Exposure to Cadmium Telluride Quantum Dots and Gene Expression Profile of Huh-7 Hepatocellular Carcinoma Cell Line.

Nanoparticles have shown promising potential for efficient drug delivery, circumventing biological interferences like immunological and renal clearance and mechanical and enzymatic destruction. However, a handful of research papers have questioned the biomedical use of metal-based nanoparticles like cadmium telluride quantum dots (CdTe-QDs) for their cytotoxic, genotoxic, and carcinogenic potential. Herein, we examined the effects of CdTe-QD NPs on gene expression profile of hepatocellular carcinoma (Huh-7) cell line. Huh-7 cells were treated with CdTe-QD NPs (10 µg/ml for 6, 12, and 24 hours, and 25 µg/ml for 6 and 12 hours), and transcriptomic analysis was performed using microarray to evaluate the global gene expression profile. Differential expressed genes (DEGs) were observed for both the doses (10 and 25 µg/ml) of CdTe-QD NPs at different time points. Gene ontology (GO) analysis revealed that genes involved in molecular function of cell cycle, organizational injury and abnormalities, cell death and survival, gene expression, cancer, organismal survival, and cellular development were differentially expressed. Overall, we have demonstrated differential expression of several genes, involved in maintaining cell survival, metabolism, and genome integrity. These findings were confirmed by RT-qPCR study for some canonical pathway genes signifying possible implication in NP toxicity-mediated cell survival and inhibition of cell death.

[Investigation of Ganciclovir Resistance in Cytomegalovirus Isolates by Phenotypic and Genotypic Methods]. / Sitomegalovirüs Izolatlarinda Gansiklovir Direncinin Fenotipik ve Genotipik Yöntemlerle Arastirilmasi.

Ganciclovir-resistant cytomegalovirus (CMV) strains are reported following long-term antiviral agent use, especially for immune-suppressive patients. In this study, it was aimed to investigate the mutations in the UL97 gene of CMV, which causes ganciclovir (GCV) resistance by genotypic and phenotypic methods in patients who developed CMV infection following hematopoietic cell (HCT) or solid organ transplantation (SOT). Thirty patients who had HCT or SOT in Mediterranean University Hospital and developed CMV infection during routine follow-up with a viral load of CMV over 1000 copies/mL were included in the study. CMV DNA was analyzed by an automated system (Cobas Ampliprep/COBAS TaqMan CMV Test, Roche Diagnostics, Germany) quantitatively. DNA sequence analysis of the regions including codons 420-664 in the UL97 gene region was done by the Sanger sequencing method to detect mutations causing antiviral resistance and compared with defined mutations. In order to investigate antiviral resistance by phenotypic methods, heparinized blood samples of the patients were collected, 'buffy coat (leukocyte layer)' was inoculated into MRC-5 cells by centrifugation method and CMV growth in these cells was controlled with monoclonal antibodies when growth was detected, virus titer was determined and plaque reduction test was applied as recommended. It was determined that 22 of the 30 patients were HCT recipients and eight were SOT (five kidney, three liver) recipients. When the CMV serology pattern of the patients was evaluated before transplantation, 29 (96.7%) patients were found to be seropositive and one (3.3%) patient was found to be seronegative. Totally, nine CMV UL97 mutations were detected in seven (23.3%) pediatric patients who had HCT, including six seropositive and one seronegative case. In addition, one mutation (D605E) not known to cause GCV resistance was detected in a seronegative recipient and three previously unidentified mutations were detected (1474T, F499S, V559A) in a seronegative recipient. Five of the mutations defined were UL97 mutations with a defined clinical resistance against GCV in each of the five recipients (C603W, C592G, H520Q, M460V, A594T). In the plaque reduction test using 3 µM, 12 µM, 48 µM and 96 µM concentrations of GCV in CMV strains, the IC50 value was determined to be ≥ 8 µM for the five CMV strains, and the phenotypic presence of GCV resistance was shown. Clinical resistance associated with CMV UL97 mutation was detected in five (22.7%) of 22 patients who had HCT. GCV resistance was also demonstrated in these patients by phenotypic methods. No UL97 mutation was detected in the patients who had SOT.

