Identification of a new gregarine parasite associated with mass mortality events of freshwater pearl mussels (Margaritifera margaritifera) in Sweden.

Freshwater bivalves play key ecological roles in lakes and rivers, largely contributing to healthy ecosystems. The freshwater pearl mussel, Margaritifera margaritifera, is found in Europe and on the East coast of North America. Once common in oxygenated streams, M. margaritifera is rapidly declining and consequently assessed as a threatened species worldwide. Deterioration of water quality has been considered the main factor for the mass mortality events affecting this species. Yet, the role of parasitic infections has not been investigated. Here, we report the discovery of three novel protist lineages found in Swedish populations of M. margaritifera belonging to one of the terrestrial groups of gregarines (Eugregarinorida, Apicomplexa). These lineages are closely related-but clearly separated-from the tadpole parasite Nematopsis temporariae. In one lineage, which is specifically associated with mortality events of M. margaritifera, we found cysts containing single vermiform zoites in the gills and other organs of diseased individuals using microscopy and in situ hybridization. This represents the first report of a parasitic infection in M. margaritifera that may be linked to the decline of this mussel species. We propose a tentative life cycle with the distribution of different developmental stages and potential exit from the host into the environment.

Predicting subcellular localization of proteins using protein-protein interaction data.

The knowledge of subcellular localization of proteins can provide useful clues about their functions. The conventional methods to determine the subcellular localization are unable to keep pace with the rate at which the new data is being generated. Thus, though sequence information is available, the localization and function of a number of proteins remains unknown. In this study, we have developed a script that makes use of the physical interactors of a protein and their localization data to predict the subcellular localization. We used the script to predict the localization of yeast proteins for which there is no localization data. Further, we experimentally verified the predicted localization for six arbitrarily chosen proteins and found our predictions to be correct for five of the proteins.

In vitro study of iron deposition in normal human brains: An Indian Scenario.

An increase in brain iron is a normal physiological process during brain development but excess accumulation is a risk factor for various neurodegenerative diseases. Thus, knowledge of the normal range of brain iron content is mandatory. The present study was planned to collect normative data on iron deposition in human brains by in vitro analysis. Iron deposition in basal ganglia was determined by Perl's staining in 31 (18 males, 13 females) nonpathological postmortem brains aged from 18 to 80 years and by inductively coupled plasma mass spectrometry (ICP-MS) in 13 of them (seven males, six females). After conventional paraffin embedding, 5 µm thick sections were prepared, fixed and stained with freshly prepared Perl's stain along with a control section. For ICP-MS analysis, approximately 12-13 mg samples of tissue from each region of interest were dried, weighed, and digested with 2 mL of concentrated nitric acid. After digestion, the samples were dissolved in ICP grade water for trace analysis and the iron concentration was determined against standards using an ICP-MS analyzer and recorded in parts per billion (ppb). Nonheme iron deposits were observed in the globus pallidus in 16.13% of cases with no significant sex difference. Iron was deposited in the perivascular area, predominantly in the tunica media and tunica adventitia. ICP-MS analysis revealed the highest iron concentration of 595 ppb (0.595 µg/g tissue) in the globus pallidus with no significant gender or age related differences. In conclusion, the present study revealed a low (16%) incidence of brain iron deposition in normal adult postmortem brains. Clin. Anat. 31:275-281, 2018. © 2017 Wiley Periodicals, Inc.

Fatal Iron Toxicity in an Adult: Clinical Profile and Review.

Acute iron toxicity is usually seen in children with accidental ingestion of iron-containing syrups. However, the literature on acute iron toxicity with suicidal intent in adults is scant. We report an instance wherein an adult committed suicide by ingestion of multiple iron tablets. Delay in treatment was there due to misdiagnosis of the intoxicating agent. She developed fulminant hepatic failure with rapid clinical deterioration. Despite aggressive supportive management, the patient succumbed to the toxic doses of iron. Clinical course and postmortem features are discussed with a review of the literature.

Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.

BACKGROUND: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations. RESULTS: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward. CONCLUSIONS: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.

Development of cleaved amplified polymorphic sequence markers and a CAPS-based genetic linkage map in watermelon (Citrullus lanatus [Thunb.] Matsum. and Nakai) constructed using whole-genome re-sequencing data.

Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). This study detected and converted SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon, for linkage map construction and quantitative trait locus (QTL) analysis. Two inbred lines, Cream of Saskatchewan (COS) and LSW-177 had been re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. 88.7% and 78.5% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. Comparative assembled genome data analysis provided 225,693 and 19,268 SNPs and indels between the two materials. 532 pairs of CAPS markers were designed with 16 restriction enzymes, among which 271 pairs of primers gave distinct bands of the expected length and polymorphic bands, via PCR and enzyme digestion, with a polymorphic rate of 50.94%. Using the new CAPS markers, an initial CAPS-based genetic linkage map was constructed with the F2 population, spanning 1836.51 cM with 11 linkage groups and 301 markers. 12 QTLs were detected related to fruit flesh color, length, width, shape index, and brix content. These newly CAPS markers will be a valuable resource for breeding programs and genetic studies of watermelon.

Algorithm for the Construction of a Global Enzymatic Network to be Used for Gene Network Reconstruction.

Relationships between genes are best represented using networks constructed from information of different types, with metabolic information being the most valuable and widely used for genetic network reconstruction. Other types of information are usually also available, and it would be desirable to systematically include them in algorithms for network reconstruction. Here, we present an algorithm to construct a global metabolic network that uses all available enzymatic and metabolic information about the organism. We construct a global enzymatic network (GEN) with a total of 4226 nodes (EC numbers) and 42723 edges representing all known metabolic reactions. As an example we use microarray data for Arabidopsis thaliana and combine it with the metabolic network constructing a final gene interaction network for this organism with 8212 nodes (genes) and 4606,901 edges. All scripts are available to be used for any organism for which genomic data is available.

Identification of functional genes in liver fibrosis based on bioinformatics analysis of a lncRNA-mediated ceRNA network.

BACKGROUND: Liver fibrosis is a major global healths problem; nevertheless, its molecular mechanism are not completely clear. This study aimed to build a lncRNA-miRNA-mRNA network, identify potentially related lncRNAs, and explore the pathogenesis of liver fibrosis. MATERIALS AND METHODS: We used the Gene Expression Omnibus databases and bioinformatics analysis to identify differentially expressed genes (DEGs) between liver fibrosis and normal tissues. The ceRNA network was constructed according to the interactions between DElncRNA, miRNA, and DEmRNA. Then, these DEGs were identified using functional enrichment analysis, and a protein-protein interaction (PPI) network was established. The critical lncRNAs were verified using the quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The ceRNA network was composed of three lncRNAs, five miRNAs, and 93 mRNAs. Gene Ontology functional enrichment analysis revealed significant enhancement in cell components, molecular function, and biological process. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed pathways associated with transcriptional misregulation in cancer, including the Rap1 signaling pathway, proteoglycans in cancer, mineral absorption, HTLV-l infection, and central carbon metabolism in cancer. According to the PPI network and the GSE84044 database, seven hub genes associated with liver fibrosis were identified. In addition, qRT-PCR revealed that lncRNA AC100861 (lncRNA TNFRSF10A-DT) was explicitly decreased in liver fibrosis tissues and activated hepatic stellate cells. CONCLUSIONS: In summary, this study preliminarily found that lncRNA TNFRSF10A-DT may be a biomarker for the diagnosis and outcome of liver fibrosis. We uncovered a novel lncRNA-mediated ceRNA regulatory mechanism in the pathogenesis of liver fibrosis.

Assessing biologic/toxicologic effects of extractables from plastic contact materials for advanced therapy manufacturing using cell painting assay and cytotoxicity screening.

Plastic components are essential in the pharmaceutical industry, encompassing container closure systems, laboratory handling equipment, and single-use systems. As part of their material qualification process, studies on interactions between plastic contact materials and process solutions or drug products are conducted. The assessment of single-use systems includes their potential impact on patient safety, product quality, and process performance. This is particularly crucial in cell and gene therapy applications since interactions with the plastic contact material may result in an adverse effect on the isolated therapeutic human cells. We utilized the cell painting assay (CPA), a non-targeted method, for profiling the morphological characteristics of U2OS human osteosarcoma cells in contact with chemicals related to plastic contact materials. Specifically, we conducted a comprehensive analysis of 45 common plastic extractables, and two extracts from single-use systems. Results of the CPA are compared with a standard cytotoxicity assay, an osteogenesis differentiation assay, and in silico toxicity predictions. The findings of this feasibility study demonstrate that the device extracts and most of the tested compounds do not evoke any measurable biological changes on the cells (induction ≤ 5%) among the 579 cell features measured at concentrations ≤ 50 µM. CPA can serve as an important assay to reveal unique information not accessible through quantitative structure-activity relationship analysis and vice versa. The results highlight the need for a combination of in vitro and in silico methods in a comprehensive assessment of single-use equipment utilized in advanced therapy medicinal products manufacturing.

