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Municipal waste management plays a crucial role in our communities and has far-reaching implications. Effective waste management contributes to environmental conservation. By reducing waste, recycling, and implementing sustainable practices, we protect our ecosystems and natural resources. Due to the increasing volume of waste and the increasingly complex administration of its collection and disposal, solid waste management is quickly becoming a demanding issue for municipalities all over the world. The objective of the paper was to describe the impacts of communal waste management on municipal budgets and to compare waste management expenditure intensity. The legislative changes, the focus on minimising waste for disposal and promoting a material recycling orientation will have a significant impact on municipal budgets. Local authorities must adjust to the standards established by the European Union. The Slovak Republic will need to progressively adopt these standards by incorporating them into national law

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Objectives: The aim of the study was to determine incidence of occult micrometastases by semiserial sectioning and immunohistochemistry with cytokeratin in patients with head and neck squamous cell carcinoma with clinically negative neck. Material and methods: Eighteen patients with squamous cell carcinoma of the head and neck who primary underwent surgical treatment of the primary tumor and elective selective neck dissection were included in the study. Lymph nodes of neck dissection specimen were evaluated by semiserial sectioning at 200 µm interval with hematoxylin-eosin staining and immunohistochemical stain method using pan-cytokeratin AE1/3. Results: Of 18 patients, 28 neck dissections were harvested 600 lymph nodes. The incidence of occult micrometastases was 22.2% per patient, 14.3% per neck and 1% per lymph node. In 4 patients was pN upgrading from pN0 to pN+, 28.6%. Conclusion: The incidence of occult micrometastases in head and neck cancer patients detected by semiserial sectioning with cytokeratin immunohistochemistry is higher than conventional pathologic techniques. Use of this technique should more accurately identify patients for postoperative adjuvant treatment. [ABSTRACT FROM AUTHOR]

In the EU new programming period 2014-2020 the Leader approach become part of community-led local development (CLLD). Under Slovak conditions, partnerships, which intend to get the legal status of local action group (LAG), are currently in the process of preparing and formulating their CLLD Strategies. Leader approach is characterised by 7 principles, which should be horizontally presented throughout the implementation process. The multilevel governance presented in the implementation of Leader approach includes the management and implementation of rural development programme, through which the Leader is implemented, as well as, formation of LAGs, as the mediators of the approach at local level. Both levels may have supporting or constraining effects on the application of Leader principles in the Leader delivery. The paper focuses on analysing the differences between theory and practice in the conditions of the Slovak Republic when answering the evaluation question: Are stakeholders in Slovakia ready for community led local development? To answer the question, six LAGs were assessed using the focus group as the assessment tool. Representatives of the national authorities were interviewed in order to complete the picture of the evaluated topic. The study pointed out several shortcomings in basic preconditions allowing smooth application of the CLLD.

Pristup LEADER je jednym zo zakladnych pilierov sučasnej politiky rozvoja vidieka realizovanej na europskej urovni. Logicky zaklad pristupu LEADER je postaveny na 7 kľučovych znakoch, ktore by mali byť pritomne prierezovo v ramci celeho implementačneho procesu. Preto cieľom prispevku bolo zodpovedanie vyskumnej otazky: do akej miery implementačny mechanizmus programu LEADER (na urovni riadiaceho organu aj miestnych akčnych skupin) v programovom obdobi 2007-2013 podporoval dodržiavanie siedmych zakladnych znakov programu?“ Pre naplnenie cieľa prispevku bolo nevyhnutne určiť jednotlive kroky implementačneho procesu na oboch urovniach, nasledne popisať jednotlive znaky a definovať kriteria uspechu - benchmarky. Vzajomnym prepojenim predchadzajucich krokov bola vytvorena implementačna matica, tvoriaca vizualny vystup študie

The aim of this study was to measure the electrical properties of dried apricots, carrot slices during drying, wheat and maize grains; poppy, Amaranth, sunflower and rape oil seeds in dependence of moisture content, moisture ratio and temperature. Moisture content and moisture ratio of samples were studied. Among the electrical properties we determined conductivity, resistance, resistivity, capacitance, relative permittivity and impedance. We found out that the moisture ratio of samples decreases with time. During drying, the moisture content of the samples decreases according to quadratic function. The resistivity with temperature decreases, and conductivity with temperature increases exponentially. The resistivity decreases with increase of the moisture content. The permittivity of samples increases with temperature linearly. The permittivity decreases with the frequency, in this frequency range, and increases with the moisture content. The change of impedance magnitude in drying time showed good correlation with the change of moisture content.