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

A molecular study of pediatric pilomyxoid and pilocytic astrocytomas: Genome-wide copy number screening, retrospective analysis of clinicopathological features and long-term clinical outcome.

Background: Pilocytic Astrocytoma (PA) is the most common pediatric brain tumors. PAs are slow-growing tumors with high survival rates. However, a distinct subgroup of tumors defined as pilomyxoid astrocytoma (PMA) presents unique histological characteristics and have more aggressive clinical course. The studies on genetics of PMA are scarce. Methods: In this study, we report one of the largest cohort of pediatric patients with pilomyxoid (PMA) and pilocytic astrocytomas (PA) in Saudi population providing a comprehensive clinical picture, retrospective analysis with long-term follow-up, genome-wide copy number changes, and clinical outcome of these pediatric tumors. We examined and compared genome-wide copy number aberrations (CNAs) and the clinical outcome of the patients with PA and PMA. Results: The median progression free survival for the whole cohort was 156 months and it was 111 months for the PMA, however, not statistically significantly different between the groups (log-rank test, P = 0.726). We have identified 41 CNAs (34 gains and 7 losses) in all tested patients. Our study yielded the previously reported KIAA1549-BRAF Fusion gene in over 88% of the tested patients (89% and 80% in PMA and PA, respectively). Besides the fusion gene, twelve patients had additional genomic CNAs. Furthermore, pathway and gene network analyses of genes in the fusion region revealed alterations in retinoic acid mediated apoptosis and MAPK signaling pathways and key hub genes that may potentially be involved in tumor growth and progression, including BRAF, LUC7L2, MKRN1, RICTOR, TP53, HIPK2, HNF4A, POU5F, and SOX4. Conclusion: Our study is the first report of a large cohort of patients with PMA and PA in the Saudi population that provides detailed clinical features, genomic copy number changes, and outcome of these pediatric tumors and may help better diagnosis and characterization of PMA.

Astrocytes derived from ASD individuals alter behavior and destabilize neuronal activity through aberrant Ca2+ signaling.

The cellular mechanisms of autism spectrum disorder (ASD) are poorly understood. Cumulative evidence suggests that abnormal synapse function underlies many features of this disease. Astrocytes regulate several key neuronal processes, including the formation of synapses and the modulation of synaptic plasticity. Astrocyte abnormalities have also been identified in the postmortem brain tissue of ASD individuals. However, it remains unclear whether astrocyte pathology plays a mechanistic role in ASD, as opposed to a compensatory response. To address this, we combined stem cell culturing with transplantation techniques to determine disease-specific properties inherent to ASD astrocytes. We demonstrate that ASD astrocytes induce repetitive behavior as well as impair memory and long-term potentiation when transplanted into the healthy mouse brain. These in vivo phenotypes were accompanied by reduced neuronal network activity and spine density caused by ASD astrocytes in hippocampal neurons in vitro. Transplanted ASD astrocytes also exhibit exaggerated Ca2+ fluctuations in chimeric brains. Genetic modulation of evoked Ca2+ responses in ASD astrocytes modulates behavior and neuronal activity deficits. Thus, this study determines that astrocytes derived from ASD iPSCs are sufficient to induce repetitive behavior as well as cognitive deficit, suggesting a previously unrecognized primary role for astrocytes in ASD.

Integrated Analysis of Transcriptomic and Genomic Data Reveals Blood Biomarkers With Diagnostic and Prognostic Potential in Non-small Cell Lung Cancer.

