Transcriptomic Analysis of Alzheimer's Disease Pathways in a Pakistani Population.

Background: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that is most prevalent in elderly individuals, especially in developed countries, and its prevalence is now increasing in developing countries like Pakistan. Objective: Our goal was to characterize key genes and their levels of expression and related molecular transcriptome networks associated with AD pathogenesis in a pilot case-control study in a Pakistani population. Methods: To obtain the spectrum of molecular networks associated with pathogenesis in AD patients in Pakistan (comparing cases and controls), we used high-throughput qRT-PCR (TaqMan Low-Density Array; n = 33 subjects) coupled with Affymetrix Arrays (n = 8) and Ingenuity Pathway Analysis (IPA) to identify signature genes associated with Amyloid processing and disease pathways. Results: We confirmed 16 differentially expressed AD-related genes, including maximum fold changes observed in CAPNS2 and CAPN1. The global gene expression study observed that 61% and 39% of genes were significantly (p-value 0.05) up- and downregulated, respectively, in AD patients compared to healthy controls. The key pathways include, e.g., Amyloid Processing, Neuroinflammation Signaling, and ErbB4 Signaling. The top-scoring networks in Diseases and Disorders Development were Neurological Disease, Organismal Injury and Abnormalities, and Psychological Disorders. Conclusions: Our pilot study offers a non-invasive and efficient way of investigating gene expression patterns by combining TLDA and global gene expression method in AD patients by utilizing whole blood. This provides valuable insights into the expression status of genes related to Amyloid Processing, which could play potential role in future studies to identify sensitive, early biomarkers of AD in general.

Analytical determination of ethylenediamine impurity in tripelennamine hydrochloride by gas chromatography-mass spectrometry using phthalaldehyde as the derivatizing agent.

In the pharmaceutical industry, effective risk management and control strategies for potential genotoxic impurities are of paramount importance. The current study utilized GC-MS to evaluate a precise, linear, and accurate analytical method for quantifying ethylenediamine present in tripelennamine hydrochloride using phthalaldehyde as a derivatizing agent. When phthalaldehyde is sonicated for 10 min at room temperature, it reacts with ethylenediamine to form (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine. This approach minimizes matrix interference issues and resolves sample preparation difficulties encountered during ethylenediamine identification in GC-MS. In this method, helium serves as the carrier gas, while methanol acts as the diluent. The stationary phase consists of a DB-5MS column (30 m × 0.25 mm × 0.25 µm) with a flow rate of 1.5 mL/min. The retention time of (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine was determined to be 6.215 min. The method validation demonstrated limits of detection and quantification for (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine at 0.4 and 1.0 ppm, respectively, with a linearity range spanning from 1 to 30 ppm concentration with respect to the specification level. System suitability, precision, linearity, and accuracy of the current method were assessed in accordance with guidelines, yielding results deemed suitable for the intended use.

Identifying novel inhibitors targeting Exportin-1 for the potential treatment of COVID-19.

The nuclear export protein 1 (XPO1) mediates the nucleocytoplasmic transport of proteins and ribonucleic acids (RNAs) and plays a prominent role in maintaining cellular homeostasis. XPO1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses. In our earlier study, we proved the inhibition of XPO1 as a therapeutic strategy for managing SARS-COV-2 and its variants. In this study, we have utilized pharmacophore-assisted computational methods to identify prominent XPO1 inhibitors. After several layers of screening, a few molecules were shortlisted for further experimental validation on the in vitro SARS-CoV-2 cell infection model. It was observed that these compounds reduced spike positivity, suggesting inhibition of SARS-COV-2 infection. The outcome of this study could be considered further for developing novel antiviral therapeutic strategies against SARS-CoV-2.

METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma.

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.

Transcriptomics of MASLD Pathobiology in African American Patients in the Washington DC Area †.

