Experiences from the ARGOS user group nuclear emergency exercise.

The Accident Reporting and Guiding Operational System (ARGOS) is a decision support system used to assist in the Emergency Preparedness and Response (EPR) to nuclear and radiological incidents. The ARGOS user group has been formed that is made up of government agencies across many countries that have a role in EPR to nuclear and radiological incidents. In 2020, a desktop exercise was organised for the members of the ARGOS user group. The exercise involved two hypothetical accidents at different times on the same date, namely a radiological release from a floating nuclear power plant (NPP) off the Norwegian coast and from the Loviisa NPP in Finland. The objectives of the exercise were to train and increase knowledge of the ARGOS system, to perform a comparison of model outputs, and to compare the recommendations of protective actions. In the case of the floating NPP the source term was provided, while in the Loviisa NPP scenario the participants were required to provide their own source term based on a description of the accident. The results on radiological consequences based on dispersion modelling, protective actions, source terms and dispersion modelling settings were collected from participants. A comparison was made between each of these reported aspects. In general, it was found that there was general agreement between the results for the floating nuclear power plant scenario in the sense of plume direction and extent, while in the case of the Loviisa NPP scenario, there was much greater variation, with the difference in source term estimates between the participants being an influencing factor. The participants acknowledged that taking part in an exercise of this nature increased their knowledge and understanding about using decision support tools such as ARGOS in planning and responding to nuclear and radiological emergencies.

Stochastic Variation in DNA Methylation Modulates Nucleosome Occupancy and Alternative Splicing in Arabidopsis thaliana.

Plants use complex gene regulatory mechanisms to overcome diverse environmental challenges. For instance, cold stress induces rapid and massive transcriptome changes via alternative splicing (AS) to confer cold tolerance in plants. In mammals, mounting evidence suggests chromatin structure can regulate co-transcriptional AS. Recent evidence also supports co-transcriptional regulation of AS in plants, but how dynamic changes in DNA methylation and the chromatin structure influence the AS process upon cold stress remains poorly understood. In this study, we used the DNA methylation inhibitor 5-Aza-2'-Deoxycytidine (5-aza-dC) to investigate the role of stochastic variations in DNA methylation and nucleosome occupancy in modulating cold-induced AS, in Arabidopsis thaliana (Arabidopsis). Our results demonstrate that 5-aza-dC derived stochastic hypomethylation modulates nucleosome occupancy and AS profiles of genes implicated in RNA metabolism, plant hormone signal transduction, and of cold-related genes in response to cold stress. We also demonstrate that cold-induced remodelling of DNA methylation regulates genes involved in amino acid metabolism. Collectively, we demonstrate that sudden changes in DNA methylation via drug treatment can influence nucleosome occupancy levels and modulate AS in a temperature-dependent manner to regulate plant metabolism and physiological stress adaptation.

If you build it, will they use it? Use of a Digital Assistant for Self-Reporting of COVID-19 Rapid Antigen Test Results during Large Nationwide Community Testing Initiative

ImportanceWide-spread distribution of rapid-antigen tests is integral to the United States strategy to address COVID-19; however, it is estimated that few rapid-antigen test results are reported to local departments of health. ObjectiveTo characterize how often individuals in six communities throughout the United States used a digital assistant to log rapid-antigen test results and report them to their local Department of Health. DesignThis prospective cohort study is based on anonymously collected data from the beneficiaries of The Say Yes! Covid Test program, which distributed 3,000,000 rapid antigen tests at no cost to residents of six communities between April and October 2021. We provide a descriptive evaluation of beneficiaries use of digital assistant for logging and reporting their rapid antigen test results. Main Outcome and MeasuresNumber and proportion of tests logged and reported to the Department of Health through the digital assistant ResultsA total of 178,785 test kits were ordered by the digital assistant, and 14,398 households used the digital assistant to log 41,465 test results. Overall, a small proportion of beneficiaries used the digital assistant (8%), but over 75% of those who used it reported their rapid antigen test results to their state public health department. The reporting behavior varied between communities and was significantly different for communities that were incentivized for reporting test results (p < 0.001). In all communities, positive tests were less reported than negative tests (60.4% vs 75.5%; p<0.001). Conclusions and RelevanceThese results indicate that app-based reporting with incentives may be an effective way to increase reporting of rapid tests for COVID-19; however, increasing the adoption of the digital assistant is a critical first step.

Targeting DNA methyltransferases in non-small-cell lung cancer.