Background: Lung cancer is the second most common cancer and the main leading cause of cancer-associated death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer diagnoses and more than 50% of all lung cancer cases are diagnosed at an advanced stage; hence have poor prognosis. Therefore, it is important to diagnose NSCLC patients reliably and as early as possible in order to reduce the risk of mortality. Methods: We identified blood-based gene markers for early NSCLC by performing a multi-omics approach utilizing integrated analysis of global gene expression and copy number alterations of NSCLC patients using array-based techniques. We also validated the diagnostic and the prognostic potential of the gene signature using independent datasets with detailed clinical information. Results: We identified 12 genes that are significantly expressed in NSCLC patients' blood, at the earliest stages of the disease, and associated with a poor disease outcome. We then validated 12-gene signature's diagnostic and prognostic value using independent datasets of gene expression profiling of over 1000 NSCLC patients. Indeed, 12-gene signature predicted disease outcome independently of other clinical factors in multivariate regression analysis (HR = 2.64, 95% CI = 1.72-4.07; p = 1.3 × 10-8). Significantly altered functions, pathways, and gene networks revealed alterations in several key genes and cancer-related pathways that may have importance for NSCLC transformation, including FAM83A, ZNF696, UBE2C, RECK, TIMM50, GEMIN7, and XPO5. Conclusion: Our findings suggest that integrated genomic and network analyses may provide a reliable approach to identify genes that are associated with NSCLC, and lead to improved diagnosis detecting the disease in early stages in patients' blood instead of using invasive techniques and also have prognostic potential for discriminating high-risk patients from the low-risk ones.

Transcriptomic data analysis coupled with copy number aberrations reveals a blood-based 17-gene signature for diagnosis and prognosis of patients with colorectal cancer.

Background: Colorectal cancer (CRC) is the third most common cancer and third leading cause of cancer-associated deaths worldwide. Diagnosing CRC patients reliably at an early and curable stage is of utmost importance to reduce the risk of mortality. Methods: We identified global differentially expressed genes with copy number alterations in patients with CRC. We then identified genes that are also expressed in blood, which resulted in a blood-based gene signature. We validated the gene signature's diagnostic and prognostic potential using independent datasets of gene expression profiling from over 800 CRC patients with detailed clinical data. Functional enrichment, gene interaction networks and pathway analyses were also performed. Results: The analysis revealed a 17-gene signature that is expressed in blood and demonstrated that it has diagnostic potential. The 17-gene SVM classifier displayed 99 percent accuracy in predicting the patients with CRC. Moreover, we developed a prognostic model and defined a risk-score using 17-gene and validated that high risk score is strongly associated with poor disease outcome. The 17-gene signature predicted disease outcome independent of other clinical factors in the multivariate analysis (HR = 2.7, 95% CI = 1.3-5.3, p = 0.005). In addition, our gene network and pathway analyses revealed alterations in oxidative stress, STAT3, ERK/MAPK, interleukin and cytokine signaling pathways as well as potentially important hub genes, including BCL2, MS4A1, SLC7A11, AURKA, IL6R, TP53, NUPR1, DICER1, DUSP5, SMAD3, and CCND1. Conclusion: Our results revealed alterations in various genes and cancer-related pathways that may be essential for CRC transformation. Moreover, our study highlights diagnostic and prognostic value of our gene signature as well as its potential use as a blood biomarker as a non-invasive diagnostic method. Integrated analysis transcriptomic data coupled with copy number aberrations may provide a reliable method to identify key biological programs associated with CRC and lead to improved diagnosis and therapeutic options.

A Network-Based Methodology to Identify Subnetwork Markers for Diagnosis and Prognosis of Colorectal Cancer.

The development of reliable methods for identification of robust biomarkers for complex diseases is critical for disease diagnosis and prognosis efforts. Integrating multi-omics data with protein-protein interaction (PPI) networks to investigate diseases may help better understand disease characteristics at the molecular level. In this study, we developed and tested a novel network-based method to detect subnetwork markers for patients with colorectal cancer (CRC). We performed an integrated omics analysis using whole-genome gene expression profiling and copy number alterations (CNAs) datasets followed by building a gene interaction network for the significantly altered genes. We then clustered the constructed gene network into subnetworks and assigned a score for each significant subnetwork. We developed a support vector machine (SVM) classifier using these scores as feature values and tested the methodology in independent CRC transcriptomic datasets. The network analysis resulted in 15 subnetwork markers that revealed several hub genes that may play a significant role in colorectal cancer, including PTP4A3, FGFR2, PTX3, AURKA, FEN1, INHBA, and YES1. The 15-subnetwork classifier displayed over 98 percent accuracy in detecting patients with CRC. In comparison to individual gene biomarkers, subnetwork markers based on integrated multi-omics and network analyses may lead to better disease classification, diagnosis, and prognosis.

