Green Plus Research Articles

Optimizing the Use of Radar when the Ship is Moving in MT. Green Plus

It is believed that the high number of collisions in narrow shipping lanes is caused by the lack of skill of sailors. Therefore, this analysis aims to determine the role of Radar in preventing collisions at sea. The research approach is qualitative, and the data will be presented using descriptive analysis. The research findings indicate that the decrease in Radar function is due to the non-implementation of Standard Operating Procedures (SOPs), the officers' lack of understanding of the manual books, and damage to the scanner. The consequences of a decrease in Radar function include navigation hazards because the Radar does not function correctly, making it difficult for the watch crew to identify other ships. To optimize the use of Radar when ships are moving at night, it is necessary to regularly check, maintain and repair navigation equipment.

The Habenula's Role in Adaptive Behaviors: Contributions From Neuroimaging.

The Habenula's Role in Adaptive Behaviors: Contributions From Neuroimaging.

Energy storage against interconnection as a balancing mechanism for a 100% renewable UK electricity grid

This study considers generation and demand challenges of a 100% renewable UK electricity grid and how this could be addressed with interconnection or energy storage. Hourly demand and electricity generation profiles for a year have been constructed: Business as Usual (BAU) with a yearly demand of 540 TWh and Green Plus (GP) with a demand of 390 TWh, Two further scenarios based on the above have been considered with electrification of heating (ASHP) and electric vehicle transportation (EV). The resultant hourly imbalances have been used to calculate the interconnection and energy storage requirements. This paper discusses the findings of the BAU scenario. The calculated interconnector capacity required was found to be 60 GW and cost £58 billion. Energy storage capacity requirements vary depending on the selected technology. Rated capacity was estimated to be 14 GW with storage capacity of 3 TWh for pumped storage, 11 GW and 2.3 TWh for liquid air, and 65 GW and 13.6 TWh for hydrogen storage, at a cost of £65, £76 and £45 billion respectively. This paper indicates that storing hydrogen in underground caverns would offer the cheapest solution. However, whilst these technological solutions can address generation and demand imbalance in a fully renewable electricity grid, there clearly remain barriers to each technology.

Clinical Implementations of Preoperative Computed Tomography Lymphography in Gastric Cancer: A Comparison with Dual Tracer Methods in Sentinel Node Navigation Surgery

Current sentinel node (SN) detection techniques require a learning period and tracers have many disadvantages for practical use. The purpose of this study was to evaluate the feasibility of preoperative computed tomography (CT) lymphography using lipiodol for detecting SNs in gastric cancer. A total of 24 patients who underwent laparoscopic surgery for early gastric cancer were enrolled in this study. Noncontrast CT images were obtained 1-2 h after endoscopic submucosal peritumoral injection of 1 mL of lipiodol the day before surgery. The final sentinel basins (SBs) were decided by the dual tracer method (indocyanine green plus (99m)Tc-antimony sulfur colloid) during laparoscopic gastrectomy. SN detection rate by preoperative CT lymphography using lipiodol and agreement between CT lymphography versus dual tracer method were evaluated. The agreement was confirmed with soft X-ray radiography of detected SBs. Technical failure of endoscopic lipiodol injection occurred in one patient. SNs were successfully detected in the remaining 23 patients (95.8 %), whereas the intraoperative SB detection rate using the dual method was 100 %. The agreement rate, defined as the concordance between two methods or inclusion of SNs detected by CT lymphography in SBs by the dual tracer method, was 87 %. Our initial experience of CT lymphography using lipiodol shows good potential in predicting SBs of gastric cancer preoperatively. However, SN detection by CT lymphography and the dual method should be applied complementarily in gastric cancer because discrepancies between these methods occur.