Utility of Perl's Prussian Blue Stain in Exfoliated Buccal Cells of Thalassemia and Sickle Cell Anemia Patients and Their Correlation With Serum Ferritin Levels.

Introduction Iron is essential for all living beings. Excess iron, on the other hand, is dangerous because it causes the creation of free radicals. As a result, iron absorption is carefully managed to maintain a balance between absorption and iron loss in the body. Due to the lack of particular excretory channels for iron in humans, iron excess in the tissues is common. It can be caused by a number of factors, including increased iron absorption, as seen in hemochromatosis, or frequent parenteral iron treatment, as seen in thalassemia and sickle cell anemia patients (a transfusional overload). Aim The study aims to demonstrate Perl's Prussian blue stain to identify iron overload at a preliminary stage and correlate with serum ferritin levels in patients with thalassemia and sickle cell anemia who frequently receive blood transfusions. Materials and methods The present study comprised 62 confirmed cases of thalassemia and sickle cell anemia patients undergoing repeated blood transfusions of a minimum of 15/more, along with 62 clinically healthy individuals between December 2016 and November 2018. The patients with thalassemia and sickle cell anemia were confirmed by hemoglobin electrophoresis (Bio-Rad D-10, Bio-Rad Laboratories, Inc, California, United States). The buccal smears were obtained from these patients along with the controls, and these slides were stained by Perl's Prussian blue stain and were examined under a light microscope. Results Sixty-two cases and 62 controls were considered in the current investigation. Forty-seven of the 62 people had thalassemia, and 15 had sickle cell anemia. Thirty-nine out of the 47 patients with thalassemia and six of the 15 individuals with sickle cell anemia had positive results for Perl's Prussian blue stain. All patients had elevated blood ferritin levels, with varying ranges associated with positive results for Perl's Prussian blue stain. Conclusion The objective of this study was to demonstrate the utility of oral exfoliative cytology in thalassemia and sickle cell anemia patients who often receive blood transfusions as a screening and diagnostic tool. The exfoliative cytology methods' acceptability and simplicity, along with their correlation with serum ferritin levels and Perl's Prussian blue reaction, make this noninvasive procedure an excellent screening and diagnostic tool for all patients who receive repeated blood transfusions.

Metallothionein I/II Expression and Metal Ion Levels in Correlation with Amyloid Beta Deposits in the Aged Feline Brain.

Brain aging has been correlated with high metallothionein I-II (MT-I/II) expression, iron and zinc dyshomeostasis, and Aß deposition in humans and experimental animals. In the present study, iron and zinc accumulation, the expression of MT-I/II and Aß42, and their potential association with aging in the feline brain were assessed. Tissue sections from the temporal and frontal grey (GM) and white (WM) matter, hippocampus, thalamus, striatum, cerebellum, and dentate nucleus were examined histochemically for the presence of age-related histopathological lesions and iron deposits and distribution. We found, using a modified Perl's/DAB method, two types of iron plaques that showed age-dependent accumulation in the temporal GM and WM and the thalamus, along with the age-dependent increment in cerebellar-myelin-associated iron. We also demonstrated an age-dependent increase in MT-I/II immunoreactivity in the feline brain. In cats over 7 years old, Aß immunoreactivity was detected in vessel walls and neuronal somata; extracellular Aß deposits were also evident. Interestingly, Aß-positive astrocytes were also observed in certain cases. ICP-MS analysis of brain content regarding iron and zinc concentrations showed no statistically significant association with age, but a mild increase in iron with age was noticed, while zinc levels were found to be higher in the Mature and Senior groups. Our findings reinforce the suggestion that cats could serve as a dependable natural animal model for brain aging and neurodegeneration; thus, they should be further investigated on the basis of metal ion concentration changes and their effects on aging.

Zirconia Toughened Alumina-Based Separator Membrane for Advanced Alkaline Water Electrolyzer.