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is becoming the most common chronic liver disease worldwide and is of concern among African Americans (AA) in the United States. This pilot study evaluated the differential gene expressions and identified the signature genes in the disease pathways of AA individuals with MASLD. Blood samples were obtained from MASLD patients (n = 23) and non-MASLD controls (n = 24) along with their sociodemographic and medical details. Whole-blood transcriptomic analysis was carried out using Affymetrix Clarion-S Assay. A validation study was performed utilizing TaqMan Arrays coupled with Ingenuity Pathway Analysis (IPA) to identify the major disease pathways. Out of 21,448 genes in total, 535 genes (2.5%) were significantly (p < 0.05) and differentially expressed when we compared the cases and controls. A significant overlap in the predominant differentially expressed genes and pathways identified in previous studies using hepatic tissue was observed. Of note, TGFB1 and E2F1 genes were upregulated, and HMBS was downregulated significantly. Hepatic fibrosis signaling is the top canonical pathway, and its corresponding biofunction contributes to the development of hepatocellular carcinoma. The findings address the knowledge gaps regarding how signature genes and functional pathways can be detected in blood samples ('liquid biopsy') in AA MASLD patients, demonstrating the potential of the blood samples as an alternative non-invasive source of material for future studies.

Electroweak Multi-Higgs Production: A Smoking Gun for the Type-I Two-Higgs-Doublet Model.

Extending the Higgs sector of the standard model (SM) by just one additional Higgs doublet field leads to the two-Higgs-doublet model (2HDM). In the type-I Z_{2}-symmetric limit of the 2HDM, all the five new physical Higgs states can be fairly light, O(100) GeV or less, without being in conflict with current data from the direct Higgs boson searches and the B-physics measurements. In this Letter, we establish that the new neutral as well as the charged Higgs bosons in this model can all be simultaneously observable in the multi-b final state. The statistical significance of the signature for each of these Higgs states, resulting from the electroweak (EW) production of their pairs, can exceed 5σ at the 13 TeV high-luminosity Large Hadron collider (HL-LHC). Since the parameter space configurations where this is achievable are precluded in the other, more extensively pursued, 2HDM types, an experimental validation of our findings would be a clear indication that the true underlying Higgs sector in nature is the type-I 2HDM.

Telomere Maintenance Mechanisms in a Cohort of High-Risk Neuroblastoma Tumors and Its Relation to Genomic Variants in the TERT and ATRX Genes.

Tumor cells are hallmarked by their capacity to undergo unlimited cell divisions, commonly accomplished either by mechanisms that activate TERT or through the alternative lengthening of telomeres pathway. Neuroblastoma is a heterogeneous pediatric cancer, and the aim of this study was to characterize telomere maintenance mechanisms in a high-risk neuroblastoma cohort. All tumor samples were profiled with SNP microarrays and, when material was available, subjected to whole genome sequencing (WGS). Telomere length was estimated from WGS data, samples were assayed for the ALT biomarker c-circles, and selected samples were subjected to methylation array analysis. Samples with ATRX aberration in this study were positive for c-circles, whereas samples with either MYCN amplification or TERT re-arrangement were negative for c-circles. Both ATRX aberrations and TERT re-arrangement were enriched in 11q-deleted samples. An association between older age at diagnosis and 1q-deletion was found in the ALT-positive group. TERT was frequently placed in juxtaposition to a previously established gene in neuroblastoma tumorigenesis or cancer in general. Given the importance of high-risk neuroblastoma, means for mitigating active telomere maintenance must be therapeutically explored.

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer.

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

Global loss of cellular m6A RNA methylation following infection with different SARS-CoV-2 variants.

Insights into host-virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N 6-Methyladenosine modification (m6A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m6A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m6A in cellular RNAs, whereas m6A is detected abundantly in viral RNA. METTL3, the m6A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m6A than did the B.1 and B.1.1.7 variants. We also observed a loss of m6A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m6A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m6A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m6A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m6A-dependent manner.

Vasoactive-ventilation-renal score and outcomes in infants and children after cardiac surgery.

Introduction: There is a need to index important clinical characteristics in pediatric cardiac surgery that can be obtained early in the postoperative period and accurately predict postoperative outcomes. Methodology: A prospective cohort study was conducted in the pediatric cardiac ICU and ward on all children aged <18 years undergoing cardiac surgery for congenital heart disease from September 2018 to October 2020. The vasoactive-ventilation-renal (VVR) score was analyzed to predict outcomes of cardiac surgeries with a comparison of postoperative variables. Results: A total of 199 children underwent cardiac surgery during the study period. The median (interquartile range) age was 2 (0.8-5) years, and the median weight was 9.3 (6-16) kg. The most common diagnoses were ventricular septal defect (46.2%) and tetralogy of Fallot (37.2%). At the 48th h, area under the curve (AUC) (95% CI) values were higher for the VVR score than those for other clinical scores measured. Similarly, at the 48th h, AUC (95% CI) values were higher for the VVR score than those for the other clinical scores measured for the length of stay and mechanical ventilation. Discussion: The VVR score at 48 h postoperation was found to best correlate with prolonged pediatric intensive care unit (PICU) stay, length of hospitalization, and ventilation duration, with the greatest AUC-receiver operating characteristic (0.715, 0.723, and 0.843, respectively). The 48-h VVR score correlates well with prolonged ICU, hospital stay, and ventilation.