Identification of Gene Signature as Diagnostic and Prognostic Blood Biomarker for Early Hepatocellular Carcinoma Using Integrated Cross-Species Transcriptomic and Network Analyses.

Background: Hepatocellular carcinoma (HCC) is considered the most common type of liver cancer and the fourth leading cause of cancer-related deaths in the world. Since the disease is usually diagnosed at advanced stages, it has poor prognosis. Therefore, reliable biomarkers are urgently needed for early diagnosis and prognostic assessment. Methods: We used genome-wide gene expression profiling datasets from human and rat early HCC (eHCC) samples to perform integrated genomic and network-based analyses, and discovered gene markers that are expressed in blood and conserved in both species. We then used independent gene expression profiling datasets for peripheral blood mononuclear cells (PBMCs) for eHCC patients and from The Cancer Genome Atlas (TCGA) database to estimate the diagnostic and prognostic performance of the identified gene signature. Furthermore, we performed functional enrichment, interaction networks and pathway analyses. Results: We identified 41 significant genes that are expressed in blood and conserved across species in eHCC. We used comprehensive clinical data from over 600 patients with HCC to verify the diagnostic and prognostic value of 41-gene-signature. We developed a prognostic model and a risk score using the 41-geneset that showed that a high prognostic index is linked to a worse disease outcome. Furthermore, our 41-gene signature predicted disease outcome independently of other clinical factors in multivariate regression analysis. Our data reveals a number of cancer-related pathways and hub genes, including EIF4E, H2AFX, CREB1, GSK3B, TGFBR1, and CCNA2, that may be essential for eHCC progression and confirm our gene signature's ability to detect the disease in its early stages in patients' biological fluids instead of invasive procedures and its prognostic potential. Conclusion: Our findings indicate that integrated cross-species genomic and network analysis may provide reliable markers that are associated with eHCC that may lead to better diagnosis, prognosis, and treatment options.

Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues.

PURPOSE: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. METHODS: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. RESULTS: In FFPE tissues, PD-L1 was positive in 7-10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20-30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13-20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. CONCLUSIONS: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.

Gene Expression and Transcriptome Profiling of Changes in a Cancer Cell Line Post-Exposure to Cadmium Telluride Quantum Dots: Possible Implications in Oncogenesis.

Cadmium telluride quantum dots (CdTe-QDs) are acquiring great interest in terms of their applications in biomedical sciences. Despite earlier sporadic studies on possible oncogenic roles and anticancer properties of CdTe-QDs, there is limited information regarding the oncogenic potential of CdTe-QDs in cancer progression. Here, we investigated the oncogenic effects of CdTe-QDs on the gene expression profiles of Chang cancer cells. Chang cancer cells were treated with 2 different doses of CdTe-QDs (10 and 25 µg/ml) at different time intervals (6, 12, and 24 h). Functional annotations helped identify the gene expression profile in terms of its biological process, canonical pathways, and gene interaction networks activated. It was found that the gene expression profiles varied in a time and dose-dependent manner. Validation of transcriptional changes of several genes through quantitative PCR showed that several genes upregulated by CdTe-QD exposure were somewhat linked with oncogenesis. CdTe-QD-triggered functional pathways that appear to associate with gene expression, cell proliferation, migration, adhesion, cell-cycle progression, signal transduction, and metabolism. Overall, CdTe-QD exposure led to changes in the gene expression profiles of the Chang cancer cells, highlighting that this nanoparticle can further drive oncogenesis and cancer progression, a finding that indicates the merit of immediate in vivo investigation.

Neurodevelopmental signatures of narcotic and neuropsychiatric risk factors in 3D human-derived forebrain organoids.