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023. Published by Elsevier B.V.)

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role., Results: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication., Conclusions: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa., (© 2023. The Author(s).)

Background: IgG4 is associated with two emerging groups of rare diseases: 1) IgG4 autoimmune diseases (IgG4-AID) and 2) IgG4-related diseases (IgG4-RLD). Anti-neuronal IgG4-AID include MuSK myasthenia gravis, LGI1- and Caspr2-encephalitis and autoimmune nodo-/paranodopathies (CNTN1/Caspr1 or NF155 antibodies). IgG4-RLD is a multiorgan disease hallmarked by tissue-destructive fibrotic lesions with lymphocyte and IgG4 plasma cell infiltrates and increased serum IgG4 concentrations. It is unclear whether IgG4-AID and IgG4-RLD share relevant clinical and immunopathological features., Methods: We collected and analyzed clinical, serological, and histopathological data in 50 patients with anti-neuronal IgG4-AID and 19 patients with IgG4-RLD., Results: A significantly higher proportion of IgG4-RLD patients had serum IgG4 elevation when compared to IgG4-AID patients (52.63% vs. 16%, p = .004). Moreover, those IgG4-AID patients with elevated IgG4 did not meet the diagnostic criteria of IgG4-RLD, and their autoantibody titers did not correlate with their serum IgG4 concentrations. In addition, patients with IgG4-RLD were negative for anti-neuronal/neuromuscular autoantibodies and among these patients, men showed a significantly higher propensity for IgG4 elevation, when compared to women ( p = .005). Last, a kidney biopsy from a patient with autoimmune paranodopathy due to CNTN1/Caspr1-complex IgG4 autoantibodies and concomitant nephrotic syndrome did not show fibrosis or IgG4 + plasma cells, which are diagnostic hallmarks of IgG4-RLD., Conclusion: Our observations suggest that anti-neuronal IgG4-AID and IgG4-RLD are most likely distinct disease entities., Competing Interests: LD received a grant from Fleury Laboratory for the Brazilian Autoimmune Encephalitis Project without personal compensation. CL served as a consultant for Roche. K-NP received a travel grant from Merck. RH reports speakers’ honoraria from Novartis and Biogen. The Medical University of Vienna (Austria; employer of Dr. Höftberger) receives payment for antibody assays and for antibody validation experiments organized by Euroimmun (Lübeck, Germany). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Endmayr, Tunc, Ergin, De Rosa, Weng, Wagner, Yu, Fichtenbaum, Perkmann, Haslacher, Kozakowski, Schwaiger, Ricken, Hametner, Klotz, Dutra, Lechner, de Simoni, Poppert, Müller, Pirker, Pirker, Angelovski, Valach, Maestri, Guida, Ricciardi, Frommlet, Sieghart, Pinter, Kircher, Artacker, Höftberger and Koneczny.)