Insights on the pathogenesis of type 2 diabetes as revealed by signature genomic classifiers in an African American population in the Washington, DC area.

AIMS: African Americans (AA) in the United States have a high risk of type 2 diabetes mellitus (T2DM) and suffer from disparities in the prevalence, mortality, and comorbidities of the disease compared to other Americans. The present study aimed to shed light on the molecular mechanisms of disease pathogenesis of T2DM among AA in the Washington, DC region. METHODS: We performed TaqMan Low Density Arrays (TLDA) on 24 genes of interest that belong to three categories: metabolic disease and disorders, cancer-related genes, and neurobehavioural disorders genes. The 18 genes, viz. ARNT, CYP2D6, IL6, INSR, RRAD, SLC2A2 (metabolic disease and disorders), APC, BCL2, CSNK1D, MYC, SOD2, TP53 (Cancer-related), APBA1, APBB2, APOC1, APOE, GSK3B, and NAE1 (neurobehavioural disorders), were differentially expressed in T2DM participants compared to controls. RESULTS: Our results suggest that factors including gender, smoking habits, and the severity or lack of control of T2DM (as indicated by HbA1c levels) were significantly associated with differential gene expression. APBA1 was significantly (p-value <0.05) downregulated in all diabetes participants. Upregulation of APOE and CYP2D6 genes and downregulation of the INSR gene were observed in the majority of diabetes patients. CONCLUSIONS: Tobacco smoking and gender were significantly associated with case-control differences in expression of the APBA1 and APOE genes (connected with Alzheimer's disease) and the INSR and CYP2D6 (associated with metabolic disorders). The results highlight the need for more effective management of T2DM and for tobacco smoking cessation interventions in this community, and further research on the associations of T2DM with other disease processes, including cancer and neurobehavioral pathways.

Peroxisome Proliferator-Activated Receptor-α (PPARα) Expression in a Clinical Population of Pakistani Patients with Type 2 Diabetes and Dyslipidemia.

Poor glycemic control and dyslipidemia are hallmarks of type 2 diabetes mellitus (T2DM), which predispose to cardiovascular diseases. Peroxisome proliferator-activated receptor-α (PPARα) has been associated with atherosclerosis, but its role in T2DM is less clear. Previously, we studied PPARα expression levels in diabetics with and without dyslipidemia (DD). In this study we described the association with fasting blood glucose, HbA1c levels and lipid levels of the study population. Patient demography and biochemical data were collected from hospitals in Islamabad, Pakistan, and RT-PCR data of PPARα expression were retrieved from our previous study from the same cohort. We performed t-tests and regression analysis to evaluate the relationships between PPARα expression and demographic and clinical variables. As expected, body mass index and HbA1c were elevated in T2DM and DD patients compared to controls. Blood lipids (total cholesterol, triglycerides, LDL and HDL) were significantly higher in the DD group compared to the other two groups. In the T2DM and DD groups, the PPARα expression was not associated with any of the physical and biochemical parameters measured in this study. Expression of the PPARα gene was independent of blood lipids and glycemic control in this study. Further research is necessary to better understand the biological parameters of PPARα expression.

CFD Investigation of Thermal Characteristics for a Dual Jet with a Parallel Co-flow.

A combined turbulent wall jet and offset jet (also known as the dual jet) with and without the presence of a parallel co-flow stream is studied. The standard k-ω turbulence model is used to predict the turbulent flow. The study focuses on the effects of the co-flow velocity (CFV) on the heat-transfer characteristics of the dual jet flow with the bottom wall maintained at a constant wall temperature. The CFV is varied up to 40% of the jet inlet velocity, and the height of the offset jet is varied from 5 to 11 times the jet width with the inlet Reynolds number taken as 15,000. The heat-transfer results reveal that the local Nusselt number (Nu x ) along the bottom wall exhibits a peak at the immediate downstream of the nozzle exit, followed by a continuous decay in the rest of the converging region before showing a small rise for a short streamwise distance in the merging region. Further downstream, in the combined region, Nu x gradually decreases with the downstream distance. Except the merging region, no influence of co-flow is observed in the other two flow zones (converging and combined regions). In the merging region, for a given offset ratio (OR), Nu x remains nearly constant for a certain axial distance, and it decreases as the CFV increases. As a result of the increase in the CFV, the average Nusselt number decreases, indicating a reduction in overall convective heat transfer for higher values of the CFV. A regression analysis among the average Nusselt number (), CFV, and OR results in a correlation function in the form of within the range OR = 5-11 and CFV = 0-40%.