It is widely accepted that narcotic use during pregnancy and specific environmental factors (e.g., maternal immune activation and chronic stress) may increase risk of neuropsychiatric illness in offspring. However, little progress has been made in defining human-specific in utero neurodevelopmental pathology due to ethical and technical challenges associated with accessing human prenatal brain tissue. Here we utilized human induced pluripotent stem cells (hiPSCs) to generate reproducible organoids that recapitulate dorsal forebrain development including early corticogenesis. We systemically exposed organoid samples to chemically defined "enviromimetic" compounds to examine the developmental effects of various narcotic and neuropsychiatric-related risk factors within tissue of human origin. In tandem experiments conducted in parallel, we modeled exposure to opiates (µ-opioid agonist endomorphin), cannabinoids (WIN 55,212-2), alcohol (ethanol), smoking (nicotine), chronic stress (human cortisol), and maternal immune activation (human Interleukin-17a; IL17a). Human-derived dorsal forebrain organoids were consequently analyzed via an array of unbiased and high-throughput analytical approaches, including state-of-the-art TMT-16plex liquid chromatography/mass-spectrometry (LC/MS) proteomics, hybrid MS metabolomics, and flow cytometry panels to determine cell-cycle dynamics and rates of cell death. This pipeline subsequently revealed both common and unique proteome, reactome, and metabolome alterations as a consequence of enviromimetic modeling of narcotic use and neuropsychiatric-related risk factors in tissue of human origin. However, of our 6 treatment groups, human-derived organoids treated with the cannabinoid agonist WIN 55,212-2 exhibited the least convergence of all groups. Single-cell analysis revealed that WIN 55,212-2 increased DNA fragmentation, an indicator of apoptosis, in human-derived dorsal forebrain organoids. We subsequently confirmed induction of DNA damage and apoptosis by WIN 55,212-2 within 3D human-derived dorsal forebrain organoids. Lastly, in a BrdU pulse-chase neocortical neurogenesis paradigm, we identified that WIN 55,212-2 was the only enviromimetic treatment to disrupt newborn neuron numbers within human-derived dorsal forebrain organoids. Cumulatively this study serves as both a resource and foundation from which human 3D biologics can be used to resolve the non-genomic effects of neuropsychiatric risk factors under controlled laboratory conditions. While synthetic cannabinoids can differ from naturally occurring compounds in their effects, our data nonetheless suggests that exposure to WIN 55,212-2 elicits neurotoxicity within human-derived developing forebrain tissue. These human-derived data therefore support the long-standing belief that maternal use of cannabinoids may require caution so to avoid any potential neurodevelopmental effects upon developing offspring in utero.

Evaluation of Aspergillus Lateral-Flow Test in Serum Samples of Pediatric Patients.

BACKGROUND: Diagnosis of invasive aspergillosis (IA) in patients with hematologic malignancies and under the risk of IA may be uncertain or may delay because of nonspecific clinical presentation of the patients and difficult application techniques of conventional methods. Early diagnosis can provide initial antifungal therapy and prevent high mortality. In this study, we investigated the performance of an Aspergillus lateral-flow device (LFD) test (OLM Diagnostics, Newcastle upon Tyne, United Kingdom) for the diagnosis of IA in pediatric febrile neutropenic patients with hematologic malignancies. METHODS: Three hundred and fourty seven serum samples of 26 febrile neutropenic episodes of 21 patients at risk for IA were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the Aspergillus LFD test at episode level and at serum level were calculated. RESULTS: According to the reference diagnostic criteria of IA, one proven and 13 probable IA episodes were defined. Twelve episodes (46.1%) did not meet the criteria for IA. The sensitivity, specificity, PPV, NPV, accuracy of the Aspergillus LFD test at episode level and at serum level were 14.3%, 100%, 100%, 50%, 53.8% and 12.1%, 100%, 100%, 50.8%, 53.9%, respectively. CONCLUSIONS: Aspergillus LFD test is an easy-to-use assay with short hands-on time; however, further study of the clinical utility in children and especially in serum samples are needed. It is a highly specific test for IA on bronchoalveolar lavage (BAL) samples but is not useful as a screening test for serum samples unless combined with galactomannan (GM) antigen test because of its potentially suboptimal sensitivity.

