakażenia. Dotychczas pule osocza do badań NAT były przygotowywane tylko z donacji seronegatywnych i z wynikami aktywności aminotransferazy alaninowej AlAT w granicach normy. Celem prezentowanej pracy była analiza badań NAT w pulach od kolejnych dawców bez względu na wyniki badań serologicznych, ze szczególnym uwzględnieniem częstości wyników niepotwierdzających się, zużycia odczynników oraz czasu potrzebnego do zwolnienia donacji. Materiał i metody: W okresie od 19 sierpnia 2008 roku do 28 lutego 2009 roku przebadano 22 794 (3799 pul) donacji ujemnych w serologicznych badaniach przeglądowych oraz z AlAT w granicach normy (algorytm 1). Kolejne 6264 próbki (1044 pule) badano równolegle metodami serologicznymi oraz NAT (algorytm 2). W obu przypadkach badania NAT wykonywano w pulach osocza po 6 donacji testem Cobas TaqScreen MPX, stosując system cobas s 201. Wyniki: W badaniach przeprowadzonych równolegle metodami serologicznymi i NAT uzyskano 12 pul dodatnich na 1044 przebadane pule (1,1%). Wszystkie pozytywne próbki pochodziły od dawców pierwszorazowych, u których jednocześnie wykryto markery serologiczne. W pozostałych próbkach, dla których otrzymano wyniki dodatnie testów serologicznych (6 HBsAg, 11 anty-HCV, 3 HIV Ag/Ab) lub, w których stwierdzono aktywność AlAT powyżej normy (n = 105), nie wykryto markerów molekularnych. W analizowanym okresie nie zidentyfikowano u żadnego dawcy zakażenia w tak zwanym okienku serologicznym HCV, HBV, HIV ani ukrytego zakażenia. Wnioski: Prowadzenie badań wedug algorytmu 2 nie spowodowało statystycznie istotnego zwiększenia wyników niepowtarzalnie dodatnich NAT w porównaniu z okresem, w którym wykonywano te badania za pomocą algorytmu 1, jednak zużyto około 12,7% odczynników więcej. Dla jednostki służby krwi wydatki zwolnienia donacji nie uległy zwiększeniu, gdyż całkowite koszty odczynników pokrywał dostawca. Zastosowanie algorytmu 2 umożliwiało znaczne skrócenie czasu zwalniania większości donacji. J. Transf. Med. 2010; 2: 55-61Background: In Polish Blood Banks molecular biology techniques (NAT) for testing hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are performed either in individual blood donations or in six-donation pools. The aim of such screening is to identify infected donors when serological markers cannot yet be detected, especially in the early infection period. Until now, pooled plasma samples for NAT testing were prepared only from sero-negative donations with AlAT values within normal range. The aim of this study was to analyze NAT testing in consecutive donor pools regardless of the serological test results. Special emphasis was put on the frequency of non-confirmed results, the reagents consumption as well as time required for donation release. Material and methods: Within the six-month period (19.08.2008 to 28.02.2009), we tested 22,794 donations (3799 pools) negative in serological screening tests; AlAT values within normal range (algorithm 1). Another 6,264 samples (1044 pools) were tested simultaneously with serological methods and NAT techniques (algorithm 2). In both algorithms, NAT analyses were performed in six-donation plasma pools using the Cobas TaqScreen MPX in the cobas s 201 system. Results: Twenty positive pools were found in the 1044 (1.1%) donation-pools tested simultaneously by NAT and serological methods. All NAT positive samples were detected in first time donors with positive results of serological markers. In the remaining samples, no molecular markers were detected either in sero positive (6 HBsAg, 11 anti-HCV, 3 HIV Ag/Ab) samples or in samples with AlAT values above reference range (n = 105). No donors within serological window for HCV, HBV, HIV or with occult infection were identified in this study period. Conclusions: Testing/screening performed according to algorithm 2 demonstrated no statistically significant increase of unrepeatable NAT positive results as compared to the period when testing/screening was performed according to algorithm 1, however more reagents were used (by about 12.7 %). The entire costs of reagents were covered by the Supplier therefore the blood bank expenditures for donation release did not increase and algorithm 2 allowed for accelerated release of most donations. J. Transf. Med. 2010; 2: 55-6