Gene expression signatures in PCB-exposed Slovak children in relation to their environmental exposures and socio-physical characteristics.

Our previous gene expression studies in a PCB-exposed cohort of young children in Slovakia revealed that early-life exposures to PCBs and other organochlorine compounds were associated with significant alterations across several pathogenetic pathways. The present study was undertaken to further explore the high-throughput qRT-PCR-based gene expression effects by using TaqMan low-density array (TLDA) for selected genes in a sample of 55 children from the cohort. We analyzed the transcriptional changes of 11 genes in relation to PCB and organochlorine pesticide exposure levels (including DDT, DDE, HCH, and HCB), and to BMI and ethnicity in this cohort. The results indicated an overall downregulation of expression of these genes. Maximum downregulation (in fold change) was observed in the ENTPD3 gene, and the minimum level of downregulation was in CYP2D6. As per our multinomial regression model study, downregulation of LEPR gene was significantly directly correlated with all the exposure variables. Downregulation of APC, ARNT, CYP2D6, LEPR, LRP12, and MYC genes was directly correlated with BMI (kg/m2) of the individuals. Gender-specific differences in gene expression were observed in CYP2D6 (p-value 0.0001) and LEPR (p-value 0.028), while downregulation of CYP2D6 (p-value 0.01), LEPR (p-value 0.02), LRP12 (p-value 0.04), and MYC (p-value 0.02) genes was consistently observed in Roma children compared to Caucasians. The investigation of such health disparities must be emphasized in future research, together with interventions to reduce the health consequences of PCB exposures. In this context, we emphasize the importance of biomarker-based approaches to future research on genetic susceptibility to the effects of these compounds.

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle.

The COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections. IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.

A patch of positively charged residues regulates the efficacy of clinical DR5 antibodies in solid tumors.

Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success.

Impact of the Double Mutants on Spike Protein of SARS-CoV-2 B.1.617 Lineage on the Human ACE2 Receptor Binding: A Structural Insight.

The recent emergence of novel SARS-CoV-2 variants has threatened the efforts to contain the COVID-19 pandemic. The emergence of these "variants of concern" has increased immune escape and has supplanted the ancestral strains. The novel variants harbored by the B.1.617 lineage (kappa and delta) carry mutations within the receptor-binding domain of spike (S) protein (L452R + E484Q and L452R + T478K), the region binding to the host receptor. The double mutations carried by these novel variants are primarily responsible for an upsurge number of COVID-19 cases in India. In this study, we thoroughly investigated the impact of these double mutations on the binding capability to the human host receptor. We performed several structural analyses and found that the studied double mutations increase the binding affinity of the spike protein to the human host receptor (ACE2). Furthermore, our study showed that these double mutants might be a dominant contributor enhancing the receptor-binding affinity of SARS-CoV-2 and consequently making it more stable. We also investigated the impact of these mutations on the binding affinity of two monoclonal antibodies (Abs) (2-15 and LY-CoV555) and found that the presence of the double mutations also hinders its binding with the studied Abs. The principal component analysis, free energy landscape, intermolecular interaction, and other investigations provided a deeper structural insight to better understand the molecular mechanism responsible for increased viral transmissibility of these variants.

Biological Effects Associated with Internal and External Contamination of Diagnostic Nuclear Medicine Sources: An In vitro Study.