Follow-up of human adenovirus viral load in pediatric hematopoietic stem cell transplant recipients.

BACKGROUND: The spectrum of human adenovirus (HAdV)-related disease is broad, and the virus acts on many organs and systems in hematopoietic stem cell transplantation (HSCT) recipients. We aimed to evaluate the effect of HAdV-DNA positivity with clinical and laboratory findings 4 months after HSCT. METHODS AND RESULTS: We retrospectively investigated HAdV-DNA in 153 HSCT recipients (≤18 years) by quantitative real-time polymerase chain reaction (RealStar; Altona Diagnostics). The results of samples from January 2014 to December 2017 are included. HAdV-DNA was positive for at least one sample type in 50 (32.67%) patients. HAdV-DNA positivity rate was 8.92% (N: 145/1625), 40.25% (N: 64/159), and 25% (N: 2/8) for plasma, stool, and urine samples, respectively. HAdV-DNA was positive in the plasma of 38 (24.83%) patients at a median 16 (range: 1-58 days) days after HSCT. The mortality rate was 23.68% and 6.95% in plasma HAdV-positive and HAdV-negative patients (p = .014). Moreover, HAdV-DNA positivity had an impact on overall survival for allogeneic-HSCT (p = .013), with the cumulative effect including graft-versus-host disease state in multivariate analysis (p = .014). CONCLUSIONS: Plasma HAdV-DNA positivity is a potential influencer that decreases survival in the early post-transplant period. Due to the high mortality rates, close monitoring is required of HAdV infections after HSCT with sensitive methods, especially at the early stage.

Incidence of BKV in the urine and blood samples of pediatric patients undergoing HSCT.

The aims were to investigate the incidence of BKV infection and the presence of HC in pediatric patients undergoing HSCT. Twenty-four children patients (M/F: 17/7) undergoing HSCT in a single center over a period of 1 year were included in the study. The presence of BKV DNA was determined by quantitative real-time PCR in plasma and urine samples at the following times: before transplantation, twice a week until engraftment time, and weekly for + 100 days. The mean age of the patients was 7.79 ± 5.03 years, the mean follow-up time was 95.6 ± 25.9 days, and the average number of samples per patient was 15.8 ± 3.2. BKV DNA was detected in at least one urine sample in 91.6% (n: 22) and at least one plasma sample in 75% (n:18) of the patients. The median time to the first BKV DNA positivity in urine and plasma samples was 11 (range: 1-80) and 32 days (range: 2-79), respectively. The median value of BKV DNA copies in urine and plasma were 1.7 × 106 (range: 2.8 × 101 -1.2 × 1014 ) and 1.9 × 103 copies/mL (range: 3-2.1 × 106 ), respectively. Thirteen patients (54.2%) had hematuria with BKV viruria; 8 (33.3%) patients had viremia. The median value of the BKV DNA copies in urine and plasma was 4.4 × 107 (range: 65-1 × 1011 ) and 2.9 × 103 (range: 7-7.8 × 104 ) copies/mL in these patients. Two (15.4%) of the 13 patients with BKV viruria and hematuria were diagnosed with BKV-related HC. BKV DNA viral load monitoring of urine and plasma in pediatric HSCT patients with a high risk for viral infections is valuable for understanding the development of BKV-related HC.

Investigation of Torque Teno Virus (TTV) DNA as an immunological and virological marker in pediatric hematopoietic stem cell transplantation (HSCT) patients.