Hydrogen is nowadays considered a favorable and attractive energy carrier fuel to replace other fuels that cause global warming problems. Water electrolysis has attracted the attention of researchers to produce green hydrogen mainly for the accumulation of renewable energy. Hydrogen can be safely used as a bridge to successfully connect the energy demand and supply divisions. An alkaline water electrolysis system owing to its low cost can efficiently use renewable energy sources on large scale. Normally organic/inorganic composite porous separator membranes have been employed as a membrane for alkaline water electrolyzers. However, the separator membranes exhibit high ionic resistance and low gas resistance values, resulting in lower efficiency and raised safety issues as well. Here, in this study, we report that zirconia toughened alumina (ZTA)-based separator membrane exhibits less ohmic resistance 0.15 Ω·cm2 and low hydrogen gas permeability 10.7 × 10-12 mol cm-1 s-1 bar-1 in 30 wt.% KOH solution, which outperforms the commercial, state-of-the-art Zirfon® PERL separator. The cell containing ZTA and advanced catalysts exhibit an excellent performance of 2.1 V at 2000 mA/cm2 at 30 wt.% KOH and 80 °C, which is comparable with PEM electrolysis. These improved results show that AWEs equipped with ZTA separators could be superior in performance to PEM electrolysis.

FrAnTK: a Frequency-based Analysis ToolKit for efficient exploration of allele sharing patterns in present-day and ancient genomic datasets.

Present-day and ancient population genomic studies from different study organisms have rapidly become accessible to diverse research groups worldwide. Unfortunately, as datasets and analyses become more complex, researchers with less computational experience often miss their chance to analyze their own data. We introduce FrAnTK, a user-friendly toolkit for computation and visualization of allele frequency-based statistics in ancient and present-day genome variation datasets. We provide fast, memory-efficient tools that allow the user to go from sequencing data to complex exploratory analyses and visual representations with minimal data manipulation. Its simple usage and low computational requirements make FrAnTK ideal for users that are less familiar with computer programming carrying out large-scale population studies.

MG2C: a user-friendly online tool for drawing genetic maps.

Genetic map is a linear arrangement of the relative positions of sites in the chromosome or genome based on the recombination frequency between genetic markers. It is the important basis for genetic analysis. Several kinds of software have been designed for genetic mapping, but all these tools require users to write or edit code, making it time-costing and difficult for researchers without programming skills to handle with. Here, MG2C, a new online tool was designed, based on PERL and SVG languages.Users can get a standard genetic map, only by providing the location of genes (or quantitative trait loci) and the length of the chromosome, without writing additional code. The operation interface of MG2C contains three sections: data input, data output and parameters. There are 33 attribute parameters in MG2C, which are further divided into 8 modules. Values of the parameters can be changed according to the users' requirements. The information submitted by users will be transformed into the genetic map in SVG file, which can be further modified by other image processing tools.MG2C is a user-friendly and time-saving online tool for drawing genetic maps, especially for those without programming skills. The tool has been running smoothly since 2015, and updated to version 2.1. It significantly lowers the technical barriers for the users, and provides great convenience for the researchers.

A newly developed free software tool set for averaging electroencephalogram implemented in the Perl programming language.

BACKGROUND: Considering the need for daily activity analysis of older adults, development of easy-to-use, free electroencephalogram (EEG) analysis tools are desired in order to decrease barriers to accessing this technology and increase the entry of a wide range of new researchers. NEW METHOD: We describe a newly developed tool set for EEG analysis, enabling import, average, waveform display and iso-potential scalp topographies, utilizing the programming language Perl. RESULTS: The basic processing, including average, display waveforms, and isopotential scalp topography was implemented in the current system. The validation was examined by making difference waveforms between the results using the current analysis system and a commercial software.Comparison with Existing Method(s): The current software tool set consists of free software. The scripts are easily editable by any user and there are no black boxes. CONCLUSIONS: The currently reported procedures provide an easy-to-begin, flexible, readable, easy-to-modify basic tool set for EEG analysis and is expected to recruit new EEG researchers.

Dtar-Finder: program for drug target identification and characterization in bacteria.