Despite the advances in treatment using chemotherapy or targeted therapies, due to static survival rates, non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths worldwide. Epigenetic-based therapies have been developed for NSCLC by targeting DNA methyltransferases (DNMTs) and histone-modifying enzymes. However, treatment using single epigenetic agents on solid tumours has been inadequate; whereas, treatment with a combination of DNMTs inhibitors with chemotherapy and immunotherapy has shown great promise. Dietary sources of phytochemicals could also inhibit DNMTs and cancer stem cells, representing a novel and promising way to prevent and treat cancer. Herein, we will discuss the different DNMTs, DNA methylation profiling in NSCLC as well as current demethylating agents in ongoing clinical trials. Therefore, providing a concise overview of future developments in the field of epigenetic therapy in NSCLC.

Epigenetic differences in an identical genetic background modulate alternative splicing in A. thaliana.

How stable and temperature-dependent variations in DNA methylation and nucleosome occupancy influence alternative splicing (AS) remains poorly understood in plants. To answer this, we generated transcriptome, whole-genome bisulfite, and MNase sequencing data for an epigenetic Recombinant Inbred Line (epiRIL) of A. thaliana at normal and cold temperature. For comparative analysis, the same data sets for the parental ecotype Columbia (Col-0) were also generated, whereas for DNA methylation, previously published high confidence methylation profiles of Col-0 were used. Significant epigenetic differences in an identical genetic background were observed between Col-0 and epiRIL lines under normal and cold temperatures. Our transcriptome data revealed that differential DNA methylation and nucleosome occupancy modulate expression levels of many genes and AS in response to cold. Collectively, DNA methylation and nucleosome levels exhibit characteristic patterns around intron-exon boundaries at normal and cold conditions, and any perturbation in them, in an identical genetic background is sufficient to modulate AS in Arabidopsis.

Differential nucleosome occupancy modulates alternative splicing in Arabidopsis thaliana.

Alternative splicing (AS) is a major gene regulatory mechanism in plants. Recent evidence supports co-transcriptional splicing in plants, hence the chromatin state can impact AS. However, how dynamic changes in the chromatin state such as nucleosome occupancy influence the cold-induced AS remains poorly understood. Here, we generated transcriptome (RNA-Seq) and nucleosome positioning (MNase-Seq) data for Arabidopsis thaliana to understand how nucleosome positioning modulates cold-induced AS. Our results show that characteristic nucleosome occupancy levels are strongly associated with the type and abundance of various AS events under normal and cold temperature conditions in Arabidopsis. Intriguingly, exitrons, alternatively spliced internal regions of protein-coding exons, exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions. Likewise, nucleosome patterns differ between exitrons and retained introns, pointing to their distinct regulation. Collectively, our data show that characteristic changes in nucleosome positioning modulate AS in plants in response to cold.

Decoding co-/post-transcriptional complexities of plant transcriptomes and epitranscriptome using next-generation sequencing technologies.

Next-generation sequencing (NGS) technologies - Illumina RNA-seq, Pacific Biosciences isoform sequencing (PacBio Iso-seq), and Oxford Nanopore direct RNA sequencing (DRS) - have revealed the complexity of plant transcriptomes and their regulation at the co-/post-transcriptional level. Global analysis of mature mRNAs, transcripts from nuclear run-on assays, and nascent chromatin-bound mRNAs using short as well as full-length and single-molecule DRS reads have uncovered potential roles of different forms of RNA polymerase II during the transcription process, and the extent of co-transcriptional pre-mRNA splicing and polyadenylation. These tools have also allowed mapping of transcriptome-wide start sites in cap-containing RNAs, poly(A) site choice, poly(A) tail length, and RNA base modifications. The emerging theme from recent studies is that reprogramming of gene expression in response to developmental cues and stresses at the co-/post-transcriptional level likely plays a crucial role in eliciting appropriate responses for optimal growth and plant survival under adverse conditions. Although the mechanisms by which developmental cues and different stresses regulate co-/post-transcriptional splicing are largely unknown, a few recent studies indicate that the external cues target spliceosomal and splicing regulatory proteins to modulate alternative splicing. In this review, we provide an overview of recent discoveries on the dynamics and complexities of plant transcriptomes, mechanistic insights into splicing regulation, and discuss critical gaps in co-/post-transcriptional research that need to be addressed using diverse genomic and biochemical approaches.