The mitoribosome, as known from studies in model organisms, deviates considerably from its ancestor, the bacterial ribosome. Deviations include substantial reduction of the mitochondrial ribosomal RNA (mt-rRNA) structure and acquisition of numerous mitochondrion-specific (M) mitoribosomal proteins (mtRPs). A broadly accepted view assumes that M-mtRPs compensate for structural destabilization of mt-rRNA resulting from its evolutionary remodeling. Since most experimental information on mitoribosome makeup comes from eukaryotes having derived mitochondrial genomes and mt-rRNAs, we tested this assumption by investigating the mitochondrial translation machinery of jakobids, a lineage of unicellular protists with the most bacteria-like mitochondrial genomes. We report here proteomics analyses of the Andalucia godoyi small mitoribosomal subunit and in silico transcriptomic and comparative genome analyses of four additional jakobids. Jakobids have mt-rRNA structures that minimally differ from their bacterial counterparts. Yet, with at least 31 small subunit and 44 large subunit mtRPs, the mitoriboproteome of Andalucia is essentially as complex as that in animals or fungi. Furthermore, the relatively high conservation of jakobid sequences has helped to clarify the identity of several mtRPs, previously considered to be lineage-specific, as divergent homologs of conserved M-mtRPs, notably mS22 and mL61. The coexistence of bacteria-like mt-rRNAs and a complex mitoriboproteome refutes the view that M-mtRPs were ancestrally recruited to stabilize deviations of mt-rRNA structural elements. We postulate instead that the numerous M-mtRPs acquired in the last eukaryotic common ancestor allowed mt-rRNAs to pursue a broad range of evolutionary trajectories across lineages: from dramatic reduction to acquisition of novel elements to structural conservatism., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one, Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination in D. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5' and 3' homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of α- and β-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging of D. papillatum genes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondria are the sandbox of evolution as exemplified most particularly by the diplonemids, a group of marine microeukaryotes. These protists are uniquely characterized by their highly multipartite mitochondrial genome and systematically fragmented genes whose pieces are spread out over several dozens of chromosomes. The type species Diplonema papillatum was the first member of this group in which the expression of fragmented mitochondrial genes was investigated experimentally. We now know that gene expression involves separate transcription of gene pieces (modules), RNA editing of module transcripts, and module joining to mature mRNAs and rRNAs. The mechanism of cognate module recognition and ligation is distinct from known intron splicing and remains to be uncovered. Here, we review the current status of research on mitochondrial genome architecture, as well as gene complement, structure, and expression modes in diplonemids. Further, we discuss the potential molecular mechanisms of posttranscriptional processing, and finally reflect on the evolutionary trajectories and trends of mtDNA evolution as seen in this protist group. © 2018 IUBMB Life, 70(12):1197-1206, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Mitochondrial genes of Euglenozoa (Kinetoplastida, Diplonemea, and Euglenida) are notorious for being barely recognizable, raising the question of whether such divergent genes actually code for functional proteins. Here we demonstrate the translation and identify the function of five previously unassigned y genes encoded by mitochondrial DNA (mtDNA) of diplonemids. As is the rule in diplonemid mitochondria, y genes are fragmented, with gene pieces transcribed separately and then trans-spliced to form contiguous mRNAs. Further, y transcripts undergo massive RNA editing, including uridine insertions that generate up to 16-residue-long phenylalanine tracts, a feature otherwise absent from conserved mitochondrial proteins. By protein sequence analyses, MS, and enzymatic assays in Diplonema papillatum , we show that these y genes encode the subunits Nad2, -3, -4L, -6, and -9 of the respiratory chain Complex I (CI; NADH:ubiquinone oxidoreductase). The few conserved residues of these proteins are essentially those involved in proton pumping across the inner mitochondrial membrane and in coupling ubiquinone reduction to proton pumping (Nad2, -3, -4L, and -6) and in interactions with subunits containing electron-transporting Fe-S clusters (Nad9). Thus, in diplonemids, 10 CI subunits are mtDNA-encoded. Further, MS of D. papillatum CI allowed identification of 26 conventional and 15 putative diplonemid-specific nucleus-encoded components. Most conventional accessory subunits are well-conserved but unusually long, possibly compensating for the streamlined mtDNA-encoded components and for missing, otherwise widely distributed, conventional subunits. Finally, D. papillatum CI predominantly exists as a supercomplex I:III:IV that is exceptionally stable, making this protist an organism of choice for structural studies., (© 2018 Valach et al.)

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering.

Diplonema papillatum is the type species of diplonemids, which are among the most abundant and diverse heterotrophic microeukaryotes in the world's oceans. Diplonemids are also known for a unique form of post-transcriptional processing in mitochondria. However, the lack of reverse genetics methodologies in these protists has hampered elucidation of their cellular and molecular biology. Here we report a protocol for D. papillatum transformation. We have identified several antibiotics to which D. papillatum is sensitive and thus are suitable selectable markers, and focus in particular on puromycin. Constructs were designed encoding antibiotic resistance markers, fluorescent tags, and additional genomic sequences from D. papillatum to facilitate vector integration into chromosomes. We established conditions for effective electroporation, and demonstrate that electroporated constructs can be stably integrated in the D. papillatum nuclear genome. In D. papillatum transformants, the heterologous puromycin resistance gene is transcribed into mRNA and translated into protein, as determined by Southern hybridization, reverse transcription, and Western blot analyses. This is the first documented case of transformation in a euglenozoan protist outside the well-studied kinetoplastids, making D. papillatum a genetically tractable organism and potentially a model system for marine microeukaryotes., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Chromosome rearrangements are important drivers in genome and gene evolution, with implications ranging from speciation to development to disease. In the flagellate Diplonema papillatum (Euglenozoa), mitochondrial genome rearrangements have resulted in nearly hundred chromosomes and a systematic dispersal of gene fragments across the multipartite genome. Maturation into functional RNAs involves separate transcription of gene pieces, joining of precursor RNAs via trans-splicing, and RNA editing by substitution and uridine additions both reconstituting crucial coding sequence. How widespread these unusual features are across diplonemids is unclear. We have analyzed the mitochondrial genomes and transcriptomes of four species from the Diplonema/Rhynchopus clade, revealing a considerable genomic plasticity. Although gene breakpoints, and thus the total number of gene pieces (~80), are essentially conserved across this group, the number of distinct chromosomes varies by a factor of two, with certain chromosomes combining up to eight unrelated gene fragments. Several internal protein-coding gene pieces overlap substantially, resulting, for example, in a stretch of 22 identical amino acids in cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 5. Finally, the variation of post-transcriptional editing patterns across diplonemids indicates compensation of two adverse trends: rapid sequence evolution and loss of genetic information through unequal chromosome segregation.