BACKGROUND: High viral loads are observed in Torque Teno Virus (TTV) infection after hematopoietic stem cell transplantation (HSCT). We aimed to analyze the kinetics of plasma TTV-DNA load in pediatric patients who received immunosuppressive therapy and developed infection complications in the first 100 days after HSCT. METHODS: As a patient group; 113 plasma samples taken from 33 pediatric HSCT recipients at a time interval after transplantation and as a control group; 38 plasma samples from 38 children without known chronic disease were included in the study. Viral nucleic acid isolation was performed by using the NucliSENS easyMAG (bioMerieux, France) system. A laboratory designed quantitative polymerase chain reaction process was performed on 7300 Real-Time PCR system (Applied Biosystems, CA, USA) with the amplification mixture containing primer and probe sequences for the UTR gene region. RESULTS: TTV-DNA was detected in all patient's samples and the median viral load was calculated as 7.67 Log10 copies/mL (range: 2.84-9.59). In the control group, the TTV-DNA median viral load was calculated as 5.51 Log10 copies/mL (range: 2.50-7.04), except for one negative sample. A significant difference was observed between the control group and the patient group in terms of TTV viral load levels. In nine patients, a median 2.15 Log10 copies/mL viral load increase was observed at 31-60 days post-transplant compared to the pre-transplant period. CONCLUSION: TTV-DNA levels should be closely monitored to understand the immune status of the first 100 days after transplantation and the effects of treatment regimens on patients with HSCT.

Evaluation of Commercially Available Real-Time Polymerase Chain Reaction Assays for the Diagnosis of Invasive Aspergillosis in Patients with Haematological Malignancies.

Early diagnosis of invasive aspergillosis (IA) is a challenge. Non-specific clinical and radiologic findings, as well as difficulties in conventional diagnostic method application, may delay correct diagnosis. Nowadays, nucleic acid-based assays have reduced the need for conventional antigen detection and culture-based methods and provided new opportunities for patient care. Aspergillus PCR is now included in the latest European Cancer Research and Treatment Organization/Mycosis Study Group definition updates. We evaluated the performance of commercial real-time polymerase chain reaction (PCR) MycAssay Aspergillus PCR and Artus Aspergillus RG PCR assays and compared the results with galactomannan enzyme immunoassay. During 41 febrile neutropenic episodes, 168 serum samples were collected from 32 patients with haematological malignancies. IA diagnosis was established according to the revised guidelines of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. Twenty-one probable episodes were identified. There were no proven IA cases in the study. In 20 episodes, patients did not fulfil the established criteria for the IA diagnosis. Artus Aspergillus RG PCR assay had a sensitivity of 47.6% and specificity of 100%, while those of MycAssay Aspergillus PCR were 61.9% and 100%, respectively. Two different PCR assays were used in this study. Although there are many studies that evaluated MycAssay Aspergillus PCR, data regarding Artus Aspergillus RG PCR assay are scarce. We found moderate sensitivity and high specificity in the diagnosis of IA in patients with haematological malignancy in both PCR methods. Our results demonstrated that commercial PCR assays can be applied for the early diagnosis and pre-emptive treatment of IA.

[Evaluation of the Two Different Real Time Polymerase Chain Reaction Methods Used for BK Virus (BKV) Quantification and BKV Genotype Assignment]. / BK Virüs (BKV) Kantitasyonunda Kullanilan Iki Farkli Gerçek Zamanli Polimeraz Zincir Reaksiyonu Yönteminin Karsilastirmasi ve BKV Genotip Tayini.