Invasive Lobular Breast Carcinoma Can Be a Challenging Diagnosis Without the Use of Tumor Markers.

Invasive lobular carcinoma is often challenging to diagnose due to the lack of physical examination findings and macrocalcifications on mammography. The cells of invasive lobular carcinoma form a distinct single file pattern that can be identified on histology slides. Often, when patients present, there is metastasis to the bones, lymph nodes, and gastrointestinal tract. Tumor markers are a valuable tool in identification, especially the loss of E-cadherin protein. However, if E-cadherin protein is not available, epidermal membrane antigen, which inhibits E-cadherin, can prove to be a significant diagnostic tool. Epidermal membrane antigen was the key tumor marker in our patient case. Other tumor markers and histology stains can drive treatment plans and help predict prognosis.

Genome-Wide Identification of Splicing Quantitative Trait Loci (sQTLs) in Diverse Ecotypes of Arabidopsis thaliana.

Alternative splicing (AS) of pre-mRNAs contributes to transcriptome diversity and enables plants to generate different protein isoforms from a single gene and/or fine-tune gene expression during different development stages and environmental changes. Although AS is pervasive, the genetic basis for differential isoform usage in plants is still emerging. In this study, we performed genome-wide analysis in 666 geographically distributed diverse ecotypes of Arabidopsis thaliana to identify genomic regions [splicing quantitative trait loci (sQTLs)] that may regulate differential AS. These ecotypes belong to different microclimatic conditions and are part of the relict and non-relict populations. Although sQTLs were spread across the genome, we observed enrichment for trans-sQTL (trans-sQTLs hotspots) on chromosome one. Furthermore, we identified several sQTL (911) that co-localized with trait-linked single nucleotide polymorphisms (SNP) identified in the Arabidopsis genome-wide association studies (AraGWAS). Many sQTLs were enriched among circadian clock, flowering, and stress-responsive genes, suggesting a role for differential isoform usage in regulating these important processes in diverse ecotypes of Arabidopsis. In conclusion, the current study provides a deep insight into SNPs affecting isoform ratios/genes and facilitates a better mechanistic understanding of trait-associated SNPs in GWAS studies. To the best of our knowledge, this is the first report of sQTL analysis in a large set of Arabidopsis ecotypes and can be used as a reference to perform sQTL analysis in the Brassicaceae family. Since whole genome and transcriptome datasets are available for these diverse ecotypes, it could serve as a powerful resource for the biological interpretation of trait-associated loci, splice isoform ratios, and their phenotypic consequences to help produce more resilient and high yield crop varieties.

Alternative Splicing and Protein Diversity: Plants Versus Animals.

Plants, unlike animals, exhibit a very high degree of plasticity in their growth and development and employ diverse strategies to cope with the variations during diurnal cycles and stressful conditions. Plants and animals, despite their remarkable morphological and physiological differences, share many basic cellular processes and regulatory mechanisms. Alternative splicing (AS) is one such gene regulatory mechanism that modulates gene expression in multiple ways. It is now well established that AS is prevalent in all multicellular eukaryotes including plants and humans. Emerging evidence indicates that in plants, as in animals, transcription and splicing are coupled. Here, we reviewed recent evidence in support of co-transcriptional splicing in plants and highlighted similarities and differences between plants and humans. An unsettled question in the field of AS is the extent to which splice isoforms contribute to protein diversity. To take a critical look at this question, we presented a comprehensive summary of the current status of research in this area in both plants and humans, discussed limitations with the currently used approaches and suggested improvements to current methods and alternative approaches. We end with a discussion on the potential role of epigenetic modifications and chromatin state in splicing memory in plants primed with stresses.

Perspective on Alternative Splicing and Proteome Complexity in Plants.

Alternative splicing (AS) generates multiple transcripts from the same gene, however, AS contribution to proteome complexity remains elusive in plants. AS is prevalent under stress conditions in plants, but it is counterintuitive why plants would invest in protein synthesis under declining energy supply. We propose that plants employ AS not only to potentially increasing proteomic complexity, but also to buffer against the stress-responsive transcriptome to reduce the metabolic cost of translating all AS transcripts. To maximise efficiency under stress, plants may make fewer proteins with disordered domains via AS to diversify substrate specificity and maintain sufficient regulatory capacity. Furthermore, we suggest that chromatin state-dependent AS engenders short/long-term stress memory to mediate reproducible transcriptional response in the future.

Does co-transcriptional regulation of alternative splicing mediate plant stress responses?

Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS.

Pediatric Orbital Primitive Neuroectodermal Tumors.

PURPOSE: To present the clinical, radiological, histopathological, immunohistochemical features and the follow-up of orbital primitive neuroectodermal tumors (PNETs) in pediatric patients along with a review of the literature. METHODS: A retrospective analysis of all diagnosed cases of orbital PNET was done. Patients' ophthalmic findings, imaging, immunohistochemistry, metastatic work-up, treatment, globe salvation, and survival were documented and a mini literature review of orbital PNET was performed. RESULTS: Four diagnosed cases of orbital PNET presented with proptosis and visual impairment were treated during the study period. The radiological imaging showed primary orbital involvement. There were three males and one female with a mean age of 63.75 months (range: 3 to 244 months). Histopathology of all studied patients showed round malignant cells with hyperchromatic nuclei, increased nuclear cytoplasmic ratio, and positive test results for CD99 and FLI-1. The studied patients underwent orbital surgery for excision of tumors followed by chemotherapy. One of the patients also had external radiation in addition to chemotherapy after a second recurrence. The follow-up period of these patients varied from 1 to 5 years. Only one child who had recurrence twice was followed up to 5 years, but was lost to follow-up after that. CONCLUSIONS: The authors believe that most orbital peripheral PNET tumors present as well-defined masses on both imaging and perioperatively and are easily removed surgically. The apparently disguised "benign profile" of orbital PNET may prove deceptive and the shorter duration of symptoms remains a strong reminder of the malignant nature of the lesion. [J Pediatr Ophthalmol Strabismus. 2018;55(2):93-99.].

Stability indicating RP-HPLC method for simultaneous determination of gatifloxacin and dexamethasone in binary combination

Abstract In this study, conditions were optimized for development of a simple RP-HPLC method for simultaneous analysis of gatifloxacin and dexamethasone in different matrices like pharmaceuticals, human serum and urine. Good separation of gatifloxacin and dexamethasone from the induced degradation products was accomplished using C8 as stationary phase; 0.02 M phosphate buffer (pH 3.0) and methanol (42:58 v/v) as mobile phase. The concentration was measured with DAD at 270 nm. Linearity was observed in the range of 0.000040-0.000280 mol/L for gatifloxacin (r2≥0.999) and 0.000013-0.000091 mol/L for dexamethasone (r2≥0.999). Both the analyte peaks were completely separated from the peaks of induced degradation products as indicated by the peak purity index (≥0.9999 for both analytes). The optimized method is recommended to be used for concurrent analysis of gatifloxacin and dexamethasone in different matrices.

Squamous Cell Carcinoma Arising in Background of Mature Cystic Teratoma.

Mature cystic teratoma of ovary is a common benign adnexal tumor in females. Malignant transformation in a mature cystic teratoma of the ovary occurs in only 1 - 2% of cases. Most common malignant transformation is squamous cell carcinoma (70-80% of cases), occurring mostly in postmenopausal women with large sized cysts. Here, we report a case of 46-year lady who presented with pain in lower abdomen. Ultrasound and CT scan revealed complex adnexal mass measuring 16x9 cm. Size and older age of the patients should be regarded as important predictors of malignant transformation in mature cystic teratoma. Adequate sampling followed by histopathological examination has important role in diagnosis of such rare tumor.

Comparative analysis of the drought-responsive transcriptome in soybean lines contrasting for canopy wilting.

Drought stress causes significant yield losses in major oil seed crops, such as soybean [Glycine max (L.) Merr]. Few soybean lines have been identified as canopy-wilting tolerant; however, the molecular mechanism conferring tolerance is not fully understood. To understand the biological process, a whole genome transcriptome analysis was performed for leaf tissues of two contrasting soybean lines: drought-susceptible (DS) Pana and drought-tolerant (DT) PI 567690. A pairwise comparison of the DS and DT lines under drought and control conditions detected 1914 and 670 genes with a greater than two-fold change in expression under drought conditions. Pairwise treatment comparison and gene enrichment analysis on the DT line showed the down-regulation of genes associated with protein binding, hydrolase activity, carbohydrate/lipid metabolism, xyloglucan endo-transglycosylases associated with cell-wall, apoplast, and chlorophyll a/b binding proteins. On the other hand, genes that were associated with the biotic stress response, ion binding and transport, the oxido-reductive process and electron carrier activity were up-regulated. Gene enrichment analysis detected UDP glucuronosyl transferase activity-encoding genes to be differentially expressed in PI 567690 under drought stress conditions. We found valuable SNPs variation in aquaporin genes of the DT line that are conserved in known slower canopy-wilting lines, this should facilitate marker-assisted selection in soybeans with improved drought tolerance.