The instructions to make proteins and structural RNAs are laid down in gene sequences. Yet, in certain instances, these primary instructions need to be modified considerably during gene expression, most often at the transcript level. Here we review a case of massive post-transcriptional revisions via trans-splicing and RNA editing, a phenomenon occurring in mitochondria of a recently recognized protist group, the diplonemids. As of now, the various post-transcriptional steps have been cataloged in detail, but how these processes function is still unknown. Since genetic manipulation techniques such as gene replacement and RNA interference have not yet been established for these organisms, alternative strategies have to be deployed. Here, we discuss the experimental and bioinformatics approaches that promise to unravel the molecular machineries of trans-splicing and RNA editing in Diplonema mitochondria.

Unrecognizable genes are an unsettling problem in genomics. Here, we survey the various types of cryptic genes and the corresponding deciphering strategies employed by cells. Encryption that renders genes substantially different from homologs in other species includes sequence substitution, insertion, deletion, fragmentation plus scrambling, and invasion by mobile genetic elements. Cells decode cryptic genes at the DNA, RNA or protein level. We will focus on a recently discovered case of unparalleled encryption involving massive gene fragmentation and nucleotide deletions and substitutions, occurring in the mitochondrial genome of a poorly understood protist group, the diplonemids. This example illustrates that comprehensive gene detection requires not only auxiliary sequence information - transcriptome and proteome data - but also knowledge about a cell's deciphering arsenal., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Gene structure and expression in diplonemid mitochondria are unparalleled. Genes are fragmented in pieces (modules) that are separately transcribed, followed by the joining of module transcripts to contiguous RNAs. Some instances of unique uridine insertion RNA editing at module boundaries were noted, but the extent and potential occurrence of other editing types remained unknown. Comparative analysis of deep transcriptome and genome data from Diplonema papillatum mitochondria reveals ∼220 post-transcriptional insertions of uridines, but no insertions of other nucleotides nor deletions. In addition, we detect in total 114 substitutions of cytosine by uridine and adenosine by inosine, amassed into unusually compact clusters. Inosines in transcripts were confirmed experimentally. This is the first report of adenosine-to-inosine editing of mRNAs and ribosomal RNAs in mitochondria. In mRNAs, editing causes mostly amino-acid additions and non-synonymous substitutions; in ribosomal RNAs, it permits formation of canonical secondary structures. Two extensively edited transcripts were compared across four diplonemids. The pattern of uridine-insertion editing is strictly conserved, whereas substitution editing has diverged dramatically, but still rendering diplonemid proteins more similar to other eukaryotic orthologs. We posit that RNA editing not only compensates but also sustains, or even accelerates, ultra-rapid evolution of genome structure and sequence in diplonemid mitochondria., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ∼ 26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events.

Objective: In this short communication we compared the data of fungaemia cases in Slovak hospitals from 1989-1998 published in 1999-2000 with data from 2005-2011., Methods: Risk factors, etiology and outcome of fungaemia between two periods (1989-1998 vs. 2005-2011) were compared and risk factors for death assessed by univariate analysis (CDC 2006 Statistical Package)., Results: In comparison to 1989-1998 when only amphotericin B and fluconazole has been used (55%), in 2005-2011 only 35.2% patients received FLU, but 26.4% received voriconazole, 22% caspofungin and anidulafungin and about 6.6% lipid formulations of Amphotericin B. In etiology, while in 1989-1998 only 37.1% (115/310) represented non-albicans Candida (NAC) and non-Candida yeasts in 2005-2011 already reached 63.7%. The significant increase of breakthrough fungaemia may be a sign of inappropriate empiric therapy.