AIM: In a Nuclear Medicine department, the risk of external and internal contamination in radiation workers is much higher than in other medical radiation facilities. The risk associated with both types of contaminations should be quantified to estimate the radiation dose received by the personal. Here, we designed an in vitro model to see the impact of internal and external contamination of F-18 and Technetium-99 m (Tc-99 m) on DNA damages. METHODOLOGY: Chinese hamster lung fibroblast V79 was used for all of the experiments. Irradiation was performed internally and externally (scenarios activity is mixed with the cell line [Internal] and activity kept at 1 cm distance from cell line [external]) using two different diagnostic radioactive sources (Tc-99 m and F-18) of known quantity 37 MBq. Total cumulated activity (MBq-min) was calculated up to one half-life of sources for both internal and external setups. An alkaline single gel electrophoresis technique (comet assay) was used for DNA damage analysis. Olive tail moment (OTM) was used to characterize DNA damage. RESULTS: We have not observed any significant difference (P > 0.05) in OTM between internal and external irradiation for cumulated activity presented before one half-life of both diagnostic isotopes. However, a significant difference in OTM was noted between internal and external irradiation for cumulated activity presented at one half-life of radioactive sources (P < 0.05). DNA damage with internal exposure was found to be 17.28% higher for F-18 and 23% higher for Tc-99 m than external exposure at one half-life of radioactive sources. Overall, we noted greater DNA damage in F-18 as compared to Tc-99 m. CONCLUSIONS: Our in vitro study practically demonstrated that internal contamination is more hazardous than external exposure.

A SIMPLE AND NOVEL APPROACH TO STUDY KINETICS AND ESTIMATE RADIATION DOSES FROM INTERNALLY ADMINISTERED RADIOPHARMACEUTICALS USING AN EXTERNAL DOSE MEASUREMENT SYSTEM.

Various methods have been reported to study radiotracer kinetics and make internal dosimetry feasible in the routine clinical nuclear medicine practice. The aim of the present study was to quantify cumulative activity and organ doses using an indigenously designed and fabricated external dose measurement system. The measurement was demonstrated on patients undergoing whole-body (WB) 18F-FDG (Fluorine-18-fluorodeoxyglucose) direct positron emission tomography/computed tomography investigations. An external dose measurement system comprising of an ionisation chamber-survey meter and the movable focussing collimator was used to quantify the uptake of 18F-FDG in liver and brain. Cumulative activity and normalised cumulative activity in these organs were calculated. The results were validated by performing measurements on a phantom uniformly filled with known activity of 18F-FDG.The difference in the absorbed dose estimated with and without collimator was statistically significant (p < 0.05). The external dose measurement technique is relatively novel, convenient and reliable for the assessment of internal absorbed dose of organs.

NBAT1/CASC15-003/USP36 control MYCN expression and its downstream pathway genes in neuroblastoma.

BACKGROUND: MYCN has been an attractive therapeutic target in neuroblastoma considering the widespread amplification of the MYCN locus in neuroblastoma, and its established role in neuroblastoma development and progression. Thus, understanding neuroblastoma-specific control of MYCN expression at the transcriptional and post-transcriptional level would lead to identification of novel MYCN-dependent oncogenic pathways and potential therapeutic strategies. METHODS: By performing loss- and gain-of-function experiments of the neuroblastoma hotspot locus 6p22.3 derived lncRNAs CASC15-003 and NBAT1, together with coimmunoprecipitation and immunoblotting of MYCN, we have shown that both lncRNAs post-translationally control the expression of MYCN through regulating a deubiquitinase enzyme USP36. USP36 oncogenic properties were investigated using cancer cell lines and in vivo models. RNA-seq analysis of loss-of-function experiments of CASC15-003/NBAT1/MYCN/USP36 and JQ1-treated neuroblastoma cells uncovered MYCN-dependent oncogenic pathways. RESULTS: We show that NBAT1/CASC15-003 control the stability of MYCN protein through their common interacting protein partner USP36. USP36 harbors oncogenic properties and its higher expression in neuroblastoma patients correlates with poor prognosis, and its downregulation significantly reduces tumor growth in neuroblastoma cell lines and xenograft models. Unbiased integration of RNA-seq data from CASC15-003, NBAT1, USP36, and MYCN knockdowns and neuroblastoma cells treated with MYCN inhibitor JQ1, identified genes that are jointly regulated by the NBAT1/CASC15-003/USP36/MYCN pathway. Functional experiments on one of the target genes, COL18A1, revealed its role in the NBAT1/CASC15-003-dependent cell adhesion feature in neuroblastoma cells. CONCLUSION: Our data show post-translational regulation of MYCN by NBAT1/CASC15-003/USP36, which represents a new regulatory layer in the complex multilayered gene regulatory network that controls MYCN expression.