Cytokeratin 18 expression pattern correlates with renal cell carcinoma progression: Relationship with Snail

Renal cell carcinoma (RCC) is the most common type of kidney cancer and recent developments in the molecular biology of RCC have identified multiple pathways associated with the development of this cancer. This study aimed at analyzing the expression pattern of cytokeratin 18 (CK18) in RCC patients and its prognostic relevance. We quantified CK18 mRNA expression and protein using real-time reverse transcription quantitative polymerase chain reaction (RT-QPCR) and immunohistochemistry, respectively, in paired tumor and non-tumor samples from 42 patients. Our data indicate that CK18 mRNA and proteins levels increased with advanced stage and grade of the disease. Using primary (RCC5) and metastatic renal cell carcinoma (RCC5 met) cell lines, we demonstrated that CK18 expression was 5-fold higher in the metastatic as compared to the primary RCC cell line and correlated with a migratory phenotype characterized by a distinct elongated morphology as revealed by Phalloidin staining. In addition, RCC5 met cells displayed an increased capacity to attach to fibronectin and collagen which was lost following CK18 knock-down. Our data also indicate that the expression of CK18 was associated with increased Snail expression which correlated positively with advanced disease in RCC patients. The present findings suggest that CK18 may play an important role in the progression of RCC and it may be used as a new predictor for RCC

Attenuation of Soft-Tissue Sarcomas Resistance to the Cytotoxic Action of TNF-α by Restoring p53 Function

BACKGROUND: Isolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect. METHODOLOGY/PRINCIPAL FINDINGS: We first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. TP53(Wt) cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, TP53(Mut) and TP53(Null) cell lines were resistant. TP53 suppression in TP53(Wt) cell lines abrogated TNF-α sensitivity and TP53 overexpression in TP53(Null) cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively TP53(Mut) and TP53(Wt)/MDM2(Ampl). In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in TP53(Mut) cells. In TP53(Wt)/MDM2(Ampl) cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-β1 and FAIM. CONCLUSION/SIGNIFICANCE: More effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment

Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC assembly

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2

FLIP: A Targetable Mediator of Resistance to Radiation in Non-Small Cell Lung Cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non-small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor, FLIP, is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were down-regulated by RNAi, IRinduced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the anti-tumor activity of IR in vivo. Therefore, FLIP down-regulation induced by HDAC inhibitors is a potential clinical strategy to radio-sensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR

Computer-aided Design of Novel CDK5 Inhibitors; Towards New Treatments of Glioblastoma

Abstract AIMS Design and statistically optimize an in silico screening protocol; apply the screening protocol to a compound database; in vitro validation of selected inhibitors (isolated CDK5 enzyme binding assay). METHOD Two large compound databases, ZINC15 and Analyticon Discovery, were used to screen for potential CDK5 ATP-binding site inhibitors. They were first filtered using a generated pharmacophore model and then virtually screened using Glide SP docking with a solved CDK5-p25 structure (PDB: 3O0G). The selected candidates were validated using enzyme binding assays to determine their inhibitory activity on CDK5, as well as against a panel of kinases. RESULTS Over 17,000 compounds were screened during molecular docking studies and of these, ~11,600 compounds returned which are predicted to bind to CDK5. The top 10% of docked compounds were analysed and 30 candidates were selected for single concentration screening at 50 µM concentrations. The 9 most potent compounds were selected for IC50 determinations and revealed 6 compounds with IC50 <10 µM. The selected novel compounds were also screened against a panel of kinases (CDK2, CDK5, CDK9, GSK-3β) with varying selective profiles observed. CONCLUSION Low micromolar potent compounds have been identified through in silico screening and validated in using in vitro binding assays. One flavonoid compound in particular, cirsiliol (IC50 = 5.90 µM), displayed best selectivity towards CDK5, a challenge in kinase inhibitor design. The identified compounds will now pass to cellular studies to determine their effect on Glioblastoma cell lines