Nucleotide Extension Genotyping by High-Resolution Melting

One limitation of small amplicon melting is the inability to genotype certain nearest-neighbor symmetric variations without manipulating the sample. We have developed a method for these exceptions: a high-resolution melting single nucleotide extension assay. Single nucleotide extension was performed in a new instrument, the LightScanner 32 (LS32), which uses capillary reaction tubes and is capable of real-time PCR and sequential high-resolution melting of 32 samples. Asymmetric PCR used Platinum Taq and LC Green Plus in the master mix for target amplification. Dideoxynucleotides and extension oligonucleotides were sequestered in the tube cap and added post-PCR, maintaining a closed system. One dideoxynucleotides was used per capillary tube. Samples were cycled five times to incorporate dideoxynucleotides into the extension products using ThermoSequenase, followed by high-resolution melting. Single nucleotide polymorphisms from the RET proto-oncogene (n = 7), hemochromatosis (HFE, n = 30), coagulation factor 2 (F2, n = 29), coagulation factor 5 (F5, n = 30), and methylenetetrahydrofolate reductase (MTHFR, n = 60) genes were genotyped. The DNA melting profiles identified the target single nucleotide polymorphisms by the lowest melting temperature transition. All genotypes had a distinctive melting pattern. The method was 100% concordant with samples previously genotyped at HFE, MTHFR, and F2 and 90% concordant with F5. F5 discordants were genotyped correctly by redesigning the assay. Our results demonstrate that although single nucleotide polymorphisms can be successfully differentiated using this methodology, the method requires careful optimization.

UTX, a Histone Demethylase, Is Inactivated through Homozygous Deletion, Mutation, and DNA Methylation in Multiple Myeloma.

Abstract 1798Poster Board I-824Histone modifications are known to mediate transcriptional regulation through changes in chromatin condensation and as such can lead to aberrant transcriptional patterns resulting in malignant transformation. Modulation of chromatin structure via histone modification is becoming recognised as an important pathogenic mechanism in myeloma and has been suggested by the over-expression of MMSET, a histone methyltransferase, by the t(4;14) chromosomal rearrangement. More recently inactivation of UTX, a histone demethylase, has also been suggested to have a role in myeloma pathogenesis and both UTX and MMSET are mediators of transcriptional repression. UTX is inactivated in a number of different cancer cell lines but importantly, mutations and deletions have been detected in myeloma cell lines and we wished to follow up on this observation in uniformly treated clinical cases. UTX is a large gene found on the X chromosome covering 240 kb of genomic DNA and consists of 29 exons encoding a protein with both JmjC-domains and tricopeptide repeats responsible for histone demethylation and polycomb protein interactions. Inactivation of UTX occurs through deletions of individual exons through to large whole gene deletions as well as by mutations scattered throughout the 29 exons. A further mechanism of UTX inactivation which has not been looked for to date is via DNA methylation of the CpG island upstream of the transcriptional start site. We set out to determine the status of UTX in our dataset which includes expression, mapping, and methylation array data from presenting myeloma samples entered into the MRC Myeloma IX clinical trial. The gene expression of UTX was measured on 272 samples using Affymetrix U133 Plus 2.0 arrays and showed that 80% of samples do not express UTX transcripts but using expression quartile analysis we could not detect an effect on overall survival. The mechanism underlying the abrogation of expression was investigated further using the Affymetrix 500K SNP mapping array on a subset of 114 samples to detect copy number alterations. UTX was hemizygously deleted in 21 (42%) female samples and was completely deleted in 1 male sample, at the resolution of the arrays. In order to determine if individual exons were deleted, at a resolution below that detectable by mapping arrays, we performed quantitative PCR coupled with high resolution melting (HRM) analysis using the Rotor-gene Q real-time cycler (Qiagen). Exons were amplified, over 40 cycles, to obtain products of ∼200 bp using LC Green Plus mastermix (Idaho Technologies) in a 10 μl reaction on the Rotor-gene Q with a final HRM step from 72-95 °C with increments of 0.1 °C. Amplification plots combined with the HRM step allows us to identify both homozygous deletions and mutations within the exons. We screened all 114 samples for micro-deletions and mutations and found homozygous deletions in ∼7% of samples and identified a significant proportion of mutations using the HRM method which accounted for a total of ∼10% of gene inactivation. In order to determine if methylation could be responsible for inactivation of the remaining allele we used the Illumina Infinium humanmethylation27 array to study the methylation status at the UTX locus. This array interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. The results of this analysis are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. In samples with a diploid copy number of UTX the methylation signals covered 2 ranges: hemi-methylated (0.35-0.55, n=7) and hyper-methylated (0.73-0.89, n=14). In samples with 1 copy of UTX, which includes all males, there were 3 ranges: hypomethylated (0.08-0.21, n=5), hemi-methylated (0.35-0.51, n=3), and hypermethylated (0.66-0.88, n=48). All of the hypomethylated samples with a single copy of UTX were male, and at least 1 of these samples contained an inactivating exonic deletion resulting in complete loss of function. These data indicate that methylation of the residual allele contributes significantly to the inactivation of UTX along with interstitial deletions and mutations. We will go on to present data on the interaction of UTX with variation at the UTY locus and how this modulates behaviour of the myeloma clone. DisclosuresNo relevant conflicts of interest to declare.