BK virus (BKV) viral load quantification has a distinct role in the clinical control of BKV nephropathy and organ rejection among renal transplant recipients. In this study, it was aimed to compare BKV DNA measurement values performed with two different real-time polymerase chain reaction (PCR) methods and to determine BKV genotypes in renal transplant recipients. Totally, 150 clinical samples tested previously in two different laboratories (Lab-1 and Lab-2) from adult and pediatric renal transplantation patients were included in the study. Fifty plasma samples of 50 different patients from Lab-1, 50 plasma and 50 urine samples of 58 different patients from Lab-2 were included in the study. Viral nucleic acid extraction was performed with automatized systems in Lab-1 and Lab-2 (EZ1, Qiagen, Germany and MagNA Pure 96, Roche Diagnostics, Germany; respectively;). Real-time PCR procedure was carried out in Lab-1 with an amplification mixture of primer, probe sequences targeting VP-1 gene region using RotorGene (Qiagen, Germany) and in Lab-2 with an amplification mixture of primer, probe sequences targeting VP-2 gene region using ABI Prism 7500 (Applied Biosystems, USA). BKV genotyping was performed with multiplex PCR using primer, probe sequences for BKV genotypes I-IV. In both of the laboratories, 82 (54.6%) of the samples were found as positive, 37(24.6%) samples were found as negative and a moderate agreement was found between qualitative results of two real-time PCR methods (ƙ= 0.56, p<0.001). Median viral load values were 4.1 x 104 copies/ml (321-6 x 109) in Lab-1 and 3.3 x 105 copies/ml (224-8.3 x 1010) in Lab-2 for positive samples. According to the lineer regression analysis of quantitative results, moderate (R2= 0.52, p<0.001) and high (R2= 0.88, p<0.001) correlation was found for plasma (n= 52) and urine (n= 30) samples, respectively. Bland-Altman analysis yielded a mean difference of -0.58 log10 for all samples. For plasma samples mean difference was -0.29 log10, while it was -1.1 log10 for urine samples. In all samples, Lab-1 measurements were lower than Lab-2 measurements. A mean difference of -1.1 log10 indicated that the measurement values of Lab-2 were more higher than Lab-1 measurments with an average of 1.1 log10. Supporting this result, 71.9% of the samples had a measurement difference more than 0.5 log 10 and 29.2% of the samples had a measurement difference more than 1 log10. Only 28.1% of the samples were measured within clinically acceptable log difference range (less than 0.5 log10). BKV genotyping was performed only for 74 different patient samples with sufficient copy numbers and genotype I (81.7%), IV (15.5%), II (1.4%), I+IV (1.4%) were detected. When the results were compared; 66.6% (n= 12) of the genotype IV samples had more than 1 log10 and 83.3% of them had more than 0.5 log10 viral load measurement difference. Correlation and linear regression analyzes were insufficient for the comparison ofthe results of the two different tests. It will be appropriate for each center to monitor patients with the same test until the international BKV standard developed by the World Health Organization is optimized. The clinical correlation of the tests is limited to the currently used test. The result of incorrect BKV quantification affects the clinical decision. Measurements less than the actual value will lead to the development of BKV nephropathy, and higher measurements will lead to unnecessary allograft biopsy and unnecessary reduction of immunosuppression.

ß1 Integrin is essential for fascin-mediated breast cancer stem cell function and disease progression.

Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, ß1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.

Deletion and Functional Analysis of Hepatitis B Virus X Protein: Evidence for an Effect on Cell Cycle Regulators.

BACKGROUND/AIMS: The hepatitis B virus X protein (HBx) is a viral trans-activator that plays a crucial role in pathogenesis of hepatocellular carcinoma (HCC) via an unknown mechanism. The role of HBx in modulating cell proliferation and programmed cell death is replete with controversies. Thus, the goal of this study was to elucidate the effect of HBx and its deletion mutants on cell cycle progression in human hepatoma cells. METHODS: Huh7 cells transfected with either full-length or truncated HBx were tested for their mitogenic potential based on their effect on the expression of key cell cycle-related proteins (p27, cyclin D1, p21, and p53) and pro-apoptotic proteins such as cleaved poly (ADP-ribose) polymerase (PARP) and Bax. Western blotting and immunofluorescence techniques were applied to detect changes in the expression levels and intracellular localization, respectively, of the investigated proteins. Also, Quantitative real-time PCR (qRT-PCR) was used to detect changes in RNA levels. RESULTS: An increased anchorage-independent growth of cells transfected with HBx-WT and its deletion mutants was observed. The cell cycle regulatory molecules were differentially modulated by full-length HBx (1-154) and its different N- and C-terminal truncated forms (HBx (31-154), HBx (61-154), HBx (1-94), and HBx (61-124)). An enhanced modulation of p27, p21, and cyclin D1 was associated with HBx (1-154), whereas p53 expression was significantly inhibited by HBx (61-124). Similarly, the expression of cleaved PARP and Bax was efficiently suppressed by HBx (1-94) and HBx (61-154). CONCLUSION: The HBx-WT and its mutants play a critical role in the pathogenesis and progression of HCC by modulating cell cycle regulatory proteins.