Stability indicating RP-HPLC method for simultaneous determination of piroxicam and ofloxacin in binary combination.

A simple and precise RP-HPLC method was developed for simultaneous determination of piroxicam and ofloxacin in pharmaceutical formulations and human serum. Optimum separations of piroxicam, ofloxacin and stress-induced degradation products were achieved by use of Hypersil BDS C8 column (250 x 4.6mm, 5 µm). The mobile phase was a mixture of acetonitrile: 0.012M K2HPO4: 0.008M sodium citrate (both buffers mixed and pH adjusted to 2.8) (50:25:25 v/v/v) delivered at flow rate of 1.5 mL min⁻¹ using DAD at 254 nm. Response was linear function of concentration over the ranges of 70-130 mg mL⁻¹ for piroxicam and ofloxacin (r² ≥ 0.999). The method efficiently separated the analytical peaks from degradation products with acceptable tailing and resolution. The developed method was successfully used for concurrent analysis of piroxicam and ofloxacin in pharmaceutical formulations, human serum and in vitro drug interaction studies.

Core clock, SUB1, and ABAR genes mediate flooding and drought responses via alternative splicing in soybean.

Circadian clocks are a great evolutionary innovation and provide competitive advantage during the day/night cycle and under changing environmental conditions. The circadian clock mediates expression of a large proportion of genes in plants, achieving a harmonious relationship between energy metabolism, photosynthesis, and biotic and abiotic stress responses. Here it is shown that multiple paralogues of clock genes are present in soybean (Glycine max) and mediate flooding and drought responses. Differential expression of many clock and SUB1 genes was found under flooding and drought conditions. Furthermore, natural variation in the amplitude and phase shifts in PRR7 and TOC1 genes was also discovered under drought and flooding conditions, respectively. PRR3 exhibited flooding- and drought-specific splicing patterns and may work in concert with PRR7 and TOC1 to achieve energy homeostasis under flooding and drought conditions. Higher expression of TOC1 also coincides with elevated levels of abscisic acid (ABA) and variation in glucose levels in the morning and afternoon, indicating that this response to abiotic stress is mediated by ABA, endogenous sugar levels, and the circadian clock to fine-tune photosynthesis and energy utilization under stress conditions. It is proposed that the presence of multiple clock gene paralogues with variation in DNA sequence, phase, and period could be used to screen exotic germplasm to find sources for drought and flooding tolerance. Furthermore, fine tuning of multiple clock gene paralogues (via a genetic engineering approach) should also facilitate the development of flooding- and drought-tolerant soybean varieties.

Understanding abiotic stress tolerance mechanisms in soybean: a comparative evaluation of soybean response to drought and flooding stress.

Many sources of drought and flooding tolerance have been identified in soybean, however underlying molecular and physiological mechanisms are poorly understood. Therefore, it is important to illuminate different plant responses to these abiotic stresses and understand the mechanisms that confer tolerance. Towards this goal we used four contrasting soybean (Glycine max) genotypes (PI 567690--drought tolerant, Pana--drought susceptible, PI 408105A--flooding tolerant, S99-2281--flooding susceptible) grown under greenhouse conditions and compared genotypic responses to drought and flooding at the physiological, biochemical, and cellular level. We also quantified these variations and tried to infer their role in drought and flooding tolerance in soybean. Our results revealed that different mechanisms contribute to reduction in net photosynthesis under drought and flooding stress. Under drought stress, ABA and stomatal conductance are responsible for reduced photosynthetic rate; while under flooding stress, accumulation of starch granules played a major role. Drought tolerant genotypes PI 567690 and PI 408105A had higher plastoglobule numbers than the susceptible Pana and S99-2281. Drought stress increased the number and size of plastoglobules in most of the genotypes pointing to a possible role in stress tolerance. Interestingly, there were seven fibrillin proteins localized within the plastoglobules that were up-regulated in the drought and flooding tolerant genotypes PI 567690 and PI 408105A, respectively, but down-regulated in the drought susceptible genotype Pana. These results suggest a potential role of Fibrillin proteins, FBN1a, 1b and 7a in soybean response to drought and flooding stress.