Candida parapsilosis species complex includes three closely related species, namely C. parapsilosis (sensu stricto), C. orthopsilosis, and C. metapsilosis. Unlike most other yeast lineages, members of this species complex possess a linear mitochondrial genome. Yet, its circularized mutant form was identified in strains of C. orthopsilosis and C. metapsilosis. To investigate the underlying variability, we performed comparative analyses of the complete mitochondrial DNA sequences in a collection of strains. Our results demonstrate that in contrast to C. parapsilosis and C. metapsilosis, C. orthopsilosis exhibits remarkably high nucleotide diversity whose pattern is consistent with intraspecific genetic exchange., (Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Ionizing radiation as a cancer therapy is associated with a variety of undesirable side effects. Consequently, radiotherapy can negatively affect neuromuscular function. Clinical observations have identified problems with swallowing and voice function. Our study aims to evaluate the impact of radiotherapy on laryngeal soft tissues using image analysis to quantify its effect on the structure of the vocalis and thyroarytenoid muscles. Case control study, retrospective analysis. We collected total laryngectomy specimens from six patients with persistent or recurrent cancer who had received preoperative radiotherapy (60-66 Gy). The control group consisted of total laryngectomy specimens from six patients who underwent surgery as primary treatment. Sampling of the specimens only included non-cancerous laryngeal tissue. Laryngeal histological slices were evaluated using digital morphometric analysis system. Percentage of fibrosis and density of muscle fibers within the thyroarytenoid muscle were evaluated in both groups. We found no significant quantitative differences in muscle fibrosis (7.92% vs. 7.52%, P > 0.1). Changes were rather qualitative and included changes in the organization of the muscular fibers. A significant reduction in muscle fibers, however, was observed in the samples from irradiated larynges (66.45% vs. 42.03%, P < 0.01). Our analysis suggests that radiotherapy is responsible for a significant reduction in muscle fibers in the thyroarytenoid muscle and that these changes occur during treatment or relatively early after its completion. Loss of muscle mass after irradiation correlates with clinical observations of muscle weakness and decreased function in patients who undergo radiotherapy.

Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.

As a part of our initiative aimed at a large-scale comparative analysis of fungal mitochondrial genomes, we determined the complete DNA sequence of the mitochondrial genome of the yeast Candida subhashii and found that it exhibits a number of peculiar features. First, the mitochondrial genome is represented by linear dsDNA molecules of uniform length (29 795 bp), with an unusually high content of guanine and cytosine residues (52.7 %). Second, the coding sequences lack introns; thus, the genome has a relatively compact organization. Third, the termini of the linear molecules consist of long inverted repeats and seem to contain a protein covalently bound to terminal nucleotides at the 5' ends. This architecture resembles the telomeres in a number of linear viral and plasmid DNA genomes classified as invertrons, in which the terminal proteins serve as specific primers for the initiation of DNA synthesis. Finally, although the mitochondrial genome of C. subhashii contains essentially the same set of genes as other closely related pathogenic Candida species, we identified additional ORFs encoding two homologues of the family B protein-priming DNA polymerases and an unknown protein. The terminal structures and the genes for DNA polymerases are reminiscent of linear mitochondrial plasmids, indicating that this genome architecture might have emerged from fortuitous recombination between an ancestral, presumably circular, mitochondrial genome and an invertron-like element.

Objectives: The aims of the study were to assess our feasibility and accuracy of sentinel lymph node radiolocalization in patients with squamous cell carcinoma of the oral cavity and oropharynx, and to determine whether the pathology of the sentinel node reflected regional disease., Material and Methods: Patients preoperatively underwent lymphoscintigraphy after peritumoral injection of a 99m Tc labeled radiocolloid. After perioperative gamma probe radiolocalization of the sentinel lymph nodes, elective neck dissection was performed. The histopathological examination of the sentinel nodes and other nodes of neck dissection specimen were compared., Results: Detection of sentinel lymph nodes by lymphoscintigraphy was feasible in all 12 patients. Also localization with a handheld gamma probe was successful in all patients. Forty sentinel nodes and 276 non-sentinel nodes were histopathologically examined. Occult metastases were confirmed in 7 sentinel nodes (4 patients). There was no false negative sentinel lymph node in our series., Conclusion: Identification of the sentinel lymph node in patients with squamous cell carcinoma of the oral cavity and oropharynx is technically feasible and accurate. This method shows to be able to predict occult metastases and select patients who would benefit from neck dissection (Fig. 1, Tab. 2, Ref. 28). Full Text in free PDF www.bmj.sk.