The drug target identification is the primary step for drug discovery. Recent development of computational techniques and availability of sequencing data has provided numerous opportunities for target identification but very few of them are fully automated. Here, we have developed a Perl program named Dtar-Finder for drug target identification and its characterization. Dtar-Finder predicts the drug targets which are essential to pathogen and non homologous to human, essential human anti-targets and gut microflora. This program is divided in 6 modules where modules 1-4 extract drug targets while module 5-6 predicts druggability and broad spectrum ability of identified candidates. The performance of this program in terms of sensitivity and specificity is calculated where specificity score was better compare to sensitivity score. Further, we have tested our script on C. botulinum (3572 proteins) and 35 potential drug targets have been identified. Out of which 16 broad spectrums candidates were predicted whereas 8 candidates are found to be druggable whiles remaining are considered to be 'novel'. These drug targets were cross-validated through literature showing 77.14% accuracy. Thus, the idea behind this work was to develop a fast, robust and generic program capable of finding drug targets in bacteria, which has been fulfilled satisfactorily.

Sharing Programming Resources Between Bio* Projects.

Open-source software encourages computer programmers to reuse software components written by others. In evolutionary bioinformatics, open-source software comes in a broad range of programming languages, including C/C++, Perl, Python, Ruby, Java, and R. To avoid writing the same functionality multiple times for different languages, it is possible to share components by bridging computer languages and Bio* projects, such as BioPerl, Biopython, BioRuby, BioJava, and R/Bioconductor.In this chapter, we compare the three principal approaches for sharing software between different programming languages: by remote procedure call (RPC), by sharing a local "call stack," and by calling program to programs. RPC provides a language-independent protocol over a network interface; examples are SOAP and Rserve. The local call stack provides a between-language mapping, not over the network interface but directly in computer memory; examples are R bindings, RPy, and languages sharing the Java virtual machine stack. This functionality provides strategies for sharing of software between Bio* projects, which can be exploited more often.Here, we present cross-language examples for sequence translation and measure throughput of the different options. We compare calling into R through native R, RSOAP, Rserve, and RPy interfaces, with the performance of native BioPerl, Biopython, BioJava, and BioRuby implementations and with call stack bindings to BioJava and the European Molecular Biology Open Software Suite (EMBOSS).In general, call stack approaches outperform native Bio* implementations, and these, in turn, outperform "RPC"-based approaches. To test and compare strategies, we provide a downloadable Docker container with all examples, tools, and libraries included.

AUSPP: A universal short-read pre-processing package.

There are many short-read aligners that can map short reads to a reference genome/sequence, and most of them can directly accept a FASTQ file as the input query file. However, the raw data usually need to be pre-processed. Few software programs specialize in pre-processing raw data generated by a variety of next-generation sequencing (NGS) technologies. Here, we present AUSPP, a Perl script-based pipeline for pre-processing and automatic mapping of NGS short reads. This pipeline encompasses quality control, adaptor trimming, collapsing of reads, structural RNA removal, length selection, read mapping, and normalized wiggle file creation. It facilitates the processing from raw data to genome mapping and is therefore a powerful tool for the steps before meta-analysis. Most importantly, since AUSPP has default processing pipeline settings for many types of NGS data, most of the time, users will simply need to provide the raw data and genome. AUSPP is portable and easy to install, and the source codes are freely available at https://github.com/highlei/AUSPP.

Identification of conserved miRNA molecules in einkorn wheat (Triticum monococcum subsp. monococcum) by using small RNA sequencing analysis.

Triticum monococcum subsp. monococcum as a first cultivated diploid wheat species possesses desirable agronomic and quality characteristics. Drought and salinity are the most dramatic environmental stress factors that have serious impact on yield and quality of crops; however, plants can use alternative defense mechanisms against these stresses. The posttranscriptional alteration of gene expression by microRNAs (miRNAs) is one of the most conserved mechanisms. In plant species including wheat genomes, miRNAs have been implicated in the management of salt and drought stress; however, studies on einkorn wheat (Triticum monococcum subsp. monococcum) are not yet available. In this study, we aimed to identify conserved miRNAs in einkorn wheat using next generation sequencing technology and bioinformatics analysis. In order to include a larger set of miRNAs, small RNA molecules from pooled plant samples grown under normal, drought, and salinity conditions were used for the library preparation and sequence analysis. After bioinformatics analysis, we identified 167 putative mature miRNA sequences belonging to 140 distinct miRNA families. We also presented a comparative analysis to propose that miRNAs and their target genes were involved in salt and drought stress control in addition to a comprehensive analysis of the scanned target genes in the T. aestivum genome.