The mitochondrial localization of RelB and NFATx in immature T cells

In order to exert their activity, transcription factors must be transported to the nucleus. Certain transcription factors have also been found on mitochondria. Here, the localization of RelB and NFATx in the mitochondrial fractions of normal thymocytes and thymic lymphoma cells is shown for the first time. CREB was only found in the nucleus, while p50 (NFkappaB) was found in both the nucleus and the cytoplasm, but outside the mitochondria. The translocation of transcription factors to the mitochondria is differentially regulated. Unlike RelB, which is always present in the mitochondrial fraction, NFATx appeared on the mitochondria in cells treated with ionomycin together with an immunosuppressant and inhibitor of calcineurin (FK506). This data reveals that the mitochondrial localization of some transcription factors is precisely controlled by a calcium signal sensitive to FK506 in T cells

Apoptosis of lymphoma cells is abolished due to blockade of cytochrome c release despite Nur77 mitochondrial targeting

Nur77 is reported to undergo translocation to mitochondria in response to apoptotic signaling in a variety of cancer cell lines. It was shown that on the mitochondrial membrane, Nur77 interacts with Bcl-2, leading to the conversion of this protein from a protector to a killer with subsequent release of cytochrome c to the cytosol. Here it is shown that in thymic lymphoma cells resistant to calcium-mediated apoptosis, cytochrome c release is abolished despite of Nur77 mitochondrial targeting. However, cytochrome c release and apoptosis can be restored by treatment with FK506. Hence, the molecular target regulation of the sensitivity of lymphoma cells to calcium signaling is associated with cytochrome c release and is FK506 sensitive. These results provide new insight into the role of FK506-sensitive factors as a critical link between calcium signaling and resistance of lymphoma cells to death

Exploring a Link Between Alzheimer’s and Glioma by Investigating SORL1 Network

Abstract AIMS Use bioinformatics methods to identify and validate associated proteins and genes in the SORL1 network. Generate a bank of patient derived cells in association with the Royal Preston Hospital BTNW tissue bank. Explore identified targets from the bioinformatics in patient derived cells. METHOD Proteins and genes associated with SORL1 and SORLA were identified on GeneCards. Connectivity mapping of known and predicted interactions linked to SORL1 was explored in STRING. For clinical validation differential expression was investigated by comparing genomic data from GTEX and TCGA, available at Xenabrowser. For survival analysis Kaplan-Meier curves were generated on cBioPortal. The five most differently expressed novel targets are taken forward for laboratory experiments using western blots for protein quantification and immunocytochemistry to identify and visualise target proteins. RESULTS A total of 73 genes (30 from GeneCards and 43 from STRING) were obtained. 63 genes from the generated SORL1-related network were shown to be differentially expressed whilst 40 were significantly different between low-surviving patients and high-surviving patients. The top five associated proteins are CKAP4, CTNND1, FN1, HSPA12A and SORCS3. CKAP4, CTNND1 and FN1 are highly expressed in both glioma and glioblastoma, but low expressed in healthy tissue. HSPA12A is low expressed in cancerous brain tissue and highly expressed in healthy samples. SORCS3 is differentially expressed in healthy samples and glioma, but significantly low expressed glioblastoma. CONCLUSION This project provides insight into SORL1 molecular relationships and function in tissue, cultured cells and serum from a patient cohort including demographics, disease progression and site

Discovery of Potent Multikinase Type-II Inhibitors Targeting CDK5 in the DFG-out Inactive State with Promising Potential against Glioblastoma

Kinases have proven valuable targets in successful cancer drug discovery projects, but not yet for malignant brain tumors where type-II inhibition of cyclin-dependent kinase 5 (CDK5) stabilizing the DFG-out inactive state has potential for design of selective and clinically efficient drug candidates. In the absence of crystallographic evidence for a CDK5 DFG-out inactive state protein–ligand complex, for the first time, a model was designed using metadynamics/molecular dynamics simulations. Glide docking of the ZINC15 biogenic database identified [pyrimidin-2-yl]amino-furo[3,2-b]-furyl-urea/amide hit chemical scaffolds. For four selected analogues (4, 27, 36, and 42), potent effects on glioblastoma cell viability in U87-MG, T98G, and U251-MG cell lines and patient-derived cultures were generally observed (IC50s ∼ 10–40 μM at 72 h). Selectivity profiling against 11 homologous kinases revealed multikinase inhibition (CDK2, CDK5, CDK9, and GSK-3α/β), most potent for GSK-3α in the nanomolar range (IC50s ∼ 0.23–0.98 μM). These compounds may therefore have diverse anticancer mechanisms of action and are of considerable interest for lead optimization