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

Mutation detection has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to design. Nongel-based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/clinical setting. In this study, we assessed the latest of the nongel-based methods, namely high-resolution melt (HRM) curve analysis. HRM is a closed-tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner System using LC Green PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of double-stranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC-rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis.

Differentiation between vaccine and wild-type varicella-zoster virus genotypes by high-resolution melt analysis of single nucleotide polymorphisms

Background The analysis of single nucleotide polymorphisms (SNPs) of varicella-zoster virus (VZV) has enabled differentiation between wild-type genotypes from the Oka vaccine strain (V-Oka). Objectives To genotype VZV strains in Australia using high-resolution melt (HRM) analysis of SNPs in five gene targets. Study design Extracted DNA from 78 samples obtained from patients with chickenpox and zoster were genotyped by HRM analysis of SNPs in five open reading frames (ORFs): 1 (685 G > A), 21 (33 725 C > T), 37 (66 288 G > A), 60 (101 464 C > A) and 62 (106 262 T > C) using a double-stranded (ds) DNA saturating dye, LC Green Plus. Results For each genotype, melt curve temperature ( T m) shifts differentiated the nucleotide present at that locus ( P < 0.0001) with melting curve shifts between alleles ranging from 0.56 °C (ORF 37) to 3.34 °C (ORF 62). The most common genotypes detected were the European Type C (59%) and B (18%) strains. This was followed by the African/Asian Type A (14%) and Japanese J1 (9%), strains, both prevalent in the Northern Territory and Western Australia. Conclusions HRM analysis of SNPs showed that the European B and C genotypes were most prevalent in Australia, with genotypes A and J strains also present. HRM analysis using a dsDNA dye provides a useful tool in classifying varicella-zoster viruses.

Mutation Scanning the GJB1 Gene with High-Resolution Melting Analysis: Implications for Mutation Scanning of Genes for Charcot-Marie-Tooth Disease

X-linked Charcot-Marie-Tooth type 1 disease has been associated with 280 mutations in the GJB1 [gap junction protein, beta 1, 32 kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene. High-resolution melting analysis with an automated instrument can be used to scan DNA for alterations, but its use in X-linked disorders has not been described. A 96-well LightScanner for high resolution melting analysis was used to scan amplicons of the GJB1 gene. All mutations reported in this study had been confirmed previously by sequence analysis. DNA samples were amplified with the double-stranded DNA-binding dye LC Green Plus. Melting curves were analyzed as fluorescence difference plots. The shift and curve shapes of melting profiles were used to distinguish controls from patient samples. The method detected each of the 23 mutations used in this study. Eighteen known mutations provided validation of the high-resolution melting method and a further 5 mutations were identified in a blind study. Altered fluorescence difference curves for all the mutations were easily distinguished from the wild-type melting profile. High-resolution melting analysis is a simple, sensitive, and cost-efficient alternative method to scan for gene mutations in the GJB1 gene. The technology has the potential to reduce sequencing burden and would be suitable for mutation screening of exons of large multiexon genes that have been discovered to be associated with Charcot Marie Tooth neuropathy.